Patent Application
20230391765
This disclosure relates to bivalent compounds (e.g., heterobifunctional compounds) which degrade and/or disrupt Eleven-Nineteen Leukemia (ENL), compositions comprising one or more of the bivalent compounds, and methods of use thereof for the treatment of ENL-mediated diseases in a subject in need thereof. The disclosure also relates to methods for designing such bivalent compounds.
Eleven-Nineteen Leukemia (ENL, also known as MLLT1 or YEATS1) is a transcriptional co-regulator that recruits transcription machinery to target genes through its chromatin reader function. ENL and its paralogue ALL1-Fused Gene From Chromosome 9 (AF9, also known as MLLT3 or YEATS3) associate with the super elongation complex (SEC) and the complex of the histone H3K79 methyltransferase DOT1L (Biswas et al., 2011; He et al., 2011), both of which play important roles in regulation of transcription elongation by RNA polymerase II (Bitoun et al., 2007; He et al., 2010; Lin et al., 2010; Mohan et al., 2010a; Mueller et al., 2007; Mueller et al., 2009; Okada et al., 2005; Yokoyama et al., 2010). Both ENL and AF9 proteins contain a N-terminal YEATS domain, which is an evolutionarily conserved domain that recognizes acylated lysine on histone H3 tail (Hsu et al., 2018; Klein et al., 2018; Li et al., 2016; Li et al., 2014; Mi et al., 2017; Shanle et al., 2015; Wan et al., 2017; Zhang et al., 2016).
ENL plays a vital role in the progression and maintenance of certain subtypes of acute leukemia, mixed lineage leukemia (MLL)-rearranged leukemia in particular (Erb et al., 2017; Wan et al., 2017). The MLL gene (also known as MLL1, ALL-4, or KMT2A) is disrupted by recurrent chromosomal rearrangements in a subgroup of high-risk acute leukemias that have unique clinical and biological features (Hess, 2004; Meyer et al., 2013; Meyer et al., 2009; Rao and Dou, 2015). MLL rearrangements account for approximately 10% of all human leukemias, most frequently in infant leukemias (Marschalek, 2015; Meyer et al., 2013). These patients have a dismal prognosis and a particularly poor response to standard treatments (Biondi et al., 2000; Pieters et al., 2007; Pui et al., 2009). Therefore, development of effective therapies for this leukemia subtype is urgently needed. Leukemogenic translocations of the MLL gene lead to in-frame fusions between the N-terminus of the MLL protein and the C-terminus of a fusion partner, and these fusion proteins are known to function as “drivers” of the diseases (Abramovich and Humphries, 2005; Armstrong et al., 2002; Artinger et al., 2013; Deshpande et al., 2012; Ferrando et al., 2002; Jude et al., 2007; Slany, 2005; Yu et al., 1995). Strikingly, among the over 70 MLL fusions characterized, a small subset of fusions accounts for most leukemogenic cases. Over 90% of MLL rearrangements in acute lymphoblastic leukemia (ALL) and 70% in acute myeloid leukemia (AML) involve only 4-5 fusion partners, all of which are subunits of the SEC and/or DOT1L complexes that ENL and AF9 reside in (Ayton and Cleary, 2001; Krivtsov and Armstrong, 2007; Meyer et al., 2013; Meyer et al., 2006; Mohan et al., 2010b). It is believed that each complex component, when fused to MLL, “hijacks” the SEC or DOT1L complex to the MLL target loci, promoting aberrant gene activation that leads to leukemogenesis (Deshpande et al., 2012). In recent studies, ENL, but not AF9, is identified as a cancer-specific acute leukemia dependency (Erb et al., 2017; Wan et al., 2017). ENL depletion or disrupting the interaction between its YEATS domain and histone acetylation leads to inhibition of oncogenic gene expression programs and suppression of leukemia progression both in vitro and in vivo (
In addition, hotspot ENL YEATS domain mutations have been identified in Wilms' tumor patients (Gadd et al., 2017; Perlman et al., 2015). The reader function of the YEATS domain is indispensable for these gain-of-function mutations to aberrantly activate the expression of genes essential for proper kidney development and derail the cell-fate decision (Wan et al., 2020).
All these studies suggest that ENL and its YEATS domain are attractive therapeutic target for certain types of human cancer. Efforts in developing ENL YEATS domain inhibitors led to the recent publications of acetyl-lysine competitive small molecules, peptide-mimic chemical probes and ligands from cell-based screen, demonstrating that the YEATS domain is pharmacologically tractable (Asiaban et al., 2020; Christott et al., 2019; Heidenreich et al., 2018; Li et al., 2018; Moustakim et al., 2018a; Ni et al., 2019). One of the recently reported ENL YEATS small molecule inhibitors, SGC-iMLLT, can effectively block the interaction between the ENL YEATS domain and acetylated histone H3 in vitro and in cells (Christott et al., 2019; Moustakim et al., 2018a). However, while SGC-iMLLT is an excellent chemical probe with nanomolar level of binding affinity to the ENL YEATS domain in vitro, it is largely ineffective in inhibiting the growth of ENL-dependent MLL-rearranged leukemia cells (Christott et al., 2019; Moustakim et al., 2018a). The lack of a significant effect by SGC-iMLLT in cells is in contrast to the effect of ENL knockout (KO) via CRISPR-Cas9 (Erb et al., 2017; Wan et al., 2017). Therefore, a new therapeutic strategy targeting ENL is needed. Here, we present small-molecule degraders of ENL, which pharmacologically degrade ENL protein in cells and tumors and more likely phenocopy the effects of ENL KO, as novel therapeutics for treating ENL-dependent diseases including cancers.
The present disclosure relates generally to bivalent compounds (e.g., bi-functional compounds), which degrade and/or disrupt ENL and to methods for the treatment of ENL-mediated diseases (i.e., a disease which depends on ENL; overexpresses ENL; depends on ENL activity; or includes elevated levels of ENL activity relative to a wild-type tissue of the same species and tissue type). It is important to note, because the ENL degraders/disruptors have dual functions (enzyme inhibition plus protein degradation/disruption), the bivalent compounds of the present disclosure can be significantly more effective therapeutic agents than currently available ENL inhibitors, which inhibit the enzymatic activity of ENL, but do not affect ENL protein levels. The present disclosure further provides methods for identifying ENL degraders/disruptors as described herein.
More specifically, the present disclosure provides a bivalent compound including an ENL ligand conjugated to a degradation/disruption tag.
In some aspects, the ENL degraders/disruptors have the form “PI-linker-EL”, as shown below:
wherein PI (protein of interest) comprises an ENL ligand and EL (E3 ligase) comprises a degradation/disruption tag (e.g., E3 ligase ligand). Exemplary ENL ligands (PI), exemplary degradation/disruption tags (EL), and exemplary linkers (Linker) are illustrated below:
ENL Ligands
In an embodiment, ENL ligands include a moiety according to FORMULA 1:
R1 is selected from H, halogen, OR5, SR5, C1-C8 alkylene NR5R6, CH2CH2NR5R6, NR5R6, C(O)R5, C(O)OR5, C(S)OR5, C(O)NR5R6, S(O)R5, S(O)2R5, S(O)2NR5R6, NR7C(O)OR6, NR7C(O)R6, NR7S(O)R6, NR7S(O)2R6, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl.
R2 is independently selected from hydrogen, halogen, oxo, CN, NO2, OR8, SR8, NR8R9, C(O)R8, C(O)OR8, C(S)OR8, C(O)NR8R9, S(O)R8, S(O)2R8, S(O)2NR8R9, NR10C(O)OR9, NR10C(O)R9, NR10S(O)R9, NR10S(O)2R9, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3-C8 heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R3 is unsubstituted or optionally substituted with one or more groups selected from hydrogen, halogen, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR11R12, C(O)R11, C(O)OR11, C(O)NR11R12, S(O)R11, S(O)2R11, S(O)2N11R12, NR13C(O)OR12, NR13C(O)R12, NR13S(O)R12, NR13S(O)2R12, optionally substituted C6-C10 aryl and optionally substituted C5-C10 heteroaryl.
each R4 is independently selected from null, hydrogen, halogen, oxo, CN, NO2, OR14, SR14, NR14R15, OCOR14, OCO2R14, OCONR14R15, COR14, CO2R15, CONR14R15, SOR14, SO2R14, SO2NR14R15, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C4-C8 heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R5, R6, R7, R8, R9, R10 R11, R12, R13 R14, R15 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R5 and R6, R6 and R7, R8 and R9, R8 and R10, R9 and R10, R11 and R12, R11 and R13, R12 and R13, R14 and R15, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 1A
R16 and R17 together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
R18, R19 are independently selected from hydrogen, halogen, CN, OH, NH2, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-8 membered heterocyclyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl;
R20 is selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3-C8 heterocyclyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl.
In an embodiment, ENL ligands include a moiety according to FORMULA 1B, 1C, 1D, 1E
R22 is unsubstituted or optionally substituted with one or more groups selected from halo, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR26R27, C1-C8NR26R27, C(O)R26, C(O)OR26, C(O)NR26R27, S(O)R26, S(O)2R26, S(O)2NR26R27, NR26C(O)OR27, NR28C(O)R27, NR28S(O)R27, NR28S(O)2R27.
R23 is unsubstituted or optionally substituted with one or more groups selected from halo, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR29R30, C(O)R29, C(O)OR29, C(O)NR29R30, S(O)R29, S(O)2R29, S(O)2NR29R30, NR31C(O)OR29, NR31C(O)R29, NR31S(O)R29, NR31S(O)2R29
R25 is unsubstituted or optionally substituted with one or more groups selected from halo, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR32R33, C(O)R32, C(O)OR32, C(O)NR32R33, S(O)R32, S(O)2R32, S(O)2NR32R33, NR34C(O)OR32, NR34C(O)R32, NR34S(O)R32, NR34S(O)2R32.
R26, R27, R28, R29, R30, R31 R32, R33, R34 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R26 and R27, R27 and R28, R29 and R30, R29 and R31, R32 and R33, R32 and R34, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 1F:
In an embodiment, ENL ligands include a moiety according to FORMULA 2.
R1 is selected from hydrogen, halogen, OR4, SR4, C1-C8 alkylene NR4R5, C(O)R4, C(O)OR4, C(S)OR4, C(O)NR4R5, S(O)R4, S(O)2R4, S(O)2NR4R5, NR6C(O)OR4, NR6C(O)R4, NR6S(O)R4, NR6S(O)2R4, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, or fused C3-C10 cycloalkyl, C3-C10 heterocyclyl.
R2 is selected from hydrogen, halogen, CN, NO2, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR7R8, C(O)R7, C(O)OR7, C(O)NR7R8, S(O)R7, S(O)2R7, S(O)2NR7R8, NR9C(O)OR7, NR9C(O)R7, NR9S(O)R7, NR9S(O)2R7, optionally substituted C6-C10 aryl and optionally substituted C5-C10 heteroaryl.
wherein
R4, R5, R6, R7, R8, R9, R10 R11, R12 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R4 and R5, R4 and R6, R7 and R8, R7 and R9, R10 and R11, R10 and R12, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 2A and 2B.
R13 is selected from hydrogen, halogen OR17, SR17, C1-C8 alkylene NR17R18, NR17R18, C(O)R17, C(O)OR17, C(S)OR17, C(O)NR17R18, S(O)R17, S(O)2R17, S(O)2NR17R18, NR19C(O)OR17, NR19C(O)R17, NR19S(O)R17, NR19S(O)2R17, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl.
each R14 is independently selected from unsubstituted or optionally substituted with one or more groups selected from hydrogen, halogen, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR20R21, C(O)R20, C(O)OR20, C(O)NR20R21, S(O)R20, S(O)2R20, S(O)2NR20R21, NR22C(O)OR20, NR22C(O)R20, NR22S(O)R20, NR22S(O)2R20, optionally substituted C6-C10 aryl and optionally substituted C5-C10 heteroaryl.
R15 is selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3-C8 heterocyclyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl.
R16 is selecy from null, hydrogen, halogen, oxo, CN, NO2, OR23, SR23, NR23R24, OCOR23, OCO2R23, OCONR23R24, COR23, CO2R23, CONR23R24, SOR23, SO2R23, SO2NR23R24, NR25C(O)OR23, NR25C(O)R23, NR25S(O)R23, NR25S(O)2R23, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C4-C8 heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
wherein
R17, R18, R19, R20, R21, R22, R23, R24, R25 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R17 and R18, R17 and R19, R20 and R21, R20 and R22, R23 and R24, R23 and R25, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 2C.
Wherein
In an embodiment, ENL ligands include a moiety according to FORMULA 3.
Wherein
In an embodiment, ENL ligands include a moiety according to FORMULA 3A.
In an embodiment, (ENL) ligands are selected from the group consisting of:
Degradation/Disruption Tags
Degradation/Disruption tags (EL) include, but are not limited to:
In an embodiment, degradation/disruption tags include a moiety according to FORMULAE 4A, 4B, 4C and 4D:
In an embodiment, degradation/disruption tags include a moiety according to one of FORMULAE 4E, 4F, 4G, 4H, and 4I:
In an embodiment, degradation/disruption tags include a moiety according to FORMULA
In an embodiment, degradation/disruption tags include a moiety according to FORMULAE 5B, 5C, 5D, 5E and 5F:
In an embodiment, degradation/disruption tags include a moiety according to FORMULA 5A:
R1, R2, R3, and R4 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted 3-7 membered heterocyclyl, optionally substituted C2-C8 alkenyl, and optionally substituted C2-C8 alkynyl.
In an embodiment, degradation/disruption tags include a moiety according to FORMULA 5B:
In an embodiment, degradation/disruption tags are selected from the group consisting of:
In any of the above-described compounds, the ENL ligand can be conjugated to the degradation/disruption tag through a linker. The linker can include, e.g., acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic, and/or carbonyl containing groups with different lengths.
In an embodiment, the linker is a moiety according to FORMULA 8:
In an embodiment, the linker is a moiety according to FORMULA 8A:
In an embodiment, the linker is a moiety according to FORMULA 8B:
In an embodiment, the linker is a moiety according to FORMULA 8C:
In an embodiment, the linker is selected from the group consisting of a ring selected from the group consisting of a 3 to 13 membered ring; a 3 to 13 membered fused ring; a 3 to 13 membered bridged ring; and a 3 to 13 membered spiro ring; and pharmaceutically acceptable salts thereof.
In an embodiment, the linker is a moiety according to one of FORMULAE C1, C2, C3, C4 and C5.
FORMULA C5; and pharmaceutically acceptable salts thereof.
In an embodiment, the bivalent compound according to the present invention is selected from the group consisting of:
In one embodiment, preferred compounds according to the present invention include:
In one embodiment, preferred compounds according to the present invention also include:
In some aspects, this disclosure provides a method of treating the ENL-mediated diseases, the method including administering to a subject in need thereof with an ENL-mediated disease one or more bivalent compounds including an ENL ligand conjugated to a degradation/disruption tag. The ENL-mediated diseases may be a disease resulting from ENL amplification. The ENL-mediated diseases can have elevated ENL enzymatic activity relative to a wild-type tissue of the same species and tissue type. Non-limiting examples of ENL-mediated diseases or diseases whose clinical symptoms could be treated by ENL degraders/disruptors-mediated therapy include: all solid and liquid cancer, chronic infections that produce exhausted immune response, infection-mediated immune suppression, age-related decline in immune response, age-related decline in cognitive function and infertility.
In any of the above-described methods, the bivalent compounds can be LQ076-46, LQ076-47, LQ076-48, LQ076-49, LQ076-50, LQ076-51, LQ076-52, LQ076-53, LQ076-54, LQ076-55, LQ076-56, LQ076-57, LQ076-58, LQ076-59, LQ076-60, LQ076-61, LQ076-62, LQ076-63, LQ076-64, LQ076-65, LQ076-66, LQ076-67, LQ076-68, LQ076-69, LQ076-70, LQ076-71, LQ076-72, LQ076-73, LQ076-74, LQ076-75, LQ076-76, LQ076-77, LQ076-78, LQ076-79, LQ076-80, LQ076-81, LQ076-82, LQ076-83, LQ076-84, LQ076-85, LQ076-86, LQ076-87, LQ076-88, LQ076-89, LQ076-90, LQ076-91, LQ076-92, LQ076-93, LQ076-94, LQ076-95, LQ076-96, LQ076-97, LQ076-98, LQ076-99, LQ076-100, LQ076-101, LQ076-102, LQ076-103, LQ076-104, LQ076-105, LQ076-106, LQ076-107, LQ076-108, LQ076-109, LQ076-110, LQ076-111, LQ076-112, LQ076-113, LQ076-114, LQ076-115, LQ076-116, LQ076-117, LQ076-118, LQ076-119, LQ076-120, LQ076-121, LQ076-122, LQ076-123, LQ076-124, LQ076-125, LQ076-126, LQ076-127, LQ076-128, LQ076-129, LQ076-130, LQ076-131, LQ076-132, LQ076-133, LQ076-134, LQ076-135, LQ076-136, LQ076-137, LQ076-138, LQ076-139, LQ076-140, LQ076-141, LQ076-142, LQ076-143, LQ076-144, LQ076-145, LQ076-146, LQ076-147, LQ076-148, LQ076-149, LQ076-150, LQ076-151, LQ076-152, LQ076-153, LQ076-154, LQ076-155, LQ076-156, LQ076-157, LQ076-158, LQ076-159, LQ076-160, LQ076-161, LQ076-162, LQ076-163, LQ081-100, LQ081-101, LQ081-102, LQ081-103, LQ081-104, LQ081-105, LQ081-108, LQ081-109, LQ081-122, LQ081-132, LQ081-133, LQ081-146, LQ081-147, LQ081-150, LQ086-31, LQ086-32, LQ086-33, LQ086-34, LQ086-35, LQ086-36, LQ086-38, LQ086-40, LQ086-41, LQ086-76, LQ086-76Na, LQ108-6, LQ108-7, LQ108-8, LQ108-9, LQ108-10, LQ108-11, LQ108-12, LQ108-146, LQ108-147, LQ108-148, LQ108-149, LQ108-150, LQ108-151, LQ108-152, LQ108-153, LQ108-154, LQ108-155, LQ108-156, LQ108-157, LQ118-23, LQ118-24, LQ118-25, LQ108-58, LQ108-60, LQ108-61, LQ108-62, LQ108-63, LQ108-64, LQ108-65, LQ108-66, LQ108-67, LQ108-68, LQ108-69, LQ108-70, LQ108-71, LQ108-72, LQ108-73, LQ108-74, LQ108-75, LQ126-46, LQ126-49, LQ126-50, LQ126-51, LQ126-52, LQ126-53, LQ126-54, LQ126-55, LQ126-56, LQ126-57, LQ126-58, LQ126-59, LQ126-60, LQ126-61, LQ126-62, LQ126-63, LQ126-77, LQ126-78, LQ126-79, LQ126-80, LQ126-81, LQ126-82, LQ126-83, LQ126-84, LQ126-85, LQ126-86, LQ126-87, LQ126-89, LQ126-90, LQ126-91, LQ126-92, LQ126-93, LQ126-94, LQ126-95, LQ126-96, LQ126-97, LQ126-98, LQ126-99, LQ126-100, LQ126-101, LQ126-102, LQ126-103, LQ126-104, LQ126-105, LQ126-106, LQ126-107, LQ126-108, LQ126-109, LQ126-110, LQ126-112, LQ126-113, LQ126-114, LQ126-115, LQ126-116, LQ126-117, LQ126-118, LQ126-120, LQ126-121, LQ126-122, LQ126-123, LQ126-124, LQ126-125, LQ126-126, LQ126-127, LQ126-128, LQ126-130, LQ126-168, LQ126-170, LQ126-171, LQ126-172, LQ126-173, LQ126-174, LQ126-175, LQ126-176, LQ126-177, LQ126-178, LQ126-180, LQ126-181, LQ126-182, LQ126-183, LQ126-184, LQ126-185, LQ126-186, LQ141-1, LQ141-2, LQ141-3, LQ141-4, LQ141-5, LQ141-6, LQ141-7, LQ141-8, LQ141-9, LQ141-10, LQ141-11, LQ141-12, LQ141-13, LQ141-14, LQ141-15, LQ141-16, LQ141-17, LQ141-18, LQ141-19, LQ141-20, LQ141-21, LQ141-22, LQ141-24, LQ141-26, LQ141-27, LQ141-28, LQ141-29, LQ141-33, LQ141-36, LQ141-37, LQ141-38, LQ141-39, LQ141-42, LQ141-43, LQ141-44, LQ141-45, LQ141-46, LQ141-47, LQ141-48, LQ141-49, LQ141-52 and LQ141-57.
In some aspects of the disclosed methods, the bivalent compounds can be administered by any of several routes of administration including, e.g., orally, parenterally, intradermally, subcutaneously, topically, and/or rectally.
Any of the above-described methods can further include treating the subject with one or more additional therapeutic regimens for treating cancer. The one or more additional therapeutic regimens for treating cancer can be, e.g., one or more of surgery, chemotherapy, radiation therapy, hormone therapy, or immunotherapy.
This disclosure additionally provides a method for identifying a bivalent compound which mediates degradation/disruption of ENL, the method including providing a heterobifunctional test compound including a ENL ligand conjugated to a degradation/disruption tag, contacting the heterobifunctional test compound with a cell (e.g., a cancer cell such as a ENL-mediated cancer cell) including a ubiquitin ligase and ENL.
As used herein, the terms “about” and “approximately” are defined as being within plus or minus 10% of a given value or state, preferably within plus or minus 5% of said value or state. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
The present disclosure is based, in part, on the discovery that novel heterobifunctional molecules which degrade ENL, ENL fusion proteins, and/or ENL mutant proteins are useful in the treatment of ENL-mediated diseases including but not limited to acute leukemia, mixed lineage leukemia (MLL)-rearranged leukemias and Wilms' tumor.
Successful strategies for selective degradation/disruption of the target protein induced by a bifunctional molecule include recruiting an E3 ubiquitin ligase and mimicking protein misfolding with a hydrophobic tag (Buckley and Crews, 2014). PROTACs (PROteolysis TArgeting Chimeras) are bivalent molecules with one moiety that binds an E3 ubiquitin ligase and another moiety that binds the protein target of interest (Buckley and Crews, 2014). The induced proximity leads to selective ubiquitination of the target followed by its degradation at the proteasome. Several types of high affinity small-molecule E3 ligase ligands have been identified/developed: They include (1) immunomodulatory drugs (IMiDs) such as thalidomide and pomalidomide, which bind cereblon (CRBN or CRL4CRBN), a component of a cullin-RING ubiquitin ligase (CRL) complex (Bondeson et al., 2015; Chamberlain et al., 2014; Fischer et al., 2014; Ito et al., 2010; Winter et al., 2015); (2) VHL-1, a hydroxyproline-containing ligand, which binds van Hippel-Lindau protein (VHL or CRL2VHL), a component of another CRL complex (Bondeson et al., 2015; Buckley et al., 2012a; Buckley et al., 2012b; Galdeano et al., 2014; Zengerle et al., 2015); (3) compound 7, which selectively binds KEAP1, a component of a CRL3 complex (Davies et al., 2016); (4) AMG232, which selectively binds MDM2, a heterodimeric RING E3 ligase (Sun et al., 2014); and (5) LCL161, which selectively binds IAP, a homodimeric RING E3 ligase (Ohoka et al., 2017; Okuhira et al, 2011; Shibata et al., 2017). The degrader technology has been successfully applied to degradation of multiple targets (Bondeson et al., 2015; Buckley et al., 2015; Lai et al., 2016; Lu et al., 2015; Winter et al., 2015; Zengerle et al., 2015), but not to degradation of ENL. In addition, a hydrophobic tagging approach, which utilizes a bulky and hydrophobic adamantyl group, has been developed to mimic protein misfolding, leading to the degradation of the target protein by proteasome (Buckley and Crews, 2014). This approach has also been successfully applied to selective degradation of the pseudokinase Her3 (Xie et al., 2014), but not to degradation of ENL proteins.
As discussed in the following examples, this disclosure provides specific examples of novel ENL degraders/disruptors, and examined the effect of exemplary degraders/disruptors on reducing ENL protein levels, and inhibiting MLL-rearranged leukemia cells proliferation. The results indicated that these novel compounds can be beneficial in treating human disease, especially acute leukemia, MLL-rearranged leukemia.
Current compounds targeting ENL generally focus on blocks the interaction between the ENL YEATS domain and acetylated histone H3, and have no effect in inhibiting the growth of ENL-dependent MLL-rearranged leukemia cells. In the present disclosure a different approach was taken: to develop compounds that they effectively degrade ENL in cells and reduce the proliferation of ENL-dependent MLL-rearranged leukemia cells in vitro and in vivo. Strategies for inducing protein degradation include recruiting E3 ubiquitin ligases, mimicking protein misfolding with hydrophobic tags, and inhibiting chaperones. For example, a thalidomide-JQ1 bivalent compound has been used to hijack the cereblon E3 ligase, inducing highly selective BET protein degradation in vitro and in vivo and resulting in a demonstrated delay in leukemia progression in mice (Winter et al., 2015). Similarly, BET protein degradation has also been induced via another E3 ligase, VHL (Zengerle et al., 2015). Partial degradation of the Her3 protein has been induced using an adamantane-modified compound (Xie et al., 2014). Such an approach, based on the use of bivalent molecules, permits more flexible regulation of protein levels in vitro and in vivo compared with techniques such as gene knockout or knockdown via RNA interference. Unlike gene knockout or knockdown, this chemical approach provides an opportunity to study dose and time dependency in a disease model by varying the concentrations and frequencies of administration of the relevant compound.
This disclosure includes all stereoisomers, geometric isomers, tautomers and isotopes of the structures depicted and compounds named herein. This disclosure also includes compounds described herein, regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.
This disclosure includes pharmaceutically acceptable salts of the structures depicted and compounds named herein.
One or more constituent atoms of the compounds presented herein can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of the hydrogen atoms in a compound can be replaced or substituted by deuterium atoms. In some embodiments, the compound includes at least one fluorine atom. In some embodiments, the compound includes two or more fluorine atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 fluorine atoms. In some embodiments, all of the hydrogen atoms in a compound can be replaced or substituted by fluorine atoms.
In some aspects, the present disclosure provides bivalent compounds, also referred to herein as degraders, comprising an ENL ligand (or targeting moiety) conjugated to a degradation tag. Linkage of the ENL ligand to the degradation tag can be direct, or indirect via a linker.
As used herein, the terms “Eleven-Nineteen Leukemia (ENL) ligand” or “ENL ligand” or “ENL targeting moiety” are to be construed broadly, and encompass a wide variety of molecules ranging from small molecules to large proteins that associate with or bind to ENL. The ENL ligand or targeting moiety can be, for example, a small molecule compound (i.e., a molecule of molecular weight less than about 1.5 kilodaltons (kDa)), a peptide or polypeptide, nucleic acid or oligonucleotide, carbohydrate such as oligosaccharides, or an antibody or fragment thereof.
The ENL ligand or targeting moiety can be derived from an ENL inhibitor (e.g., SGC-iMLLT), which can block the interaction between the ENL YEATS domain and acetylated histone H3 in vitro and in cells. As used herein, an “inhibitor” refers to an agent that restrains, retards, or otherwise causes inhibition of a physiological, chemical or enzymatic action or function. As used herein an inhibitor causes a decrease in enzyme activity of at least 5%. An inhibitor can also or alternatively refer to a drug, compound, or agent that prevents or reduces the expression, transcription, or translation of a gene or protein. An inhibitor can reduce or prevent the function of a protein, e.g., by binding to or activating/inactivating another protein or receptor.
Exemplary ENL ligands include, but are not limited to, the compounds listed below:
As used herein, the term “degradation/disruption tag” refers to a compound, which associates with or binds to a ubiquitin ligase for recruitment of the corresponding ubiquitination machinery to ENL or induces ENL protein misfolding and subsequent degradation at the proteasome or loss of function.
In some aspects, the degradation/disruption tags of the present disclosure include, e.g., thalidomide, pomalidomide, lenalidomide, VHL-1, adamantane, 1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112, RG7338, AMG232, AA-115, bestatin, MV-1, LCL161, FK506, rapamycin and/or analogs thereof.
As used herein, a “linker” is a bond, molecule, or group of molecules that binds two separate entities to one another. Linkers can provide for optimal spacing of the two entities. The term “linker” in some aspects refers to any agent or molecule that bridges the ENL ligand to the degradation/disruption tag. One of ordinary skill in the art recognizes that sites on the ENL ligand or the degradation/disruption tag, which are not necessary for the function of the degraders of the present disclosure, are ideal sites for attaching a linker, provided that the linker, once attached to the conjugate of the present disclosure, does not interfere with the function of the degrader, i.e., its ability to target ENL and its ability to recruit a ubiquitin ligase.
The length of the linker of the bivalent compound can be adjusted to minimize the molecular weight of the disruptors/degraders and avoid any potential clash of the ENL ligand or targeting moiety with either the ubiquitin ligase or the induction of ENL misfolding by the hydrophobic tag at the same time.
In some aspects, the degradation/disruption tags of the present disclosure include, for example, thalidomide, pomalidomide, lenalidomide, VHL-1, adamantane, 1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112, RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161, FK506, rapamycin and analogs thereof. The degradation/disruption tags can be attached to any portion of the structure of an ENL ligand or targeting moiety (SGC-iMLLT) with linkers of different types and lengths in order to generate effective bivalent compounds. In particular, attaching VHL1, pomalidomide, to any portion of the molecule can recruit the E3 ligase to ENL.
The bivalent compounds disclosed herein can selectively reduce the proliferation of ENL-mediated disease cells in vitro and in vivo.
Additional bivalent compounds (i.e., ENL degraders/disruptors) can be developed using the principles and methods disclosed herein. For example, other linkers, degradation tags, and ENL binding/inhibiting moieties can be synthesized and tested. Non-limiting examples of ENL disruptors/degraders (e.g., bivalent compounds) are shown in Table 1 (below). The left portion of each ENL disruptors/degrader compound as shown binds to ENL (as SGC-iMLLT do), and the right portion of each compound recruits for the ubiquitination machinery to ENL, which induces the poly-ubiquitination and degradation of ENL at the proteasome.
More specifically, the present disclosure provides a bivalent compound including an ENL ligand conjugated to a degradation/disruption tag.
In some aspects, the ENL degraders/disruptors have the form “PI-linker-EL”, as shown below:
wherein PI (protein of interest) comprises an ENL ligand and EL (E3 ligase) comprises a degradation/disruption tag (e.g., E3 ligase ligand). Exemplary ENL ligands (PI), exemplary degradation/disruption tags (EL), and exemplary linkers (Linker) are illustrated below:
ENL Ligands
In an embodiment, ENL ligands include a moiety according to FORMULA 1:
R1 is selected from H, halogen, OR5, SR5, C1-C8 alkylene NR5R6, CH2CH2NR5R6, NR5R6, C(O)R5, C(O)OR5, C(S)OR5, C(O)NR5R6, S(O)R5, S(O)2R5, S(O)2NR5R6, NR7C(O)OR6, NR7C(O)R6, NR7S(O)R6, NR7S(O)2R6, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl.
R2 is independently selected from hydrogen, halogen, oxo, CN, NO2, OR8, SR8, NR8R9, C(O)R8, C(O)OR8, C(S)OR8, C(O)NR8R9, S(O)R8, S(O)2R8, S(O)2NR8R9, NR10C(O)OR9, NR10C(O)R9, NR10S(O)R9, NR10S(O)2R9, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3-C8 heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R3 is unsubstituted or optionally substituted with one or more groups selected from hydrogen, halogen, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR11R12, C(O)R11, C(O)OR11, C(O)NR11R12, S(O)R11, S(O)2R11, S(O)2NR11R12, NR13C(O)OR12, NR13C(O)R12, NR13S(O)R12, NR13S(O)2R12, optionally substituted C6-C10 aryl and optionally substituted C5-C10 heteroaryl.
each R4 is independently selected from null, hydrogen, halogen, oxo, CN, NO2, OR14, SR14, NR14R15, OCOR14, OCO2R14, OCONR14R15, COR14, CO2R15, CONR14R15, SOR14, SO2R14, SO2NR14R15, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C4-C8 heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
R5, R6, R7, R8, R9, R10 R11, R12, R13 R14, R15 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R5 and R6, R6 and R7, R8 and R9, R8 and R10, R9 and R10, R11 and R12, R11 and R13, R12 and R13, R14 and R15, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 1A
R16, R17 is selected from hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C6-C10 aryl, C5-C10 heteroaryl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, C(O)C6-C10 aryl, C(O)C5-C10 heteroaryl
or
R16 and R17 together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
R18, R19 are independently selected from hydrogen, halogen, CN, OH, NH2, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted 3-8 membered heterocyclyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl;
R20 is selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3-C8 heterocyclyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl.
In an embodiment, ENL ligands include a moiety according to FORMULA 1B, 1C, 1D, 1E
R22 is unsubstituted or optionally substituted with one or more groups selected from halo, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR26R27, C1-C8NR26R27, C(O)R26, C(O)OR26, C(O)NR26R27, S(O)R26, S(O)2R26, S(O)2NR26R27, NR26C(O)OR27, NR28C(O)R27, NR28S(O)R27, NR28S(O)2R27.
R23 is unsubstituted or optionally substituted with one or more groups selected from halo, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR29R30, C(O)R29, C(O)OR29, C(O)NR29R30, S(O)R29, S(O)2R29, S(O)2NR29R30, NR31C(O)OR29, NR31C(O)R29, NR31S(O)R29, NR31S(O)2R29.
R25 is unsubstituted or optionally substituted with one or more groups selected from halo, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR32R33, C(O)R32, C(O)OR32, C(O)NR32R33, S(O)R32, S(O)2R32, S(O)2NR32R33, NR34C(O)OR32, NR34C(O)R32, NR34S(O)R32, NR34S(O)2R32.
R26, R27, R28, R29, R30, R31 R32, R33, R34 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R26 and R27, R27 and R28, R29 and R30, R29 and R31, R32 and R33, R32 and R34, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 1F:
In an embodiment, ENL ligands include a moiety according to FORMULA 2.
R1 is selected from hydrogen, halogen, OR4, SR4, C1-C8 alkylene NR4R5, C(O)R4, C(O)OR4, C(S)OR4, C(O)NR4R5, S(O)R4, S(O)2R4, S(O)2NR4R5, NR6C(O)OR4, NR6C(O)R4, NR6S(O)R4, NR6S(O)2R4, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, or fused C3-C10 cycloalkyl, C3-C10 heterocyclyl.
R2 is selected from hydrogen, halogen, CN, NO2, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR7R8, C(O)R7, C(O)OR7, C(O)NR7R8, S(O)R7, S(O)2R7, S(O)2NR7R8, NR9C(O)OR7, NR9C(O)R7, NR9S(O)R7, NR9S(O)2R7, optionally substituted C6-C10 aryl and optionally substituted C5-C10 heteroaryl.
R4, R5, R6, R7, R8, R9, R10 R11, R12 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R4 and R5, R4 and R6, R7 and R8, R7 and R9, R10 and R11, R10 and R12, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 2A and 2B.
R13 is selected from hydrogen, halogen OR17, SR17, C1-C8 alkylene NR17R18, NR17R18, C(O)R17, C(O)OR17, C(S)OR17, C(O)NR17R18, S(O)R17, S(O)2R17, S(O)2NR17R15, NR19C(O)OR17, NR19C(O)R17, NR19S(O)R17, NR19S(O)2R17, or unsubstituted or optionally substituted C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl.
each R14 is independently selected from unsubstituted or optionally substituted with one or more groups selected from hydrogen, halogen, CN, NO2, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O)C1-C8 alkyl, C(O)C1-C8 haloalkyl, C(O)C1-C8 hydroxyalkyl, C(O)C3-C10 cycloalkyl, C(O)C3-C10 heterocyclyl, NR20R21, C(O)R20, C(O)OR20, C(O)NR20R21, S(O)R20, S(O)2R20, S(O)2NR20R21, NR22C(O)OR20, NR22C(O)R20, NR22S(O)R20, NR22S(O)2R20, optionally substituted C6-C10 aryl and optionally substituted C5-C10 heteroaryl.
R15 is selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C3-C8 heterocyclyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8 alkoxyalkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 alkylamino, and optionally substituted C1-C8 alkylaminoC1-C8 alkyl.
R16 is selecy from null, hydrogen, halogen, oxo, CN, NO2, OR23, SR23, NR23R24, OCOR23, OCO2R23, OCONR23R24, COR23, CO2R23, CONR23R24, SOR23, SO2R23, SO2NR23R24, NR25C(O)OR23, NR25C(O)R23, NR25S(O)R23, NR25S(O)2R23, optionally substituted C1-C8 alkyl, optionally substituted C2-C8 alkenyl, optionally substituted C2-C8 alkynyl, optionally substituted C1-C8 alkoxy, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkoxy, optionally substituted C4-C8 heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
wherein
R17, R18, R19, R20, R21, R22, R23, R24, R25 are independently selected from H, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C(O) C1-C8 alkyl, C(O) C1-C8 haloalkyl, C(O) C1-C8 hydroxyalkyl, C(O) C3-C10 cycloalkyl, C(O) C3-C10 heterocyclyl, optionally substituted C6-C10 aryl or C5-C10 heteroaryl.
R17 and Rig, R17 and R19, R20 and R21, R20 and R22, R23 and R24, R23 and R25, together with the nitrogen atom to which they connected can independently form optionally substituted C3-C13 heterocyclyl rings, optionally substituted C3-C13 fused cycloalkyl ring, optionally substituted C3-C13 fused heterocyclyl ring, optionally substituted C3-C13 bridged cycloalkyl ring, optionally substituted C3-C13 bridged heterocyclyl ring, optionally substituted C3-C13 spiro cycloalkyl ring, and optionally substituted C3-C13 spiro heterocyclyl ring.
In an embodiment, ENL ligands include a moiety according to FORMULA 2C.
In an embodiment, ENL ligands include a moiety according to FORMULA 3.
In an embodiment, ENL ligands include a moiety according to FORMULA 3A.
In an embodiment, (ENL) ligands are selected from the group consisting of:
Degradation/Disruption Tags
Degradation/Disruption tags (EL) include, but are not limited to:
In an embodiment, degradation/disruption tags include a moiety according to FORMULAE 4A, 4B, 4C and 4D:
In an embodiment, degradation/disruption tags include a moiety according to one of FORMULAE 4E, 4F, 4G, 4H, and 4I:
In an embodiment, degradation/disruption tags include a moiety according to FORMULA 5A:
wherein
R1 and R2 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C1-C8 aminoalkyl, optionally substituted C1-C8alkylaminoC1-C8alkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted 3-7 membered heterocyclyl, optionally substituted C2-C8 alkenyl, and optionally substituted C2-C8 alkynyl; and
R3 is hydrogen, optionally substituted C(O)C1-C8 alkyl, optionally substituted C(O)C1-C8alkoxyC1-C8alkyl, optionally substituted C(O)C1-C8 haloalkyl, optionally substituted C(O)C1-C8 hydroxyalkyl, optionally substituted C(O)C1-C8 aminoalkyl, optionally substituted C(O)C1-C8alkylaminoC1-C8alkyl, optionally substituted C(O)C3-C7 cycloalkyl, optionally substituted C(O)(3-7 membered heterocyclyl), optionally substituted C(O)C2-C8 alkenyl, optionally substituted C(O)C2-C8 alkynyl, optionally substituted C(O)OC1-C8alkoxyC1-C8alkyl, optionally substituted C(O)OC1-C8 haloalkyl, optionally substituted C(O)OC1-C8 hydroxyalkyl, optionally substituted C(O)OC1-C8 aminoalkyl, optionally substituted C(O)OC1-C8alkylaminoC1-C8alkyl, optionally substituted C(O)OC3-C7 cycloalkyl, optionally substituted C(O)O(3-7 membered heterocyclyl), optionally substituted C(O)OC2-C8 alkenyl, optionally substituted C(O)OC2-C8 alkynyl, optionally substituted C(O)NC1-C8alkoxyC1-C8alkyl, optionally substituted C(O)NC1-C8 haloalkyl, optionally substituted C(O)NC1-C8 hydroxyalkyl, optionally substituted C(O)NC1-C8 aminoalkyl, optionally substituted C(O)NC1-C8alkylaminoC1-C8alkyl, optionally substituted C(O)NC3-C7 cycloalkyl, optionally substituted C(O)N(3-7 membered heterocyclyl), optionally substituted C(O)NC2-C8 alkenyl, optionally substituted C(O)NC2-C8 alkynyl, optionally substituted P(O)(OH)2, optionally substituted P(O)(OC1-C8 alkyl)2, and optionally substituted P(O)(OC1-C8 aryl)2.
In an embodiment, degradation/disruption tags include a moiety according to FORMULAE 5B, 5C, 5D, 5E and 5F:
In an embodiment, degradation/disruption tags include a moiety according to FORMULA 5A:
R1, R2, R3, and R4 are independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8alkoxyC1-C8alkyl, optionally substituted C1-C8 haloalkyl, optionally substituted C1-C8 hydroxyalkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted 3-7 membered heterocyclyl, optionally substituted C2-C8 alkenyl, and optionally substituted C2-C8 alkynyl.
In an embodiment, degradation/disruption tags include a moiety according to FORMULA 5B:
In an embodiment, degradation/disruption tags are selected from the group consisting of:
and pharmaceutically acceptable salts thereof.
In any of the above-described compounds, the ENL ligand can be conjugated to the degradation/disruption tag through a linker. The linker can include, e.g., acyclic or cyclic saturated or unsaturated carbon, ethylene glycol, amide, amino, ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic, and/or carbonyl containing groups with different lengths.
In an embodiment, the linker is a moiety according to FORMULA 8:
In an embodiment, the linker is a moiety according to FORMULA 8A:
In an embodiment, the linker is a moiety according to FORMULA 8B:
In an embodiment, the linker is a moiety according to FORMULA 8C:
In an embodiment, the linker is selected from the group consisting of a ring selected from the group consisting of a 3 to 13 membered ring; a 3 to 13 membered fused ring; a 3 to 13 membered bridged ring; and a 3 to 13 membered spiro ring; and pharmaceutically acceptable salts thereof.
In an embodiment, the linker is a moiety according to one of FORMULAE C1, C2, C3, C4 and C5.
FORMULA C5; and pharmaceutically acceptable salts thereof.
The binding affinity of novel synthesized bivalent compounds (i.e., ENL degraders/disruptors) can be assessed using standard biophysical assays known in the art (e.g., isothermal titration calorimetry (ITC)). Cellular assays can then be used to assess the bivalent compound's ability to induce ENL degradation and inhibit cancer cell proliferation. Suitable cell lines for use in any or all of these steps are known in the art and include, e.g. MV4; 11, Jurkat, MOLM13. Suitable mouse models for use in any or all of these steps are known in the art and include MV4; 11 and MOLM13 xenograft model.
By way of non-limiting example, detailed synthesis protocols are described in the Examples for specific exemplary ENL degraders/disruptors.
Pharmaceutically acceptable isotopic variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate isotopic variations of those reagents). Specifically, an isotopic variation is a compound in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Useful isotopes are known in the art and include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary isotopes thus include, e.g., 2H, 3H, 13C, 14C, 15N, 17O, 18O, 32P, 35S, 18F, and 36Cl.
Isotopic variations (e.g., isotopic variations containing 2H) can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements. In addition, certain isotopic variations (particularly those containing a radioactive isotope) can be used in drug or substrate tissue distribution studies. The radioactive isotopes tritium (3H) and carbon-14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Pharmaceutically acceptable solvates of the compounds disclosed herein are contemplated. A solvate can be generated, e.g., by substituting a solvent used to crystallize a compound disclosed herein with an isotopic variation (e.g., D2O in place of H2O, d6-acetone in place of acetone, or d6-DMSO in place of DMSO).
Pharmaceutically acceptable fluorinated variations of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (substituting appropriate reagents with appropriate fluorinated variations of those reagents). Specifically, a fluorinated variation is a compound in which at least one hydrogen atom is replaced by a fluoro atom. Fluorinated variations can provide therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements
Pharmaceutically acceptable prodrugs of the compounds disclosed herein are contemplated and can be synthesized using conventional methods known in the art or methods corresponding to those described in the Examples (e.g., concerting hydroxyl groups to ester groups or sodium phosphate salt). As used herein, a “prodrug” refers to a compound that can be converted via some chemical or physiological process (e.g., enzymatic process and metabolic hydrolysis) to a therapeutic agent. Thus, the term “prodrug” also refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in an organism. The term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a subject. Prodrugs of an active compound may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound. Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
Specific exemplary ENL degraders/disruptors were firstly characterized in ENL-dependent leukemia MV4; 11 cells to evaluate their concentration-dependent ability in cell growth suppression (
Treatment of cells with ENL degraders LQ076-122 and LQ081-108 suppressed ENL target gene expression in a concentration- and time-dependent manner in both MOLM13 and MV4; 11 cells (
The plasma concentrations of ENL degrader LQ076-122 was measured over 12 h following a single 50 mg/kg IP injection in a mouse pharmacokinetic (PK) study. The concentrations of LQ076-122 in plasma were maintained above 2 μM for 6 h with the maximum plasma concentration of about 6 μM (
Furthermore, specific exemplary ENL degraders/disruptors were firstly characterized in ENL-dependent leukemia MV4; 11 cells stably expressing 3Flag-HA-tagged ENL to evaluate their ability in inducing degradation of ectopically expressed 3Flag-HA-ENL protein at 1 μM and 10 μM doses (
As used herein, the terms “comprising” and “including” are used in their open, non-limiting sense.
“Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation. An alkyl may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkyl comprises one to fifteen carbon atoms (e.g., C1-C15 alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C1-C8 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C5-C8 alkyl). The alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), pentyl, 3-methylhexyl, 2-methylhexyl, and the like.
“Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond. An alkenyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkenyl comprises two to twelve carbon atoms (e.g., C2-C12 alkenyl). In certain embodiments, an alkenyl comprises two to eight carbon atoms (e.g., C2-C8 alkenyl). In certain embodiments, an alkenyl comprises two to six carbon atoms (e.g., C2-C6 alkenyl). In other embodiments, an alkenyl comprises two to four carbon atoms (e.g., C2-C4 alkenyl). The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.
The term “allyl,” as used herein, means a —CH2CH═CH2 group.
As used herein, the term “alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond. An alkynyl may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen carbon atoms. In certain embodiments, an alkynyl comprises two to twelve carbon atoms (e.g., C2-C12 alkynyl). In certain embodiments, an alkynyl comprises two to eight carbon atoms (e.g., C2-C8 alkynyl). In other embodiments, an alkynyl has two to six carbon atoms (e.g., C2-C6 alkynyl). In other embodiments, an alkynyl has two to four carbon atoms (e.g., C2-C4 alkynyl). The alkynyl is attached to the rest of the molecule by a single bond. Examples of such groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and the like.
The term “alkoxy”, as used herein, means an alkyl group as defined herein which is attached to the rest of the molecule via an oxygen atom. Examples of such groups include, but are not limited to, methoxy, ethoxy, n-propyloxy, iso-propyloxy, n-butoxy, iso-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
The term “aryl”, as used herein, “refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon atoms. An aryl may comprise from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system in accordance with the Hückel theory. In certain embodiments, an aryl comprises six to fourteen carbon atoms (C6-C14 aryl). In certain embodiments, an aryl comprises six to ten carbon atoms (C6-C10 aryl). Examples of such groups include, but are not limited to, phenyl, fluorenyl and naphthyl. The terms “Ph” and “phenyl,” as used herein, mean a —C6H5 group.
The term “heteroaryl”, refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system in accordance with the Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
Examples of such groups include, but not limited to, pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, and the like. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a ring carbon atom. In certain embodiments, an heteroaryl is attached to the rest of the molecule via a nitrogen atom (N-attached) or a carbon atom (C-attached). For instance, a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached).
The term “heterocyclyl”, as used herein, means a non-aromatic, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 atoms in its ring system, and containing from 3 to 12 carbon atoms and from 1 to 4 heteroatoms each independently selected from O, S and N, and with the proviso that the ring of said group does not contain two adjacent O atoms or two adjacent S atoms. A heterocyclyl group may include fused, bridged or spirocyclic ring systems. In certain embodiments, a heterocyclyl group comprises 3 to 10 ring atoms (3-10 membered heterocyclyl). In certain embodiments, a heterocyclyl group comprises 3 to 8 ring atoms (3-8 membered heterocyclyl). In certain embodiments, a heterocyclyl group comprises 4 to 8 ring atoms (4-8 membered heterocyclyl). In certain embodiments, a heterocyclyl group comprises 3 to 6 ring atoms (3-6 membered heterocyclyl). A heterocyclyl group may contain an oxo substituent at any available atom that will result in a stable compound. For example, such a group may contain an oxo atom at an available carbon or nitrogen atom. Such a group may contain more than one oxo substituent if chemically feasible. In addition, it is to be understood that when such a heterocyclyl group contains a sulfur atom, said sulfur atom may be oxidized with one or two oxygen atoms to afford either a sulfoxide or sulfone. An example of a 4 membered heterocyclyl group is azetidinyl (derived from azetidine). An example of a 5 membered cycloheteroalkyl group is pyrrolidinyl. An example of a 6 membered cycloheteroalkyl group is piperidinyl. An example of a 9 membered cycloheteroalkyl group is indolinyl. An example of a 10 membered cycloheteroalkyl group is 4H-quinolizinyl. Further examples of such heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, quinolizinyl, 3-oxopiperazinyl, 4-methylpiperazinyl, 4-ethylpiperazinyl, and 1-oxo-2,8,diazaspiro[4.5]dec-8-yl. A heteroaryl group may be attached to the rest of molecular via a carbon atom (C-attached) or a nitrogen atom (N-attached). For instance, a group derived from piperazine may be piperazin-1-yl (N-attached) or piperazin-2-yl (C-attached).
The term “cycloalkyl” means a saturated, monocyclic, bicyclic, tricyclic, or tetracyclic radical having a total of from 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms in its ring system. A cycloalkyl may be fused, bridged or spirocyclic. In certain embodiments, a cycloalkyl comprises 3 to 8 carbon ring atoms (C3-C8 cycloalkyl). In certain embodiments, a cycloalkyl comprises 3 to 6 carbon ring atoms (C3-C6 cycloalkyl). Examples of such groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
The term “cycloalkylene” is a bidentate radical obtained by removing a hydrogen atom from a cycloalkyl ring as defined above. Examples of such groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclopentenylene, cyclohexylene, cycloheptylene, and the like.
The term “spirocyclic” as used herein has its conventional meaning, that is, any ring system containing two or more rings wherein two of the rings have one ring carbon in common. Each ring of the spirocyclic ring system, as herein defined, independently comprises 3 to 20 ring atoms. Preferably, they have 3 to 10 ring atoms. Non-limiting examples of a spirocyclic system include spiro[3.3]heptane, spiro[3.4]octane, and spiro[4.5]decane.
The term cyano” refers to a —C≡N group.
An “aldehyde” group refers to a —C(O)H group.
An “alkoxy” group refers to both an —O-alkyl, as defined herein.
An “alkoxycarbonyl” refers to a —C(O)-alkoxy, as defined herein.
An “alkylaminoalkyl” group refers to an -alkyl-NR-alkyl group, as defined herein.
An “alkylsulfonyl” group refer to a —SO2alkyl, as defined herein.
An “amino” group refers to an optionally substituted —NH2.
An “aminoalkyl” group refers to an -alky-amino group, as defined herein.
An “aminocarbonyl” refers to a —C(O)-amino, as defined herein.
An “arylalkyl” group refers to -alkylaryl, where alkyl and aryl are defined herein.
An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group, as defined herein.
An “aryloxycarbonyl” refers to —C(O)-aryloxy, as defined herein.
An “arylsulfonyl” group refers to a —SO2aryl, as defined herein.
A “carbonyl” group refers to a —C(O)— group, as defined herein.
A “carboxylic acid” group refers to a —C(O)OH group.
A “cycloalkoxy” refers to a —O-cycloalkyl group, as defined herein.
A “halo” or “halogen” group refers to fluorine, chlorine, bromine or iodine.
A “haloalkyl” group refers to an alkyl group substituted with one or more halogen atoms.
A “hydroxy” group refers to an —OH group.
A “nitro” group refers to a —NO2 group.
An “oxo” group refers to the ═O substituent.
A “trihalomethyl” group refers to a methyl substituted with three halogen atoms.
The term “substituted,” means that the specified group or moiety bears one or more substituents independently selected from C1-C4 alkyl, aryl, heteroaryl, aryl-C1-C4 alkyl-, heteroaryl-C1-C4 alkyl-, C1-C4 haloalkyl, —OC1-C4 alkyl, —OC1-C4 alkylphenyl, —C1-C4 alkyl-OH, —OC1-C4 haloalkyl, halo, —OH, —NH2, —C1-C4 alkyl-NH2, —N(C1-C4 alkyl)(C1-C4 alkyl), —NH(C1-C4 alkyl), —N(C1-C4 alkyl)(C1-C4 alkylphenyl), —NH(C1-C4 alkylphenyl), cyano, nitro, oxo, —CO2H, —C(O)OC1-C4 alkyl, —CON(C1-C4 alkyl)(C1-C4 alkyl), —CONH(C1-C4 alkyl), —CONH2, —NHC(O)(C1-C4 alkyl), —NHC(O)(phenyl), —N(C1-C4 alkyl)C(O)(C1-C4 alkyl), —N(C1-C4 alkyl)C(O)(phenyl), —C(O)C1-C4 alkyl, —C(O)C1-C4 alkylphenyl, —C(O)C1-C4 haloalkyl, —OC(O)C1-C4 alkyl, —SO2(C1-C4 alkyl), —SO2(phenyl), —SO2(C1-C4 haloalkyl), —SO2NH2, —SO2NH(C1-C4 alkyl), —SO2NH(phenyl), —NHSO2(C1-C4 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C4 haloalkyl).
The term “optionally substituted” means that the specified group may be either unsubstituted or substituted by one or more substituents as defined herein. It is to be understood that in the compounds of the present invention when a group is said to be “unsubstituted,” or is “substituted” with fewer groups than would fill the valencies of all the atoms in the compound, the remaining valencies on such a group are filled by hydrogen. For example, if a C6 aryl group, also called “phenyl” herein, is substituted with one additional substituent, one of ordinary skill in the art would understand that such a group has 4 open positions left on carbon atoms of the C6 aryl ring (6 initial positions, minus one at which the remainder of the compound of the present invention is attached to and an additional substituent, remaining 4 positions open). In such cases, the remaining 4 carbon atoms are each bound to one hydrogen atom to fill their valencies. Similarly, if a C6 aryl group in the present compounds is said to be “disubstituted,” one of ordinary skill in the art would understand it to mean that the C6 aryl has 3 carbon atoms remaining that are unsubstituted. Those three unsubstituted carbon atoms are each bound to one hydrogen atom to fill their valencies.
“Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the bivalent compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
“Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 66:1-19 (1997), which is hereby incorporated by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
“Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.
In some aspects, the compositions and methods described herein include the manufacture and use of pharmaceutical compositions and medicaments that include one or more bivalent compounds as disclosed herein. Also included are the pharmaceutical compositions themselves.
In some aspects, the compositions disclosed herein can include other compounds, drugs, or agents used for the treatment of cancer. For example, in some instances, pharmaceutical compositions disclosed herein can be combined with one or more (e.g., one, two, three, four, five, or less than ten) compounds. Such additional compounds can include, e.g., conventional chemotherapeutic agents known in the art. When co-administered, ENL degraders/disruptors disclosed herein can operate in conjunction with conventional chemotherapeutic agents to produce mechanistically additive or synergistic therapeutic effects.
In some aspects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the ENL degraders/disruptor or its delivery form.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier, adjuvant, or vehicle. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable carrier, adjuvant, or vehicle is a composition that can be administered to a patient, together with a compound of the invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Exemplary conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles include saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
In particular, pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also be advantageously used to enhance delivery of compounds of the formulae described herein.
As used herein, the ENL degraders/disruptors disclosed herein are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester, or other derivative of a compound or agent disclosed herein, which upon administration to a recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds disclosed herein when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group that enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol. 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.
The ENL degraders/disruptors disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated derivative thereof.
In particular, pharmaceutically acceptable salts of the ENL degraders/disruptors disclosed herein include, e.g., those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate, trifluoromethylsulfonate, and undecanoate. Salts derived from appropriate bases include, e.g., ENL alkali metal (e.g., sodium), ENL alkaline earth metal (e.g., magnesium), ammonium and N-(ENLyl)4+ salts. The invention also envisions the quaternization of any basic nitrogen-containing groups of the ENL degraders/disruptors disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization.
In some aspects, the pharmaceutical compositions disclosed herein can include an effective amount of one or more ENL degraders/disruptors. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).
In some aspects, pharmaceutical compositions can further include one or more additional compounds, drugs, or agents used for the treatment of cancer (e.g., conventional chemotherapeutic agents) in amounts effective for causing an intended effect or physiological outcome (e.g., treatment or prevention of cell growth, cell proliferation, or cancer).
In some aspects, the pharmaceutical compositions disclosed herein can be formulated for sale in the United States, import into the United States, or export from the United States.
The pharmaceutical compositions disclosed herein can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (DSM) (available at http://www.fda.gov/Drugs/DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs). In particular, the pharmaceutical compositions can be formulated for and administered via oral, parenteral, or transdermal delivery. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
For example, the pharmaceutical compositions disclosed herein can be administered, e.g., topically, rectally, nasally (e.g., by inhalation spray or nebulizer), buccally, vaginally, subdermally (e.g., by injection or via an implanted reservoir), or ophthalmically.
For example, pharmaceutical compositions of this invention can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.
For example, the pharmaceutical compositions of this invention can be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
For example, the pharmaceutical compositions of this invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, or other solubilizing or dispersing agents known in the art.
For example, the pharmaceutical compositions of this invention can be administered by injection (e.g., as a solution or powder). Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, e.g., olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens, Spans, or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.
In some aspects, an effective dose of a pharmaceutical composition of this invention can include, but is not limited to, e.g., about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, or 10000 mg/kg/day, or according to the requirements of the particular pharmaceutical composition.
When the pharmaceutical compositions disclosed herein include a combination of a compound of the formulae described herein (e.g., a ENL degraders/disruptors) and one or more additional compounds (e.g., one or more additional compounds, drugs, or agents used for the treatment of cancer or any other condition or disease, including conditions or diseases known to be associated with or caused by cancer), both the compound and the additional compound should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents can be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
In some aspects, the pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration.
The methods disclosed herein contemplate administration of an effective amount of a compound or composition to achieve the desired or stated effect. Typically, the compounds or compositions of the invention will be administered from about 1 to about 6 times per day or, alternately or in addition, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations can contain from about 20% to about 80% active compound.
In some aspects, the present disclosure provides methods for using a composition comprising an ENL degrader/disruptor, including pharmaceutical compositions (indicated below as ‘X’) disclosed herein in the following methods:
Substance X for use as a medicament in the treatment of one or more diseases or conditions disclosed herein (e.g., cancer, referred to in the following examples as ‘Y’). Use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment of Y.
In some aspects, the methods disclosed include the administration of a therapeutically effective amount of one or more of the compounds or compositions described herein to a subject (e.g., a mammalian subject, e.g., a human subject) who is in need, or who has been determined to be in need of, such treatment. In some aspects, the methods disclosed include selecting a subject and administering to the subject an effective amount of one or more of the compounds or compositions described herein, and optionally repeating administration as required for the prevention or treatment of cancer.
In some aspects, subject selection can include obtaining a sample from a subject (e.g., a candidate subject) and testing the sample for an indication that the subject is suitable for selection.
In some aspects, the subject can be confirmed or identified, e.g. by a health care professional, as having had or having a condition or disease. In some aspects, suitable subjects include, for example, subjects who have or had a condition or disease but that resolved the disease or an aspect thereof, present reduced symptoms of disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), or that survive for extended periods of time with the condition or disease (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease), e.g., in an asymptomatic state (e.g., relative to other subjects (e.g., the majority of subjects) with the same condition or disease). In some aspects, exhibition of a positive immune response towards a condition or disease can be made from patient records, family history, or detecting an indication of a positive immune response. In some aspects, multiple parties can be included in subject selection. For example, a first party can obtain a sample from a candidate subject and a second party can test the sample. In some aspects, subjects can be selected or referred by a medical practitioner (e.g., a general practitioner). In some aspects, subject selection can include obtaining a sample from a selected subject and storing the sample or using the in the methods disclosed herein. Samples can include, e.g., cells or populations of cells.
In some aspects, methods of treatment can include a single administration, multiple administrations, and repeating administration of one or more compounds disclosed herein as required for the prevention or treatment of the disease or condition from which the subject is suffering (e.g., an ENL-mediated cancer). In some aspects, methods of treatment can include assessing a level of disease in the subject prior to treatment, during treatment, or after treatment. In some aspects, treatment can continue until a decrease in the level of disease in the subject is detected.
The term “subject,” as used herein, refers to any animal. In some instances, the subject is a mammal. In some instances, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).
The terms “administer,” “administering,” or “administration,” as used herein, refer to implanting, ingesting, injecting, inhaling, or otherwise absorbing a compound or composition, regardless of form. For example, the methods disclosed herein include administration of an effective amount of a compound or composition to achieve the desired or stated effect.
The terms “treat”, “treating,” or “treatment,” as used herein, refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder (e.g., cancer) refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present invention. In some aspects, treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average/mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject's symptoms prior to treatment.
As used herein, the term “treating cancer” means causing a partial or complete decrease in the rate of growth of a tumor, and/or in the size of the tumor and/or in the rate of local or distant tumor metastasis, and/or the overall tumor burden in a subject, and/or any decrease in tumor survival, in the presence of a degrader/disruptor (e.g., an ENL degrader/disruptor) described herein.
The terms “prevent,” “preventing,” and “prevention,” as used herein, shall refer to a decrease in the occurrence of a disease or decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that the occurrence of the disease or pathological cells in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the present invention. Exemplary ENL-mediated diseases that can be treated with ENL degraders/disruptors include acute leukemia, mixed lineage leukemia (MLL)-rearranged leukemias, Wilms' tumor and other diseases that are dependent on ENL.
As used herein, the term “preventing a disease” (e.g., preventing cancer) in a subject means for example, to stop the development of one or more symptoms of a disease in a subject before they occur or are detectable, e.g., by the patient or the patient's doctor. Preferably, the disease (e.g., cancer) does not develop at all, i.e., no symptoms of the disease are detectable. However, it can also mean delaying or slowing of the development of one or more symptoms of the disease. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.
Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.
An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. Moreover, treatment of a subject with a therapeutically effective amount of the compounds or compositions described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.
Following administration, the subject can be evaluated to detect, assess, or determine their level of disease. In some instances, treatment can continue until a change (e.g., reduction) in the level of disease in the subject is detected. Upon improvement of a patient's condition (e.g., a change (e.g., decrease) in the level of disease in the subject), a maintenance dose of a compound, or composition disclosed herein can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, e.g., as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
The ENL degraders/disruptors disclosed herein include pure enantiomers, mixtures of enantiomers, pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates, mixtures of diastereoisomeric racemates and the meso-form and pharmaceutically acceptable salts, solvent complexes, morphological forms, or deuterated and fluoro derivatives thereof.
The following Examples describe the synthesis of exemplary ENL degrader/disrupter compounds according to the present invention.
A solution of intermediate 1 (Moustakim et al., 2018b) (211 mg, 1 mmol) and Methyl 3-pyrrolidinylacetate hydrochloride (198 mg, 1.1 mmol) in 5 mL of DMF was treated with K2CO3 (276 mg, 2 mmol). The resulting mixture was stirred overnight at RT. After the reaction was completed, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The resulting residue was purified by silica gel flash chromatography to give the compound as yellow oil (222 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.58 (d, J=2.2 Hz, 1H), 8.24 (dd, J=8.9, 2.2 Hz, 1H), 7.77 (d, J=9.0 Hz, 1H), 4.84 (d, J=1.3 Hz, 2H), 3.98-3.86 (m, 1H), 3.77-3.62 (m, 5H), 3.43-3.34 (m, 1H), 2.94-2.84 (m, 1H), 2.71-2.60 (m, 2H), 2.46-2.37 (m, 1H), 1.94-1.84 (m, 1H). MS (ESI): m/z 319.2 [M+H]+.
10% Pd on carbon (20 mg) was added to a solution of intermediate 2 (220 mg, 0.69 mmol) in MeOH, and the mixture was stirred under H2 atmosphere overnight. The catalyst was removed by filtration through a pad of celite, the solvent was removed in vacuo and the residue was used in next step without further purification. The obtained intermediate was dissolved in dichloromethane and treated with 1-Methyl-1H-indazole-5-carboxylic acid (121 mg, 0.69 mmol), HATU (293 mg, 0.76 mmol) and DIEA (155 μL, 1.1 mmol). After being stirring 1 h at room temperature, the reaction mixture was washed with brine, dried and concentrated. The resulting residue was purified by silica gel flash chromatography to give the compound as yellow solid (223 mg, 72% for two steps). 1H NMR (600 MHz, Methanol-d4) δ 8.48 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.19 (s, 1H), 8.06 (dd, J=8.8, 1.7 Hz, 1H), 7.72-7.66 (m, 2H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 4.70 (s, 2H), 4.15 (s, 3H), 3.85-3.79 (m, 1H), 3.71 (s, 3H), 3.64-3.54 (m, 2H), 3.25 (t, J=10.3 Hz, 1H), 2.90-2.82 (m, 1H), 2.69-2.58 (m, 2H), 2.43-2.34 (m, 1H), 1.89-1.80 (m, 1H). MS (ESI): m/z 447.3 [M+H]+.
To a solution of intermediate 3 (300 mg, 0.67 mmol) in 5 mL MeOH, 5 mL H2O, and 5 mL THF, LiOH (30 mg, 1 mmol) was added. The mixture was stirred at RT overnight. Then the mixture was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 4 as white solid in TFA salt form (486 mg, 89%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (dd, J=1.7, 0.8 Hz, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.17 (d, J=0.9 Hz, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.66 (m, 2H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 4.76 (s, 2H), 4.13 (s, 3H), 3.83 (dd, J=11.5, 8.1 Hz, 1H), 3.66-3.55 (m, 2H), 3.29 (dd, J=11.5, 8.8 Hz, 1H), 2.90-2.81 (m, 1H), 2.65-2.55 (m, 2H), 2.43-2.36 (m, 1H), 1.91-1.82 (m, 1H). MS (ESI): m/z 433.4 [M+H]+.
To a solution of Intermediate 4 (12 mg, 0.02 mmol) in DMSO (1 mL) were added (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11.4 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ076-46 as white solid in TFA salt form (19.3 mg, 81%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.44 (s, 1H), 8.30-8.24 (m, 1H), 8.03 (dd, J=8.9, 1.6 Hz, 1H), 7.68 (d, J=8.8 Hz, 1H), 7.66-7.61 (m, 1H), 7.53-7.50 (m, 1H), 7.46-7.37 (m, 4H), 4.73-4.64 (m, 2H), 4.60-4.49 (m, 4H), 4.41-4.30 (m, 1H), 4.13 (s, 3H), 4.07-3.95 (m, 1H), 3.95-3.87 (m, 1H), 3.84-3.77 (m, 1H), 3.77-3.68 (m, 1H), 3.67-3.50 (m, 3H), 3.29-3.22 (m, 1H), 2.87-2.77 (m, 1H), 2.61-2.50 (m, 3H), 2.45 (d, J=7.8 Hz, 3H), 2.43-2.40 (m, 1H), 2.39-2.20 (m, 3H), 2.14-2.06 (m, 2H), 1.91-1.78 (m, 1H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C49H60N11O7S+ 946.4392, found 946.4385.
LQ076-47 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(3-(2-aminoethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-47 was obtained as white solid in TFA salt form (20.2 mg, 85%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.47-8.45 (m, 1H), 8.34-8.33 (m, 1H), 8.19-8.17 (m, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.69 (dd, J=8.8, 2.0 Hz, 2H), 7.61-7.59 (m, 1H), 7.47-7.40 (m, 4H), 4.77 (s, 2H), 4.68-4.66 (m, 1H), 4.63-4.57 (m, 1H), 4.53-4.47 (m, 2H), 4.42-4.38 (m, 1H), 4.14 (s, 3H), 3.91 (d, J=11.0 Hz, 1H), 3.82 (dd, J=11.0, 3.8 Hz, 1H), 3.78-3.69 (m, 3H), 3.65-3.51 (m, 4H), 3.31-3.26 (m, 1H), 2.86-2.78 (m, 1H), 2.57-2.41 (m, 11H), 2.38-2.30 (m, 1H), 2.28-2.23 (m, 1H), 2.12-2.06 (m, 1H), 1.89-1.81 (m, 1H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C50H62N11O7S+ 960.4549, found 960.4576.
LQ076-48 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (13 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-48 was obtained as white solid in TFA salt form (21.1 mg, 88%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 8.46 (s, 1H), 8.37-8.33 (m, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.66 (m, 2H), 7.62-7.59 (m, 1H), 7.47-7.37 (m, 4H), 4.81-4.74 (m, 3H), 4.64-4.56 (m, 1H), 4.54-4.47 (m, 2H), 4.39 (d, J=15.3 Hz, 1H), 4.13 (s, 3H), 4.05-3.97 (m, 2H), 3.91-3.80 (m, 2H), 3.76-3.48 (m, 11H), 3.30-3.24 (m, 1H), 2.87-2.79 (m, 1H), 2.55-2.40 (m, 5H), 2.35-2.25 (m, 2H), 2.12-2.06 (m, 1H), 1.90-1.79 (m, 1H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O8S+ 990.4655, found 990.4740.
LQ076-49 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (16.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-49 was obtained as white solid in TFA salt form (17.2 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.46 (d, J=1.4 Hz, 1H), 8.34 (s, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.67 (m, 2H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 7.48-7.40 (m, 4H), 4.78 (s, 2H), 4.67-4.65 (m, 1H), 4.60-4.56 (m, 1H), 4.54-4.49 (m, 2H), 4.40-4.35 (m, 1H), 4.13 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.83-3.70 (m, 4H), 3.67-3.51 (m, 9H), 3.38-3.34 (m, 2H), 3.31-3.27 (m, 1H), 2.87-2.80 (m, 1H), 2.60-2.55 (m, 1H), 2.52-2.40 (m, 6H), 2.38-2.32 (m, 1H), 2.27-2.21 (m, 1H), 2.12-2.05 (m, 1H), 1.90-1.82 (m, 1H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C52H66N11O8S+ 1004.4811, found 1004.4790.
LQ076-50 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-50 was obtained as white solid in TFA salt form (18.9 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.46 (d, J=1.5 Hz, 1H), 8.36 (d, J=2.0 Hz, 1H), 8.17 (d, J=0.9 Hz, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.66 (m, 2H), 7.63-7.61 (m, 1H), 7.48-7.41 (m, 4H), 4.80 (s, 2H), 4.68 (d, J=4.4 Hz, 1H), 4.61-4.57 (m, 1H), 4.55-4.49 (m, 2H), 4.40-4.34 (m, 1H), 4.13 (s, 3H), 4.08-4.04 (m, 2H), 3.90-3.86 (m, 1H), 3.82-3.75 (m, 2H), 3.72-3.54 (m, 11H), 3.51 (t, J=5.4 Hz, 2H), 3.37-3.34 (m, 2H), 2.87-2.81 (m, 1H), 2.51-2.46 (m, 4H), 2.42 (dd, J=14.9, 8.0 Hz, 1H), 2.38-2.31 (m, 1H), 2.27-2.22 (m, 1H), 2.12-2.06 (m, 1H), 1.89-1.82 (m, 1H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O9S+ 1034.4917, found 1034.4932.
LQ076-51 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-1-amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-51 was obtained as white solid in TFA salt form (18.3 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 9.11 (s, 1H), 8.48-8.45 (m, 1H), 8.36 (d, J=2.0 Hz, 1H), 8.18 (d, J=0.9 Hz, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.73-7.66 (m, 2H), 7.63 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.41 (m, 4H), 4.80 (s, 2H), 4.66 (d, J=2.8 Hz, 1H), 4.62-4.56 (m, 1H), 4.55-4.49 (m, 2H), 4.40-4.35 (m, 1H), 4.13 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.83-3.69 (m, 4H), 3.67-3.56 (m, 10H), 3.53 (t, J=5.4 Hz, 2H), 3.39-3.35 (m, 2H), 3.31-3.29 (m, OH), 2.88-2.81 (m, 1H), 2.62-2.55 (m, 1H), 2.52-2.41 (m, 6H), 2.39-2.32 (m, 1H), 2.27-2.22 (m, 1H), 2.12-2.06 (m, 1H), 1.90-1.82 (m, 1H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H70N11O9S+ 1048.5073, found 1048.5066.
LQ076-52 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-52 was obtained as white solid in TFA salt form (17.9 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.09 (s, 1H), 8.48-8.46 (m, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.18 (d, J=0.9 Hz, 1H), 8.05 (dd, J=8.8, 1.6 Hz, 1H), 7.71-7.67 (m, 2H), 7.62 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.41 (m, 4H), 4.80 (s, 2H), 4.67-4.64 (m, 1H), 4.61-4.48 (m, 3H), 4.40-4.35 (m, 1H), 4.13 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.83-3.69 (m, 4H), 3.67-3.57 (m, 14H), 3.53 (t, J=5.4 Hz, 2H), 3.39-3.35 (m, 2H), 3.32-3.28 (m, 1H), 2.88-2.81 (m, 1H), 2.61-2.55 (m, 1H), 2.53-2.41 (m, 6H), 2.39-2.32 (m, 1H), 2.26-2.21 (m, 1H), 2.11-2.06 (m, 1H), 1.90-1.83 (m, 1H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C56H74N11O10S+ 1092.5335, found 1092.5349.
LQ076-53 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-1-amino-20-(tert-butyl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (18 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-53 was obtained as white solid in TFA salt form (17.7 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.13 (s, 1H), 8.48-8.45 (m, 1H), 8.37 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.73-7.67 (m, 2H), 7.63 (dd, J=8.8, 1.9 Hz, 1H), 7.50-7.42 (m, 4H), 4.81 (s, 2H), 4.66-4.64 (m, 1H), 4.62-4.50 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.13 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.82-3.69 (m, 4H), 3.66-3.56 (m, 18H), 3.53 (t, J=5.4 Hz, 2H), 3.39-3.35 (m, 2H), 3.32-3.30 (m, 1H), 2.88-2.81 (m, 1H), 2.61-2.55 (m, 1H), 2.53-2.42 (m, 6H), 2.39-2.33 (m, 1H), 2.26-2.21 (m, 1H), 2.12-2.06 (m, 1H), 1.90-1.83 (m, 1H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C58H78N11O11S+ 1136.5597, found 1136.5645.
LQ076-54 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(2-aminoacetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-54 was obtained as white solid in TFA salt form (18.5 mg, 82%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 8.47-8.44 (m, 1H), 8.36 (d, J=1.9 Hz, 1H), 8.18 (s, 1H), 8.06-8.02 (m, 1H), 7.71-7.67 (m, 2H), 7.63-7.60 (m, 1H), 7.46-7.39 (m, 4H), 4.79 (s, 2H), 4.61 (d, J=3.4 Hz, 1H), 4.59-4.55 (m, 1H), 4.52-4.46 (m, 2H), 4.37 (dd, J=15.5, 4.1 Hz, 1H), 4.13 (s, 3H), 3.94-3.83 (m, 3H), 3.81-3.75 (m, 2H), 3.69-3.62 (m, 1H), 3.60-3.54 (m, 1H), 3.40-3.35 (m, 1H), 2.91-2.84 (m, 1H), 2.61-2.57 (m, 1H), 2.52-2.46 (m, 4H), 2.41-2.35 (m, 1H), 2.26-2.21 (m, 1H), 2.11-2.05 (m, 1H), 1.97-1.89 (m, 1H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C47H56N11O6S+ 902.4130, found 902.4128.
LQ076-55 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-55 was obtained as white solid in TFA salt form (17.4 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (d, J=2.3 Hz, 1H), 8.46 (s, 1H), 8.34-8.31 (m, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.66 (m, 2H), 7.61-7.58 (m, 1H), 7.48-7.36 (m, 4H), 4.82-4.75 (m, 2H), 4.62 (d, J=2.4 Hz, 1H), 4.59-4.55 (m, 1H), 4.53-4.50 (m, 1H), 4.49-4.46 (m, 1H), 4.41-4.36 (m, 1H), 4.14 (s, 3H), 3.98-3.94 (m, 1H), 3.83-3.75 (m, 2H), 3.67-3.61 (m, 1H), 3.60-3.54 (m, 1H), 3.53-3.46 (m, 1H), 3.43-3.37 (m, 1H), 3.31-3.27 (m, 1H), 2.85-2.78 (m, 1H), 2.56-2.44 (m, 6H), 2.43-2.31 (m, 2H), 2.30-2.24 (m, 1H), 2.13-2.07 (m, 1H), 1.89-1.81 (m, 1H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C48H58N11O6S+ 916.4287, found 916.4319.
LQ076-56 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-56 was obtained as white solid in TFA salt form (16.9 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.07-9.05 (m, 1H), 8.47 (s, 1H), 8.33 (d, J=1.8 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.69 (dd, J=8.9, 5.1 Hz, 2H), 7.61-7.59 (m, 1H), 7.48-7.39 (m, 4H), 4.77 (s, 2H), 4.65 (d, J=18.6 Hz, 1H), 4.61-4.57 (m, 1H), 4.53-4.48 (m, 2H), 4.38 (dd, J=15.5, 4.5 Hz, 1H), 4.14 (s, 3H), 3.95-3.91 (m, 1H), 3.85-3.61 (m, 3H), 3.59-3.52 (m, 1H), 3.25-3.19 (m, 2H), 2.86-2.80 (m, 1H), 2.55-2.41 (m, 5H), 2.39-2.22 (m, 4H), 2.12-2.07 (m, 1H), 1.91-1.76 (m, 3H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C49H60N11O6S+ 930.4443, found 930.4528.
LQ076-57 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-57 was obtained as white solid in TFA salt form (16.2 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.47 (s, 1H), 8.33 (s, 1H), 8.18 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.71-7.67 (m, 2H), 7.61-7.58 (m, 1H), 7.49-7.40 (m, 4H), 4.77 (s, 2H), 4.64-4.49 (m, 4H), 4.38 (d, J=15.4 Hz, 1H), 4.14 (s, 3H), 3.91 (d, J=11.0 Hz, 1H), 3.83-3.75 (m, 2H), 3.67-3.54 (m, 2H), 3.32-3.27 (m, 1H), 3.22-3.16 (m, 2H), 2.87-2.81 (m, 1H), 2.51-2.46 (m, 4H), 2.44-2.21 (m, 6H), 2.12-2.06 (m, 1H), 1.88-1.81 (m, 1H), 1.66-1.58 (m, 2H), 1.55-1.49 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C50H62N11O6S+ 944.4600, found 944.4664.
LQ076-58 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(6-aminohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-58 was obtained as white solid in TFA salt form (18.3 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.47 (s, 1H), 8.33 (d, J=8.3 Hz, 1H), 8.18 (s, 1H), 8.07-8.03 (m, 1H), 7.70-7.67 (m, 2H), 7.59 (d, J=8.8 Hz, 1H), 7.49-7.40 (m, 4H), 4.75 (s, 2H), 4.64 (d, J=2.7 Hz, 1H), 4.62-4.57 (m, 1H), 4.55-4.49 (m, 2H), 4.38 (d, J=15.4 Hz, 1H), 4.14 (s, 3H), 3.92 (d, J=11.0 Hz, 1H), 3.83-3.80 (m, 1H), 3.76 (t, J=9.9 Hz, 1H), 3.67-3.60 (m, 1H), 3.59-3.53 (m, 1H), 3.30-3.25 (m, 1H), 3.17 (t, J=7.0 Hz, 2H), 2.85-2.78 (m, 1H), 2.51-2.46 (m, 4H), 2.44-2.21 (m, 5H), 2.12-2.07 (m, 1H), 1.88-1.80 (m, 1H), 1.66-1.59 (m, 2H), 1.55-1.48 (m, 2H), 1.38-1.31 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O6S+ 958.4756, found 958.4768.
LQ076-59 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(7-aminoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-59 was obtained as white solid in TFA salt form (19.2 mg, 80%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.47 (d, J=1.5 Hz, 1H), 8.32 (d, J=6.5 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.9, 1.7 Hz, 1H), 7.71-7.66 (m, 2H), 7.58 (dd, J=8.8, 1.9 Hz, 1H), 7.49-7.40 (m, 4H), 4.73 (s, 2H), 4.64 (d, J=1.7 Hz, 1H), 4.61-4.57 (m, 1H), 4.55-4.49 (m, 2H), 4.40-4.36 (m, 1H), 4.14 (s, 3H), 3.92 (d, J=10.9 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.78-3.73 (m, 1H), 3.66-3.60 (m, 1H), 3.59-3.54 (m, 1H), 3.29-3.25 (m, 1H), 3.17 (t, J=7.0 Hz, 2H), 2.85-2.79 (m, 1H), 2.51-2.46 (m, 4H), 2.44-2.21 (m, 5H), 2.12-2.06 (m, 1H), 1.88-1.81 (m, 1H), 1.64-1.57 (m, 2H), 1.52-1.47 (m, 2H), 1.38-1.31 (m, 4H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C52H66N11O6S+ 972.4913, found 972.4952.
LQ076-60 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(8-aminooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-60 was obtained as white solid in TFA salt form (18 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 8.47 (d, J=1.5 Hz, 1H), 8.33 (s, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.9, 1.7 Hz, 1H), 7.70-7.67 (m, 2H), 7.60 (dd, J=8.7, 1.9 Hz, 1H), 7.49-7.41 (m, 4H), 4.76 (s, 2H), 4.65-4.63 (m, 1H), 4.61-4.55 (m, 1H), 4.54-4.49 (m, 2H), 4.37 (d, J=15.5 Hz, 1H), 4.13 (s, 3H), 3.92 (d, J=11.0 Hz, 1H), 3.83-3.73 (m, 2H), 3.67-3.54 (m, 2H), 3.30-3.26 (m, 1H), 3.17 (t, J=7.1 Hz, 2H), 2.86-2.80 (m, 1H), 2.51-2.45 (m, 4H), 2.44-2.21 (m, 5H), 2.12-2.06 (m, 1H), 1.88-1.81 (m, 1H), 1.64-1.55 (m, 2H), 1.53-1.45 (m, 2H), 1.37-1.28 (m, 6H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O6S+ 986.5069, found 986.5115.
LQ076-61 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(9-aminononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-61 was obtained as white solid in TFA salt form (19.1 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.47 (s, 1H), 8.30 (s, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.65 (m, 2H), 7.57 (dd, J=8.8, 1.9 Hz, 1H), 7.49-7.40 (m, 4H), 4.72 (s, 2H), 4.66-4.63 (m, 1H), 4.61-4.57 (m, 1H), 4.56-4.49 (m, 2H), 4.37 (d, J=15.5 Hz, 1H), 4.13 (s, 3H), 3.94-3.89 (m, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.75 (t, J=9.9 Hz, 1H), 3.66-3.53 (m, 2H), 3.31-3.25 (m, 1H), 3.16 (t, J=7.1 Hz, 2H), 2.86-2.79 (m, 1H), 2.51-2.45 (m, 4H), 2.44-2.39 (m, 1H), 2.38-2.20 (m, 4H), 2.12-2.06 (m, 1H), 1.88-1.80 (m, 1H), 1.64-1.55 (m, 2H), 1.52-1.46 (m, 2H), 1.36-1.27 (m, 8H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C54H70N11O6S+ 1000.5226, found 1000.5241.
LQ076-62 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(10-aminodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (16.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-62 was obtained as white solid in TFA salt form (15.6 mg, 63%). 1H NMR (800 MHz, Methanol-d4) δ 8.96 (s, 1H), 8.47 (s, 1H), 8.30 (s, 1H), 8.19 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.58 (d, J=8.7 Hz, 1H), 7.49-7.41 (m, 4H), 4.73 (s, 2H), 4.66-4.64 (m, 1H), 4.62-4.58 (m, 1H), 4.56-4.50 (m, 2H), 4.38 (d, J=15.4 Hz, 1H), 4.14 (s, 3H), 3.92 (d, J=11.0 Hz, 1H), 3.82 (dd, J=10.9, 4.0 Hz, 1H), 3.78-3.74 (m, 1H), 3.66-3.54 (m, 2H), 3.31-3.26 (m, 1H), 3.17 (t, J=7.1 Hz, 2H), 2.86-2.80 (m, 1H), 2.51-2.46 (m, 4H), 2.44-2.40 (m, 1H), 2.38-2.33 (m, 1H), 2.32-2.28 (m, 1H), 2.27-2.21 (m, 2H), 2.13-2.08 (m, 1H), 1.88-1.82 (m, 1H), 1.64-1.56 (m, 2H), 1.52-1.46 (m, 2H), 1.36-1.26 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H72N11O6S+ 1014.5382, found 1014.5252.
LQ076-63 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (13 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-63 was obtained as white solid in TFA salt form (18.3 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.48-8.46 (m, 1H), 8.34 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.9, 1.7 Hz, 1H), 7.71-7.67 (m, 2H), 7.62 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.41 (m, 4H), 4.78 (s, 2H), 4.66-4.64 (m, 1H), 4.61-4.55 (m, 1H), 4.54-4.49 (m, 2H), 4.37 (d, J=15.6 Hz, 1H), 4.13 (s, 3H), 3.92 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 4.0 Hz, 1H), 3.79-3.74 (m, 1H), 3.68-3.54 (m, 2H), 3.31-3.27 (m, 1H), 3.16 (t, J=7.1 Hz, 2H), 2.86-2.80 (m, 1H), 2.51-2.46 (m, 4H), 2.44-2.20 (m, 5H), 2.12-2.06 (m, 1H), 1.89-1.81 (m, 1H), 1.64-1.55 (m, 2H), 1.52-1.44 (m, 2H), 1.35-1.25 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C56H74N11O6S+ 1028.5539, found 1028.5552.
LQ076-64 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-64 was obtained as yellow solid in TFA salt form (13.2 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (dd, J=1.7, 0.8 Hz, 1H), 8.26 (s, OH), 8.18 (d, J=0.9 Hz, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.68 (d, J=8.6 Hz, 1H), 7.66-7.63 (m, 1H), 7.55-7.51 (m, 2H), 7.06-7.01 (m, 2H), 5.08-5.03 (m, 1H), 4.68 (s, 2H), 4.14 (s, 3H), 3.76-3.70 (m, 1H), 3.70-3.64 (m, 2H), 3.62-3.49 (m, 4H), 3.47-3.36 (m, 4H), 3.29-3.21 (m, 1H), 2.91-2.65 (m, 4H), 2.54-2.42 (m, 1H), 2.42-2.36 (m, 1H), 2.34-2.26 (m, 1H), 2.16-2.08 (m, 1H), 1.89-1.77 (m, 1H). HRMS m/z [M+H]+ calcd for C40H43N10O7+ 775.3311, found 775.3346.
LQ076-65 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-65 was obtained as yellow solid in TFA salt form (14 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (dd, J=1.7, 0.9 Hz, 1H), 8.27 (d, J=2.0 Hz, 1H), 8.17 (s, 1H), 8.06-8.01 (m, 1H), 7.69-7.63 (m, 2H), 7.55 (dd, J=8.7, 2.0 Hz, 1H), 7.51 (dd, J=8.5, 7.1 Hz, 1H), 7.03 (dd, J=11.9, 7.8 Hz, 2H), 5.08-5.03 (m, 1H), 4.70 (s, 2H), 4.13 (s, 3H), 3.76-3.68 (m, 3H), 3.66-3.58 (m, 5H), 3.58-3.51 (m, 3H), 3.47 (t, J=5.2 Hz, 2H), 3.39-3.34 (m, 2H), 3.30-3.24 (m, 1H), 2.89-2.65 (m, 4H), 2.50-2.42 (m, 1H), 2.42-2.36 (m, 1H), 2.36-2.26 (m, 1H), 2.15-2.07 (m, 1H), 1.87-1.77 (m, 1H). HRMS m/z [M+H]+ calcd for C42H47N10O8+ 819.3573, found 819.3590.
LQ076-66 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-66 was obtained as yellow solid in TFA salt form (14.6 mg, 67%).
1H NMR (600 MHz, Methanol-d4) δ 8.47-8.44 (m, 1H), 8.27 (d, J=1.9 Hz, 1H), 8.17 (d, J=0.9 Hz, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.63 (m, 2H), 7.56 (dd, J=8.7, 2.0 Hz, 1H), 7.51 (dd, J=8.6, 7.1 Hz, 1H), 7.05-7.00 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.71 (s, 2H), 4.13 (s, 3H), 3.76-3.68 (m, 4H), 3.67-3.60 (m, 8H), 3.59-3.54 (m, 3H), 3.51 (t, J=5.4 Hz, 2H), 3.46 (t, J=5.2 Hz, 2H), 3.38-3.34 (m, 2H), 3.31-3.26 (m, 1H), 2.89-2.77 (m, 2H), 2.76-2.65 (m, 2H), 2.48 (dd, J=15.0, 6.1 Hz, 1H), 2.41 (dd, J=15.0, 7.9 Hz, 1H), 2.37-2.28 (m, 1H), 2.15-2.07 (m, 1H), 1.88-1.79 (m, 1H). HRMS m/z [M+H]+ calcd for C44H51N10O9+ 863.3835, found 863.3878.
LQ076-67 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((14-amino-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-67 was obtained as yellow solid in TFA salt form (15.6 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (dd, J=1.7, 0.8 Hz, 1H), 8.28 (d, J=2.0 Hz, 1H), 8.18 (d, J=0.9 Hz, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.63 (m, 2H), 7.56-7.50 (m, 2H), 7.06-7.02 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.69 (s, 2H), 4.14 (s, 3H), 3.79-3.69 (m, 3H), 3.67-3.58 (m, 11H), 3.58-3.50 (m, 5H), 3.47 (t, J=5.2 Hz, 2H), 3.40-3.34 (m, 2H), 3.31-3.24 (m, 1H), 2.90-2.66 (m, 4H), 2.50 (dd, J=15.0, 6.1 Hz, 1H), 2.42 (dd, J=15.0, 7.9 Hz, 1H), 2.38-2.29 (m, 1H), 2.15-2.08 (m, 1H), 1.89-1.79 (m, 1H). HRMS m/z [M+H]+ calcd for C46H55N10O10+ 907.4097, found 907.4127.
dioxopiperidin-3-yl)isoindoline-1,3-dione (12.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-68 was obtained as yellow solid in TFA salt form (15 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (d, J=1.4 Hz, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.9, 1.7 Hz, 1H), 7.71-7.66 (m, 2H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 7.51 (dd, J=8.6, 7.1 Hz, 1H), 7.04-7.00 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.80 (s, 2H), 4.13 (s, 3H), 3.72-3.49 (m, 23H), 3.45 (t, J=5.2 Hz, 2H), 3.40-3.34 (m, 2H), 3.32-3.26 (m, 1H), 2.89-2.82 (m, 2H), 2.77-2.67 (m, 2H), 2.50 (dd, J=15.0, 6.0 Hz, 1H), 2.42 (dd, J=15.0, 8.0 Hz, 1H), 2.38-2.31 (m, 1H), 2.14-2.09 (m, 1H), 1.89-1.82 (m, 1H). HRMS m/z [M+H]+ calcd for C48H59N10O11+ 951.4359, found 951.4397.
LQ076-69 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-69 was obtained as yellow solid in TFA salt form (13 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (d, J=1.7 Hz, 1H), 8.29-8.27 (m, 1H), 8.17 (s, 1H), 8.04 (dd, J=8.9, 1.7 Hz, 1H), 7.69-7.65 (m, 2H), 7.57-7.51 (m, 2H), 7.09 (dd, J=8.6, 6.7 Hz, 1H), 7.05-7.01 (m, 1H), 5.06-5.02 (m, 1H), 4.76-4.71 (m, 2H), 4.13 (s, 3H), 3.75-3.70 (m, 1H), 3.63-3.57 (m, 1H), 3.55-3.43 (m, 5H), 3.31-3.27 (m, 1H), 2.88-2.64 (m, 4H), 2.51-2.28 (m, 3H), 2.12-2.06 (m, 1H), 1.84-1.75 (m, 1H). HRMS m/z [M+H]+ calcd for C38H39N10O6+ 731.3049, found 731.3080.
LQ076-70 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((3-aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-70 was obtained as yellow solid in TFA salt form (14.5 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.33-8.30 (m, 1H), 8.19 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.71-7.65 (m, 2H), 7.58-7.54 (m, 1H), 7.53-7.49 (m, 1H), 7.02-6.99 (m, 2H), 5.05 (dd, J=12.4, 5.6 Hz, 1H), 4.77-4.73 (m, 2H), 4.14 (s, 3H), 3.76-3.70 (m, 1H), 3.68-3.62 (m, 1H), 3.59-3.52 (m, 1H), 3.37 (t, J=6.5 Hz, 2H), 3.34-3.33 (m, 2H), 3.32-3.30 (m, 1H), 2.88-2.80 (m, 2H), 2.77-2.67 (m, 2H), 2.54-2.42 (m, 3H), 2.38-2.32 (m, 1H), 2.13-2.07 (m, 1H), 1.86-1.80 (m, 3H). HRMS m/z [M+H]+ calcd for C39H41N10O6+ 745.3205, found 745.3233.
LQ076-71 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-71 was obtained as yellow solid in TFA salt form (13.6 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.45-8.43 (m, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.17 (s, 1H), 8.03 (dd, J=8.8, 1.7 Hz, 1H), 7.68-7.65 (m, 2H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.50 (dd, J=8.6, 7.1 Hz, 1H), 7.01-6.97 (m, 2H), 5.03 (dd, J=12.8, 5.5 Hz, 1H), 4.74 (s, 2H), 4.13 (s, 3H), 3.77-3.71 (m, 1H), 3.65-3.59 (m, 1H), 3.57-3.51 (m, 1H), 3.32-3.27 (m, 3H), 3.26-3.21 (m, 2H), 2.87-2.79 (m, 2H), 2.76-2.65 (m, 2H), 2.50-2.45 (m, 1H), 2.41 (dd, J=15.0, 7.9 Hz, 1H), 2.35-2.30 (m, 1H), 2.12-2.07 (m, 1H), 1.86-1.78 (m, 1H), 1.69-1.58 (m, 4H). HRMS m/z [M+H]+ calcd for C40H43N10O6+ 759.3362, found 759.3369.
LQ076-72 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-72 was obtained as yellow solid in TFA salt form (15.4 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 8.45-8.43 (m, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.17 (s, 1H), 8.03 (dd, J=8.8, 1.7 Hz, 1H), 7.68-7.65 (m, 2H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.50 (dd, J=8.6, 7.1 Hz, 1H), 7.01-6.97 (m, 2H), 5.03 (dd, J=12.8, 5.5 Hz, 1H), 4.74 (s, 2H), 4.13 (s, 3H), 3.77-3.71 (m, 1H), 3.65-3.59 (m, 1H), 3.57-3.51 (m, 1H), 3.32-3.27 (m, 3H), 3.26-3.21 (m, 2H), 2.87-2.79 (m, 2H), 2.76-2.65 (m, 2H), 2.50-2.45 (m, 1H), 2.41 (dd, J=15.0, 7.9 Hz, 1H), 2.35-2.30 (m, 1H), 2.12-2.07 (m, 1H), 1.86-1.78 (m, 1H), 1.69-1.58 (m, 4H). HRMS m/z [M+H]+ calcd for C41H45N10O6+ 773.3518, found 773.3555.
LQ076-73 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((6-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (8.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-73 was obtained as yellow solid in TFA salt form (13.8 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.17 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.69-7.65 (m, 2H), 7.56 (dd, J=8.7, 2.0 Hz, 1H), 7.51 (dd, J=8.5, 7.1 Hz, 1H), 7.01-6.97 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.71 (s, 2H), 4.13 (s, 3H), 3.76-3.71 (m, 1H), 3.65-3.60 (m, 1H), 3.58-3.52 (m, 1H), 3.31-3.26 (m, 3H), 3.21-3.15 (m, 2H), 2.89-2.79 (m, 2H), 2.77-2.67 (m, 2H), 2.48 (dd, J=15.0, 6.0 Hz, 1H), 2.43-2.31 (m, 2H), 2.14-2.08 (m, 1H), 1.87-1.80 (m, 1H), 1.66-1.61 (m, 2H), 1.55-1.49 (m, 2H), 1.46-1.35 (m, 4H). HRMS m/z [M+H]+ calcd for C42H47N10O6+ 787.3675, found 787.3702.
LQ076-74 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((7-aminoheptyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-74 was obtained as yellow solid in TFA salt form (15.6 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (s, 1H), 8.32 (d, J=1.9 Hz, 1H), 8.17 (s, 1H), 8.03 (dd, J=8.9, 1.7 Hz, 1H), 7.68 (d, J=3.3 Hz, 1H), 7.67 (d, J=3.4 Hz, 1H), 7.58 (dd, J=8.8, 2.0 Hz, 1H), 7.51 (dd, J=8.6, 7.0 Hz, 1H), 7.01-6.97 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.74 (s, 2H), 4.13 (s, 3H), 3.76-3.71 (m, 1H), 3.65-3.60 (m, 1H), 3.58-3.53 (m, 1H), 3.30-3.24 (m, 3H), 3.20-3.13 (m, 2H), 2.89-2.80 (m, 2H), 2.77-2.67 (m, 2H), 2.48 (dd, J=15.0, 6.1 Hz, 1H), 2.40 (dd, J=15.0, 8.0 Hz, 1H), 2.37-2.31 (m, 1H), 2.13-2.08 (m, 1H), 1.88-1.81 (m, 1H), 1.66-1.60 (m, 2H), 1.52-1.47 (m, 2H), 1.43-1.32 (m, 6H). HRMS m/z [M+H]+ calcd for C43H49N10O6+ 801.3831, found 801.3872.
LQ076-75 was synthesized following the standard procedure for preparing LQ076-46 from intermediate 4 (12 mg, 0.02 mmol), 4-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.0 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-75 was obtained as yellow solid in TFA salt form (13.5 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.17 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.69-7.66 (m, 2H), 7.56 (dd, J=8.7, 1.9 Hz, 1H), 7.52 (dd, J=8.5, 7.1 Hz, 1H), 7.00 (dd, J=11.2, 7.8 Hz, 2H), 5.06 (dd, J=12.7, 5.4 Hz, 1H), 4.71 (s, 2H), 4.13 (s, 3H), 3.76-3.71 (m, 1H), 3.66-3.60 (m, 1H), 3.58-3.53 (m, 1H), 3.30-3.26 (m, 3H), 3.19-3.14 (m, 2H), 2.89-2.79 (m, 2H), 2.77-2.67 (m, 2H), 2.49 (dd, J=15.0, 6.1 Hz, 1H), 2.43-2.33 (m, 2H), 2.14-2.09 (m, 1H), 1.87-1.82 (m, 1H), 1.66-1.61 (m, 2H), 1.52-1.47 (m, 2H), 1.44-1.39 (m, 2H), 1.37-1.32 (m, 6H). HRMS m/z [M+H]+ calcd for C44H51N10O6+ 815.3988, found 815.4024.
Intermediate 5 was synthesized according to the procedures for the preparation of intermediate 2 as a yellow solid in 90% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.55 (d, J=2.2 Hz, 1H), 8.21 (dd, J=8.9, 2.2 Hz, 1H), 7.75 (d, J=8.9 Hz, 1H), 4.83 (s, 2H), 3.93-3.56 (m, 3H), 3.17-3.06 (m, 2H), 2.59-2.49 (m, 1H), 2.43-2.33 (m, 1H), 1.89-1.80 (m, 1H), 1.75-1.64 (m, 2H), 1.46-1.38 (m, 10H). MS (ESI): m/z 390.3 [M+H]+.
Intermediate 6 was synthesized according to the procedures for the preparation of intermediate 3 as a white solid in 76%. 1H NMR (600 MHz, Methanol-d4) δ 8.47 (s, 1H), 8.32 (d, J=2.0 Hz, 1H), 8.17 (d, J=0.9 Hz, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.66 (m, 2H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 4.78 (s, 2H), 4.13 (s, 3H), 3.77 (dd, J=11.4, 7.9 Hz, 1H), 3.66-3.57 (m, 2H), 3.22-3.16 (m, 1H), 3.14-3.07 (m, 2H), 2.56-2.47 (m, 1H), 2.40-2.32 (m, 1H), 1.86-1.78 (m, 1H), 1.72-1.65 (m, 2H), 1.43 (s, 9H). MS (ESI): m/z 518.3 [M+H]+.
Intermediate 6 (100 mg, 0.19 mmol) was dissolved in 1 mL DCM, to the resulting solution was added 1 mL TFA. After being stirred for 1 h at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 7 as white solid in TFA salt form (77 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (dd, J=1.7, 0.8 Hz, 1H), 8.33 (d, J=1.9 Hz, 1H), 8.17 (d, J=0.9 Hz, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.72-7.62 (m, 3H), 4.80 (s, 2H), 4.13 (s, 3H), 3.77 (dd, J=11.3, 7.9 Hz, 1H), 3.67-3.55 (m, 2H), 3.25-3.19 (m, 1H), 3.03-2.96 (m, 2H), 2.60-2.54 (m, 1H), 2.41-2.34 (m, 1H), 1.94-1.80 (m, 3H). MS (ESI): m/z 418.4 [M+H]+.
To a solution of Intermediate 7 (13 mg, 0.02 mmol) in DMSO (1 mL) were added 2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)acetic acid (10.9 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ076-76 as white solid in TFA salt form (20.6 mg, 88%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.47 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.8, 1.6 Hz, 1H), 7.71-7.66 (m, 2H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.48-7.44 (m, 2H), 7.43-7.40 (m, 2H), 4.75-4.71 (m, 3H), 4.61-4.57 (m, 1H), 4.54-4.49 (m, 2H), 4.38 (d, J=15.5 Hz, 1H), 4.15-4.05 (m, 6H), 3.93-3.89 (m, 1H), 3.83 (dd, J=11.0, 3.7 Hz, 1H), 3.80-3.75 (m, 1H), 3.66-3.56 (m, 2H), 3.23-3.17 (m, 1H), 2.54-2.46 (m, 4H), 2.39-2.33 (m, 1H), 2.29-2.23 (m, 1H), 2.13-2.08 (m, 1H), 1.84-1.73 (m, 3H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C49H60N11O7S+ 946.4392, found 946.4428.
LQ076-77 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 3-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)propanoic acid (11.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-77 was obtained as white solid in TFA salt form (20.4 mg, 85%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.46 (s, 1H), 8.34-8.32 (m, 1H), 8.18 (s, 1H), 8.04 (d, J=8.8 Hz, 1H), 7.70-7.67 (m, 2H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.47-7.44 (m, 2H), 7.43-7.39 (m, 2H), 4.76 (s, 2H), 4.65-4.63 (m, 1H), 4.61-4.56 (m, 1H), 4.54-4.49 (m, 2H), 4.38 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.91-3.86 (m, 1H), 3.82-3.67 (m, 5H), 3.64-3.58 (m, 2H), 3.28-3.16 (m, 3H), 2.58-2.42 (m, 9H), 2.38-2.32 (m, 1H), 2.27-2.22 (m, 1H), 2.12-2.06 (m, 1H), 1.85-1.79 (m, 1H), 1.73-1.68 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O7S+ 946.4705, found 974.4784.
LQ076-78 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(3-(4-(4-methylthiazol-5-yl)phenyl)propanoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)acetic acid (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-78 was obtained as white solid in TFA salt form (19.5 mg, 80%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.46 (s, 1H), 8.33 (s, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.70-7.66 (m, 2H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.47-7.40 (m, 4H), 4.75 (s, 2H), 4.72-4.69 (m, 1H), 4.61-4.56 (m, 2H), 4.52-4.48 (m, 1H), 4.47-4.42 (m, 1H), 4.15-4.00 (m, 6H), 3.88 (d, J=11.1 Hz, 1H), 3.83-3.69 (m, 6H), 3.65-3.56 (m, 2H), 3.32-3.27 (m, 2H), 3.22-3.15 (m, 1H), 2.52-2.44 (m, 5H), 2.38-2.31 (m, 1H), 2.29-2.23 (m, 1H), 2.11-2.05 (m, 1H), 1.84-1.69 (m, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O8S+ 990.4655, found 990.4723.
LQ076-79 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 3-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)propanoic acid (12.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-79 was obtained as white solid in TFA salt form (19.4 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.46 (s, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.72-7.67 (m, 2H), 7.62 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.40 (m, 4H), 4.79 (s, 2H), 4.66-4.64 (m, 1H), 4.62-4.57 (m, 1H), 4.55-4.49 (m, 2H), 4.40-4.35 (m, 1H), 4.13 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.82-3.69 (m, 6H), 3.67-3.56 (m, 6H), 3.30-3.18 (m, 3H), 2.60-2.40 (m, 8H), 2.38-2.31 (m, 1H), 2.27-2.21 (m, 1H), 2.12-2.06 (m, 1H), 1.86-1.78 (m, 1H), 1.73-1.68 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O8S+ 1018.4968, found 1018.5060.
LQ076-80 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecanoic acid (12.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-80 was obtained as white solid in TFA salt form (18 mg, 71%). 1H NMR (800 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.47 (s, 1H), 8.32 (s, 1H), 8.19 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.71-7.67 (m, 2H), 7.57 (d, J=8.7 Hz, 1H), 7.48-7.41 (m, 4H), 4.74-4.68 (m, 3H), 4.60-4.50 (m, 3H), 4.37 (d, J=15.3 Hz, 1H), 4.15 (s, 3H), 4.11-4.03 (m, 2H), 4.02-3.92 (m, 2H), 3.88 (d, J=11.1 Hz, 1H), 3.82-3.57 (m, 12H), 3.32-3.26 (m, 2H), 3.22-3.16 (m, 1H), 2.50-2.45 (m, 4H), 2.38-2.33 (m, 1H), 2.25 (dd, J=13.2, 7.6 Hz, 1H), 2.13-2.08 (m, 1H), 1.84-1.79 (m, 1H), 1.75-1.71 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O9S+ 1034.4917, found 1034.4890.
LQ076-81 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), (S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoic acid (13.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-81 was obtained as white solid in TFA salt form (18.4 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.47 (s, 1H), 8.34 (d, J=1.9 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.9, 1.7 Hz, 1H), 7.71-7.67 (m, 2H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.40 (m, 4H), 4.79 (s, 2H), 4.66-4.64 (m, 1H), 4.61-4.56 (m, 1H), 4.55-4.49 (m, 2H), 4.37 (d, J=15.5 Hz, 1H), 4.13 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.82-3.69 (m, 6H), 3.66-3.56 (m, 11H), 3.29-3.17 (m, 3H), 2.60-2.55 (m, 1H), 2.53-2.42 (m, 6H), 2.38-2.32 (m, 1H), 2.24 (dd, J=13.3, 7.7 Hz, 1H), 2.12-2.06 (m, 1H), 1.85-1.78 (m, 1H), 1.73-1.68 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H72N11O9S+ 1062.5230, found 1062.5310.
LQ076-82 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), (S)-18-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-19,19-dimethyl-16-oxo-4,7,10,13-tetraoxa-17-azaicosanoic acid (14.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL).
LQ076-82 was obtained as white solid in TFA salt form (17.2 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 8.47 (s, 1H), 8.35 (s, 1H), 8.19 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.72-7.68 (m, 2H), 7.60 (dd, J=8.7, 2.0 Hz, 1H), 7.50-7.41 (m, 4H), 4.77 (s, 2H), 4.66-4.64 (m, 1H), 4.60-4.49 (m, 3H), 4.38 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.83-3.69 (m, 6H), 3.66-3.57 (m, 15H), 3.29-3.17 (m, 3H), 2.60-2.55 (m, 1H), 2.52-2.42 (m, 5H), 2.39-2.33 (m, 1H), 2.26-2.21 (m, 1H), 2.12-2.06 (m, 1H), 1.86-1.79 (m, 1H), 1.74-1.69 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C57H76N11O10S+ 1106.5492, found 1106.5516.
LQ076-83 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), (S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosanoic acid (15.0 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-83 was obtained as white solid in TFA salt form (18.9 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 8.46 (s, 1H), 8.33 (s, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.67 (m, 2H), 7.59 (dd, J=8.7, 2.0 Hz, 1H), 7.48-7.40 (m, 4H), 4.75 (s, 2H), 4.69-4.67 (m, 1H), 4.62-4.57 (m, 1H), 4.56-4.49 (m, 2H), 4.37 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 4.06-4.03 (m, 2H), 3.99-3.96 (m, 2H), 3.91-3.87 (m, 1H), 3.83-3.76 (m, 2H), 3.71-3.58 (m, 18H), 3.32-3.27 (m, 2H), 3.23-3.17 (m, 1H), 2.52-2.45 (m, 4H), 2.40-2.32 (m, 1H), 2.27-2.22 (m, 1H), 2.12-2.07 (m, 1H), 1.86-1.80 (m, 1H), 1.77-1.72 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C57H76N11O11S+ 1122.5441, found 1122.5517.
LQ076-84 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), (S)-21-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-22,22-dimethyl-19-oxo-4,7,10,13,16-pentaoxa-20-azatricosanoic acid (15.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-84 was obtained as white solid in TFA salt form (20 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.02 (s, 1H), 8.47 (s, 1H), 8.34-8.31 (m, 1H), 8.19 (s, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.71-7.67 (m, 2H), 7.60-7.57 (m, 1H), 7.49-7.41 (m, 4H), 4.75 (s, 2H), 4.66-4.64 (m, 1H), 4.61-4.49 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.82-3.56 (m, 25H), 3.30-3.17 (m, 3H), 2.60-2.41 (m, 7H), 2.39-2.32 (m, 1H), 2.26-2.21 (m, 1H), 2.12-2.06 (m, 1H), 1.85-1.78 (m, 1H), 1.74-1.69 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C59H80N11O11S+ 1150.5754, found 1150.5834.
LQ076-85 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (10.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-85 was obtained as white solid in TFA salt form (17.8 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.47 (s, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.71-7.67 (m, 2H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 7.48-7.40 (m, 4H), 4.76 (s, 2H), 4.61-4.47 (m, 4H), 4.40-4.35 (m, 1H), 4.14 (s, 3H), 3.93-3.88 (m, 1H), 3.83-3.75 (m, 2H), 3.66-3.56 (m, 2H), 3.30-3.17 (m, 3H), 2.66-2.46 (m, 8H), 2.35 (d, 1H), 2.26-2.20 (m, 1H), 2.12-2.06 (m, 1H), 1.85-1.77 (m, 1H), 1.73-1.67 (m, 2H), 1.08-1.03 (m, 9H). HRMS m/z [M+H]+ calcd for C49H60N11O6S+ 930.4443, found 930.4498.
LQ076-86 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (11.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-86 was obtained as white solid in TFA salt form (18.6 mg, 79%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.47 (s, 1H), 8.31 (s, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.8, 1.6 Hz, 1H), 7.71-7.66 (m, 2H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.48-7.39 (m, 4H), 4.74 (s, 2H), 4.64-4.56 (m, 2H), 4.54-4.49 (m, 2H), 4.38 (d, J=15.7 Hz, 1H), 4.14 (s, 3H), 3.94 (d, J=10.9 Hz, 1H), 3.84-3.74 (m, 2H), 3.68-3.56 (m, 2H), 3.29-3.15 (m, 3H), 2.50-2.46 (m, 4H), 2.38-2.19 (m, 6H), 2.12-2.07 (m, 1H), 1.94-1.86 (m, 2H), 1.84-1.77 (m, 1H), 1.74-1.66 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C50H62N11O6S+ 944.4600, found 944.4639.
LQ076-87 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (11.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-87 was obtained as white solid in TFA salt form (19.4 mg, 82%). 1H NMR (600 MHz, Methanol-d4) δ 9.11 (s, 1H), 8.46 (s, 1H), 8.36 (s, 1H), 8.17 (s, 1H), 8.06-8.03 (m, 1H), 7.72-7.66 (m, 2H), 7.63 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.40 (m, 4H), 4.81 (s, 2H), 4.63-4.48 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 4.13 (s, 3H), 3.90 (dd, J=11.1, 4.7 Hz, 1H), 3.81-3.75 (m, 2H), 3.67-3.59 (m, 2H), 3.29-3.18 (m, 3H), 2.54-2.47 (m, 4H), 2.39-2.16 (m, 7H), 2.12-2.06 (m, 1H), 1.86-1.79 (m, 1H), 1.74-1.56 (m, 6H), 1.08-1.02 (m, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O6S+ 958.4756, found 958.4833.
LQ076-88 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoic acid (11.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-88 was obtained as white solid in TFA salt form (19.8 mg, 82%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 8.47 (s, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.67 (m, 2H), 7.59 (dd, J=8.7, 2.0 Hz, 1H), 7.49-7.40 (m, 4H), 4.76 (s, 2H), 4.65-4.62 (m, 1H), 4.61-4.49 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.90 (d, J=11.0 Hz, 1H), 3.83-3.74 (m, 2H), 3.66-3.58 (m, 2H), 3.27-3.17 (m, 3H), 2.52-2.45 (m, 4H), 2.39-2.21 (m, 4H), 2.20-2.15 (m, 2H), 2.12-2.07 (m, 1H), 1.86-1.78 (m, 1H), 1.74-1.67 (m, 2H), 1.65-1.58 (m, 4H), 1.37-1.32 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C52H66N11O6S+ 972.4913, found 972.4950.
LQ076-89 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctanoic acid (12.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-89 was obtained as white solid in TFA salt form (21.1 mg, 87%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.47 (s, 1H), 8.35-8.33 (m, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.9, 1.6 Hz, 1H), 7.69 (dd, J=8.7, 2.3 Hz, 2H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.41 (m, 4H), 4.76 (s, 2H), 4.63 (s, 1H), 4.61-4.49 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.93-3.89 (m, 1H), 3.83-3.75 (m, 2H), 3.66-3.57 (m, 2H), 3.28-3.16 (m, 3H), 2.52-2.44 (m, 4H), 2.39-2.20 (m, 4H), 2.20-2.15 (m, 2H), 2.12-2.06 (m, 1H), 1.86-1.78 (m, 1H), 1.74-1.67 (m, 2H), 1.64-1.56 (m, 4H), 1.38-1.32 (m, 4H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O6S+ 986.5069, found 986.5139.
LQ076-90 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoic acid (12.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-90 was obtained as white solid in TFA salt form (18.7 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.47 (s, 1H), 8.31 (s, 1H), 8.18 (s, 1H), 8.05 (dd, J=8.9, 1.6 Hz, 1H), 7.70-7.66 (m, 2H), 7.58 (dd, J=8.8, 1.9 Hz, 1H), 7.49-7.41 (m, 4H), 4.73 (s, 2H), 4.65-4.62 (m, 1H), 4.61-4.49 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.91 (d, J=10.9 Hz, 1H), 3.82-3.74 (m, 2H), 3.66-3.56 (m, 2H), 3.28-3.16 (m, 3H), 2.51-2.44 (m, 4H), 2.38-2.14 (m, 8H), 2.12-2.06 (m, 1H), 1.85-1.78 (m, 1H), 1.74-1.67 (m, 2H), 1.63-1.55 (m, 4H), 1.36-1.32 (m, 4H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C54H70N11O6S+ 1000.5226, found 1000.5303.
LQ076-91 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 10-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecanoic acid (12.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-91 was obtained as white solid in TFA salt form (18.8 mg, 76%). 1H NMR (600 MHz, Methanol-d/4) δ 8.99 (s, 1H), 8.47 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.18 (d, J=0.9 Hz, 1H), 8.05 (dd, J=8.9, 1.7 Hz, 1H), 7.71-7.66 (m, 2H), 7.57 (dd, J=8.8, 1.9 Hz, 1H), 7.50-7.46 (m, 2H), 7.45-7.41 (m, 2H), 4.73 (s, 2H), 4.66-4.63 (m, 1H), 4.61-4.50 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.91 (d, J=11.0 Hz, 1H), 3.83-3.74 (m, 2H), 3.66-3.56 (m, 2H), 3.26-3.16 (m, 3H), 2.51-2.45 (m, 4H), 2.38-2.14 (m, 7H), 2.12-2.06 (m, 1H), 1.85-1.78 (m, 1H), 1.73-1.67 (m, 2H), 1.64-1.55 (N, 4H), 1.34-1.29 (m, 7H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C55H72N11O6S+ 1014.5382, found 1014.5464.
LQ076-92 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 11-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoic acid (13 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-92 was obtained as white solid in TFA salt form (20 mg, 79%). 1H NMR (600 MHz, Methanol-d4) δ 9.09 (s, 1H), 8.47 (s, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.19 (s, 1H), 8.05 (dd, J=8.9, 1.7 Hz, 1H), 7.72-7.68 (m, 2H), 7.61 (dd, J=8.7, 1.9 Hz, 1H), 7.50-7.42 (m, 4H), 4.77 (s, 2H), 4.65-4.63 (m, 1H), 4.61-4.49 (m, 3H), 4.38 (d, J=15.5 Hz, 1H), 4.14 (s, 3H), 3.91 (d, J=11.0 Hz, 1H), 3.83-3.74 (m, 2H), 3.66-3.57 (m, 2H), 3.27-3.17 (m, 3H), 2.52-2.45 (m, 4H), 2.38-2.15 (m, 7H), 2.12-2.06 (m, 1H), 1.86-1.78 (m, 1H), 1.73-1.68 (m, 2H), 1.63-1.54 (m, 4H), 1.31-1.28 (m, 9H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C56H74N11O6S+ 1028.5539, found 1028.5597.
LQ076-93 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine (6.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-93 was obtained as yellow solid in TFA salt form (12 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.31-8.29 (m, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.9, 1.6 Hz, 1H), 7.71-7.66 (m, 2H), 7.59-7.55 (m, 2H), 7.11 (d, J=7.1 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 5.08 (dd, J=12.6, 5.4 Hz, 1H), 4.72 (s, 2H), 4.14 (s, 3H), 4.00 (s, 2H), 3.78-3.73 (m, 1H), 3.64-3.54 (m, 2H), 3.31-3.24 (m, 2H), 3.21-3.15 (m, 1H), 2.90-2.83 (m, 1H), 2.78-2.67 (m, 2H), 2.47-2.41 (m, 1H), 2.35-2.28 (m, 1H), 2.14-2.08 (m, 1H), 1.83-1.76 (m, 1H), 1.72-1.67 (m, 2H). HRMS m/z [M+H]+ calcd for C38H39N10O6+ 731.3049, found 731.3090.
LQ076-94 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanoic acid (7.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-94 was obtained as yellow solid in TFA salt form (13.2 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.9, 1.7 Hz, 1H), 7.71-7.67 (m, 2H), 7.58-7.54 (m, 2H), 7.10 (d, J=8.6 Hz, 1H), 7.05 (d, J=7.0 Hz, 1H), 5.04 (dd, J=12.5, 5.6 Hz, 1H), 4.73 (s, 2H), 4.14 (s, 3H), 3.75-3.50 (m, 5H), 3.29-3.14 (m, 3H), 2.87-2.78 (m, 1H), 2.75-2.66 (m, 2H), 2.56-2.50 (m, 2H), 2.46-2.39 (m, 1H), 2.32-2.25 (m, 1H), 2.12-2.07 (m, 1H), 1.78-1.71 (m, 1H), 1.65-1.59 (m, 2H). HRMS m/z [M+H]+ calcd for C39H41N10O6+ 745.3205, found 745.3248.
LQ076-95 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoic acid (7.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-95 was obtained as yellow solid in TFA salt form (15.6 mg, 79%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.33 (d, J=1.9 Hz, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.9, 1.6 Hz, 1H), 7.71-7.67 (m, 2H), 7.58 (dd, J=8.7, 1.9 Hz, 1H), 7.53 (dd, J=8.6, 7.0 Hz, 1H), 7.06-7.01 (m, 2H), 5.05 (dd, J=12.7, 5.5 Hz, 1H), 4.74 (s, 2H), 4.14 (s, 3H), 3.77-3.72 (m, 1H), 3.63-3.55 (m, 2H), 3.37-3.34 (m, 2H), 3.24-3.16 (m, 3H), 2.89-2.82 (m, 1H), 2.77-2.67 (m, 2H), 2.49-2.43 (m, 1H), 2.36-2.28 (m, 3H), 2.13-2.08 (m, 1H), 1.97-1.91 (m, 2H), 1.83-1.77 (m, 1H), 1.69-1.65 (m, 2H). HRMS m/z [M+H]+ calcd for C40H43N10O6+ 759.3362, found 759.3401.
LQ076-96 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentanoic acid (7.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-96 was obtained as yellow solid in TFA salt form (15.4 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.17 (s, 1H), 8.03 (dd, J=8.9, 1.7 Hz, 1H), 7.69-7.65 (m, 2H), 7.56 (dd, J=8.7, 2.0 Hz, 1H), 7.52 (dd, J=8.6, 7.1 Hz, 1H), 7.03-7.01 (m, 1H), 7.01-6.99 (m, 1H), 5.03 (dd, J=12.8, 5.4 Hz, 1H), 4.71 (s, 2H), 4.13 (s, 3H), 3.76-3.72 (m, 1H), 3.63-3.54 (m, 2H), 3.31-3.29 (m, 2H), 3.25-3.15 (m, 3H), 2.87-2.80 (m, 1H), 2.76-2.65 (m, 2H), 2.49-2.43 (m, 1H), 2.35-2.29 (m, 1H), 2.23 (t, J=7.2 Hz, 2H), 2.11-2.06 (m, 1H), 1.83-1.77 (m, 1H), 1.74-1.62 (m, 6H). HRMS m/z [M+H]+ calcd for C41H45N10O6+ 773.3518, found 773.3530.
LQ076-97 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoic acid (7.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-97 was obtained as yellow solid in TFA salt form (14.9 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (s, 1H), 8.29 (d, J=1.9 Hz, 1H), 8.17 (d, J=0.9 Hz, 1H), 8.03 (dd, J=8.8, 1.7 Hz, 1H), 7.69-7.64 (m, 2H), 7.55 (dd, J=8.7, 2.0 Hz, 1H), 7.50 (dd, J=8.5, 7.1 Hz, 1H), 7.02-6.97 (m, 2H), 5.05 (dd, J=12.5, 5.7 Hz, 1H), 4.70 (s, 2H), 4.13 (s, 3H), 3.77-3.71 (m, 1H), 3.64-3.53 (m, 2H), 3.29 (t, J=6.9 Hz, 2H), 3.25-3.20 (m, 2H), 3.20-3.13 (m, 1H), 2.90-2.81 (m, 1H), 2.78-2.66 (m, 2H), 2.50-2.41 (m, 1H), 2.37-2.28 (m, 1H), 2.19 (t, J=7.4 Hz, 2H), 2.14-2.07 (m, 1H), 1.84-1.74 (m, 1H), 1.71-1.59 (m, 6H), 1.46-1.38 (m, 2H). HRMS m/z [M+H]+ calcd for C42H47N10O6+ 787.3675, found 787.3710.
LQ076-98 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptanoic acid (8.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-98 was obtained as yellow solid in TFA salt form (16.1 mg, 78%). 1H NMR (800 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.30 (s, 1H), 8.17 (s, 1H), 8.04 (d, J=8.8 Hz, 1H), 7.67 (dd, J=8.7, 3.8 Hz, 2H), 7.57 (d, J=8.6 Hz, 1H), 7.53-7.50 (m, 1H), 7.02-6.98 (m, 2H), 5.06 (dd, J=12.7, 5.6 Hz, 1H), 4.71 (s, 2H), 4.13 (s, 3H), 3.75 (t, J=9.9 Hz, 1H), 3.64-3.55 (m, 2H), 3.30-3.15 (m, 5H), 2.89-2.83 (m, 1H), 2.77-2.69 (m, 2H), 2.49-2.45 (m, 1H), 2.36-2.32 (m, 1H), 2.19-2.16 (m, 2H), 2.13-2.10 (m, 1H), 1.83-1.79 (m, 1H), 1.72-1.58 (m, 6H), 1.45-1.40 (m, 2H), 1.39-1.35 (m, 2H). HRMS m/z [M+H]+ calcd for C43H49N10O6+ 801.3831, found 801.3799.
LQ076-99 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoic acid (8.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-99 was obtained as yellow solid in TFA salt form (14.3 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.45 (s, 1H), 8.33 (d, J=1.6 Hz, 1H), 8.16 (s, 1H), 8.03 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.64 (m, 2H), 7.60 (dd, J=8.7, 2.0 Hz, 1H), 7.50 (dd, J=8.6, 7.1 Hz, 1H), 7.01-6.96 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.76 (s, 2H), 4.12 (s, 3H), 3.78-3.72 (m, 1H), 3.65-3.55 (m, 2H), 3.29-3.16 (m, 5H), 2.89-2.81 (m, 1H), 2.77-2.66 (m, 2H), 2.51-2.43 (m, 1H), 2.37-2.30 (m, 1H), 2.16 (t, J=7.5 Hz, 2H), 2.13-2.07 (m, 1H), 1.85-1.76 (m, 1H), 1.71-1.67 (m, 2H), 1.66-1.55 (m, 4H), 1.44-1.28 (m, 6H). HRMS m/z [M+H]+ calcd for C44H51N10O6+ 815.3988, found 815.4019.
LQ076-100 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoic acid (8.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-100 was obtained as yellow solid in TFA salt form (15.1 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.7 Hz, 1H), 7.70-7.66 (m, 2H), 7.56 (dd, J=8.7, 1.9 Hz, 1H), 7.54-7.50 (m, 1H), 7.07 (d, J=8.6 Hz, 1H), 7.02 (d, J=7.1 Hz, 1H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.70 (s, 2H), 4.14 (s, 3H), 3.77-3.66 (m, 5H), 3.61-3.52 (m, 2H), 3.47 (t, J=5.1 Hz, 2H), 3.24-3.11 (m, 3H), 2.89-2.82 (m, 1H), 2.78-2.66 (m, 2H), 2.45 (t, J=5.8 Hz, 3H), 2.32-2.25 (m, 1H), 2.14-2.09 (m, 1H), 1.79-1.72 (m, 1H), 1.65-1.60 (m, 2H). HRMS m/z [M+H]+ calcd for C41H45N10O7+ 789.3467, found 789.3511.
LQ076-101 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanoic acid (9.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-101 was obtained as yellow solid in TFA salt form (16.1 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.17 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.70-7.65 (m, 2H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.53-7.49 (m, 1H), 7.05-7.01 (m, 2H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.72 (s, 2H), 4.13 (s, 3H), 3.75-3.69 (m, 5H), 3.66-3.52 (m, 6H), 3.46 (t, J=5.2 Hz, 2H), 3.26-3.12 (m, 3H), 2.89-2.80 (m, 1H), 2.77-2.66 (m, 2H), 2.49-2.40 (m, 3H), 2.33-2.26 (m, 1H), 2.14-2.09 (m, 1H), 1.80-1.73 (m, 1H), 1.69-1.64 (m, 2H). HRMS m/z [M+H]+ calcd for C43H49N10O8+ 833.3729, found 833.3785.
LQ076-102 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoic acid (9.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-102 was obtained as yellow solid in TFA salt form (15.7 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.17 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.70-7.65 (m, 2H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.51 (dd, J=8.5, 7.1 Hz, 1H), 7.04-7.00 (m, 2H), 5.05 (dd, J=12.9, 5.5 Hz, 1H), 4.73 (s, 2H), 4.13 (s, 3H), 3.78-3.54 (m, 15H), 3.45 (t, J=5.1 Hz, 2H), 3.29-3.15 (m, 3H), 2.89-2.82 (m, 1H), 2.77-2.66 (m, 2H), 2.51-2.45 (m, 1H), 2.41 (t, J=6.0 Hz, 2H), 2.36-2.29 (m, 1H), 2.14-2.08 (m, 1H), 1.82-1.76 (m, 1H), 1.71-1.65 (m, 2H). HRMS m/z [M+H]+ calcd for C45H53N10O9+ 877.3991, found 877.4037.
LQ076-103 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecan-15-oic acid (10.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-103 was obtained as yellow solid in TFA salt form (16.2 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.32 (s, 1H), 8.18 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.70-7.66 (m, 2H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.54-7.48 (m, 1H), 7.05-7.00 (m, 2H), 5.05 (dd, J=12.8, 5.4 Hz, 1H), 4.74 (s, 2H), 4.13 (s, 3H), 3.75 (t, J=9.8 Hz, 1H), 3.72-3.68 (m, 4H), 3.66-3.55 (m, 14H), 3.45 (t, J=5.2 Hz, 2H), 3.28-3.15 (m, 3H), 2.89-2.82 (m, 1H), 2.77-2.67 (m, 2H), 2.49 (s, 1H), 2.41 (t, J=6.0 Hz, 2H), 2.36-2.30 (m, 1H), 2.14-2.08 (m, 1H), 1.83-1.76 (m, 1H), 1.71-1.66 (m, 2H). HRMS m/z [M+H]+ calcd for C47H57N10O10+ 921.4524, found 921.4546.
LQ076-104 was synthesized following the standard procedure for preparing LQ076-76 from intermediate 7 (13 mg, 0.02 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (11.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-104 was obtained as yellow solid in TFA salt form (16.7 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.36 (d, J=1.9 Hz, 1H), 8.17 (s, 1H), 8.04 (dd, J=8.8, 1.6 Hz, 1H), 7.72-7.64 (m, 3H), 7.62 (dd, J=8.7, 2.1 Hz, 1H), 7.53-7.47 (m, 1H), 7.04-7.00 (m, 2H), 5.05 (dd, J=12.9, 5.5 Hz, 1H), 4.80 (s, 2H), 4.13 (s, 3H), 3.83-3.75 (m, 1H), 3.73-3.67 (m, 4H), 3.67-3.51 (m, 18H), 3.45 (t, J=5.2 Hz, 2H), 3.29-3.17 (m, 3H), 2.90-2.82 (m, 1H), 2.78-2.66 (m, 2H), 2.55-2.47 (m, 1H), 2.42 (t, J=6.2 Hz, 2H), 2.38-2.31 (m, 1H), 2.15-2.09 (m, 1H), 1.86-1.77 (m, 1H), 1.74-1.67 (m, 2H). HRMS m/z [M+H]+ calcd for C49H61N10O11+ 965.4516, found 965.4554.
A solution of intermediate 8 (Moustakim et al., 2018b) (579 mg, 3.2 mmol) was dissolved in DMF and treated with 4-(Methoxycarbonyl)benzoic acid (740 mg, 3.2 mmol), HATU (1.4 g, 3.8 mmol) and DIEA (845 μL, 4.8 mmol). After being stirring 1 h at room temperature, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The resulting residue was purified by silica gel flash chromatography to give the compound as grey solid (880 mg, 54%).
1H NMR (600 MHz, Methanol-d4) δ 8.31 (d, J=2.0 Hz, 1H), 8.19-8.14 (m, 2H), 8.08-8.03 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.7, 2.0 Hz, 1H), 4.84 (d, J=14.6 Hz, 1H), 4.61 (d, J=14.6 Hz, 1H), 3.97 (s, 3H), 3.79-3.70 (m, 2H), 3.50-3.43 (m, 1H), 2.44-2.35 (m, 1H), 2.19-2.06 (m, 2H), 1.88-1.78 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). MS (ESI): m/z 393.3 [M+H]+.
Intermediate 10 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 88% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=1.9 Hz, 1H), 8.20-8.15 (m, 2H), 8.08-8.03 (m, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.57 (dd, J=8.8, 2.0 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.79-3.70 (m, 2H), 3.50-3.43 (m, 1H), 2.44-2.35 (m, 1H), 2.20-2.05 (m, 2H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). MS (ESI): m/z 379.3 [M+H]+.
To a solution of Intermediate 10 (10 mg, 0.02 mmol) in DMSO (1 mL) were added (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11.5 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ076-105 as white solid in TFA salt form (19.2 mg, 86%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 8.30 (d, J=1.6 Hz, 1H), 8.04-8.01 (m, 4H), 7.68 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.47-7.38 (m, 4H), 4.83 (d, J=14.6 Hz, 1H), 4.75-4.72 (m, 1H), 4.62-4.50 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 4.15-4.05 (m, 2H), 3.85-3.63 (m, 8H), 3.51-3.42 (m, 1H), 2.45 (s, 3H), 2.42-2.36 (m, 1H), 2.29-2.23 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C47H58N9O7S+ 892.4174, found 892.4202.
LQ076-106 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(3-(2-aminoethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-106 was obtained as white solid in TFA salt form (20 mg, 88%). 1H NMR (600 MHz, Methanol-d4) δ 9.13 (s, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.06-7.97 (m, 4H), 7.72 (d, J=8.8 Hz, 1H), 7.63 (dd, J=8.8, 2.0 Hz, 1H), 7.48-7.39 (m, 4H), 4.90 (d, J=14.7 Hz, 1H), 4.68-4.57 (m, 3H), 4.54-4.49 (m, 2H), 4.37 (d, J=15.6 Hz, 1H), 3.91 (d, J=11.0 Hz, 1H), 3.83-3.57 (m, 11H), 3.50-3.43 (m, 1H), 2.63-2.52 (m, 2H), 2.48 (s, 3H), 2.44-2.36 (m, 1H), 2.28-2.23 (m, 1H), 2.19-2.06 (m, 3H), 1.88-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C48H60N9O7S+ 906.4331, found 906.4353.
LQ076-107 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-107 was obtained as white solid in TFA salt form (18.1 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.32 (d, J=2.0 Hz, 1H), 8.07-7.93 (m, 4H), 7.70 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.7, 2.0 Hz, 1H), 7.47-7.38 (m, 4H), 4.85 (d, J=14.6 Hz, 1H), 4.78-4.72 (m, 1H), 4.64-4.48 (m, 4H), 4.39-4.31 (m, 1H), 4.08-3.98 (m, 2H), 3.92-3.81 (m, 2H), 3.78-3.55 (m, 11H), 3.50-3.44 (m, 1H), 2.48 (s, 3H), 2.43-2.37 (m, 1H), 2.30-2.24 (m, 1H), 2.20-2.07 (m, 3H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C49H62N9O8S+ 936.4437, found 936.4454.
LQ076-108 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (16.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-108 was obtained as white solid in TFA salt form (19.7 mg, 83%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.07-8.03 (m, 2H), 7.99-7.96 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.7, 2.0 Hz, 1H), 7.48-7.40 (m, 4H), 4.85 (d, J=14.6 Hz, 1H), 4.69-4.66 (m, 1H), 4.63-4.57 (m, 2H), 4.54-4.49 (m, 2H), 4.36 (d, J=15.5 Hz, 1H), 3.91 (d, J=11.0 Hz, 1H), 3.83-3.60 (m, 14H), 3.50-3.44 (m, 1H), 2.58-2.52 (m, 1H), 2.49 (s, 3H), 2.43-2.37 (m, 1H), 2.23 (d, J=13.1, 7.6 Hz, 1H), 2.18-2.06 (m, 3H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.6 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C50H64N9O8S+ 950.4593, found 950.4599.
LQ076-109 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-109 was obtained as white solid in TFA salt form (20.3 mg, 84%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 8.34 (d, J=1.9 Hz, 1H), 8.06-8.02 (m, 2H), 7.99-7.95 (m, 2H), 7.70 (d, J=8.6 Hz, 1H), 7.60 (dd, J=8.7, 2.0 Hz, 1H), 7.49-7.42 (m, 4H), 4.86 (d, J=15.2 Hz, 1H), 4.74-4.70 (m, 1H), 4.65-4.49 (m, 4H), 4.36 (d, J=15.4 Hz, 1H), 4.06-3.94 (m, 2H), 3.90 (d, J=11.0 Hz, 1H), 3.84-3.79 (m, 1H), 3.78-3.56 (m, 14H), 3.50-3.44 (m, 1H), 2.50 (s, 3H), 2.44-2.36 (m, 1H), 2.28-2.23 (m, 1H), 2.19-2.06 (m, 3H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H66N9O9S+ 980.4699, found 980.4730.
LQ076-110 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-1-amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-110 was obtained as white solid in TFA salt form (19.1 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.34 (d, J=1.9 Hz, 1H), 8.07-8.04 (m, 2H), 8.00-7.96 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.41 (m, 4H), 4.86 (d, J=14.7 Hz, 1H), 4.67-4.48 (m, 5H), 4.37 (d, J=15.6 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.81-3.58 (m, 17H), 3.49-3.43 (m, 1H), 2.59-2.53 (m, 1H), 2.49 (s, 3H), 2.48-2.37 (m, 2H), 2.26-2.21 (m, 1H), 2.18-2.06 (m, 3H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C52H68N9O9S+ 994.4855, found 994.4898.
LQ076-111 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-111 was obtained as white solid in TFA salt form (18.2 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 9.02 (s, 1H), 8.32 (d, J=2.0 Hz, 1H), 8.08-8.04 (m, 2H), 8.01-7.98 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.41 (m, 4H), 4.83 (d, J=14.7 Hz, 1H), 4.67-4.49 (m, 5H), 4.37 (d, J=15.6 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.82-3.58 (m, 21H), 3.50-3.43 (m, 1H), 2.60-2.54 (m, 1H), 2.49 (s, 3H), 2.48-2.36 (m, 2H), 2.26-2.21 (m, 1H), 2.19-2.05 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O10S+ 1038.5117, found 1038.55152.
LQ076-112 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-1-amino-20-(tert-butyl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (19.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-112 was obtained as white solid in TFA salt form (20.3 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 8.34 (d, J=2.0 Hz, 1H), 8.08-8.05 (m, 2H), 8.01-7.98 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.42 (m, 4H), 4.85 (d, J=14.6 Hz, 1H), 4.67-4.49 (m, 5H), 4.37 (d, J=15.5 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.83-3.57 (m, 25H), 3.50-3.43 (m, 1H), 2.60-2.54 (m, 1H), 2.50 (s, 3H), 2.49-2.37 (m, 2H), 2.26-2.21 (m, 1H), 2.18-2.06 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C56H76N9O11S+ 1082.5380, found 1082.5399.
LQ076-113 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(2-aminoacetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-113 was obtained as white solid in TFA salt form (17.6 mg, 82%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.08-8.00 (m, 4H), 7.70 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.51-7.48 (m, 2H), 7.45-7.41 (m, 2H), 4.85 (d, J=14.6 Hz, 1H), 4.72-4.69 (m, 1H), 4.65-4.50 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 4.20-4.10 (m, 2H), 3.93 (d, J=11.0 Hz, 1H), 3.83 (dd, J=10.9, 3.8 Hz, 1H), 3.79-3.71 (m, 2H), 3.50-3.44 (m, 1H), 2.50 (s, 3H), 2.44-2.37 (m, 1H), 2.28-2.22 (m, 1H), 2.19-2.07 (m, 3H), 1.87-1.79 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C45H54N9O6S+ 848.3912, found 848.3970.
LQ076-114 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-114 was obtained as white solid in TFA salt form (16.3 mg, 84%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.05-8.01 (m, 2H), 7.99-7.95 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.50-7.47 (m, 2H), 7.44-7.40 (m, 2H), 4.84 (d, J=14.6 Hz, 1H), 4.68-4.65 (m, 1H), 4.63-4.51 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.97 (d, J=11.0 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.78-3.71 (m, 3H), 3.68-3.61 (m, 1H), 3.49-3.44 (m, 1H), 2.69-2.61 (m, 2H), 2.48 (s, 3H), 2.43-2.37 (m, 1H), 2.28-2.22 (m, 1H), 2.19-2.06 (m, 3H), 1.88-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C46H56N9O6S+ 862.4069, found 862.4082.
LQ076-115 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-115 was obtained as white solid in TFA salt form (15.3 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 8.33 (d, J=1.9 Hz, 1H), 8.07-8.04 (m, 2H), 8.00-7.97 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.51-7.48 (m, 2H), 7.46-7.42 (m, 2H), 4.85 (d, J=14.7 Hz, 1H), 4.66-4.51 (m, 5H), 4.38 (d, J=15.5 Hz, 1H), 3.95 (d, J=11.0 Hz, 1H), 3.83 (dd, J=10.9, 3.9 Hz, 1H), 3.78-3.71 (m, 2H), 3.50-3.41 (m, 3H), 2.50 (s, 3H), 2.44-2.36 (m, 3H), 2.27-2.22 (m, 1H), 2.17-2.07 (m, 3H), 1.98-1.91 (m, 2H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.6 Hz, 3H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C47H58N9O6S+ 876.4225, found 876.4252.
LQ076-116 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-116 was obtained as white solid in TFA salt form (15.7 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.05 (d, J=8.3 Hz, 2H), 7.98 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.49 (s, 2H), 7.46-7.42 (m, 2H), 4.82 (d, J=14.6 Hz, 1H), 4.66-4.64 (m, 1H), 4.61-4.51 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.93 (d, J=11.0 Hz, 1H), 3.83 (dd, J=11.0, 4.0 Hz, 1H), 3.78-3.71 (m, 2H), 3.49-3.41 (m, 3H), 2.50 (s, 3H), 2.43-2.33 (m, 3H), 2.26-2.21 (m, 1H), 2.17-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.75-1.66 (m, 4H), 1.51 (d, J=6.6 Hz, 3H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C48H60N9O6S+ 890.4382, found 890.4414.
LQ076-117 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(6-aminohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-117 was obtained as white solid in TFA salt form (14.9 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.02 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.05 (d, J=8.2 Hz, 2H), 7.97 (d, J=8.4 Hz, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.51-7.47 (m, 2H), 7.45-7.42 (m, 2H), 4.82 (d, J=14.7 Hz, 1H), 4.67-4.64 (m, 1H), 4.62-4.51 (m, 4H), 4.37 (d, J=15.6 Hz, 1H), 3.93 (d, J=11.0 Hz, 1H), 3.82 (dd, J=10.9, 3.9 Hz, 1H), 3.78-3.70 (m, 2H), 3.50-3.41 (m, 3H), 2.50 (s, 3H), 2.43-2.21 (m, 4H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.73-1.64 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.48-1.42 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C49H62N9O6S+ 904.4538, found 904.4587.
LQ076-118 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(7-aminoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-118 was obtained as white solid in TFA salt form (15.4 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.05 (d, J=8.2 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.47 (m, 2H), 7.46-7.42 (m, 2H), 4.82 (d, J=14.7 Hz, 1H), 4.67-4.65 (m, 1H), 4.63-4.50 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.93 (d, J=11.1 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.77-3.70 (m, 2H), 3.49-3.40 (m, 3H), 2.50 (s, 3H), 2.43-2.27 (m, 3H), 2.26-2.21 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.69-1.64 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.46-1.39 (m, 4H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C50H64N9O6S+ 918.4695, found 918.4592.
LQ076-119 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(8-aminooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (16.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-119 was obtained as white solid in TFA salt form (17.3 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.04 (d, J=8.1 Hz, 2H), 7.96 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.46 (m, 2H), 7.45-7.41 (m, 2H), 4.81 (d, J=14.6 Hz, 1H), 4.67-4.65 (m, 1H), 4.62-4.50 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.82 (dd, J=10.9, 3.9 Hz, 1H), 3.77-3.71 (m, 2H), 3.50-3.39 (m, 3H), 2.49 (s, 3H), 2.43-2.20 (m, 4H), 2.18-2.06 (m, 3H), 1.87-1.78 (m, 1H), 1.69-1.61 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.45-1.35 (m, 6H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H66N9O6S+ 932.4851, found 932.4872.
LQ076-120 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(9-aminononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (13.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-120 was obtained as white solid in TFA salt form (17.7 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.05 (d, J=8.3 Hz, 2H), 7.96 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.50-7.47 (m, 2H), 7.45-7.42 (m, 2H), 4.83 (d, J=14.6 Hz, 1H), 4.67-4.64 (m, 1H), 4.62-4.50 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.77-3.71 (m, 2H), 3.50-3.40 (m, 3H), 2.50 (s, 3H), 2.43-2.37 (m, 1H), 2.34-2.21 (m, 3H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.68-1.60 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.44-1.33 (m, 8H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C52H68N9O6S+ 946.5008, found 946.4933.
LQ076-121 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(10-aminodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (17.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-121 was obtained as white solid in TFA salt form (16.3 mg, 69%). 1H NMR (800 MHz, Methanol-d4) δ 8.98 (s, 1H), 8.30 (s, 1H), 8.05 (d, J=8.0 Hz, 2H), 7.97 (d, J=7.9 Hz, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.58 (d, J=8.7 Hz, 1H), 7.50-7.47 (m, 2H), 7.45-7.41 (m, 2H), 4.82 (d, J=14.6 Hz, 1H), 4.67-4.65 (m, 1H), 4.62-4.50 (m, 4H), 4.38 (d, J=15.4 Hz, 1H), 3.93 (d, J=11.0 Hz, 1H), 3.82 (dd, J=10.9, 3.9 Hz, 1H), 3.78-3.71 (m, 2H), 3.49-3.44 (m, 1H), 3.44-3.38 (m, 2H), 2.50 (s, 3H), 2.43-2.37 (m, 1H), 2.34-2.29 (m, 1H), 2.28-2.22 (m, 2H), 2.18-2.07 (m, 3H), 1.86-1.80 (m, 1H), 1.68-1.59 (m, 4H), 1.51 (d, J=6.6 Hz, 3H), 1.44-1.35 (m, 10H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C53H70N9O6S+ 960.5164, found 960.5074.
LQ076-122 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-122 was obtained as white solid in TFA salt form (16.7 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.30 (d, J=1.7 Hz, 1H), 8.05 (d, J=8.1 Hz, 2H), 7.97 (d, J=8.3 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.56 (dd, J=8.7, 2.0 Hz, 1H), 7.50-7.46 (m, 2H), 7.45-7.42 (m, 2H), 4.80 (d, J=14.6 Hz, 1H), 4.66-4.64 (m, 1H), 4.61-4.50 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.77-3.71 (m, 2H), 3.49-3.39 (m, 3H), 2.49 (s, 3H), 2.43-2.36 (m, 1H), 2.34-2.21 (m, 3H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.69-1.58 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.45-1.30 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O6S+ 974.5321, found 974.5359.
LQ076-123 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-123 was obtained as yellow solid in TFA salt form (11 mg, 58%). 1H NMR (600 MHz, Methanol-d4) δ 8.31 (d, J=2.0 Hz, 1H), 8.00 (d, J=8.1 Hz, 2H), 7.90 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.58 (dd, J=8.6, 1.9 Hz, 1H), 7.49 (dd, J=8.6, 7.1 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 6.97 (d, J=7.0 Hz, 1H), 5.00 (dd, J=12.9, 5.5 Hz, 1H), 4.82 (d, J=14.6 Hz, 1H), 4.58 (d, J=14.6 Hz, 1H), 3.79-3.71 (m, 6H), 3.63 (t, J=5.3 Hz, 2H), 3.55-3.51 (m, 2H), 3.50-3.44 (m, 1H), 2.89-2.82 (m, 1H), 2.74-2.63 (m, 2H), 2.43-2.36 (m, 1H), 2.17-2.05 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C38H41N8O7+ 721.3461, found 721.3495.
LQ076-124 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-124 was obtained as yellow solid in TFA salt form (12.4 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=2.0 Hz, 1H), 7.97 (d, J=8.3 Hz, 2H), 7.93 (d, J=8.3 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.55 (dd, J=8.7, 1.9 Hz, 1H), 7.51 (dd, J=8.5, 7.1 Hz, 1H), 7.03 (d, J=8.6 Hz, 1H), 6.99 (d, J=7.1 Hz, 1H), 5.03 (dd, J=12.8, 5.5 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.79-3.70 (m, 10H), 3.65-3.61 (m, 2H), 3.50-3.43 (m, 3H), 2.86-2.79 (m, 1H), 2.74-2.63 (m, 2H), 2.44-2.37 (m, 1H), 2.18-2.06 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C40H45N8O8+ 765.3355, found 765.3350.
LQ076-125 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-125 was obtained as yellow solid in TFA salt form (13.4 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=2.0 Hz, 1H), 8.04-8.00 (m, 2H), 7.98-7.94 (m, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.55 (dd, J=8.7, 2.0 Hz, 1H), 7.49 (dd, J=8.6, 7.0 Hz, 1H), 7.03 (d, J=8.6 Hz, 1H), 6.98 (d, J=7.0 Hz, 1H), 5.03 (dd, J=12.8, 5.5 Hz, 1H), 4.83 (d, J=14.7 Hz, 1H), 4.59 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.70-3.59 (m, 14H), 3.50-3.42 (m, 3H), 2.87-2.79 (m, 1H), 2.75-2.64 (m, 2H), 2.43-2.37 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C42H49N8O9+ 809.3617, found 809.3636.
LQ076-126 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((14-amino-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (13.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-126 was obtained as yellow solid in TFA salt form (15.0 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.28 (d, J=1.9 Hz, 1H), 8.04 (d, J=8.1 Hz, 2H), 7.98 (d, J=8.3 Hz, 2H), 7.66 (d, J=8.8 Hz, 1H), 7.54 (dd, J=8.8, 2.0 Hz, 1H), 7.49 (dd, J=8.6, 7.1 Hz, 1H), 7.03 (d, J=8.6 Hz, 1H), 6.99 (d, J=7.1 Hz, 1H), 5.04 (dd, J=12.7, 5.5 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.78-3.59 (m, 20H), 3.48-3.44 (m, 3H), 2.88-2.80 (m, 1H), 2.76-2.65 (m, 2H), 2.43-2.36 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.78 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C44H53N8O10+ 853.3879, found 853.3920.
LQ076-127 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((17-amino-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (13.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-127 was obtained as yellow solid in TFA salt form (15.8 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=2.0 Hz, 1H), 8.06-8.03 (m, 2H), 8.00-7.96 (m, 2H), 7.67 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.8, 2.0 Hz, 1H), 7.50 (dd, J=8.6, 7.1 Hz, 1H), 7.03 (d, J=8.6 Hz, 1H), 7.00 (d, J=7.1 Hz, 1H), 5.04 (dd, J=12.8, 5.5 Hz, 1H), 4.83 (d, J=14.6 Hz, 1H), 4.59 (d, J=14.6 Hz, 1H), 3.78-3.58 (m, 24H), 3.48-3.43 (m, 3H), 2.88-2.81 (m, 1H), 2.76-2.66 (m, 2H), 2.43-2.37 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C46H57N8O11+ 897.4141, found 897.4174.
LQ076-128 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-128 was obtained as yellow solid in TFA salt form (11.7 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=2.1 Hz, 1H), 8.02 (d, J=8.2 Hz, 2H), 7.92 (d, J=8.3 Hz, 2H), 7.85-7.81 (m, 1H), 7.68 (d, J=8.6 Hz, 1H), 7.58-7.48 (m, 2H), 7.20 (d, J=8.6 Hz, 1H), 7.04 (d, J=7.0 Hz, 1H), 5.06 (dd, J=12.8, 5.5 Hz, 1H), 4.83 (d, J=14.6 Hz, 1H), 4.59 (d, J=14.6 Hz, 1H), 3.78-3.70 (m, 3H), 3.68-3.61 (m, 3H), 3.49-3.43 (m, 1H), 2.89-2.82 (m, 1H), 2.77-2.67 (m, 2H), 2.43-2.36 (m, 1H), 2.18-2.06 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C36H37N8O6+ 677.2831, found 677.2857.
LQ076-129 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((3-aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (9.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-129 was obtained as yellow solid in TFA salt form (12.3 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=2.0 Hz, 1H), 8.04 (d, J=8.1 Hz, 2H), 7.96 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.58-7.53 (m, 2H), 7.09 (d, J=8.6 Hz, 1H), 7.05 (d, J=7.1 Hz, 1H), 5.05 (dd, J=12.7, 5.5 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.57 (t, J=6.7 Hz, 2H), 3.50-3.45 (m, 3H), 2.90-2.83 (m, 1H), 2.77-2.66 (m, 2H), 2.43-2.37 (m, 1H), 2.18-2.06 (m, 3H), 2.03-1.97 (m, 2H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.6 Hz, 3H). HRMS m/z [M+H]+ calcd for C37H39N8O6+ 691.2987, found 691.3031.
LQ076-130 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-130 was obtained as yellow solid in TFA salt form (14.9 mg, 80%). 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=2.0 Hz, 1H), 8.04 (d, J=8.3 Hz, 2H), 7.95 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.57-7.53 (m, 2H), 7.08 (d, J=8.6 Hz, 1H), 7.04 (d, J=7.1 Hz, 1H), 5.07 (dd, J=12.8, 5.5 Hz, 1H), 4.80 (d, J=14.6 Hz, 1H), 4.56 (d, J=14.6 Hz, 1H), 3.77-3.71 (m, 2H), 3.51-3.41 (m, 5H), 2.90-2.83 (m, 1H), 2.78-2.68 (m, 2H), 2.43-2.37 (m, 1H), 2.18-2.08 (m, 3H), 1.85-1.76 (m, 5H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C38H41N8O6+ 705.3144, found 705.3162.
LQ076-131 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-131 was obtained as yellow solid in TFA salt form (14.3 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.31 (d, J=2.0 Hz, 1H), 8.03 (d, J=8.3 Hz, 2H), 7.95-7.91 (m, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.58 (dd, J=8.8, 2.0 Hz, 1H), 7.53 (dd, J=8.6, 7.1 Hz, 1H), 7.04 (d, J=8.6 Hz, 1H), 7.01 (d, J=7.0 Hz, 1H), 5.05 (dd, J=12.8, 5.5 Hz, 1H), 4.82 (d, J=14.6 Hz, 1H), 4.58 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.48-3.43 (m, 3H), 3.36 (t, J=6.9 Hz, 2H), 2.90-2.82 (m, 1H), 2.76-2.66 (m, 2H), 2.43-2.36 (m, 1H), 2.18-2.06 (m, 3H), 1.86-1.79 (m, 1H), 1.77-1.70 (m, 4H), 1.58-1.53 (m, 2H), 1.51 (d, J=6.6 Hz, 3H). HRMS m/z [M+H]+ calcd for C39H43N8O6+ 719.3300, found 719.3340.
LQ076-132 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((6-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-132 was obtained as yellow solid in TFA salt form (13.7 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 8.31 (d, J=1.9 Hz, 1H), 8.04 (d, J=8.2 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.59-7.53 (m, 2H), 7.06-7.02 (m, 2H), 5.06 (dd, J=12.5, 5.5 Hz, 1H), 4.82 (d, J=14.6 Hz, 1H), 4.58 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.50-3.41 (m, 3H), 3.37-3.34 (m, 2H), 2.90-2.83 (m, 1H), 2.78-2.67 (m, 2H), 2.43-2.36 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.75-1.67 (m, 4H), 1.55-1.47 (m, 7H). HRMS m/z [M+H]+ calcd for C40H45N8O6+ 733.3457, found 733.3479.
LQ076-133 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((7-aminoheptyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-133 was obtained as yellow solid in TFA salt form (14.6 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.31 (d, J=2.0 Hz, 1H), 8.04 (d, J=8.2 Hz, 2H), 7.97-7.94 (m, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.54 (dd, J=8.5, 7.0 Hz, 1H), 7.05-7.00 (m, 2H), 5.05 (dd, J=12.7, 5.5 Hz, 1H), 4.83 (d, J=14.6 Hz, 1H), 4.59 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.50-3.40 (m, 3H), 3.34-3.33 (m, 2H), 2.88-2.80 (m, 1H), 2.76-2.67 (m, 2H), 2.43-2.37 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.71-1.64 (m, 4H), 1.53-1.42 (m, 9H). HRMS m/z [M+H]+ calcd for C41H47N8O6+ 747.3613, found 747.3639.
LQ076-134 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 4-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-134 was obtained as yellow solid in TFA salt form (15.1 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=2.0 Hz, 1H), 8.05 (d, J=8.1 Hz, 2H), 7.96 (d, J=8.3 Hz, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.58-7.52 (m, 2H), 7.05-7.01 (m, 2H), 5.06 (dd, J=12.4, 5.5 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.50-3.40 (m, 3H), 3.32-3.29 (m, 2H), 2.90-2.82 (m, 1H), 2.78-2.68 (m, 2H), 2.43-2.36 (m, 1H), 2.18-2.07 20 (m, 3H), 1.86-1.78 (m, 1H), 1.67 (q, J=7.5 Hz, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.49-1.39 (m, 8H). HRMS m/z [M+H]+ calcd for C42H49N8O6+ 761.3770, found 761.3802.
Methyl 1H-indazole-5-carboxylate (0.87 g, 4.9 mmol) and 18-crown-6 (20 mg) were added to 20 5 mL dry THF. Sodium bis(trimethylsilyl)amide (7.3 mL, 7.3 mmol, 1.0 M in THF) was added via syringe, followed by tert-Butyl (2-bromoethyl)carbamate (1.4 g, 6.4 mmol). The reaction was heated at reflux for 24 hr, cooled, and concentrated under vacuum. The residue was partitioned between ethyl acetate and water, separated, and the aqueous layer extracted with ethyl acetate. The combined organics were washed with brine, dried over sodium sulfate and concentrated. The resulting residue was purified by silica gel flash chromatography to give the two separate products. The one is intermediate 11 (750 mg, 48%). 1H NMR (600 MHz, Methanol-d4) δ 8.53 (s, 1H), 8.20 (s, 1H), 8.04 (d, J=9.0 Hz, 1H), 7.62 (d, J=8.9 Hz, 1H), 4.54 (t, J=5.9 Hz, 2H), 3.95 (s, 3H), 3.52 (t, J=5.9 Hz, 2H), 1.32 (s, 9H). MS (ESI): m/z 320.1 [M+H]+. The others is 2-substitute products. 1H NMR (600 MHz, Methanol-d4) δ 8.54 (s, 1H), 8.41 (s, 1H), 7.89 (d, J=9.1 Hz, 1H), 7.66 (d, J=9.1 Hz, 1H), 4.56 (t, J=5.9 Hz, 2H), 3.94 (s, 3H), 3.62 (t, J=5.9 Hz, 2H), 1.38 (s, 9H).
Intermediate 12 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 85% yield. MS (ESI): m/z 306.0 [M+H]+.
Intermediate 13 was synthesized according to the procedures for the preparation of intermediate 9 as a white solid in 69% yield. MS (ESI): m/z 518.3 [M+H]+.
Intermediate 13 (700 mg, 1.35 mmol) was dissolved in 5 mL DCM, to the resulting solution was added 3 mL TFA. After being stirred for 1 h at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 14 as white solid in TFA salt form (600 mg, 86%). 1H NMR (600 MHz, Methanol-d4) δ 8.52 (d, J=1.6 Hz, 1H), 8.32-8.29 (m, 2H), 8.11 (dd, J=8.8, 1.7 Hz, 1H), 7.76 (d, J=8.9 Hz, 1H), 7.70 (d, J=8.7 Hz, 1H), 7.61 (dd, J=8.7, 2.0 Hz, 1H), 4.84 (d, J=14.5 Hz, 1H), 4.77 (t, J=5.8 Hz, 2H), 4.61 (d, J=14.6 Hz, 1H), 3.80-3.70 (m, 2H), 3.59 (t, J=5.8 Hz, 2H), 3.51-3.43 (m, 1H), 2.44-2.36 (m, 1H), 2.21-2.05 (m, 2H), 1.88-1.79 (m, 1H), 1.51 (d, J=6.4 Hz, 3H). MS (ESI): m/z 418.4 [M+H]+.
To a solution of Intermediate 14 (13 mg, 0.02 mmol) in DMSO (1 mL) were added 2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)acetic acid (11.3 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ076-135 as white solid in TFA salt form (19.2 mg, 83%). 1H NMR (800 MHz, Methanol-d4) δ 8.95 (s, 1H), 8.44 (s, 1H), 8.31 (s, 1H), 8.22 (s, 1H), 8.03 (d, J=8.9 Hz, 1H), 7.71-7.66 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.47-7.38 (m, 4H), 4.82 (d, J=14.7 Hz, 1H), 4.73-4.70 (m, 1H), 4.68-4.63 (m, 3H), 4.60-4.52 (m, 3H), 4.34 (d, J=15.3 Hz, 1H), 3.96-3.82 (m, 6H), 3.79-3.71 (m, 4H), 3.49-3.44 (m, 1H), 2.46 (s, 3H), 2.42-2.37 (m, 1H), 2.29-2.25 (m, 1H), 2.18-2.08 (m, 4H), 1.86-1.81 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C49H60N11O7S+ 946.4392, found 946.4411.
LQ076-136 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 3-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)propanoic acid (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-136 was obtained as white solid in TFA salt form (14.9 mg, 62%). 1H NMR (800 MHz, Methanol-d4) δ 8.96 (s, 1H), 8.47 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.71-7.66 (m, 2H), 7.59 (d, J=8.8 Hz, 1H), 7.47 (d, J=7.8 Hz, 2H), 7.41 (d, J=7.8 Hz, 2H), 4.81 (d, J=14.8 Hz, 1H), 4.67-4.65 (m, 1H), 4.63-4.49 (m, 6H), 4.37 (d, J=15.3 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.80 (dd, J=10.9, 4.0 Hz, 1H), 3.77-3.68 (m, 4H), 3.65-3.55 (m, 4H), 3.49-3.44 (m, 1H), 2.48-2.38 (m, 6H), 2.36-2.30 (m, 2H), 2.26-2.22 (m, 1H), 2.18-2.08 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O7S+ 974.4705, found 974.4701.
LQ076-137 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(3-(4-(4-methylthiazol-5-yl)phenyl)propanoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)acetic acid (12.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-137 was obtained as white solid in TFA salt form (15.7 mg, 65%). 1H NMR (800 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.46 (s, 1H), 8.32 (s, 1H), 8.22 (s, 1H), 8.03 (d, J=8.6 Hz, 1H), 7.71-7.67 (m, 2H), 7.61 (d, J=8.5 Hz, 1H), 7.47-7.36 (m, 4H), 4.83 (d, J=14.7 Hz, 1H), 4.76-4.73 (m, 1H), 4.70-4.58 (m, 4H), 4.55-4.44 (m, 2H), 4.37 (d, J=15.3 Hz, 1H), 4.05-3.99 (m, 1H), 3.95-3.88 (m, 3H), 3.85-3.80 (m, 2H), 3.79-3.70 (m, 4H), 3.65-3.54 (m, 3H), 3.51-3.44 (m, 2H), 2.47 (s, 3H), 2.43-2.38 (m, 1H), 2.31-2.27 (m, 1H), 2.19-2.07 (m, 3H), 1.87-1.81 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O8S+ 990.4655, found 990.4668.
LQ076-138 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 3-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)propanoic acid (12.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-138 was obtained as white solid in TFA salt form (17.1 mg, 69%). 1H NMR (800 MHz, Methanol-d4) δ 9.01 (s, 1H), 8.48 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.71-7.67 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.51-7.39 (m, 4H), 4.85 (d, J=14.7 Hz, 1H), 4.68-4.65 (m, 1H), 4.63-4.50 (m, 6H), 4.40-4.36 (m, 1H), 3.93-3.89 (m, 1H), 3.84-3.80 (m, 1H), 3.78-3.66 (m, 6H), 3.64-3.57 (m, 3H), 3.56-3.44 (m, 4H), 2.61-2.38 (m, 6H), 2.32 (t, J=6.3 Hz, 2H), 2.26-2.22 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O8S+ 1018.4968, found 1018.4990.
LQ076-139 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecanoic acid (13 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-139 was obtained as white solid in TFA salt form (16.5 mg, 65%). 1H NMR (800 MHz, Methanol-d4) δ 8.98 (s, 1H), 8.47 (s, 1H), 8.32 (s, 1H), 8.22 (s, 1H), 8.05 (d, J=8.7 Hz, 1H), 7.71-7.67 (m, 2H), 7.59 (d, J=8.7 Hz, 1H), 7.48-7.39 (m, 4H), 4.83 (d, J=14.7 Hz, 1H), 4.73-4.70 (m, 1H), 4.67-4.50 (m, 6H), 4.35 (d, J=15.2 Hz, 1H), 4.07-3.97 (m, 2H), 3.91-3.71 (m, 8H), 3.68-3.55 (m, 6H), 3.53-3.44 (m, 3H), 2.47 (s, 3H), 2.43-2.38 (m, 1H), 2.25 (dd, J=13.1, 7.6 Hz, 1H), 2.18-2.08 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O9S+ 1034.4917, found 1034.4919.
LQ076-140 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), (S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoic acid (13.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-140 was obtained as white solid in TFA salt form (18.2 mg, 71%). 1H NMR (800 MHz, Methanol-d4) δ 8.98 (s, 1H), 8.48 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.71-7.66 (m, 2H), 7.59 (d, J=8.7 Hz, 1H), 7.49-7.38 (m, 4H), 4.83 (d, J=14.7 Hz, 1H), 4.67-4.49 (m, 7H), 4.37 (d, J=15.4 Hz, 1H), 3.90 (d, J=10.9 Hz, 1H), 3.82-3.65 (m, 7H), 3.62-3.44 (m, 11H), 2.57-2.52 (m, 1H), 2.50-2.38 (m, 5H), 2.32 (t, J=6.2 Hz, 2H), 2.26-2.22 (m, 1H), 2.18-2.07 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C55H72N11O9S+ 1062.5230, found 1062.5218.
LQ076-141 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), (S)-18-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-19,19-dimethyl-16-oxo-4,7,10,13-tetraoxa-17-azaicosanoic acid (14.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-141 was obtained as white solid in TFA salt form (20 mg, 75%). 1H NMR (800 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.49 (s, 1H), 8.32 (s, 1H), 8.24 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.72-7.67 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.51-7.39 (m, 4H), 4.83 (d, J=14.7 Hz, 1H), 4.67-4.50 (m, 7H), 4.37 (d, J=15.4 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.78-3.65 (m, 6H), 3.63-3.44 (m, 15H), 2.58-2.53 (m, 1H), 2.50-2.38 (m, 5H), 2.33 (t, J=6.2 Hz, 2H), 2.26-2.22 (m, 1H), 2.19-2.07 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.6 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C57H76N11O10S+ 1106.5492, found 1106.5511.
LQ076-142 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), (S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosanoic acid (15.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-142 was obtained as white solid in TFA salt form (20.2 mg, 75%). 1H NMR (800 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.48 (s, 1H), 8.32 (s, 1H), 8.22 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.73-7.68 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.49-7.39 (m, 4H), 4.83 (d, J=14.7 Hz, 1H), 4.71-4.69 (m, 1H), 4.67-4.51 (m, 6H), 4.37 (d, J=15.4 Hz, 1H), 4.05-3.97 (m, 2H), 3.89 (d, J=11.0 Hz, 1H), 3.85-3.80 (m, 3H), 3.79-3.71 (m, 4H), 3.69-3.61 (m, 8H), 3.58-3.44 (m, 9H), 2.48 (s, 3H), 2.43-2.38 (m, 1H), 2.27-2.23 (m, 1H), 2.18-2.08 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C57H76N11O11S+ 1122.5441, found 1122.5440.
LQ076-143 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), (S)-21-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-22,22-dimethyl-19-oxo-4,7,10,13,16-pentaoxa-20-azatricosanoic acid (15.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-143 was obtained as white solid in TFA salt form (19.5 mg, 70%). 1H NMR (800 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.49 (s, 1H), 8.32 (s, 1H), 8.24 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.73-7.67 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.50-7.38 (m, 4H), 4.83 (d, J=14.6 Hz, 1H), 4.67-4.65 (m, 1H), 4.63-4.49 (m, 6H), 4.37 (d, J=15.4 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.78-3.44 (m, 25H), 2.58-2.54 (m, 1H), 2.50-2.44 (m, 4H), 2.43-2.38 (m, 1H), 2.33 (t, J=6.2 Hz, 2H), 2.26-2.22 (m, 1H), 2.19-2.07 (m, 3H), 1.87-1.81 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C59H80N11O11S+ 1150.5754, found 1150.5782.
LQ076-144 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (10.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-144 was obtained as white solid in TFA salt form (14.6 mg, 63%). 1H NMR (800 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.47 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.9 Hz, 1H), 7.71-7.65 (m, 2H), 7.60 (d, J=8.6 Hz, 1H), 7.49-7.37 (m, 4H), 4.82 (d, J=14.7 Hz, 1H), 4.63-4.47 (m, 7H), 4.37 (d, J=15.6 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.74 (s, 2H), 3.71-3.63 (m, 2H), 3.49-3.44 (m, 1H), 2.53-2.32 (m, 8H), 2.26-2.21 (m, 1H), 2.18-2.06 (m, 3H), 1.86-1.81 (m, 1H), 1.52 (d, J=6.6 Hz, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C49H60N11O6S+ 930.4443, found 930.4458.
LQ076-145 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (11.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-145 was obtained as white solid in TFA salt form (17.1 mg, 73%). 1H NMR (800 MHz, Methanol-d4) δ 8.96 (s, 1H), 8.47 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.47 (d, J=7.8 Hz, 2H), 7.40 (d, J=7.9 Hz, 2H), 4.81 (d, J=14.6 Hz, 1H), 4.64-4.49 (m, 7H), 4.36 (d, J=15.4 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.80-3.63 (m, 5H), 3.49-3.44 (m, 1H), 2.47 (s, 3H), 2.43-2.36 (m, 1H), 2.26-2.05 (m, 8H), 1.86-1.80 (m, 1H), 1.78-1.73 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C50H62N11O6S+ 944.4600, found 944.4622.
LQ076-146 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (11.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-146 was obtained as white solid in TFA salt form (15.5 mg, 65%). 1H NMR (800 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.47 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.61 (d, J=8.7 Hz, 1H), 7.47 (d, J=7.8 Hz, 2H), 7.41 (d, J=7.9 Hz, 2H), 4.83 (d, J=14.7 Hz, 1H), 4.65-4.49 (m, 7H), 4.38 (d, J=15.4 Hz, 1H), 3.93 (d, J=11.0 Hz, 1H), 3.82-3.64 (m, 5H), 3.49-3.44 (m, 1H), 2.48 (s, 3H), 2.43-2.38 (m, 1H), 2.26-2.03 (m, 8H), 1.86-1.80 (m, 1H), 1.53-1.42 (m, 7H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H64N11O6S+ 958.4756, found 958.4755.
LQ076-147 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoic acid (12.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-147 was obtained as white solid in TFA salt form (17.3 mg, 72%). 1H NMR (800 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.48 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.59 (d, J=8.6 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H), 7.41 (d, J=7.8 Hz, 2H), 4.81 (d, J=14.8 Hz, 1H), 4.65-4.49 (m, 7H), 4.38 (d, J=15.4 Hz, 1H), 3.91 (d, J=11.0 Hz, 1H), 3.80 (dd, J=10.9, 3.9 Hz, 1H), 3.77-3.68 (m, 4H), 3.49-3.43 (m, 1H), 2.48 (s, 3H), 2.42-2.37 (m, 1H), 2.28-2.02 (m, 8H), 1.86-1.80 (m, 1H), 1.59-1.42 (m, 7H), 1.23-1.17 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C52H66N11O6S+ 972.4913, found 972.4936.
LQ076-148 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctanoic acid (12.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-148 was obtained as white solid in TFA salt form (16.7 mg, 69%). 1H NMR (800 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.50 (s, 1H), 8.35 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.71-7.65 (m, 2H), 7.61 (d, J=8.5 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H), 7.41 (d, J=7.8 Hz, 2H), 4.84 (d, J=14.7 Hz, 1H), 4.66-4.50 (m, 7H), 4.38 (d, J=15.5 Hz, 1H), 3.93 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 4.0 Hz, 1H), 3.78-3.66 (m, 4H), 3.49-3.44 (m, 1H), 2.47 (s, 3H), 2.43-2.38 (m, 1H), 2.29-2.20 (m, 3H), 2.18-2.07 (m, 3H), 2.03 (t, J=7.5 Hz, 2H), 1.86-1.80 (m, 1H), 1.56-1.49 (m, 5H), 1.43-1.38 (m, 2H), 1.26-1.20 (m, 2H), 1.19-1.13 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C53H68N11O6S+ 986.5069, found 986.5060.
LQ076-149 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoic acid (13.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-149 was obtained as white solid in TFA salt form (17.7 mg, 72%). 1H NMR (800 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.50 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.71-7.65 (m, 2H), 7.60 (d, J=8.7 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H), 7.42 (d, J=7.8 Hz, 2H), 4.83 (d, J=14.6 Hz, 1H), 4.66-4.64 (m, 1H), 4.62-4.49 (m, 6H), 4.39 (d, J=15.5 Hz, 1H), 3.93 (d, J=10.9 Hz, 1H), 3.82 (dd, J=10.9, 4.0 Hz, 1H), 3.78-3.65 (m, 4H), 3.49-3.44 (m, 1H), 2.48 (s, 3H), 2.43-2.37 (m, 1H), 2.30-2.20 (m, 3H), 2.18-2.07 (m, 3H), 2.03 (t, J=7.6 Hz, 2H), 1.86-1.80 (m, 1H), 1.57 (d, J=6.9 Hz, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.44-1.38 (m, 2H), 1.28-1.20 (m, 4H), 1.18-1.12 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H70N11O6S+ 1000.5226, found 1000.5273.
LQ076-150 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 10-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecanoic acid (13.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-150 was obtained as white solid in TFA salt form (14.8 mg, 59%). 1H NMR (800 MHz, Methanol-d4) δ 8.98 (s, 1H), 8.50 (s, 1H), 8.33 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.59 (d, J=8.7 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H), 7.42 (d, J=7.8 Hz, 2H), 4.82 (d, J=14.6 Hz, 1H), 4.66-4.64 (m, 1H), 4.62-4.50 (m, 6H), 4.39 (d, J=15.3 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.82 (dd, J=10.9, 4.0 Hz, 1H), 3.77-3.67 (m, 4H), 3.49-3.44 (m, 1H), 2.48 (s, 3H), 2.42-2.38 (m, 1H), 2.29-2.08 (m, 6H), 2.02 (t, J=7.6 Hz, 2H), 1.86-1.80 (m, 1H), 1.61-1.54 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.42-1.37 (m, 2H), 1.31-1.19 (m, 6H), 1.18-1.11 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H72N11O6S+ 1014.5382, found 1014.5381.
LQ076-151 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 11-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoic acid (13.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-151 was obtained as white solid in TFA salt form (18.9 mg, 75%). 1H NMR (800 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.50 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.58 (d, J=8.7 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H), 7.42 (d, J=7.8 Hz, 2H), 4.81 (d, J=14.6 Hz, 1H), 4.66-4.63 (m, 1H), 4.62-4.49 (m, 6H), 4.38 (d, J=15.4 Hz, 1H), 3.93 (d, J=10.9 Hz, 1H), 3.82 (dd, J=10.9, 4.0 Hz, 1H), 3.77-3.68 (m, 4H), 3.50-3.44 (m, 1H), 2.49 (s, 3H), 2.43-2.38 (m, 1H), 2.28-2.09 (m, 6H), 2.04-2.00 (m, 2H), 1.86-1.80 (m, 1H), 1.60-1.53 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.41-1.36 (m, 2H), 1.32-1.11 (m, 10H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C56H74N11O6S+ 1028.5539, found 1028.5529.
LQ076-152 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine (6.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-152 was obtained as yellow solid in TFA salt form (11.2 mg, 58%). 1H NMR (800 MHz, Methanol-d4) δ 8.42 (s, 1H), 8.29 (s, 1H), 8.17 (s, 1H), 7.97 (d, J=8.8 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 7.61 (d, J=8.8 Hz, 1H), 7.57 (d, J=8.7 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.01 (d, J=7.1 Hz, 1H), 6.61 (d, J=8.5 Hz, 1H), 5.05 (dd, J=12.8, 5.6 Hz, 1H), 4.80 (d, J=14.6 Hz, 1H), 4.61-4.55 (m, 3H), 3.84 (s, 2H), 3.79-3.72 (m, 4H), 3.50-3.45 (m, 1H), 2.86-2.79 (m, 1H), 2.74-2.67 (m, 2H), 2.43-2.38 (m, 1H), 2.19-2.08 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C38H39N10O6+ 731.3049, found 731.3050.
LQ076-153 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanoic acid (7.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-153 was obtained as yellow solid in TFA salt form (13.3 mg, 68%). 1H NMR (800 MHz, Methanol-d4) δ 8.40 (s, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 7.92 (d, J=8.7 Hz, 1H), 7.68 (d, J=8.7 Hz, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.50 (t, J=7.8 Hz, 1H), 6.98-6.94 (m, 2H), 5.06 (dd, J=12.8, 5.6 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.61-4.54 (m, 3H), 3.78-3.67 (m, 4H), 3.50-3.45 (m, 1H), 3.41 (t, J=6.6 Hz, 2H), 2.86-2.80 (m, 1H), 2.74-2.67 (m, 2H), 2.43-2.33 (m, 3H), 2.19-2.07 (m, 3H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C39H41N10O6+ 745.3205, found 745.3204.
LQ076-154 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoic acid (8.0 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-154 was obtained as yellow solid in TFA salt form (14.7 mg, 74%). 1H NMR (800 MHz, Methanol-d4) δ 8.41 (s, 1H), 8.26 (s, 1H), 8.20 (s, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.67 (d, J=8.7 Hz, 2H), 7.54-7.49 (m, 2H), 6.96 (d, J=7.0 Hz, 1H), 6.93 (d, J=8.5 Hz, 1H), 4.96 (dd, J=13.7, 5.5 Hz, 1H), 4.82 (d, J=14.8 Hz, 1H), 4.63-4.56 (m, 3H), 3.78-3.69 (m, 4H), 3.51-3.45 (m, 1H), 3.08 (t, J=7.3 Hz, 2H), 2.78-2.71 (m, 1H), 2.68-2.57 (m, 2H), 2.44-2.38 (m, 1H), 2.19-2.07 (m, 4H), 2.02-1.98 (m, 1H), 1.87-1.81 (m, 1H), 1.74-1.68 (m, 2H), 1.53 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C40H43N10O6+ 759.3362, found 759.3334.
LQ076-155 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentanoic acid (8.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-155 was obtained as yellow solid in TFA salt form (13.9 mg, 69%). 1H NMR (800 MHz, Methanol-d4) δ 8.42 (s, 1H), 8.20 (d, J=11.8 Hz, 1H), 8.02 (d, J=8.8 Hz, 1H), 7.68-7.62 (m, 2H), 7.51 (d, J=8.7 Hz, 1H), 7.43 (t, J=7.8 Hz, 1H), 6.92 (d, J=8.5 Hz, 1H), 6.86 (d, J=7.0 Hz, 1H), 5.03 (dd, J=12.8, 5.6 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.64-4.55 (m, 3H), 3.78-3.69 (m, 4H), 3.50-3.44 (m, 1H), 3.12 (t, J=7.1 Hz, 2H), 2.84-2.78 (m, 1H), 2.73-2.65 (m, 2H), 2.43-2.38 (m, 1H), 2.19-2.05 (m, 5H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.47-1.40 (m, 2H), 1.37-1.30 (m, 2H). HRMS m/z [M+H]+ calcd for C41H45N10O6+ 773.3518, found 773.3535.
LQ076-156 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoic acid (8.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-156 was obtained as yellow solid in TFA salt form (15.7 mg, 77%). 1H NMR (800 MHz, Methanol-d4) δ 8.47 (s, 1H), 8.23 (d, J=4.3 Hz, 1H), 8.05 (d, J=8.7 Hz, 1H), 7.68 (d, J=8.9 Hz, 1H), 7.64 (d, J=8.7 Hz, 1H), 7.53 (d, J=8.7 Hz, 1H), 7.45 (t, J=7.8 Hz, 1H), 6.94 (d, J=8.5 Hz, 1H), 6.88 (d, J=7.1 Hz, 1H), 5.01 (dd, J=12.9, 5.5 Hz, 1H), 4.80 (d, J=14.6 Hz, 1H), 4.63-4.54 (m, 3H), 3.77-3.69 (m, 4H), 3.49-3.44 (m, 1H), 3.17 (t, J=7.1 Hz, 2H), 2.85-2.80 (m, 1H), 2.75-2.70 (m, 1H), 2.70-2.62 (m, 1H), 2.43-2.38 (m, 1H), 2.19-2.09 (m, 2H), 2.09-2.02 (m, 3H), 1.86-1.80 (m, 1H), 1.56-1.50 (m, 5H), 1.45-1.40 (m, 2H), 1.22-1.17 (m, 2H). HRMS m/z [M+H]+ calcd for C42H47N10O6+ 787.3675, found 787.3680.
LQ076-157 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptanoic acid (9.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-157 was obtained as yellow solid in TFA salt form (14.4 mg, 70%). 1H NMR (800 MHz, Methanol-d4) δ 8.48 (s, 1H), 8.25 (s, 1H), 8.23 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.66 (d, J=8.8 Hz, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 6.90-6.84 (m, 2H), 5.05 (dd, J=12.8, 5.6 Hz, 1H), 4.80 (d, J=14.7 Hz, 1H), 4.63-4.54 (m, 3H), 3.77-3.69 (m, 4H), 3.48-3.43 (m, 1H), 3.18 (t, J=7.1 Hz, 2H), 2.88-2.82 (m, 1H), 2.77-2.66 (m, 2H), 2.43-2.37 (m, 1H), 2.18-2.07 (m, 3H), 2.01 (t, J=7.5 Hz, 2H), 1.86-1.80 (m, 1H), 1.53-1.47 (m, 5H), 1.35-1.24 (m, 4H), 1.15-1.10 (m, 2H). HRMS m/z [M+H]+ calcd for C43H49N10O6+ 801.3831, found 801.3786.
LQ076-158 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoic acid (9.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-158 was obtained as yellow solid in TFA salt form (15.2 mg, 730). 1H NMR (800 MHz, Methanol-d4) δ 8.49 (s, 1H), 8.26 (s, 1H), 8.23 (s, 1H), 8.06 (d, J=8.8 Hz, 1H), 7.66 (d, J=8.9 Hz, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.42 (t, J=7.8 Hz, 1H), 6.91 (d, J=7.0 Hz, 1H), 6.88 (d, J=8.6 Hz, 1H), 5.07 (dd, J=12.7, 5.6 Hz, 1H), 4.78-4.75 (m, 1H), 4.61 (t, J=5.8 Hz, 2H), 4.53 (d, J=14.7 Hz, 1H), 3.76-3.68 (m, 4H), 3.47-3.42 (m, 1H), 3.20 (t, J=7.2 Hz, 2H), 2.89-2.82 (m, 1H), 2.78-2.68 (m, 2H), 2.42-2.36 (m, 1H), 2.16-2.07 (m, 3H), 2.01 (t, J=7.4 Hz, 2H), 1.85-1.79 (m, 1H), 1.58-1.54 (m, 2H), 1.50 (d, J=6.6 Hz, 3H), 1.36-1.19 (m, 6H), 1.10-1.05 (m, 2H). HRMS m/z [M+H]+ calcd for C44H51N10O6+ 815.3988, found 815.3991.
LQ076-159 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)propanoic acid (7.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-159 was obtained as yellow solid in TFA salt form (12.4 mg, 61%). 1H NMR (800 MHz, Methanol-d4) δ 8.42 (s, 1H), 8.27 (s, 1H), 8.19 (s, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.70-7.65 (m, 2H), 7.53 (d, J=8.6 Hz, 1H), 7.50 (t, J=7.8 Hz, 1H), 7.03 (d, J=8.5 Hz, 1H), 6.96 (d, J=7.0 Hz, 1H), 4.97 (dd, J=12.9, 5.6 Hz, 1H), 4.80 (d, J=14.7 Hz, 1H), 4.60 (t, J=6.1 Hz, 2H), 4.56 (d, J=14.7 Hz, 1H), 3.78-3.73 (m, 2H), 3.72-3.69 (m, 2H), 3.65-3.59 (m, 4H), 3.50-3.45 (m, 1H), 3.41 (t, J=5.2 Hz, 2H), 2.81-2.75 (m, 1H), 2.71-2.67 (m, 1H), 2.66-2.60 (m, 1H), 2.44-2.38 (m, 1H), 2.36 (t, J=6.0 Hz, 2H), 2.19-2.08 (m, 2H), 2.05-2.00 (m, 1H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C41H45N10O7+ 789.3467, found 789.3501.
LQ076-160 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanoic acid (8.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-160 was obtained as yellow solid in TFA salt form (14.2 mg, 67%). 1H NMR (800 MHz, Methanol-d4) δ 8.43 (s, 1H), 8.27 (s, 1H), 8.19 (s, 1H), 8.01 (d, J=8.8 Hz, 1H), 7.68-7.63 (m, 2H), 7.56 (d, J=8.8 Hz, 1H), 7.45 (t, J=7.8 Hz, 1H), 6.99-6.93 (m, 2H), 5.03 (dd, J=12.7, 5.6 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.61-4.53 (m, 3H), 3.77-3.71 (m, 2H), 3.70-3.63 (m, 4H), 3.62-3.56 (m, 4H), 3.55-3.51 (m, 2H), 3.49-3.44 (m, 1H), 3.42 (t, J=5.3 Hz, 2H), 2.92-2.78 (m, 1H), 2.77-2.65 (m, 2H), 2.43-2.37 (m, 1H), 2.33 (t, J=6.1 Hz, 2H), 2.19-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C43H49N10O8+ 833.3729, found 833.3760.
LQ076-161 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoic acid (9.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-161 was obtained as yellow solid in TFA salt form (15.3 mg, 72%). 1H NMR (800 MHz, Methanol-d4) δ 8.44 (s, 1H), 8.28 (s, 1H), 8.20 (s, 1H), 8.03 (d, J=8.7 Hz, 1H), 7.69-7.65 (m, 2H), 7.57 (d, J=8.7 Hz, 1H), 7.47 (t, J=7.8 Hz, 1H), 7.00-6.96 (m, 2H), 5.04 (dd, J=12.6, 5.6 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.61-4.54 (m, 3H), 3.77-3.65 (m, 6H), 3.64-3.52 (m, 8H), 3.51-3.44 (m, 3H), 3.42 (t, J=5.3 Hz, 2H), 2.87-2.81 (m, 1H), 2.75-2.65 (m, 2H), 2.42-2.37 (m, 1H), 2.31 (t, J=6.1 Hz, 2H), 2.18-2.07 (m, 3H), 1.86-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C45H53N10O9+ 877.3991, found 877.4050.
LQ076-162 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecan-15-oic acid (10.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-162 was obtained as yellow solid in TFA salt form (14.5 mg, 63%). 1H NMR (800 MHz, Methanol-d4) δ 8.46 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 8.04 (d, J=8.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.57 (d, J=8.7 Hz, 1H), 7.49 (t, J=7.8 Hz, 1H), 7.03-6.98 (m, 2H), 5.04 (dd, J=12.7, 5.6 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.62-4.55 (m, 3H), 3.77-3.72 (m, 2H), 3.70 (t, J=6.0 Hz, 2H), 3.67 (t, J=5.2 Hz, 2H), 3.64-3.55 (m, 10H), 3.54-3.51 (m, 2H), 3.50-3.45 (m, 3H), 3.43 (t, J=5.3 Hz, 2H), 2.88-2.82 (m, 1H), 2.76-2.67 (m, 2H), 2.43-2.38 (m, 1H), 2.31 (t, J=6.2 Hz, 2H), 2.18-2.08 (m, 3H), 1.86-1.80 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C47H57N10O10+ 921.4254, found 921.4290.
LQ076-163 was synthesized following the standard procedure for preparing LQ076-135 from intermediate 14 (13 mg, 0.02 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ076-163 was obtained as yellow solid in TFA salt form (16.6 mg, 69%). 1H NMR (800 MHz, Methanol-d4) δ 8.47 (s, 1H), 8.30 (s, 1H), 8.22 (s, 1H), 8.05 (d, J=8.8 Hz, 1H), 7.71-7.65 (m, 2H), 7.59 (d, J=8.6 Hz, 1H), 7.52-7.47 (m, 1H), 7.04-6.99 (m, 2H), 5.05 (dd, J=12.7, 5.5 Hz, 1H), 4.82 (d, J=14.7 Hz, 1H), 4.64-4.55 (m, 3H), 3.77-3.66 (m, 5H), 3.63-3.45 (m, 20H), 3.44 (t, J=5.3 Hz, 2H), 2.89-2.81 (m, 1H), 2.76-2.67 (m, 2H), 2.43-2.38 (m, 1H), 2.35-2.28 (m, 2H), 2.19-2.07 (m, 3H), 1.87-1.80 (m, 1H), 1.51 (d, J=6.3 Hz, 3H). HRMS m/z [M+H]+ calcd for C49H61N10O11+ 965.4516, found 965.4540.
LQ081-100 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((8-aminooctyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-100 was obtained as white solid in TFA salt form (17.6 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.06 (d, J=8.4 Hz, 2H), 7.97 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.52-7.45 (m, 4H), 5.33 (dd, J=8.5, 5.9 Hz, 1H), 4.82 (d, J=14.6 Hz, 1H), 4.75 (d, J=9.2 Hz, 1H), 4.63-4.55 (m, 2H), 4.49-4.44 (m, 1H), 3.87-3.82 (m, 1H), 3.80-3.70 (m, 3H), 3.51-3.43 (m, 1H), 3.38 (t, J=7.2 Hz, 2H), 3.17-3.09 (m, 1H), 3.09-3.02 (m, 1H), 2.86 (dd, J=14.0, 5.8 Hz, 1H), 2.78-2.72 (m, 1H), 2.50 (s, 3H), 2.44-2.35 (m, 1H), 2.26-2.19 (m, 1H), 2.18-2.06 (m, 2H), 2.01-1.94 (m, 1H), 1.88-1.78 (m, 1H), 1.59 (p, J=7.4 Hz, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.42-1.23 (m, 12H), 1.21-1.15 (m, 2H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C57H74FN10O7S+ 1061.5441, found 1061.5461.
LQ081-101 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((10-aminodecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (17 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-101 was obtained as white solid in TFA salt form (18.1 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.82 (s, 1H), 8.17 (d, J=1.9 Hz, 1H), 7.94 (d, J=8.4 Hz, 2H), 7.86 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.7 Hz, 1H), 7.43 (dd, J=8.7, 2.0 Hz, 1H), 7.40-7.32 (m, 4H), 5.21 (dd, J=8.5, 5.8 Hz, 1H), 4.67 (d, J=14.6 Hz, 1H), 4.64 (d, J=9.3 Hz, 1H), 4.50-4.46 (m, 1H), 4.44 (d, J=14.7 Hz, 1H), 4.36-4.33 (m, 1H), 3.76-3.70 (m, 1H), 3.69-3.59 (m, 3H), 3.38-3.33 (m, 1H), 3.30 (t, J=7.2 Hz, 2H), 3.05-2.97 (m, 1H), 2.96-2.89 (m, 1H), 2.74 (dd, J=14.0, 5.8 Hz, 1H), 2.64 (dd, J=14.0, 8.6 Hz, 1H), 2.38 (s, 3H), 2.33-2.25 (m, 1H), 2.13-2.07 (m, 1H), 2.06-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.76-1.67 (m, 1H), 1.52 (p, J=7.3 Hz, 2H), 1.39 (d, J=6.5 Hz, 3H), 1.32-1.08 (m, 14H), 1.07-1.00 (m, 2H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C59H78FN10O7S+ 1089.5754, found 1089.5825.
LQ081-102 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((8-aminooctyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-102 was obtained as white solid in TFA salt form (16.4 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 8.98 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.06 (d, J=8.2 Hz, 2H), 7.97 (d, J=8.1 Hz, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.42 (m, 4H), 5.33 (dd, J=8.5, 5.9 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.69-4.65 (m, 1H), 4.63-4.54 (m, 2H), 4.47-4.44 (m, 1H), 3.81 (d, J=11.1 Hz, 1H), 3.78-3.70 (m, 3H), 3.51-3.43 (m, 1H), 3.38 (t, J=7.2 Hz, 2H), 3.18-3.10 (m, 1H), 3.09-3.01 (m, 1H), 2.86 (dd, J=14.1, 5.9 Hz, 1H), 2.80-2.72 (m, 1H), 2.50 (s, 3H), 2.44-2.36 (m, 1H), 2.23-2.08 (m, 3H), 2.00-1.93 (m, 1H), 1.87-1.78 (m, 1H), 1.68-1.55 (m, 6H), 1.51 (d, J=6.5 Hz, 3H), 1.40-1.23 (m, 8H), 1.21-1.13 (m, 2H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C58H74N11O7S+ 1068.5488, found 1068.5527.
LQ081-103 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((10-aminodecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-cyanocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (16.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-103 was obtained as white solid in TFA salt form (17.9 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 8.30 (d, J=1.7 Hz, 1H), 8.06 (d, J=8.1 Hz, 2H), 7.97 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.55 (dd, J=8.8, 2.0 Hz, 1H), 7.48-7.44 (m, 3H), 7.44-7.36 (m, 1H), 5.32 (dd, J=8.6, 5.7 Hz, 1H), 4.79 (d, J=14.5 Hz, 1H), 4.67 (d, J=8.8 Hz, 1H), 4.62-4.49 (m, 2H), 4.47-4.44 (m, 1H), 3.81 (d, J=11.2 Hz, 1H), 3.78-3.72 (m, 3H), 3.50-3.44 (m, 1H), 3.41 (t, J=7.1 Hz, 2H), 3.17-3.10 (m, 1H), 3.08-3.00 (m, 1H), 2.86 (dd, J=14.0, 5.9 Hz, 1H), 2.76 (dd, J=14.0, 8.6 Hz, 1H), 2.50 (s, 3H), 2.44-2.36 (m, 1H), 2.24-2.07 (m, 2H), 2.00-1.93 (m, 1H), 1.87-1.79 (m, 1H), 1.68-1.55 (m, 9H), 1.51 (d, J=6.5 Hz, 3H), 1.42-1.20 (m, 10H), 1.18-1.11 (m, 2H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C60H78N11O7S+ 1096.5801, found 1096.5721.
LQ081-104 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((8-aminooctyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (13.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-104 was obtained as white solid in TFA salt form (14.4 mg, 58%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.33 (s, 1H), 8.05 (d, J=8.3 Hz, 2H), 7.97 (d, J=8.1 Hz, 2H), 7.70 (d, J=8.7 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.51-7.38 (m, 4H), 6.28-6.19 (m, 1H), 5.38-5.26 (m, 1H), 4.84 (d, J=14.5 Hz, 1H), 4.63-4.55 (m, 1H), 4.52-4.43 (m, 2H), 3.93-3.86 (m, 1H), 3.82-3.65 (m, 3H), 3.61 (d, J=10.6 Hz, 1H), 3.51-3.43 (m, 1H), 3.41-3.35 (m, 2H), 3.15-3.02 (m, 2H), 2.89-2.70 (m, 2H), 2.51 (s, 3H), 2.46-2.36 (m, 2H), 2.28-2.07 (m, 5H), 2.01-1.94 (m, 1H), 1.87-1.79 (m, 1H), 1.64-1.56 (m, 2H), 1.52 (d, J=6.5 Hz, 3H), 1.39-1.14 (m, 9H), 1.09-1.05 (m, 3H), 0.88 (dd, J=18.8, 6.7 Hz, 3H). HRMS m/z [M+H]+ calcd for C56H71N10O7S+ 1027.5222, found 1027.5257.
LQ081-105 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((10-aminodecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (15.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-105 was obtained as white solid in TFA salt form (16.3 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.05 (d, J=8.0 Hz, 2H), 7.97 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.50-7.37 (m, 5H), 6.28-6.22 (m, 1H), 5.36-5.27 (m, 1H), 4.82 (d, J=14.7 Hz, 1H), 4.61-4.43 (m, 3H), 3.93-3.86 (m, 1H), 3.82-3.59 (m, 5H), 3.50-3.38 (m, 3H), 3.14-2.99 (m, 2H), 2.89-2.82 (m, 1H), 2.80-2.70 (m, 1H), 2.50 (s, 3H), 2.47-2.35 (m, 2H), 2.28-2.22 (m, 3H), 2.19-2.07 (m, 2H), 2.01-1.94 (m, 1H), 1.87-1.78 (m, 1H), 1.63 (q, J=7.3 Hz, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.41-1.10 (m, 9H), 1.07 (dd, J=6.6, 2.6 Hz, 3H), 0.88 (dd, J=18.9, 6.7 Hz, 3H). HRMS m/z [M+H]+ calcd for C58H75N10O7S+ 1055.5535, found 1055.5540.
LQ081-106 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2R,4S)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (13.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-106 was obtained as white solid in TFA salt form (18.4 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.98 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.05 (d, J=8.3 Hz, 2H), 7.96 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.54-7.51 (m, 2H), 7.47-7.44 (m, 2H), 5.06-5.00 (m, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.60-4.54 (m, 2H), 4.51-4.49 (m, 1H), 4.49-4.44 (m, 1H), 3.96 (dd, J=10.8, 5.0 Hz, 1H), 3.78-3.68 (m, 3H), 3.50-3.38 (m, 3H), 2.51 (s, 3H), 2.44-2.35 (m, 1H), 2.34-2.26 (m, 1H), 2.24-2.07 (m, 4H), 1.86-1.78 (m, 1H), 1.57 (dd, J=70.8, 7.0 Hz, 8H), 1.46 (d, J=7.0 Hz, 3H), 1.42-1.25 (m, 12H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5487.
LQ081-107 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2R,4S)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (12.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-107 was obtained as white solid in TFA salt form (17.7 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 8.34 (d, J=2.0 Hz, 1H), 8.05 (d, J=8.3 Hz, 2H), 7.97 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.47-7.43 (m, 2H), 7.42-7.38 (m, 2H), 4.84 (d, J=14.6 Hz, 1H), 4.62-4.57 (m, 2H), 4.56-4.49 (m, 2H), 4.47-4.44 (m, 1H), 4.35 (d, J=15.6 Hz, 1H), 4.02 (dd, J=10.9, 4.9 Hz, 1H), 3.78-3.70 (m, 3H), 3.50-3.43 (m, 1H), 3.40 (t, J=7.1 Hz, 2H), 2.51 (s, 3H), 2.43-2.37 (m, 1H), 2.31-2.26 (m, 1H), 2.23-2.07 (m, 3H), 2.05-1.99 (m, 1H), 1.86-1.79 (m, 1H), 1.63 (p, J=7.2 Hz, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.48-1.42 (m, 1H), 1.41-1.17 (m, 14H), 1.09 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O6S+ 974.5321, found 974.5351.
LQ081-108 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (13.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-108 was obtained as white solid in TFA salt form (18.5 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.08-8.03 (m, 2H), 7.99-7.94 (m, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.48-7.42 (m, 4H), 5.01 (q, J=6.9 Hz, 1H), 4.82 (d, J=14.5 Hz, 1H), 4.65-4.62 (m, 1H), 4.62-4.55 (m, 2H), 4.46-4.43 (m, 1H), 3.89 (d, J=11.1 Hz, 1H), 3.79-3.70 (m, 3H), 3.50-3.37 (m, 3H), 2.50 (s, 3H), 2.43-2.36 (m, 1H), 2.34-2.07 (m, 4H), 2.00-1.93 (m, 1H), 1.86-1.79 (m, 1H), 1.69-1.57 (m, 5H), 1.54-1.49 (m, 6H), 1.45-1.30 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5487.
LQ081-109 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(12-aminododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (13.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-109 was obtained as white solid in TFA salt form (17.3 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.32 (d, J=2.0 Hz, 1H), 8.08-8.03 (m, 2H), 7.99-7.94 (m, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.58 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.42 (m, 4H), 5.01 (q, J=6.9 Hz, 1H), 4.83 (d, J=14.6 Hz, 1H), 4.65-4.56 (m, 3H), 4.46-4.43 (m, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.79-3.70 (m, 3H), 3.49-3.39 (m, 3H), 2.50 (s, 3H), 2.43-2.37 (m, 1H), 2.35-2.19 (m, 2H), 2.18-2.08 (m, 1H), 1.99-1.94 (m, 1H), 1.86-1.79 (m, 1H), 1.69-1.57 (m, 5H), 1.54-1.49 (m, 6H), 1.45-1.30 (m, 14H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C56H76N9O6S+ 1002.5634, found 1002.5669.
LQ081-122 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(12-aminododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (13.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-122 was obtained as white solid in TFA salt form (15.9 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.07-8.03 (m, 2H), 7.99-7.94 (m, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.50-7.41 (m, 4H), 4.81 (d, J=14.7 Hz, 1H), 4.66-4.64 (m, 1H), 4.62-4.49 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.77-3.70 (m, 2H), 3.48-3.40 (m, 3H), 2.49 (s, 3H), 2.44-2.35 (m, 1H), 2.34-2.21 (m, 3H), 2.17-2.07 (m, 2H), 1.82 (s, 1H), 1.68-1.57 (m, 3H), 1.51 (d, J=6.6 Hz, 3H), 1.44-1.30 (m, 16H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5481.
A solution of intermediate 8 (Moustakim et al., 2018) (100 mg, 0.43 mmol) was dissolved in DMF and treated with 4-((tert-Butoxycarbonyl)amino)benzoic acid (103 mg, 0.43 mmol), HATU (196 mg, 0.52 mmol) and DIEA (220 μL, 1.3 mmol). After being stirring 1 h at room temperature, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The resulting residue was purified by silica gel flash chromatography to give the compound as yellow oil. The obtained oil was dissolved in 2 mL DCM, to the resulting solution was added 1 mL TFA. After being stirred for 1 h at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 15 as white solid in TFA salt form (135 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 8.25 (d, J=2.0 Hz, 1H), 7.92-7.89 (m, 2H), 7.66 (d, J=8.8 Hz, 1H), 7.54 (dd, J=8.8, 2.0 Hz, 1H), 7.04-7.00 (m, 2H), 4.80 (d, J=14.7 Hz, 1H), 4.56 (d, J=14.6 Hz, 1H), 3.77-3.69 (m, 2H), 3.48-3.42 (m, 1H), 2.43-2.36 (m, 1H), 2.19-2.06 (m, 2H), 1.86-1.78 (m, 1H), 1.50 (d, J=6.5 Hz, 3H). MS (ESI): m/z 350.3 [M+H]+.
To a solution of Intermediate 15 (13 mg, 0.02 mmol) in DMSO (1 mL) were added 12-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecanoic acid (13.3 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ081-132 as white solid in TFA salt form (19.2 mg, 80%). 1H NMR (600 MHz, Methanol-d4) δ 8.96 (s, 1H), 8.27 (d, J=1.9 Hz, 1H), 7.96 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.6 Hz, 2H), 7.66 (d, J=8.7 Hz, 1H), 7.53 (dd, J=8.8, 2.0 Hz, 1H), 7.49-7.42 (m, 4H), 4.79 (d, J=14.6 Hz, 1H), 4.68-4.64 (m, 1H), 4.62-4.49 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.76-3.70 (m, 1H), 3.49-3.43 (m, 1H), 3.37 (s, 1H), 2.49 (s, 3H), 2.45-2.36 (m, 2H), 2.34-2.21 (m, 3H), 2.17-2.06 (m, 1H), 1.85-1.79 (m, 1H), 1.76-1.69 (m, 1H), 1.66-1.57 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.44-1.30 (m, 14H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O6S+ 974.5321, found 974.5312.
LQ081-133 was synthesized following the standard procedure for preparing LQ081-132 from intermediate 15 (13 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(12-aminododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (13.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-133 was obtained as white solid in TFA salt form (18.6 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 8.93 (s, 1H), 8.26 (d, J=1.9 Hz, 1H), 7.96 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.6 Hz, 2H), 7.66 (d, J=8.7 Hz, 1H), 7.53 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.41 (m, 4H), 4.78 (d, J=14.7 Hz, 1H), 4.67-4.64 (m, 1H), 4.62-4.50 (m, 4H), 4.37 (d, J=15.4 Hz, 1H), 3.92 (d, J=11.1 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.76-3.70 (m, 2H), 3.49-3.42 (m, 1H), 2.49 (s, 3H), 2.45-2.36 (m, 2H), 2.34-2.20 (m, 3H), 2.18-2.06 (m, 1H), 1.85-1.78 (m, 1H), 1.76-1.70 (m, 2H), 1.66-1.57 (m, 2H), 1.50 (d, J=6.6 Hz, 3H), 1.44-1.28 (m, 17H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5505.
Intermediate 16 was synthesized according to the procedures for the preparation of intermediate 10 as a white solid in 67% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.63 (s, 1H), 8.31 (d, J=1.9 Hz, 1H), 8.25 (d, J=7.8 Hz, 1H), 8.20 (d, J=7.9 Hz, 1H), 7.72-7.64 (m, 2H), 7.60 (dd, J=8.7, 2.0 Hz, 1H), 4.84 (d, J=14.6 Hz, 1H), 4.61 (d, J=14.6 Hz, 1H), 3.80-3.69 (m, 2H), 3.50-3.42 (m, 1H), 2.44-2.35 (m, 1H), 2.20-2.05 (m, 2H), 1.87-1.79 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). MS (ESI): m/z 379.3 [M+H]+.
To a solution of Intermediate 16 (10 mg, 0.02 mmol) in DMSO (1 mL) were added (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.3 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ081-146 as white solid in TFA salt form (17.7 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.43 (t, J=1.9 Hz, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.12 (d, J=7.7 Hz, 1H), 8.03 (dt, J=7.7, 1.4 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.64 (t, J=7.8 Hz, 1H), 7.58 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.42 (m, 4H), 4.84 (d, J=14.6 Hz, 1H), 4.66-4.63 (m, 1H), 4.62-4.49 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.77-3.71 (m, 2H), 3.49-3.39 (m, 3H), 2.50 (s, 3H), 2.43-2.36 (m, 1H), 2.33-2.20 (m, 3H), 2.18-2.06 (m, 2H), 1.87-1.79 (m, 1H), 1.69-1.57 (m, 3H), 1.51 (d, J=6.5 Hz, 3H), 1.45-1.30 (m, 14H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O6S+ 974.5321, found 974.5337.
LQ081-147 was synthesized following the standard procedure for preparing LQ081-132 from intermediate 15 (13 mg, 0.02 mmol), 11-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoic acid (13.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-147 was obtained as white solid in TFA salt form (16.4 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.27 (d, J=1.9 Hz, 1H), 7.98-7.93 (m, 2H), 7.76 (d, J=8.7 Hz, 2H), 7.67 (d, J=8.7 Hz, 1H), 7.53 (dd, J=8.8, 2.0 Hz, 1H), 7.50-7.46 (m, 2H), 7.45-7.42 (m, 2H), 4.79 (d, J=14.5 Hz, 1H), 4.66-4.64 (m, 1H), 4.61-4.50 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.1 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.77-3.70 (m, 1H), 3.49-3.43 (m, 1H), 2.49 (s, 3H), 2.45-2.37 (m, 2H), 2.34-2.21 (m, 3H), 2.18-2.07 (m, 2H), 1.86-1.80 (m, 1H), 1.75-1.70 (m, 2H), 1.66-1.58 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.44-1.32 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C53H70N9O6S+ 960.5164, found 960.5212.
Intermediate 17 was synthesized according to the procedures for the preparation of intermediate 10 as a white solid in 77% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.01-7.95 (m, 2H), 7.93-7.87 (m, 2H), 7.80-7.75 (m, 2H), 7.41 (dd, J=8.6, 1.9 Hz, 1H), 4.84 (d, J=14.6 Hz, 1H), 4.61 (d, J=14.6 Hz, 1H), 3.82-3.74 (m, 2H), 3.53-3.46 (m, 1H), 2.45-2.36 (m, 1H), 2.21-2.05 (m, 2H), 1.88-1.79 (m, 1H), 1.52 (d, J=6.5 Hz, 3H). MS (ESI): m/z 379.2 [M+H]+.
To a solution of Intermediate 17 (10 mg, 0.02 mmol) in DMSO (1 mL) were added (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (14.4 mg, 0.02 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (6.1 mg, 0.06 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-60% acetonitrile/0.1% TFA in H2O) to afford LQ081-150 as white solid in TFA salt form (18.2 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.31 (s, 1H), 7.70 (d, J=6.0 Hz, 1H), 7.66-7.58 (m, 3H), 7.51-7.42 (m, 6H), 4.80 (d, J=14.8 Hz, 1H), 4.66-4.63 (m, 1H), 4.60-4.50 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.82 (dd, J=11.0, 3.7 Hz, 1H), 3.76-3.70 (m, 2H), 3.49-3.42 (m, 1H), 3.35-3.33 (m, 2H), 2.50 (s, 3H), 2.43-2.36 (m, 1H), 2.32-2.20 (m, 3H), 2.18-2.07 (m, 1H), 1.85-1.78 (m, 1H), 1.65-1.52 (m, 4H), 1.50 (d, J=6.5 Hz, 3H), 1.41-1.19 (m, 14H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O6S+ 974.5321, found 974.5343.
LQ081-158 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2R,4S)-1-((S)-2-(12-aminododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (14.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ081-158 was obtained as white solid in TFA salt form (19.1 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.08-8.03 (m, 2H), 7.99-7.94 (m, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.60-7.55 (m, 1H), 7.54-7.50 (m, 2H), 7.49-7.45 (m, 2H), 5.07-5.01 (m, 1H), 4.81 (d, J=14.7 Hz, 1H), 4.60-4.54 (m, 2H), 4.52-4.48 (m, 1H), 4.48-4.44 (m, 1H), 3.99-3.93 (m, 1H), 3.78-3.67 (m, 3H), 3.50-3.43 (m, 1H), 3.41 (t, J=7.2 Hz, 2H), 2.51 (s, 3H), 2.44-2.36 (m, 1H), 2.34-2.27 (m, 1H), 2.25-2.06 (m, 5H), 1.87-1.78 (m, 1H), 1.69-1.53 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.46 (d, J=7.0 Hz, 3H), 1.43-1.25 (m, 14H), 1.08 (s, 9H). HRMS m/z [M+H]+ calcd for C56H76N9O6S+ 1002.5634, found 1002.5642.
LQ086-31 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((2-aminoethyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-31 was obtained as white solid in TFA salt form (16.7 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.94 (s, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.05-8.00 (m, 2H), 7.95-7.91 (m, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.48-7.44 (m, 2H), 7.42-7.37 (m, 2H), 5.40 (t, J=7.1 Hz, 1H), 4.81 (d, J=14.7 Hz, 1H), 4.78-4.72 (m, 1H), 4.66-4.55 (m, 2H), 4.50-4.45 (m, 1H), 3.90-3.85 (m, 1H), 3.82-3.71 (m, 3H), 3.54-3.37 (m, 6H), 2.87 (dd, J=14.4, 7.0 Hz, 1H), 2.80 (dd, J=14.3, 7.3 Hz, 1H), 2.47 (s, 3H), 2.44-2.36 (m, 1H), 2.26-2.20 (m, 1H), 2.18-2.06 (m, 1H), 2.01-1.95 (m, 1H), 1.87-1.78 (m, 1H), 1.51 (d, J=6.5 Hz, 3H), 1.40-1.20 (m, 4H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C51H62FN10O7S+ 977.4502, found 977.4488.
LQ086-32 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((3-aminopropyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-32 was obtained as white solid in TFA salt form (16.2 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 8.80 (s, 1H), 8.18 (d, J=1.9 Hz, 1H), 7.96-7.91 (m, 2H), 7.86-7.80 (m, 2H), 7.57 (d, J=8.7 Hz, 1H), 7.44 (dd, J=8.7, 2.0 Hz, 1H), 7.41-7.32 (m, 4H), 5.26 (dd, J=8.2, 6.2 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.64 (dd, J=9.4, 1.2 Hz, 1H), 4.51 (dd, J=9.3, 7.6 Hz, 1H), 4.45 (d, J=14.6 Hz, 1H), 4.37-4.32 (m, 1H), 3.76-3.71 (m, 1H), 3.70-3.59 (m, 3H), 3.39-3.31 (m, 1H), 3.21-3.01 (m, 5H), 2.79 (dd, J=14.1, 6.2 Hz, 1H), 2.69 (dd, J=14.2, 8.3 Hz, 1H), 2.36 (s, 3H), 2.33-2.24 (m, 1H), 2.13-2.08 (m, 1H), 2.07-1.95 (m, 1H), 1.90-1.83 (m, 1H), 1.76-1.66 (m, 1H), 1.62-1.54 (m, 2H), 1.40 (d, J=6.5 Hz, 3H), 1.30-1.14 (m, 4H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C52H64FN10O7S+ 991.4659, found 991.4624.
LQ086-33 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((4-aminobutyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-33 was obtained as white solid in TFA salt form (17.2 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.81 (s, 1H), 8.18 (d, J=2.0 Hz, 1H), 7.94-7.90 (m, 2H), 7.85-7.80 (m, 2H), 7.57 (d, J=8.8 Hz, 1H), 7.45 (dd, J=8.8, 2.0 Hz, 1H), 7.38-7.31 (m, 4H), 5.23 (dd, J=8.2, 6.2 Hz, 1H), 4.69 (d, J=14.6 Hz, 1H), 4.64 (d, J=8.7 Hz, 1H), 4.52-4.42 (m, 2H), 4.38-4.32 (m, 1H), 3.76-3.71 (m, 1H), 3.69-3.59 (m, 3H), 3.39-3.31 (m, 1H), 3.25 (t, J=6.6 Hz, 2H), 3.14-3.00 (m, 2H), 2.75 (dd, J=14.1, 6.2 Hz, 1H), 2.66 (dd, J=14.1, 8.3 Hz, 1H), 2.36 (s, 3H), 2.32-2.25 (m, 1H), 2.13-2.07 (m, 1H), 2.07-1.95 (m, 2H), 1.89-1.83 (m, 1H), 1.76-1.66 (m, 1H), 1.45-1.36 (m, 7H), 1.30-1.13 (m, 4H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C53H66FN10O7S+ 1005.4815, found 1005.4822.
LQ086-34 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((5-aminopentyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-34 was obtained as white solid in TFA salt form (17.6 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 8.92 (s, 1H), 8.29 (d, J=2.0 Hz, 1H), 8.08-8.03 (m, 2H), 7.98-7.93 (m, 2H), 7.67 (d, J=8.8 Hz, 1H), 7.55 (dd, J=8.7, 2.0 Hz, 1H), 7.52-7.44 (m, 4H), 5.33 (dd, J=8.2, 6.3 Hz, 1H), 4.81 (d, J=14.7 Hz, 1H), 4.75 (dd, J=9.3, 1.3 Hz, 1H), 4.63-4.55 (m, 2H), 4.48-4.44 (m, 1H), 3.87-3.82 (m, 1H), 3.80-3.71 (m, 3H), 3.51-3.43 (m, 1H), 3.38-3.34 (m, 2H), 3.22-3.15 (m, 1H), 3.15-3.08 (m, 1H), 2.85 (dd, J=14.2, 6.3 Hz, 1H), 2.75 (dd, J=14.2, 8.2 Hz, 1H), 2.48 (s, 3H), 2.44-2.37 (m, 1H), 2.26-2.19 (m, 1H), 2.19-2.06 (m, 2H), 2.03-1.94 (m, 1H), 1.87-1.79 (m, 1H), 1.64-1.56 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.49-1.42 (m, 2H), 1.41-1.26 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C54H68FN10O7S+ 1019.4972, found 1019.4964.
LQ086-35 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((6-aminohexyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-35 was obtained as white solid in TFA salt form (18.1 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 8.93 (s, 1H), 8.30 (d, J=2.0 Hz, 1H), 8.08-8.03 (m, 2H), 7.98-7.94 (m, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.52-7.44 (m, 4H), 5.33 (dd, J=8.4, 6.0 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.75 (dd, J=9.3, 1.3 Hz, 1H), 4.63-4.55 (m, 2H), 4.48-4.43 (m, 1H), 3.87-3.82 (m, 1H), 3.80-3.70 (m, 3H), 3.51-3.43 (m, 1H), 3.36 (t, J=7.1 Hz, 2H), 3.19-3.13 (m, 1H), 3.12-3.06 (m, 1H), 2.86 (dd, J=14.1, 5.9 Hz, 1H), 2.77 (dd, J=14.1, 8.4 Hz, 1H), 2.49 (s, 3H), 2.44-2.36 (m, 1H), 2.26-2.19 (m, 1H), 2.18-2.06 (m, 2H), 2.01-1.95 (m, 1H), 1.87-1.79 (m, 1H), 1.64-1.55 (m, 2H), 1.51 (d, J=6.5 Hz, 3H), 1.45-1.22 (m, 10H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C55H70FN10O7S+ 1033.5128, found 1033.5138.
LQ086-36 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((7-aminoheptyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-36 was obtained as white solid in TFA salt form (16.3 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 8.83 (s, 1H), 8.18 (d, J=1.9 Hz, 1H), 7.94 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.5 Hz, 2H), 7.56 (d, J=8.8 Hz, 1H), 7.45-7.42 (m, 1H), 7.35 (s, 4H), 5.21 (dd, J=8.4, 6.0 Hz, 1H), 4.68 (d, J=14.6 Hz, 1H), 4.63 (d, J=9.5 Hz, 1H), 4.50-4.42 (m, 2H), 4.36-4.33 (m, 1H), 3.75-3.70 (m, 1H), 3.68-3.60 (m, 3H), 3.39-3.32 (m, 1H), 3.28-3.24 (m, 2H), 3.07-2.99 (m, 1H), 2.98-2.93 (m, 1H), 2.74 (dd, J=14.1, 6.0 Hz, 1H), 2.64 (dd, J=14.1, 8.4 Hz, 1H), 2.38 (s, 3H), 2.32-2.24 (m, 1H), 2.13-2.07 (m, 1H), 2.07-1.95 (m, 2H), 1.89-1.83 (m, 1H), 1.75-1.67 (m, 1H), 1.48 (p, J=7.2 Hz, 2H), 1.40 (d, J=6.5 Hz, 3H), 1.30-1.16 (m, 10H), 1.13-1.06 (m, 2H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C56H72FN10O7S+ 1047.5285, found 1047.5291.
LQ086-38 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((9-aminononyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (16.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-38 was obtained as white solid in TFA salt form (19.1 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 8.84 (s, 1H), 8.18 (d, J=1.9 Hz, 1H), 7.94 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.7 Hz, 1H), 7.44 (dd, J=8.7, 2.0 Hz, 1H), 7.41-7.33 (m, 4H), 5.21 (dd, J=8.5, 5.9 Hz, 1H), 4.69 (d, J=14.6 Hz, 1H), 4.64 (d, J=9.3 Hz, 1H), 4.51-4.43 (m, 2H), 4.37-4.32 (m, 1H), 3.75-3.70 (m, 1H), 3.69-3.59 (m, 3H), 3.39-3.31 (m, 1H), 3.28 (t, J=7.2 Hz, 2H), 3.05-2.97 (m, 1H), 2.97-2.89 (m, 1H), 2.74 (dd, J=14.0, 5.9 Hz, 1H), 2.67-2.61 (m, 1H), 2.38 (s, 3H), 2.32-2.24 (m, 1H), 2.13-2.07 (m, 1H), 2.07-1.95 (m, 2H), 1.90-1.83 (m, 1H), 1.76-1.66 (m, 1H), 1.50 (p, J=7.2 Hz, 2H), 1.39 (d, J=6.5 Hz, 3H), 1.31-1.09 (m, 14H), 1.07-1.00 (m, 2H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C55H76FN10O7S+ 1075.5598, found 1075.5607.
LQ086-40 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((11-aminoundecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (16.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-40 was obtained as white solid in TFA salt form (20 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.83 (s, 1H), 8.17 (d, J=2.0 Hz, 1H), 7.94 (d, J=8.4 Hz, 2H), 7.86 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.7 Hz, 1H), 7.44 (dd, J=8.7, 2.0 Hz, 1H), 7.41-7.32 (m, 4H), 5.21 (dd, J=8.5, 5.8 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.64 (d, J=9.3 Hz, 1H), 4.51-4.43 (m, 2H), 4.37-4.32 (m, 1H), 3.75-3.70 (m, 1H), 3.69-3.60 (m, 3H), 3.39-3.32 (m, 1H), 3.30 (t, J=7.2 Hz, 2H), 3.04-2.98 (m, 1H), 2.96-2.89 (m, 1H), 2.74 (dd, J=14.0, 5.8 Hz, 1H), 2.64 (dd, J=14.0, 8.6 Hz, 1H), 2.38 (s, 3H), 2.32-2.25 (m, 1H), 2.13-2.07 (m, 1H), 2.06-1.95 (m, 2H), 1.89-1.83 (m, 1H), 1.75-1.67 (m, 1H), 1.53 (p, J=7.3 Hz, 2H), 1.39 (d, J=6.5 Hz, 3H), 1.34-1.07 (m, 16H), 1.06-0.99 (m, 2H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C60H80FN10O7S+ 1103.5911, found 1103.5898.
LQ086-41 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N—((S)-3-((12-aminododecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (17.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-41 was obtained as white solid in TFA salt form (19.4 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 8.84 (s, 1H), 8.18 (d, J=2.0 Hz, 1H), 7.94 (d, J=8.5 Hz, 2H), 7.85 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.8 Hz, 1H), 7.44 (dd, J=8.7, 2.0 Hz, 1H), 7.40-7.32 (m, 4H), 5.21 (dd, J=8.5, 5.8 Hz, 1H), 4.69 (d, J=14.6 Hz, 1H), 4.64 (d, J=9.5 Hz, 1H), 4.50-4.42 (m, 2H), 4.36-4.33 (m, 1H), 3.75-3.71 (m, 1H), 3.69-3.59 (m, 3H), 3.39-3.33 (m, 1H), 3.31 (t, J=7.2 Hz, 2H), 3.04-2.98 (m, 1H), 2.96-2.89 (m, 1H), 2.74 (dd, J=14.1, 5.9 Hz, 1H), 2.64 (dd, J=14.0, 8.5 Hz, 1H), 2.38 (s, 3H), 2.32-2.24 (m, 1H), 2.13-2.07 (m, 1H), 2.06-1.95 (m, 2H), 1.89-1.83 (m, 1H), 1.75-1.67 (m, 1H), 1.54 (p, J=7.2 Hz, 2H), 1.39 (d, J=6.5 Hz, 3H), 1.34-1.06 (m, 20H), 1.05-0.99 (m, 2H), 0.96 (s, 9H). HRMS m/z [M+H]+ calcd for C61H82FN10O7S+ 1117.6017, found 1117.6005.
An ice bath cooled solution of POCl3 (40 μL, 0.35 mmol) in 0.5 mL dry pyridine was slowly added to a cooled solution of intermediate 18 (100 mg, 0.14 mmol) in 1 mL dry pyridine. The reaction mixture was keeping stirred at ice bath until intermediate 18 was disappeared. Then water was added. After being stirred for 10 mins, the reaction mixture was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford a coler less oil. The obtained oil was dissolved in 0.5 mL DCM, to the resulting solution was added 0.3 mL TFA. After being stirred for 1 h at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 19 as white solid in TFA salt form (78 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.11 (s, 1H), 7.51-7.48 (m, 2H), 7.46-7.43 (m, 2H), 4.63-4.54 (m, 3H), 4.38 (d, J=15.5 Hz, 1H), 4.26-4.20 (m, 1H), 3.95-3.90 (m, 1H), 2.92 (t, J=7.7 Hz, 2H), 2.58-2.50 (m, 4H), 2.37-2.30 (m, 1H), 2.29-2.20 (m, 2H), 1.70-1.57 (m, 4H), 1.44-1.31 (m, 13H), 1.06 (s, 9H). MS (ESI): m/z 694.4 [M+H]+.
LQ086-76 was synthesized following the similar procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), intermediate 19 (16.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ086-76 was obtained as white solid in free base (16.2 mg, 77%).
1H NMR (600 MHz, Methanol-d4) δ 8.88 (s, 1H), 8.21 (s, 1H), 8.09-8.02 (m, 2H), 7.97-7.91 (m, 2H), 7.66 (d, J=8.8 Hz, 1H), 7.61 (dd, J=8.7, 1.9 Hz, 1H), 7.48-7.42 (m, 2H), 7.41-7.38 (m, 2H), 4.99-4.93 (m, 1H), 4.77 (d, J=14.5 Hz, 1H), 4.63-4.57 (m, 2H), 4.56-4.49 (m, 2H), 4.35 (dd, J=15.5, 4.8 Hz, 1H), 4.19-4.13 (m, 1H), 3.90-3.86 (m, 1H), 3.74-3.62 (m, 2H), 3.46-3.35 (m, 4H), 2.55-2.49 (m, 1H), 2.42-2.33 (m, 1H), 2.31-2.22 (m, 1H), 2.21-2.06 (m, 3H), 1.88-1.79 (m, 1H), 1.67-1.60 (m, 2H), 1.59-1.46 (m, 5H), 1.43-1.23 (m, 12H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C54H73N9O9PS+ 1054.4984, found 1054.4997.
LQ076-76 (42 mg, 0.039 mmol) was dissolved in methanol. After the reaction mixture was cooled to ice bath, two equivalent of MeONa (0.5 M in methanol) was added, the mixture was stirred at RT for 1 h. Then evaporated the solvent to give the desired product as white solid. 1H NMR (600 MHz, Methanol-d4) δ 8.76 (s, 1H), 7.98 (d, J=2.0 Hz, 1H), 7.94 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.6 Hz, 1H), 7.38-7.33 (m, 3H), 7.32-7.28 (m, 2H), 4.53-4.50 (m, 1H), 4.50-4.44 (m, 1H), 4.42 (d, J=15.5 Hz, 1H), 4.26 (d, J=15.4 Hz, 1H), 4.06 (d, J=14.3 Hz, 1H), 4.02 (d, J=11.1 Hz, 1H), 3.74-3.69 (m, 1H), 3.55 (d, J=14.3 Hz, 1H), 3.30 (t, J=7.1 Hz, 2H), 3.07-2.99 (m, 3H), 2.99-2.92 (m, 1H), 2.54-2.46 (m, 1H), 2.37 (s, 3H), 2.34-2.26 (m, 2H), 2.21-2.15 (m, 1H), 2.15-2.09 (m, 1H), 2.00-1.88 (m, 1H), 1.73-1.62 (m, 1H), 1.60-1.45 (m, 4H), 1.44-1.34 (m, 1H), 1.35-1.18 (m, 8H), 1.09 (d, J=6.1 Hz, 3H), 0.93 (s, 9H). HRMS m/z [M+H]+ calcd for C54H73N9O9PS+ 1054.4984, found 1054.5027.
A solution of (4-(4-methylthiazol-5-yl)phenyl)methanamine (500 mg, 1.55 mmol) was dissolved in DMF and treated with (2S,4R)-4-(benzyloxy)-1-[(tert-butoxy)carbonyl]pyrrolidine-2-carboxylic acid (500 mg, 1.55 mmol), HATU (707 mg, 1.8 mmol) and DIEA (845 μL, 4.8 mmol). After being stirred 1 h at room temperature, the reaction mixture was poured into ice water, aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine twice, dried and concentrated. The resulting residue was purified by silica gel flash chromatography to give the compound as yellow solid. The obtained solid was dissolved in 5 mL DCM, to the resulting solution was added 3 mL TFA. After being stirred for 1 h at room temperature, the reaction mixture was concentrated and the residue was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 20 as white solid in TFA salt form (500 mg, 79% yield for 2 steps). 1H NMR (600 MHz, Methanol-d4) δ 8.93 (s, 1H), 7.46-7.43 (m, 2H), 7.42-7.39 (m, 2H), 7.36-7.31 (m, 4H), 7.29-7.25 (m, 1H), 4.56 (d, J=3.4 Hz, 2H), 4.51-4.44 (m, 3H), 4.43-4.40 (m, 1H), 3.56-3.52 (m, 1H), 3.46 (dd, J=12.6, 3.9 Hz, 1H), 2.74-2.68 (m, 1H), 2.46 (s, 3H), 2.10-2.04 (m, 1H). MS (ESI): m/z 408.3 [M+H]+.
Intermediate 21 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 79% yield. 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 7.51-7.48 (m, 2H), 7.45-7.42 (m, 2H), 7.39-7.33 (m, 4H), 7.32-7.28 (m, 1H), 4.68 (dd, J=9.7, 7.5 Hz, 1H), 4.61-4.55 (m, 3H), 4.41-4.34 (m, 2H), 4.13 (s, 1H), 4.11-4.06 (m, 1H), 3.75 (dd, J=11.5, 3.7 Hz, 1H), 2.59-2.54 (m, 1H), 2.50 (s, 3H), 2.13-2.07 (m, 1H), 1.16 (s, 9H). MS (ESI): m/z 521.3 [M+H]+.
Intermediate 22 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 83% yield. MS (ESI): m/z 704.4 [M+H]+.
LQ108-4 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), intermediate 22 (16.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-4 was obtained as white solid in TFA salt form (17.9 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.16 (s, 1H), 8.36 (d, J=1.9 Hz, 1H), 8.05 (d, J=8.5 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H), 7.72 (d, J=8.8 Hz, 1H), 7.63 (dd, J=8.8, 2.0 Hz, 1H), 7.51-7.42 (m, 4H), 7.35-7.29 (m, 4H), 7.29-7.24 (m, 1H), 4.89 (d, J=14.7 Hz, 1H), 4.74-4.71 (m, 1H), 4.67-4.48 (m, 5H), 4.38 (d, J=15.5 Hz, 1H), 4.32-4.27 (m, 2H), 3.80-3.70 (m, 3H), 3.49-3.44 (m, 1H), 3.41 (t, J=7.2 Hz, 2H), 2.51 (s, 3H), 2.44-2.37 (m, 2H), 2.33-2.20 (m, 2H), 2.19-2.07 (m, 3H), 1.87-1.80 (m, 1H), 1.67-1.55 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.43-1.26 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C61H78N9O6S+ 1064.5790, found 1064.5825.
Intermediate 23 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 65% yield. 1H NMR (600 MHz, Methanol-d4) δ 9.12 (s, 1H), 7.50-7.43 (m, 4H), 4.52 (d, J=2.3 Hz, 2H), 4.46 (dd, J=10.8, 7.4 Hz, 1H), 4.23 (t, J=4.0 Hz, 1H), 3.55-3.51 (m, 1H), 3.46 (dd, J=12.6, 3.8 Hz, 1H), 3.37 (s, 3H), 2.73-2.67 (m, 1H), 2.51 (s, 3H), 2.10-2.03 (m, 1H). MS (ESI): m/z 331.2 [M+H]+.
Intermediate 24 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 83% yield. 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.51-7.48 (m, 2H), 7.46-7.42 (m, 2H), 4.63-4.55 (m, 2H), 4.38 (d, J=15.5 Hz, 1H), 4.16-4.11 (m, 2H), 4.07-4.02 (m, 1H), 3.69 (dd, J=11.6, 3.6 Hz, 1H), 3.37 (s, 3H), 2.52-2.45 (m, 4H), 2.10-2.03 (m, 1H), 1.16 (s, 9H). MS (ESI): m/z 445.3 [M+H]+.
Intermediate 25 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 77% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.91 (s, 1H), 7.44-7.36 (m, 4H), 4.61 (s, 1H), 4.49 (d, J=15.5 Hz, 1H), 4.42 (dd, J=9.4, 7.5 Hz, 1H), 4.32 (d, J=15.4 Hz, 1H), 4.13-4.09 (m, 1H), 4.06-4.03 (m, 1H), 3.66 (dd, J=11.3, 3.7 Hz, 1H), 3.28 (s, 3H), 2.86 (t, J=7.7 Hz, 2H), 2.44 (s, 3H), 2.34-2.16 (m, 3H), 2.04-1.99 (m, 1H), 1.63-1.52 (m, 5H), 1.39-1.25 (m, 12H), 0.99 (s, 9H). MS (ESI): m/z 628.8 [M+H]+.
LQ108-5 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), intermediate 25 (14.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-5 was obtained as white solid in TFA salt form (17.7 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.12 (s, 1H), 8.35 (d, J=1.9 Hz, 1H), 8.05 (d, J=8.4 Hz, 2H), 7.96 (d, J=8.5 Hz, 2H), 7.71 (d, J=8.8 Hz, 1H), 7.62 (dd, J=8.8, 1.9 Hz, 1H), 7.50-7.46 (m, 2H), 7.45-7.41 (m, 2H), 4.88 (d, J=14.6 Hz, 1H), 4.69-4.62 (m, 2H), 4.54 (d, J=15.5 Hz, 1H), 4.50 (dd, J=9.4, 7.5 Hz, 1H), 4.38 (d, J=15.5 Hz, 1H), 4.18 (d, J=11.6 Hz, 1H), 4.12-4.07 (m, 1H), 3.80-3.68 (m, 3H), 3.50-3.39 (m, 3H), 2.50 (s, 3H), 2.44-2.22 (m, 4H), 2.19-2.04 (m, 3H), 1.88-1.79 (m, 1H), 1.68-1.57 (m, 5H), 1.51 (d, J=6.5 Hz, 3H), 1.44-1.29 (m, 10H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5576.
LQ108-6 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N-(2-(2-((2-aminoethyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.1 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-6 was obtained as white solid in TFA salt form (18.2 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.06-8.01 (m, 2H), 7.95-7.90 (m, 2H), 7.71 (d, J=8.8 Hz, 1H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 7.52-7.45 (m, 2H), 7.09 (dd, J=7.8, 1.6 Hz, 1H), 7.01 (d, J=1.7 Hz, 1H), 4.87 (d, J=14.7 Hz, 1H), 4.75-4.72 (m, 1H), 4.70-4.58 (m, 5H), 4.52-4.46 (m, 2H), 3.89-3.72 (m, 4H), 3.66-3.56 (m, 4H), 3.51-3.43 (m, 1H), 2.50 (s, 3H), 2.45-2.37 (m, 1H), 2.24-2.05 (m, 4H), 1.88-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.39-1.24 (m, 4H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C51H62FN10O8S+ 993.4451, found 933.4523.
LQ108-7 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol (2S,4R)—N-(2-(2-((3-aminopropyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-7 was obtained as white solid in TFA salt form (17.6 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 8.32 (d, J=1.9 Hz, 1H), 8.05-8.02 (m, 2H), 7.99-7.96 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.53 (d, J=7.7 Hz, 1H), 7.48 (dd, J=9.4, 3.3 Hz, 1H), 7.12 (dd, J=7.7, 1.6 Hz, 1H), 7.01 (d, J=1.6 Hz, 1H), 4.85 (d, J=14.6 Hz, 1H), 4.75-4.71 (m, 1H), 4.67-4.64 (m, 2H), 4.64-4.58 (m, 3H), 4.55-4.48 (m, 2H), 3.88-3.82 (m, 1H), 3.81-3.71 (m, 3H), 3.51-3.41 (m, 6H), 2.51 (s, 3H), 2.44-2.37 (m, 1H), 2.23-2.06 (m, 3H), 1.93-1.87 (m, 2H), 1.86-1.80 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.40-1.23 (m, 4H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C52H64FN10O8S+ 1007.4608, found 1007.4653.
LQ108-8 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N-(2-(2-((4-aminobutyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-8 was obtained as white solid in TFA salt form (18.2 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.33 (d, J=1.9 Hz, 1H), 8.07-8.02 (m, 2H), 7.99-7.94 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.48 (dd, J=9.4, 3.3 Hz, 1H), 7.11 (dd, J=7.7, 1.6 Hz, 1H), 7.00 (d, J=1.6 Hz, 1H), 4.85 (d, J=14.7 Hz, 1H), 4.76-4.72 (m, 1H), 4.65-4.57 (m, 5H), 4.51-4.45 (m, 2H), 3.86-3.71 (m, 4H), 3.50-3.35 (m, 6H), 2.51 (s, 3H), 2.44-2.37 (m, 1H), 2.24-2.05 (m, 3H), 1.87-1.80 (m, 1H), 1.71-1.64 (m, 4H), 1.52 (d, J=6.5 Hz, 3H), 1.40-1.24 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C53H66FN10O8S+ 1021.4764, found 1021.4816.
LQ108-9 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N-(2-(2-((5-aminopentyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (14.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-9 was obtained as white solid in TFA salt form (20 mg, 79%). 1H NMR (600 MHz, Methanol-d4) δ 9.02 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.05-8.00 (m, 2H), 7.98-7.93 (m, 2H), 7.69 (d, J=8.7 Hz, 1H), 7.57 (dd, J=8.7, 2.0 Hz, 1H), 7.51-7.46 (m, 2H), 7.11 (dd, J=7.7, 1.6 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 4.83 (d, J=14.7 Hz, 1H), 4.76-4.72 (m, 1H), 4.62-4.57 (m, 5H), 4.52-4.48 (m, 1H), 4.44 (d, J=15.0 Hz, 1H), 3.88-3.83 (m, 1H), 3.82-3.72 (m, 3H), 3.50-3.44 (m, 1H), 3.43-3.34 (m, 5H), 2.51 (s, 3H), 2.44-2.37 (m, 1H), 2.25-2.05 (m, 3H), 1.87-1.79 (m, 1H), 1.71-1.62 (m, 4H), 1.52 (d, J=6.5 Hz, 3H), 1.47-1.25 (m, 6H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C54H68FN10O8S+ 1035.4921, found 1035.4963.
LQ108-10 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N-(2-(2-((6-aminohexyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-10 was obtained as white solid in TFA salt form (18.9 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 8.34 (d, J=1.9 Hz, 1H), 8.07-8.02 (m, 2H), 7.99-7.94 (m, 2H), 7.71 (d, J=8.8 Hz, 1H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 7.50 (dd, J=21.8, 7.0 Hz, 2H), 7.12 (dd, J=7.7, 1.6 Hz, 1H), 7.00 (d, J=1.6 Hz, 1H), 4.86 (d, J=14.6 Hz, 1H), 4.76-4.72 (m, 1H), 4.65-4.58 (m, 5H), 4.52-4.45 (m, 2H), 3.88-3.83 (m, 1H), 3.81-3.71 (m, 3H), 3.50-3.44 (m, 1H), 3.40 (t, J=7.1 Hz, 2H), 3.32-3.28 (m, 1H), 2.52 (s, 3H), 2.45-2.36 (m, 1H), 2.25-2.05 (m, 4H), 1.88-1.80 (m, 1H), 1.66-1.57 (m, 4H), 1.52 (d, J=6.5 Hz, 3H), 1.45-1.24 (m, 9H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C55H70FN10O8S+ 1049.5077, found 1049.5140.
LQ108-11 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N-(2-(2-((7-aminoheptyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-11 was obtained as white solid in TFA salt form (18.3 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 8.32 (d, J=1.9 Hz, 1H), 8.07-8.02 (m, 2H), 7.99-7.94 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.58 (dd, J=8.8, 2.0 Hz, 1H), 7.52-7.46 (m, 2H), 7.11 (dd, J=7.7, 1.6 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 4.84 (d, J=14.6 Hz, 1H), 4.77-4.72 (m, 1H), 4.65-4.57 (m, 5H), 4.52-4.45 (m, 2H), 3.89-3.83 (m, 1H), 3.82-3.72 (m, 3H), 3.51-3.43 (m, 1H), 3.41 (t, J=7.2 Hz, 2H), 3.32-3.27 (m, 3H), 2.51 (s, 3H), 2.45-2.36 (m, 1H), 2.25-2.06 (m, 3H), 1.87-1.79 (m, 1H), 1.67-1.55 (m, 4H), 1.52 (d, J=6.5 Hz, 3H), 1.42-1.25 (m, 10H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C56H72FN10O8S+ 1063.5234, found 1063.5276.
LQ108-12 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), (2S,4R)—N-(2-(2-((8-aminooctyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (15.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-12 was obtained as white solid in TFA salt form (17.9 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.07-8.02 (m, 2H), 7.99-7.94 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.52 (d, J=7.7 Hz, 1H), 7.49 (dd, J=9.4, 3.4 Hz, 1H), 7.12 (dd, J=7.7, 1.6 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 4.85 (d, J=14.7 Hz, 1H), 4.77-4.73 (m, 1H), 4.65-4.56 (m, 5H), 4.52-4.45 (m, 2H), 3.89-3.83 (m, 1H), 3.82-3.71 (m, 3H), 3.51-3.43 (m, 1H), 3.41 (t, J=7.2 Hz, 2H), 3.32-3.26 (m, 3H), 2.52 (s, 3H), 2.44-2.37 (m, 1H), 2.26-2.20 (m, 1H), 2.18-2.06 (m, 3H), 1.83 (s, 1H), 1.64 (p, J=7.2 Hz, 2H), 1.57 (p, J=7.1 Hz, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.44-1.26 (m, 12H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C57H74FN10O8S+ 1077.5390, found 1077.5443.
Intermediate 26 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 63% yield. MS (ESI): m/z 422.6 [M+H]+.
Intermediate 27 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 74% yield. 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.49-7.43 (m, 4H), 7.39-7.32 (m, 4H), 7.32-7.27 (m, 1H), 5.03 (q, J=7.1 Hz, 1H), 4.69 (dd, J=9.6, 7.6 Hz, 1H), 4.57 (s, 2H), 4.29 (t, J=4.0 Hz, 1H), 4.12 (s, 1H), 4.08-4.02 (m, 1H), 3.69 (dd, J=11.6, 3.7 Hz, 1H), 2.58-2.52 (m, 1H), 2.50 (s, 3H), 1.98-1.92 (m, 1H), 1.52 (d, J=7.1 Hz, 3H), 1.17 (s, 9H). MS (ESI): m/z 535.4 [M+H]+.
Intermediate 28 was synthesized according to the procedures for the preparation of intermediate 20 as a white solid in 69% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.93 (s, 1H), 7.48-7.42 (m, 4H), 7.36-7.30 (m, 4H), 7.29-7.25 (m, 1H), 5.02 (q, J=6.9 Hz, 1H), 4.72 (s, 1H), 4.62-4.47 (m, 3H), 4.29-4.24 (m, 2H), 3.71 (dd, J=11.6, 3.9 Hz, 1H), 2.92 (t, J=7.7 Hz, 2H), 2.50 (s, 3H), 2.44-2.36 (m, 1H), 2.35-2.20 (m, 2H), 2.02-1.96 (m, 1H), 1.70-1.56 (m, 5H), 1.52 (d, J=7.0 Hz, 3H), 1.44-1.29 (m, 12H), 1.07 (s, 9H). MS (ESI): m/z 718.3 [M+H]+.
LQ108-141 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), intermediate 28 (16.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-141 was obtained as white solid in TFA salt form (20.4 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 9.09 (s, 1H), 8.34 (d, J=2.0 Hz, 1H), 8.08-8.02 (m, 2H), 7.98-7.94 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 7.48-7.42 (m, 4H), 7.34-7.29 (m, 4H), 7.29-7.24 (m, 1H), 5.01 (q, J=7.0 Hz, 1H), 4.86 (d, J=14.6 Hz, 1H), 4.72 (s, 1H), 4.65-4.55 (m, 3H), 4.51-4.46 (m, 1H), 4.29-4.22 (m, 2H), 3.79-3.66 (m, 3H), 3.49-3.38 (m, 4H), 2.50 (s, 3H), 2.43-2.36 (m, 2H), 2.33-2.20 (m, 2H), 2.19-2.05 (m, 2H), 2.01-1.95 (m, 1H), 1.87-1.79 (m, 1H), 1.70-1.54 (m, 5H), 1.53-1.49 (m, 6H), 1.45-1.26 (m, 11H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C62H80N9O6S+ 1078.5947, found 1078.5958.
Intermediate 29 was synthesized according to the procedure for the preparation of intermediate 20 as a white solid in 77% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.86 (s, 1H), 7.41-7.35 (m, 4H), 7.29-7.24 (m, 4H), 7.23-7.19 (m, 1H), 4.96 (q, J=6.8 Hz, 1H), 4.66 (s, 1H), 4.55-4.41 (m, 3H), 4.23-4.17 (m, 2H), 3.64 (dd, J=11.6, 3.9 Hz, 1H), 2.86 (t, J=7.7 Hz, 2H), 2.43 (s, 3H), 2.37-2.30 (m, 1H), 2.27-2.14 (m, 2H), 1.96-1.89 (m, 1H), 1.63-1.49 (m, 3H), 1.46 (d, J=7.0 Hz, 3H), 1.37-1.21 (m, 14H), 1.00 (s, 9H). MS (ESI): m/z 732.7 [M+H]+.
LQ108-142 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), intermediate 29 (17 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-142 was obtained as white solid in TFA salt form (20.8 mg, 79%). 1H NMR (600 MHz, Methanol-d4) δ 9.09 (s, 1H), 8.34 (d, J=2.0 Hz, 1H), 8.07-8.02 (m, 2H), 7.99-7.94 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.61 (dd, J=8.8, 2.0 Hz, 1H), 7.48-7.42 (m, 4H), 7.34-7.28 (m, 5H), 7.28-7.24 (m, 1H), 5.01 (q, J=7.0 Hz, 1H), 4.86 (d, J=14.6 Hz, 1H), 4.71 (s, 1H), 4.65-4.55 (m, 3H), 4.51-4.45 (m, 1H), 4.29-4.23 (m, 2H), 3.79-3.66 (m, 3H), 3.49-3.39 (m, 3H), 2.50 (s, 3H), 2.43-2.36 (m, 2H), 2.33-2.20 (m, 2H), 2.18-2.06 (m, 2H), 2.01-1.95 (m, 1H), 1.87-1.79 (m, 1H), 1.68-1.55 (m, 3H), 1.51 (d, J=7.5 Hz, 4H), 1.45-1.26 (m, 16H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C63H82N9O6S+ 1092.6103, found 1092.6113.
LQ108-146 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (10.9 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-146 was obtained as yellow solid in TFA salt form (14.8 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 8.16 (d, J=1.9 Hz, 1H), 7.93-7.89 (m, 2H), 7.84-7.78 (m, 2H), 7.55 (d, J=8.8 Hz, 1H), 7.46-7.40 (m, 2H), 6.97 (d, J=2.2 Hz, 1H), 6.82 (dd, J=8.4, 2.2 Hz, 1H), 4.92 (dd, J=12.6, 5.5 Hz, 1H), 4.68 (d, J=14.6 Hz, 1H), 4.44 (d, J=14.6 Hz, 1H), 3.67-3.59 (m, 2H), 3.55 (t, J=6.3 Hz, 2H), 3.43 (t, J=6.3 Hz, 2H), 3.37-3.31 (m, 1H), 2.77-2.69 (m, 1H), 2.64-2.53 (m, 2H), 2.32-2.23 (m, 1H), 2.07-1.93 (m, 3H), 1.75-1.66 (m, 1H), 1.39 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C36H37N8O6+ 677.2831, found 677.2797.
LQ108-147 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((3-aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-147 was obtained as yellow solid in TFA salt form (13.9 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 8.17 (d, J=1.9 Hz, 1H), 7.96-7.91 (m, 2H), 7.87-7.83 (m, 2H), 7.57-7.53 (m, 1H), 7.48-7.42 (m, 2H), 6.90 (d, J=2.2 Hz, 1H), 6.76 (dd, J=8.4, 2.2 Hz, 1H), 4.93 (dd, J=12.5, 5.5 Hz, 1H), 4.67 (d, J=14.6 Hz, 1H), 4.44 (d, J=14.7 Hz, 1H), 3.66-3.59 (m, 2H), 3.45 (t, J=6.8 Hz, 2H), 3.38-3.31 (m, 1H), 3.24 (t, J=6.9 Hz, 2H), 2.78-2.69 (m, 1H), 2.66-2.55 (m, 2H), 2.32-2.24 (m, 1H), 2.09-1.95 (m, 3H), 1.89 (p, J=6.9 Hz, 2H), 1.75-1.66 (m, 1H), 1.39 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C37H39N8O6+ 691.2987, found 691.2999.
LQ108-148 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((4-aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-148 was obtained as yellow solid in TFA salt form (12.8 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=2.0 Hz, 1H), 8.06-8.01 (m, 2H), 7.95-7.91 (m, 2H), 7.70-7.66 (m, 1H), 7.58-7.52 (m, 2H), 6.99 (d, J=2.2 Hz, 1H), 6.85 (dd, J=8.4, 2.2 Hz, 1H), 5.04 (dd, J=12.6, 5.5 Hz, 1H), 4.79 (d, J=14.6 Hz, 1H), 4.55 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.51-3.43 (m, 3H), 3.31 (t, J=6.5 Hz, 2H), 2.87-2.79 (m, 1H), 2.75-2.66 (m, 2H), 2.44-2.37 (m, 1H), 2.19-2.05 (m, 2H), 1.87-1.75 (m, 6H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C38H41N8O6+ 705.3144, found 705.3141.
LQ108-149 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((5-aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-149 was obtained as yellow solid in TFA salt form (14.3 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=1.9 Hz, 1H), 8.06-8.01 (m, 2H), 7.96-7.91 (m, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.57-7.52 (m, 2H), 6.98 (d, J=2.2 Hz, 1H), 6.84 (dd, J=8.4, 2.2 Hz, 1H), 5.03 (dd, J=12.4, 5.5 Hz, 1H), 4.79 (d, J=14.7 Hz, 1H), 4.55 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.50-3.43 (m, 3H), 3.27 (t, J=6.9 Hz, 2H), 2.86-2.77 (m, 1H), 2.74-2.64 (m, 2H), 2.44-2.36 (m, 1H), 2.19-2.05 (m, 2H), 1.86-1.70 (m, 6H), 1.60-1.53 (m, 2H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C39H43N8O6+ 719.3300, found 719.3295.
LQ108-150 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((6-aminohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-150 was obtained as yellow solid in TFA salt form (15.6 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 8.30 (d, J=2.0 Hz, 1H), 8.07-8.01 (m, 2H), 7.97-7.92 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.59-7.52 (m, 2H), 6.97 (d, J=2.2 Hz, 1H), 6.83 (dd, J=8.4, 2.2 Hz, 1H), 5.03 (dd, J=12.6, 5.5 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.51-3.42 (m, 3H), 3.23 (t, J=7.0 Hz, 2H), 2.87-2.78 (m, 1H), 2.75-2.65 (m, 2H), 2.45-2.36 (m, 1H), 2.19-2.05 (m, 2H), 1.88-1.78 (m, 1H), 1.75-1.65 (m, 4H), 1.57-1.46 (m, 8H). HRMS m/z [M+H]+ calcd for C40H45N8O6+ 733.3457, found 733.3485.
LQ108-151 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((7-aminoheptyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12.2 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-151 was obtained as yellow solid in TFA salt form (17.3 mg, 80%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=1.9 Hz, 1H), 8.07-8.02 (m, 2H), 7.98-7.93 (m, 2H), 7.68 (d, J=8.8 Hz, 1H), 7.57-7.53 (m, 2H), 6.97 (d, J=2.2 Hz, 1H), 6.83 (dd, J=8.4, 2.2 Hz, 1H), 5.04 (dd, J=12.4, 5.5 Hz, 1H), 4.80 (d, J=14.7 Hz, 1H), 4.56 (d, J=14.6 Hz, 1H), 3.78-3.70 (m, 2H), 3.50-3.41 (m, 3H), 3.22 (t, J=7.1 Hz, 2H), 2.88-2.79 (m, 1H), 2.75-2.65 (m, 2H), 2.44-2.36 (m, 1H), 2.19-2.06 (m, 2H), 1.87-1.78 (m, 1H), 1.72-1.64 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.50-1.43 (m, 7H). HRMS m/z [M+H]+ calcd for C41H47N8O6+ 747.3613, found 747.3638.
LQ108-152 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12.6 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-152 was obtained as yellow solid in TFA salt form (16.4 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=2.0 Hz, 1H), 8.07-8.02 (m, 2H), 7.98-7.94 (m, 2H), 7.68 (dd, J=8.7, 0.6 Hz, 1H), 7.57-7.53 (m, 2H), 6.97 (d, J=2.2 Hz, 1H), 6.83 (dd, J=8.4, 2.2 Hz, 1H), 5.04 (dd, J=12.7, 5.5 Hz, 1H), 4.80 (d, J=14.6 Hz, 1H), 4.56 (d, J=14.6 Hz, 1H), 3.78-3.71 (m, 2H), 3.50-3.40 (m, 3H), 3.21 (t, J=7.1 Hz, 2H), 2.88-2.80 (m, 1H), 2.76-2.65 (m, 2H), 2.45-2.36 (m, 1H), 2.20-2.06 (m, 2H), 1.86-1.79 (m, 1H), 1.71-1.64 (m, 4H), 1.51 (d, J=6.5 Hz, 3H), 1.49-1.40 (m, 9H). HRMS m/z [M+H]+ calcd for C42H49N8O6+ 761.3770, found 761.3764.
LQ108-153 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((2-(2-aminoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (11.8 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-153 was obtained as yellow solid in TFA salt form (15.8 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 8.18 (d, J=1.9 Hz, 1H), 7.90-7.86 (m, 2H), 7.79-7.75 (m, 2H), 7.58-7.54 (m, 1H), 7.46 (dd, J=8.7, 2.0 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 6.87 (d, J=2.2 Hz, 1H), 6.74 (dd, J=8.4, 2.2 Hz, 1H), 4.85 (dd, J=12.4, 5.4 Hz, 1H), 4.68 (d, J=14.6, 1H), 4.44 (d, J=14.6, 1H), 3.68-3.58 (m, 6H), 3.54-3.48 (m, 2H), 3.39-3.30 (m, 3H), 2.63-2.43 (m, 3H), 2.33-2.24 (m, 1H), 2.09-1.89 (m, 3H), 1.75-1.67 (m, 1H), 1.40 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C38H41N8O7+ 721.3093, found 721.3121.
LQ108-154 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12.7 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-154 was obtained as yellow solid in TFA salt form (16.6 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.28 (d, J=2.0 Hz, 1H), 8.03-7.97 (m, 2H), 7.97-7.92 (m, 2H), 7.67 (d, J=8.7 Hz, 1H), 7.55 (dd, J=8.8, 2.0 Hz, 1H), 7.51 (d, J=8.3 Hz, 1H), 6.97 (d, J=2.2 Hz, 1H), 6.83 (dd, J=8.4, 2.2 Hz, 1H), 4.97 (dd, J=12.6, 5.5 Hz, 1H), 4.81 (d, J=14.7 Hz, 1H), 4.57 (d, J=14.7, 1H), 3.79-3.68 (m, 11H), 3.63 (t, J=5.4 Hz, 2H), 3.51-3.43 (m, 1H), 3.37 (t, J=5.3 Hz, 2H), 2.78-2.70 (m, 1H), 2.66-2.58 (m, 2H), 2.44-2.37 (m, 1H), 2.20-2.07 (m, 2H), 2.05-1.99 (m, 1H), 1.87-1.79 (m, 1H), 1.52 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C40H45N8O8+ 765.3355, found 765.3390.
LQ108-155 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (13.5 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-155 was obtained as yellow solid in TFA salt form (17.8 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 8.28 (d, J=1.9 Hz, 1H), 8.04-8.01 (m, 2H), 7.98-7.93 (m, 2H), 7.68-7.65 (m, 1H), 7.54 (dd, J=8.7, 2.0 Hz, 1H), 7.49 (d, J=8.3 Hz, 1H), 6.97 (d, J=2.2 Hz, 1H), 6.83 (dd, J=8.4, 2.2 Hz, 1H), 5.01 (dd, J=12.7, 5.5 Hz, 1H), 4.80 (d, J=14.6 Hz, 1H), 4.56 (d, J=14.7 Hz, 1H), 3.78-3.60 (m, 17H), 3.51-3.43 (m, 1H), 3.37 (t, J=5.4 Hz, 2H), 2.86-2.77 (m, 1H), 2.73-2.62 (m, 2H), 2.45-2.36 (m, 1H), 2.20-2.03 (m, 2H), 1.87-1.78 (m, 1H), 1.52 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C42H49N8O8+ 809.3617, found 809.3643.
LQ108-156 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((14-amino-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (14.4 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-156 was obtained as yellow solid in TFA salt form (16.9 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=2.0 Hz, 1H), 8.06-8.03 (m, 2H), 7.99-7.95 (m, 2H), 7.67 (d, J=8.8 Hz, 1H), 7.56 (dd, J=8.8, 2.0 Hz, 1H), 7.49 (dd, J=8.3, 1.1 Hz, 1H), 6.98 (dd, J=2.2, 1.0 Hz, 1H), 6.84-6.80 (m, 1H), 5.03 (dd, J=12.7, 5.5 Hz, 1H), 4.81 (d, J=14.7, 1H), 4.57 (d, J=14.6, 1H), 3.79-3.71 (m, 2H), 3.71-3.59 (m, 19H), 3.50-3.43 (m, 1H), 3.39-3.34 (m, 2H), 2.88-2.80 (m, 1H), 2.75-2.64 (m, 2H), 2.44-2.37 (m, 1H), 2.19-2.05 (m, 2H), 1.87-1.78 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C44H53N8O10+ 853.3879, found 853.3871.
LQ108-157 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 10 (10 mg, 0.02 mmol), 5-((17-amino-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (15.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-157 was obtained as yellow solid in TFA salt form (16.3 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 8.29 (d, J=1.9 Hz, 1H), 8.07-8.02 (m, 2H), 8.01-7.96 (m, 2H), 7.66 (d, J=8.8 Hz, 1H), 7.55 (dd, J=8.7, 2.0 Hz, 1H), 7.51 (dd, J=8.6, 7.1 Hz, 1H), 7.04 (d, J=8.5 Hz, 1H), 7.01 (d, J=7.1 Hz, 1H), 5.04 (dd, J=12.8, 5.5 Hz, 1H), 4.81 (d, J=14.6 Hz, 1H), 4.57 (d, J=14.6 Hz, 1H), 3.78-3.57 (m, 25H), 3.49-3.43 (m, 3H), 2.89-2.81 (m, 1H), 2.76-2.66 (m, 2H), 2.43-2.36 (m, 1H), 2.20-2.07 (m, 2H), 1.87-1.78 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). HRMS m/z [M+H]+ calcd for C46H57N8O11+ 897.4141, found 8974174.
Intermediate 30 was synthesized according to the procedures for the preparation of intermediate 10 as a white solid in yield. 1H NMR (600 MHz, Methanol-d4) δ 8.32 (d, J=2.0 Hz, 1H), 8.20-8.14 (m, 2H), 8.08-8.02 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.60 (dd, J=8.8, 2.0 Hz, 1H), 4.85 (d, J=14.6 Hz, 1H), 4.62 (d, J=14.6 Hz, 1H), 3.80-3.69 (m, 2H), 3.50-3.43 (m, 1H), 2.44-2.35 (m, 1H), 2.20-2.05 (m, 2H), 1.88-1.78 (m, 1H), 1.51 (d, J=6.5 Hz, 3H). MS (ESI): m/z 379.7 [M+H]+.
LQ118-23 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 30 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ118-23 was obtained as white solid in TFA salt form (16.3 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 8.31 (d, J=2.0 Hz, 1H), 8.08-8.03 (m, 2H), 7.98-7.95 (m, 2H), 7.69 (d, J=8.8 Hz, 1H), 7.58 (dd, J=8.7, 2.0 Hz, 1H), 7.51-7.46 (m, 2H), 7.45-7.42 (m, 2H), 4.82 (d, J=14.6 Hz, 1H), 4.65 (s, 1H), 4.62-4.50 (m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.95-3.90 (m, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.77-3.71 (m, 2H), 3.50-3.39 (m, 3H), 2.50 (s, 3H), 2.43-2.36 (m, 1H), 2.34-2.21 (m, 3H), 2.18-2.07 (m, 2H), 1.87-1.79 (m, 1H), 1.69-1.58 (m, 5H), 1.51 (d, J=6.5 Hz, 3H), 1.46-1.31 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H72N9O6S+ 974.5321, found 974.5323.
Sodium hydride (60 mg, 1.5 mmol, 60% in mineral oil) was added in portions to a solution of intermediate 1 (211 mg, 1 mmol) in dry dimethylformamide (3 mL) at ice bath. The mixture was stirred for 30 min at the same temperature, then iodomethane (63 μL, 1 mmol) was added. The resultant mixture was stirred for 1 h at room temperature. After cooling with ice bath, water was added slowly to quench the excess of sodium hydride. The mixture was extracted with ethyl acetate. Combined organic phases was washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified to by silica gel flash chromatography yield the title compound as yellow solid (136 mg, 61%). MS (ESI): m/z 226.1 [M+H]+.
Intermediate 32 was synthesized according to the procedures for the preparation of intermediate 10 as a white solid in 69% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.15 (d, J=1.9 Hz, 1H), 8.05-8.00 (m, 2H), 7.95-7.89 (m, 2H), 7.55 (dd, J=8.8, 2.0 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 4.84 (d, J=15.5 Hz, 1H), 4.58 (d, J=15.4 Hz, 1H), 3.83 (s, 3H), 3.79-3.68 (m, 2H), 3.38-3.30 (m, 1H), 2.35-2.26 (m, 1H), 2.12-1.97 (m, 2H), 1.79-1.71 (m, 1H), 1.43 (d, J=6.6 Hz, 3H). MS (ESI): m/z 393.1 [M+H]+.
LQ118-24 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 32 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ118-24 was obtained as white solid in TFA salt form (16.3 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 8.26 (dd, J=7.8, 1.9 Hz, 1H), 8.09-8.03 (m, 2H), 8.00-7.94 (m, 2H), 7.71 (d, J=8.7 Hz, 1H), 7.67-7.60 (m, 1H), 7.52-7.42 (m, 4H), 4.97-4.91 (m, 3H), 4.70-4.63 (m, 2H), 4.62-4.49 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 3.96-3.86 (m, 6H), 3.82 (dd, J=10.9, 3.9 Hz, 1H), 3.49-3.39 (m, 4H), 2.50 (s, 3H), 2.46-2.39 (m, 1H), 2.35-2.07 (m, 5H), 1.91-1.83 (m, 1H), 1.70-1.58 (m, 4H), 1.54 (dd, J=6.6, 2.3 Hz, 3H), 1.46-1.32 (m, 12H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5465.
Intermediate 33 was synthesized according to the procedures for the preparation of Intermediate 10 as a white solid in 78% yield. 1H NMR (600 MHz, Methanol-d4) δ 9.53 (d, J=1.7 Hz, 1H), 8.56 (s, 1H), 8.20 (dd, J=9.3, 1.7 Hz, 1H), 7.93 (dd, J=9.8, 2.8 Hz, 1H), 7.86-7.82 (m, 1H), 7.37-7.31 (m, 5H), 7.08 (d, J=9.8 Hz, 1H), 4.54 (s, 2H), 3.46 (t, J=7.3 Hz, 2H), 3.43-3.39 (m, 5H), 3.11 (t, J=7.2 Hz, 2H). MS (ESI): m/z 393.4 [M+H]+.
LQ118-25 was synthesized following the standard procedure for preparing LQ076-105 from intermediate 33 (10 mg, 0.02 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (15.3 mg, 0.02 mmol, 1.0 equiv), EDCI (5.8 mg, 0.03 mmol, 1.5 equiv), HOAt (4.1 mg, 0.03 mmol, 1.5 equiv), and NMM (6.1 mg, 0.06 mmol, 3.0 equiv) in DMSO (1 mL). LQ118-25 was obtained as white solid in TFA salt form (16.3 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 8.33 (d, J=2.0 Hz, 1H), 8.07-8.04 (m, 2H), 7.99-7.95 (m, 2H), 7.70 (d, J=8.5 Hz, 1H), 7.59 (dd, J=8.8, 2.0 Hz, 1H), 7.51-7.43 (m, 4H), 4.65 (s, 1H), 4.62-4.50 (m, 5H), 4.38 (d, J=15.5 Hz, 1H), 3.94-3.90 (m, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.69-3.64 (m, 2H), 3.42 (t, J=7.2 Hz, 2H), 3.20-3.13 (m, 2H), 2.50 (s, 3H), 2.35-2.21 (m, 3H), 2.12-2.07 (m, 1H), 2.00-1.95 (m, 2H), 1.79-1.71 (m, 1H), 1.70-1.50 (m, 5H), 1.46-1.31 (m, 16H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H74N9O6S+ 988.5477, found 988.5471.
Intermediate 34 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 78% yield. 1H NMR (600 MHz, Methanol-d4) δ 7.97 (d, J=8.1 Hz, 1H), 7.50-7.45 (m, 2H), 2.60 (s, 3H). MS (ESI): m/z 179.0 [M−H]+.
Intermediate 35 was synthesized according to the procedures for the preparation of intermediate 3 as a white solid in 76%. 1H NMR (600 MHz, Methanol-d4) δ 7.57-7.51 (m, 1H), 7.45 (s, 1H), 7.33-7.28 (m, 1H), 3.02 (d, J=103.3 Hz, 6H), 2.58 (s, 1H). MS (ESI): m/z 208.3 [M+H]+.
To a solution of intermediate 35 (400 mg, 1.93 mmol) was added imidazole (263 mg, 3.86 mmol) and triisopropylsilyl chloride (667 mg, 3.47 mmol). The resulting mixture was stirred 6 h at RT.
After the reaction was completed, the reaction mixture was poured into water, aqueous phase was extracted with DCM. The combined organic phase was washed with brine, dried and concentrated. The resulting residue was purified by silica gel flash chromatography to give the compound as yellow oil (525 mg, 75%). 1H NMR (600 MHz, Chloroform-d) δ 7.54 (dd, J=7.8, 1.5 Hz, 1H), 7.43 (d, J=1.5 Hz, 1H), 7.32 (d, J=7.8 Hz, 1H), 3.08 (s, 3H), 2.86 (s, 3H), 2.57 (s, 3H), 1.35-1.27 (m, 3H), 1.13-1.03 (m, 18H). MS (ESI): m/z 364.5 [M+H]+.
A solution of intermediate 36 (560 mg, 1.54 mmol) and dimethyl oxalate (182 mg, 1.54 mmol) in Et2O was added sodium methoxide solution (0.5 M, 4 mL) slowly. The resulting mixture was stirred overnight at RT. After the reaction was completed, the mixture was purified by reverse phase C18 column (10%-100% acetonitrile/0.1% TFA in water) to afford intermediate 37 as white solid (85 mg, 19%). 1H NMR (600 MHz, Methanol-d4) δ 7.58 (d, J=7.8 Hz, 1H), 7.51 (s, 1H), 7.34 (d, J=7.8 Hz, 1H), 7.07 (s, 1H), 3.92 (s, 3H), 3.11 (s, 3H), 2.94 (s, 3H). MS (ESI): m/z 294.2 [M+H]+.
The intermediate 37 (97 mg, 0.33 mmol) was dissolved in methanol and treated with hydroxylamine hydrochloride (69 mg, 1 mmol) The resulting mixture was heated to 55° C. overnight. Then the mixture was purified by reverse phase C18 column (10%-100% methanol/0.1% TFA in water) to afford intermediate 38 as white solid (57 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 7.43 (dd, J=7.9, 1.5 Hz, 1H), 7.36 (d, J=1.5 Hz, 1H), 7.34 (d, J=7.9 Hz, 1H), 7.18 (s, 1H), 3.98 (s, 3H), 3.04 (d, J=79.3 Hz, 6H). MS (ESI): m/z 291.3 [M+H]+.
Intermediate 39 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 34% yield. 1H NMR (600 MHz, Methanol-d4) δ 7.48-7.41 (m, 1H), 7.40-7.32 (m, 2H), 7.19-7.13 (m, 1H), 3.06 (d, J=76.5 Hz, 6H). MS (ESI): m/z 277.1 [M+H]+.
Intermediate 40 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 66% yield. 1H NMR (600 MHz, Methanol-d4) δ 7.98 (s, 1H), 7.94 (dd, J=7.9, 1.6 Hz, 1H), 7.44 (dd, J=7.9, 1.6 Hz, 1H), 7.40-7.36 (m, 2H), 7.35 (d, J=7.9 Hz, 1H), 7.16 (s, 1H), 5.70 (t, J=7.7 Hz, 1H), 3.37 (s, 1H), 3.18-2.94 (m, 8H), 2.70-2.63 (m, 1H), 2.18-2.11 (m, 1H). MS (ESI): m/z 435.9 [M+H]+.
To a solution of Intermediate 40 (5 mg, 0.01 mmol) in DMSO (1 mL) were added (2S,4R)—N-(2-(2-((2-aminoethyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.5 mg, 0.01 mmol, 1.0 equiv), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (1-hydroxy-7-azabenzo-triazole) (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (N-Methylmorpholine) (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-70% acetonitrile/0.1% TFA in H2O) to afford LQ108-58 as white solid (8 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 9.12 (s, 1H), 7.75 (s, 1H), 7.70 (dd, J=8.0, 1.7 Hz, 1H), 7.54-7.44 (m, 3H), 7.37-7.32 (m, 3H), 7.18 (s, 1H), 6.96 (dd, J=5.4, 1.6 Hz, 2H), 5.69 (t, J=7.9 Hz, 1H), 4.75-4.71 (m, 1H), 4.66-4.43 (m, 6H), 3.87-3.75 (m, 2H), 3.60-3.48 (m, 4H), 3.18-2.93 (m, 8H), 2.70-2.63 (m, 1H), 2.46 (s, 3H), 2.21-2.10 (m, 2H), 2.09-2.00 (m, 2H), 1.41-1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C53H61FN9O11S+ 1050.4190, found 1050.4182.
LQ108-60 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((4-aminobutyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-60 was obtained as white solid (8.4 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 8.98 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J=7.9, 1.7 Hz, 1H), 7.47-7.39 (m, 3H), 7.38-7.30 (m, 3H), 7.15 (s, 1H), 7.06 (dd, J=7.7, 1.6 Hz, 1H), 6.95 (d, J=1.6 Hz, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.71-4.67 (m, 1H), 4.63-4.53 (m, 4H), 4.47-4.40 (m, 2H), 3.78 (d, J=11.1 Hz, 1H), 3.73 (dd, J=11.1, 3.8 Hz, 1H), 3.37-3.32 (m, 2H), 3.17-2.93 (m, 8H), 2.69-2.61 (m, 1H), 2.47 (s, 3H), 2.19-2.09 (m, 2H), 2.05-1.99 (m, 1H), 1.65-1.57 (m, 4H), 1.38-1.22 (m, 6H), 0.98 (s, 9H). HRMS m/z [M+H]+ calcd for C55H65FN9O11S+ 1078.4503, found 1078.4501.
LQ108-61 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((5-aminopentyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-61 was obtained as white solid (7.6 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.02 (s, 1H), 7.80 (s, 1H), 7.74 (dd, J=7.8, 1.7 Hz, 1H), 7.50-7.41 (m, 3H), 7.38-7.33 (m, 3H), 7.16 (s, 1H), 7.08 (dd, J=7.7, 1.6 Hz, 1H), 6.96 (d, J=1.6 Hz, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.74-4.71 (m, 1H), 4.64-4.52 (m, 4H), 4.48 (dd, J=4.8, 2.5 Hz, 1H), 4.43 (d, J=15.0 Hz, 1H), 3.84 (d, J=10.9 Hz, 1H), 3.77 (dd, J=11.0, 3.8 Hz, 1H), 3.40-3.34 (m, 2H), 3.18-2.94 (m, 8H), 2.68-2.62 (m, 1H), 2.50 (s, 3H), 2.23-2.03 (m, 3H), 1.65-1.57 (m, 4H), 1.42-1.24 (m, 8H), 1.00 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O11S+ 1092.4659, found 1092.4648.
LQ108-62 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((6-aminohexyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-62 was obtained as white solid (7.9 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 9.02 (s, 1H), 7.79 (s, 1H), 7.73 (dd, J=7.9, 1.7 Hz, 1H), 7.49-7.43 (m, 2H), 7.40 (dd, J=7.9, 1.6 Hz, 1H), 7.38-7.31 (m, 3H), 7.14 (s, 1H), 7.07 (dd, J=7.7, 1.6 Hz, 1H), 6.95 (d, J=1.6 Hz, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.72-4.69 (m, 1H), 4.62-4.53 (m, 4H), 4.48-4.42 (m, 2H), 3.82 (d, J=11.1 Hz, 1H), 3.75 (dd, J=11.1, 3.8 Hz, 1H), 3.36-3.32 (m, 2H), 3.27 (t, J=7.0 Hz, 2H), 3.16-2.91 (m, 8H), 2.68-2.61 (m, 1H), 2.48 (s, 3H), 2.21-2.16 (m, 1H), 2.14-2.09 (m, 1H), 2.07-2.01 (m, 1H), 1.57-1.50 (m, 4H), 1.40-1.23 (m, 8H), 0.99 (s, 9H). HRMS m/z [M+H]+ calcd for C57H69FN9O11S+ 1106.4816, found 1106.4813.
LQ108-63 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((7-aminoheptyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-63 was obtained as white solid (7.3 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 7.80 (s, 1H), 7.74 (dd, J=8.0, 1.7 Hz, 1H), 7.50-7.46 (m, 2H), 7.43 (dd, J=7.9, 1.6 Hz, 1H), 7.40-7.33 (m, 3H), 7.16 (s, 1H), 7.09 (dd, J=7.8, 1.6 Hz, 1H), 6.97 (d, J=1.6 Hz, 1H), 5.70 (t, J=7.9 Hz, 1H), 4.76-4.71 (m, 1H), 4.64-4.54 (m, 4H), 4.52-4.45 (m, 2H), 3.85 (d, J=11.1 Hz, 1H), 3.79 (dd, J=11.0, 3.8 Hz, 1H), 3.37-3.34 (m, 2H), 3.30-3.26 (m, 2H), 3.18-2.94 (m, 8H), 2.70-2.63 (m, 1H), 2.50 (s, 3H), 2.24-2.19 (m, 1H), 2.17-2.04 (m, 2H), 1.62-1.51 (m, 4H), 1.41-1.24 (m, 10H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O11S+ 1120.4972, found 1120.4970.
LQ108-64 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((8-aminooctyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-64 was obtained as white solid (7.9 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J=8.0, 1.7 Hz, 1H), 7.50-7.45 (m, 2H), 7.41 (dd, J=7.9, 1.6 Hz, 1H), 7.38-7.31 (m, 3H), 7.14 (s, 1H), 7.08 (dd, J=7.8, 1.6 Hz, 1H), 6.95 (d, J=1.6 Hz, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.74-4.70 (m, 1H), 4.62-4.53 (m, 4H), 4.50-4.43 (m, 2H), 3.83 (d, J=10.8 Hz, 1H), 3.77 (dd, J=11.1, 3.8 Hz, 1H), 3.36-3.32 (m, 2H), 3.25 (t, J=7.0 Hz, 2H), 3.16-2.92 (m, 8H), 2.68-2.61 (m, 1H), 2.48 (s, 3H), 2.23-2.17 (m, 1H), 2.15-2.02 (m, 2H), 1.60-1.47 (m, 4H), 1.39-1.22 (m, 12H), 1.00 (s, 9H). HRMS m/z [M+H]+ calcd for C59H73FN9O11S+ 1134.5129, found 1134.5119.
LQ108-65 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((2-aminoethyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-65 was obtained as white solid (6.2 mg, 60%).
1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 7.66 (s, 1H), 7.56 (dd, J=7.9, 1.7 Hz, 1H), 7.38-7.29 (m, 3H), 7.27-7.19 (m, 5H), 7.05 (s, 1H), 5.56 (t, J=7.9 Hz, 1H), 5.23 (t, J=7.0 Hz, 1H), 4.64-4.59 (m, 1H), 4.49 (dd, J=9.3, 7.6 Hz, 1H), 4.35-4.31 (m, 1H), 3.72 (d, J=11.2 Hz, 1H), 3.65 (dd, J=11.1, 3.8 Hz, 1H), 3.32-3.26 (m, 4H), 3.06-2.81 (m, 8H), 2.71 (dd, J=14.2, 6.8 Hz, 1H), 2.64 (dd, J=14.2, 7.3 Hz, 1H), 2.56-2.49 (m, 1H), 2.35 (s, 3H), 2.12-2.07 (m, 1H), 2.04-1.97 (m, 1H), 1.87-1.81 (m, 1H), 1.30-1.10 (m, 4H), 0.93 (s, 9H). HRMS m/z [M+H]+ calcd for C53H61FN9O10S+ 1034.4241, found 1034.4243.
LQ108-66 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((3-aminopropyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-66 was obtained as white solid (6.9 mg, 66%).
1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.77 (s, 1H), 7.70 (dd, J=8.0, 1.7 Hz, 1H), 7.48-7.45 (m, 2H), 7.44-7.39 (m, 3H), 7.37-7.31 (m, 2H), 7.18 (s, 1H), 5.70 (t, J=8.0 Hz, 1H), 5.33 (dd, J=8.1, 6.0 Hz, 1H), 4.75-4.70 (m, 1H), 4.63 (dd, J=9.2, 7.6 Hz, 1H), 4.45-4.41 (m, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.25-3.18 (m, 1H), 3.17-2.91 (m, 11H), 2.85 (dd, J=14.1, 6.0 Hz, 1H), 2.75 (dd, J=14.1, 8.2 Hz, 1H), 2.69-2.62 (m, 1H), 2.44 (s, 3H), 2.25-2.09 (m, 2H), 1.98-1.92 (m, 1H), 1.64-1.56 (m, 2H), 1.40-1.23 (m, 4H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H63FN9O10S+ 1048.4397, found 1048.4402.
LQ108-67 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((4-aminobutyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-67 was obtained as white solid (7.3 mg, 69%).
1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.75 (s, 1H), 7.69 (dd, J=7.9, 1.7 Hz, 1H), 7.50-7.38 (m, 5H), 7.37-7.32 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 5.30 (dd, J=8.3, 6.1 Hz, 1H), 4.74-4.71 (m, 1H), 4.60-4.55 (m, 1H), 4.45-4.41 (m, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.75 (dd, J=11.1, 3.8 Hz, 1H), 3.27 (t, J=6.7 Hz, 2H), 3.19-2.92 (m, 11H), 2.82 (dd, J=14.1, 6.1 Hz, 1H), 2.73 (dd, J=14.1, 8.4 Hz, 1H), 2.68-2.61 (m, 1H), 2.44 (s, 3H), 2.21-2.09 (m, 2H), 1.98-1.92 (m, 1H), 1.47-1.23 (m, 7H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H65FN9O10S+ 1062.4554, found 1062.4547.
LQ108-68 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((5-aminopentyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-68 was obtained as white solid (8.1 mg, 75%).
1H NMR (600 MHz, Methanol-d4) δ 8.88 (s, 1H), 7.67 (s, 1H), 7.62 (dd, J=7.9, 1.7 Hz, 1H), 7.37 (dd, J=9.4, 3.4 Hz, 1H), 7.34-7.21 (m, 7H), 7.06 (s, 1H), 5.59 (t, J=8.0 Hz, 1H), 5.17 (dd, J=8.5, 6.0 Hz, 1H), 4.65-4.60 (m, 1H), 4.48 (dd, J=9.3, 7.7 Hz, 1H), 4.36-4.31 (m, 1H), 3.72 (d, J=10.9 Hz, 1H), 3.66 (dd, J=11.1, 3.8 Hz, 1H), 3.16 (t, J=7.1 Hz, 2H), 3.07-2.82 (m, 11H), 2.72 (dd, J=14.1, 6.0 Hz, 1H), 2.61 (dd, J=14.1, 8.5 Hz, 1H), 2.58-2.50 (m, 1H), 2.36 (s, 3H), 2.11-2.00 (m, 2H), 1.88-1.82 (m, 1H), 1.45-1.38 (m, 2H), 1.33-1.05 (m, 7H), 0.95 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O10S+ 1076.4710, found 1076.4715.
LQ108-69 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((6-aminohexyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-69 was obtained as white solid (7.8 mg, 72%).
1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.77 (s, 1H), 7.72 (dd, J=7.9, 1.7 Hz, 1H), 7.51-7.39 (m, 5H), 7.37-7.31 (m, 3H), 7.15 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.29 (dd, J=8.4, 5.8 Hz, 1H), 4.75-4.71 (m, 1H), 4.61-4.55 (m, 1H), 4.46-4.41 (m, 1H), 3.85-3.80 (m, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.26 (t, J=7.1 Hz, 2H), 3.17-2.92 (m, 11H), 2.83 (dd, J=14.1, 5.9 Hz, 1H), 2.73 (dd, J=14.1, 8.4 Hz, 1H), 2.68-2.61 (m, 1H), 2.47 (s, 3H), 2.21-2.08 (m, 2H), 1.98-1.92 (m, 1H), 1.54-1.46 (m, 2H), 1.40-1.24 (m, 7H), 1.21-1.13 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C57H69FN9O10S+ 1090.4867, found 1090.4860.
LQ108-70 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((7-aminoheptyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.0 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-70 was obtained as white solid (7.4 mg, 67%).
1H NMR (600 MHz, Methanol-d4) δ 9.16 (s, 1H), 7.77 (s, 1H), 7.72 (dd, J=7.9, 1.7 Hz, 1H), 7.49-7.40 (m, 5H), 7.37-7.31 (m, 3H), 7.15 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.30 (dd, J=8.5, 5.9 Hz, 1H), 4.75-4.71 (m, 1H), 4.58 (dd, J=9.4, 7.7 Hz, 1H), 4.46-4.41 (m, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.30-3.26 (m, 1H), 3.16-2.93 (m, 11H), 2.83 (dd, J=14.1, 5.9 Hz, 1H), 2.73 (dd, J=14.0, 8.5 Hz, 1H), 2.67-2.61 (m, 1H), 2.49 (s, 3H), 2.22-2.10 (m, 2H), 1.98-1.92 (m, 1H), 1.56-1.49 (m, 2H), 1.41-1.20 (m, 9H), 1.16-1.10 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O10S+ 1104.5023, found 1104.5018.
LQ108-71 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((8-aminooctyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-71 was obtained as white solid (7 mg, 63%).
1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J=7.9, 1.7 Hz, 1H), 7.50-7.39 (m, 5H), 7.38-7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J=8.0 Hz, 1H), 5.30 (dd, J=8.5, 5.8 Hz, 1H), 4.76-4.71 (m, 1H), 4.58 (dd, J=9.3, 7.7 Hz, 1H), 4.46-4.42 (m, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.30-3.26 (m, 1H), 3.17-2.91 (m, 11H), 2.83 (dd, J=14.0, 5.8 Hz, 1H), 2.72 (dd, J=14.0, 8.6 Hz, 1H), 2.68-2.61 (m, 1H), 2.48 (s, 3H), 2.22-2.10 (m, 2H), 1.98-1.92 (m, 1H), 1.51 (p, J=7.2 Hz, 2H), 1.41-1.15 (m, 11H), 1.14-1.08 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C59H73FN9O10S+ 1118.5180, found 1118.5183.
LQ108-72 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((9-aminononyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-72 was obtained as white solid (7.7 mg, 68%).
1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 7.79 (s, 1H), 7.73 (dd, J=7.8, 1.7 Hz, 1H), 7.50-7.39 (m, 5H), 7.36 (d, J=7.9 Hz, 1H), 7.35-7.29 (m, 2H), 7.14 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.30 (dd, J=8.5, 5.8 Hz, 1H), 4.76-4.71 (m, 1H), 4.58 (dd, J=9.3, 7.7 Hz, 1H), 4.47-4.42 (m, 1H), 3.83 (d, J=11.0 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.34-3.32 (m, 2H), 3.16-2.91 (m, 11H), 2.83 (dd, J=14.0, 5.8 Hz, 1H), 2.76-2.70 (m, 1H), 2.68-2.61 (m, 1H), 2.48 (s, 3H), 2.24-2.08 (m, 2H), 1.99-1.92 (m, 1H), 1.58-1.50 (m, 2H), 1.40-1.21 (m, 7H), 1.19-1.15 (m, 6H), 1.11-1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O10S+ 1132.5336, found 1132.5329.
LQ108-73 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((10-aminodecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-73 was obtained as white solid (8.1 mg, 71%).
1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J=8.0, 1.7 Hz, 1H), 7.51-7.39 (m, 5H), 7.38-7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.30 (dd, J=8.5, 5.8 Hz, 1H), 4.76-4.72 (m, 1H), 4.58 (dd, J=9.2, 7.5 Hz, 1H), 4.46-4.42 (m, 1H), 3.83 (d, J=11.0 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.36-3.32 (m, 2H), 3.16-2.92 (m, 11H), 2.83 (dd, J=14.0, 5.8 Hz, 1H), 2.77-2.70 (m, 1H), 2.69-2.61 (m, 1H), 2.48 (s, 3H), 2.23-2.16 (m, 1H), 2.16-2.07 (m, 1H), 1.98-1.92 (m, 1H), 1.56 (p, J=7.2 Hz, 2H), 1.38-1.13 (m, 15H), 1.10-1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C61H77FN9O10S+ 1146.5493, found 1146.5490.
LQ108-74 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((11-aminoundecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-74 was obtained as white solid (8.9 mg, 77%).
1H NMR (600 MHz, Methanol-d4) δ 9.11 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J=8.0, 1.7 Hz, 1H), 7.51-7.40 (m, 5H), 7.38-7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.31 (dd, J=8.5, 5.8 Hz, 1H), 4.76-4.71 (m, 1H), 4.58 (dd, J=9.2, 7.6 Hz, 1H), 4.46-4.42 (m, 1H), 3.83 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.36-3.32 (m, 2H), 3.16-2.94 (m, 11H), 2.84 (dd, J=14.0, 5.8 Hz, 1H), 2.73 (dd, J=14.0, 8.6 Hz, 1H), 2.68-2.61 (m, 1H), 2.49 (s, 3H), 2.23-2.16 (m, 1H), 2.15-2.07 (m, 1H), 1.99-1.92 (m, 1H), 1.58 (p, J=7.2 Hz, 2H), 1.41-1.25 (m, 9H), 1.23-1.12 (m, 8H), 1.11-1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C62H79FN9O10S+ 1160.5649, found 1160.5652.
LQ108-75 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((12-aminododecyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ108-75 was obtained as white solid (8.6 mg, 73%).
1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J=7.9, 1.7 Hz, 1H), 7.52-7.40 (m, 5H), 7.38-7.31 (m, 3H), 7.14 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.31 (dd, J=8.5, 5.8 Hz, 1H), 4.74 (dd, J=9.3, 1.3 Hz, 1H), 4.58 (dd, J=9.2, 7.5 Hz, 1H), 4.47-4.42 (m, 1H), 3.85-3.80 (m, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.37-3.32 (m, 2H), 3.16-2.94 (m, 11H), 2.84 (dd, J=14.0, 5.8 Hz, 1H), 2.74 (dd, J=14.0, 8.5 Hz, 1H), 2.68-2.60 (m, 1H), 2.48 (s, 3H), 2.23-2.16 (m, 1H), 2.15-2.07 (m, 1H), 1.99-1.92 (m, 1H), 1.59 (p, J=7.2 Hz, 2H), 1.42-1.13 (m, 19H), 1.12-1.07 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C63H81FN9O10S+ 1174.5806, found 1174.5795.
LQ126-46 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-46 was obtained as white solid (6.3 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.14 (s, 1H), 7.81 (s, 1H), 7.78-7.75 (m, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.43-7.37 (m, 3H), 7.35-7.28 (m, 3H), 7.14 (s, 1H), 5.64 (t, J=7.8 Hz, 1H), 4.70-4.66 (m, 1H), 4.62-4.56 (m, 2H), 4.51-4.48 (m, 1H), 4.34 (d, J=15.7 Hz, 1H), 4.09-3.97 (m, 2H), 3.86 (d, J=11.0 Hz, 1H), 3.79 (dd, J=11.0, 3.8 Hz, 1H), 3.76-3.55 (m, 3H), 3.18-2.91 (m, 8H), 2.91-2.82 (m, 1H), 2.60-2.54 (m, 1H), 2.47 (s, 3H), 2.27-2.20 (m, 1H), 2.13-2.03 (m, 2H), 0.98 (s, 9H). HRMS m/z [M+H]+ calcd for C49H57N8O10S+ 949.3913, found 949.3911.
LQ126-47 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(3-(2-aminoethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-47 was obtained as white solid (6.7 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 7.80-7.78 (m, 1H), 7.76-7.73 (m, 1H), 7.46-7.31 (m, 8H), 7.15 (s, 1H), 5.67 (t, J=7.9 Hz, 1H), 4.64-4.60 (m, 1H), 4.58-4.46 (m, 3H), 4.33 (d, J=15.6 Hz, 1H), 3.86 (d, J=10.9 Hz, 1H), 3.80-3.69 (m, 3H), 3.65-3.51 (m, 4H), 3.16-2.90 (m, 8H), 2.67-2.59 (m, 1H), 2.57-2.44 (m, 5H), 2.24-2.17 (m, 1H), 2.14-2.03 (m, 2H), 0.99 (s, 9H). HRMS m/z [M+H]+ calcd for C50H59N8O10S+ 963.4069, found 963.4070.
LQ126-49 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-49 was obtained as white solid (6.5 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.78 (s, 1H), 7.76-7.71 (m, 1H), 7.47-7.31 (m, 8H), 7.18 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.63 (s, 1H), 4.58-4.46 (m, 3H), 4.33 (d, J=15.5 Hz, 1H), 3.86 (d, J=10.8 Hz, 1H), 3.77 (dd, J=11.0, 3.9 Hz, 1H), 3.72-3.66 (m, 2H), 3.64-3.57 (m, 6H), 3.55-3.49 (m, 2H), 3.16-2.90 (m, 8H), 2.67-2.59 (m, 1H), 2.53-2.40 (m, 5H), 2.21 (dd, J=13.1, 7.8 Hz, 1H), 2.16-2.03 (m, 2H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C52H63N8O11S+ 1007.4332, found 1007.4335.
LQ126-50 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-50 was obtained as white solid (6.3 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 9.14 (s, 1H), 7.77 (s, 1H), 7.74-7.70 (m, 1H), 7.48-7.39 (m, 5H), 7.37-7.32 (m, 3H), 7.16 (s, 1H), 5.67 (t, J=8.0 Hz, 1H), 4.68 (s, 1H), 4.61-4.47 (m, 3H), 4.34 (d, J=15.5 Hz, 1H), 4.00-3.95 (m, 1H), 3.91-3.83 (m, 2H), 3.78 (dd, J=11.0, 3.8 Hz, 1H), 3.68-3.57 (m, 10H), 3.55-3.50 (m, 2H), 3.17-2.92 (m, 8H), 2.67-2.59 (m, 1H), 2.48 (s, 3H), 2.26-2.19 (m, 1H), 2.15-2.04 (m, 2H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O12S+ 1037.4437, found 1037.4432.
LQ126-51 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-1-amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-51 was obtained as white solid (5.8 mg, 55%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 7.78 (s, 1H), 7.74 (dd, J=7.9, 1.7 Hz, 1H), 7.48-7.45 (m, 2H), 7.44-7.39 (m, 3H), 7.37-7.32 (m, 3H), 7.17 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.63 (s, 1H), 4.59-4.51 (m, 2H), 4.50-4.47 (m, 1H), 4.34 (d, J=15.5 Hz, 1H), 3.87 (d, J=11.0 Hz, 1H), 3.78 (dd, J=10.9, 3.9 Hz, 1H), 3.70-3.50 (m, 14H), 3.16-2.91 (m, 8H), 2.68-2.59 (m, 1H), 2.53-2.45 (m, 4H), 2.45-2.36 (m, 1H), 2.24-2.18 (m, 1H), 2.14-2.03 (m, 2H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C54H67N8O12S+ 1051.4594, found 1051.4594.
LQ126-52 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-52 was obtained as white solid (7.3 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 9.16 (s, 1H), 7.79 (s, 1H), 7.75 (dd, J=7.9, 1.7 Hz, 1H), 7.50-7.46 (m, 2H), 7.44-7.40 (m, 3H), 7.38-7.32 (m, 3H), 7.16 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.64 (s, 1H), 4.59-4.52 (m, 2H), 4.50-4.47 (m, 1H), 4.35 (d, J=15.5 Hz, 1H), 3.91-3.85 (m, 1H), 3.79 (dd, J=11.0, 3.9 Hz, 1H), 3.73-3.51 (m, 18H), 3.17-2.92 (m, 8H), 2.68-2.60 (m, 1H), 2.57-2.50 (m, 1H), 2.49 (s, 3H), 2.47-2.42 (m, 1H), 2.25-2.19 (m, 1H), 2.15-2.04 (m, 2H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C56H71N8O13S+ 1095.4856, found 1095.4853.
LQ126-53 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-1-amino-20-(tert-butyl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-53 was obtained as white solid (6.4 mg, 62%). 1H NMR (600 MHz, Methanol-d4) δ 9.21 (s, 1H), 7.79 (s, 1H), 7.76-7.73 (m, 1H), 7.51-7.47 (m, 2H), 7.45-7.41 (m, 3H), 7.38-7.32 (m, 3H), 7.16 (s, 1H), 5.69 (t, J=7.9 Hz, 1H), 4.64 (s, 1H), 4.59-4.52 (m, 2H), 4.51-4.47 (m, 1H), 4.35 (d, J=15.6 Hz, 1H), 3.90-3.86 (m, 1H), 3.79 (dd, J=11.0, 3.9 Hz, 1H), 3.74-3.67 (m, 2H), 3.65-3.51 (m, 20H), 3.16-2.92 (m, 8H), 2.67-2.60 (m, 1H), 2.59-2.52 (m, 1H), 2.50 (s, 3H), 2.48-2.42 (m, 1H), 2.24-2.18 (m, 1H), 2.15-2.03 (m, 2H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C58H75N8O14S+ 1139.5118, found 1139.5113.
LQ126-54 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(2-aminoacetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-54 was obtained as white solid (6.7 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 9.09 (s, 1H), 7.86-7.77 (m, 2H), 7.49-7.45 (m, 2H), 7.43-7.31 (m, 6H), 7.14 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.63 (s, 1H), 4.58-4.52 (m, 2H), 4.50-4.45 (m, 1H), 4.34 (d, J=15.6 Hz, 1H), 4.05 (d, J=2.3 Hz, 2H), 3.87 (d, J=11.0 Hz, 1H), 3.78 (dd, J=11.0, 3.8 Hz, 1H), 3.17-2.92 (m, 8H), 2.68-2.60 (m, 1H), 2.47 (s, 3H), 2.23-2.17 (m, 1H), 2.15-2.03 (m, 2H), 1.00 (s, 9H). HRMS m/z [M+H]+ calcd for C47H53N8O9S+ 905.3651, found 905.3638.
LQ126-55 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-55 was obtained as white solid (6.2 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 7.98-7.92 (m, 1H), 7.76-7.72 (m, 1H), 7.49-7.31 (m, 8H), 7.16-7.15 (m, 1H), 5.66 (t, J=7.6 Hz, 1H), 4.59 (s, 1H), 4.57-4.46 (m, 3H), 4.34 (d, J=15.5 Hz, 1H), 3.90 (d, J=10.9 Hz, 1H), 3.76 (dd, J=11.0, 3.9 Hz, 1H), 3.67-3.53 (m, 2H), 3.18-2.88 (m, 8H), 2.68-2.52 (m, 3H), 2.48 (s, 3H), 2.20 (dd, J=13.3, 7.7 Hz, 1H), 2.17-2.03 (m, 2H), 0.97 (s, 9H). HRMS m/z [M+H]+ calcd for C48H55N8O9S+ 919.3807, found 919.3798.
LQ126-56 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-56 was obtained as white solid (5.5 mg, 59%). 1H NMR (600 MHz, Methanol-d4) δ 9.17 (s, 1H), 7.79 (s, 1H), 7.75 (dd, J=7.9, 1.7 Hz, 1H), 7.48 (d, J=8.0 Hz, 2H), 7.44-7.39 (m, 3H), 7.38-7.31 (m, 3H), 7.13 (s, 1H), 5.68 (t, J=8.0 Hz, 1H), 4.62-4.47 (m, 4H), 4.38-4.32 (m, 1H), 3.90 (d, J=11.1 Hz, 1H), 3.79 (dd, J=11.0, 4.0 Hz, 1H), 3.44-3.34 (m, 2H), 3.16-2.91 (m, 8H), 2.68-2.61 (m, 1H), 2.49 (s, 3H), 2.39-2.31 (m, 2H), 2.20 (dd, J=12.8, 8.0 Hz, 1H), 2.15-2.04 (m, 2H), 1.92-1.84 (m, 2H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C49H57N8O9S+ 933.3964, found 933.3954.
LQ126-57 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-57 was obtained as white solid (6.1 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 9.16 (s, 1H), 7.77 (s, 1H), 7.75-7.72 (m, 1H), 7.48 (d, J=8.2 Hz, 2H), 7.44-7.40 (m, 3H), 7.37-7.32 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.60 (s, 1H), 4.54 (dd, J=16.0, 8.4 Hz, 2H), 4.50-4.46 (m, 1H), 4.35 (d, J=15.6 Hz, 1H), 3.91-3.86 (m, 1H), 3.78 (dd, J=10.9, 3.9 Hz, 1H), 3.39-3.33 (m, 2H), 3.16-2.90 (m, 8H), 2.67-2.59 (m, 1H), 2.49 (s, 3H), 2.37-2.26 (m, 2H), 2.20 (dd, J=13.2, 7.7 Hz, 1H), 2.14-2.03 (m, 2H), 1.73-1.58 (m, 4H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C50H59N8O9S+ 947.4120, found 947.4125.
LQ126-58 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(6-aminohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-58 was obtained as white solid (6.1 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 8.94 (s, 1H), 7.77 (s, 1H), 7.73 (dd, J=7.8, 1.8 Hz, 1H), 7.47-7.44 (m, 2H), 7.43-7.39 (m, 3H), 7.38-7.31 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.61 (s, 1H), 4.58-4.50 (m, 2H), 4.50-4.47 (m, 1H), 4.35 (d, J=15.5 Hz, 1H), 3.88 (d, J=11.0 Hz, 1H), 3.78 (dd, J=11.0, 3.9 Hz, 1H), 3.15-2.93 (m, 8H), 2.68-2.60 (m, 1H), 2.47 (s, 3H), 2.33-2.17 (m, 3H), 2.14-2.04 (m, 2H), 1.73-1.58 (m, 4H), 1.43-1.25 (m, 4H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C51H61N8O9S+ 961.4277, found 961.4275.
LQ126-59 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(7-aminoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-59 was obtained as white solid (6.5 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 7.76 (s, 1H), 7.73-7.71 (m, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.44-7.39 (m, 3H), 7.37-7.31 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.61 (s, 1H), 4.59-4.47 (m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.92-3.87 (m, 1H), 3.79 (dd, J=10.9, 3.9 Hz, 1H), 3.37-3.32 (m, 2H), 3.16-2.92 (m, 8H), 2.67-2.60 (m, 1H), 2.48 (s, 3H), 2.32-2.18 (m, 3H), 2.15-2.03 (m, 2H), 1.65-1.56 (m, 4H), 1.42-1.32 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C52H63N8O9S+ 975.4433, found 975.4428.
LQ126-60 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(8-aminooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-60 was obtained as white solid (7 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.76 (s, 1H), 7.72 (dd, J=7.7, 1.7 Hz, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.43-7.40 (m, 3H), 7.37-7.31 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.62 (s, 1H), 4.59-4.47 (m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.89 (d, J=10.9 Hz, 1H), 3.79 (dd, J=10.9, 3.9 Hz, 1H), 3.39-3.32 (m, 2H), 3.16-2.93 (m, 8H), 2.68-2.60 (m, 1H), 2.48 (s, 3H), 2.32-2.17 (m, 3H), 2.15-2.03 (m, 2H), 1.65-1.55 (m, 4H), 1.41-1.28 (m, 6H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O9S+ 989.4590, found 989.4589.
LQ126-61 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(9-aminononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-61 was obtained as white solid (6.5 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.76 (s, 1H), 7.72 (dd, J=7.9, 1.7 Hz, 1H), 7.49-7.45 (m, 2H), 7.44-7.40 (m, 3H), 7.37-7.32 (m, 3H), 7.16 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.62 (s, 1H), 4.59-4.47 (m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.92-3.87 (m, 1H), 3.79 (dd, J=10.9, 3.9 Hz, 1H), 3.37-3.32 (m, 2H), 3.18-2.92 (m, 8H), 2.68-2.60 (m, 1H), 2.48 (s, 3H), 2.32-2.18 (m, 3H), 2.16-2.03 (m, 2H), 1.65-1.54 (m, 4H), 1.40-1.28 (m, 8H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C54H67N8O9S+ 1003.4746, found 1003.4752.
LQ126-62 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(10-aminodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-62 was obtained as white solid (6.8 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.76 (s, 1H), 7.74-7.69 (m, 1H), 7.47 (d, J=8.2 Hz, 2H), 7.44-7.39 (m, 3H), 7.37-7.32 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.62 (s, 1H), 4.59-4.52 (m, 2H), 4.50-4.47 (m, 1H), 4.35 (d, J=15.5 Hz, 1H), 3.89 (d, J=10.9 Hz, 1H), 3.79 (dd, J=11.0, 3.9 Hz, 1H), 3.36-3.31 (m, 2H), 3.15-2.93 (m, 8H), 2.68-2.61 (m, 1H), 2.48 (s, 3H), 2.30-2.18 (m, 3H), 2.15-2.03 (m, 2H), 1.62-1.53 (m, 4H), 1.39-1.26 (m, 10H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C55H69N8O9S+ 1017.4903, found 1017.4900.
LQ126-63 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-63 was obtained as white solid (6.3 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 9.15 (s, 1H), 7.76 (s, 1H), 7.72 (dd, J=8.0, 1.7 Hz, 1H), 7.50-7.46 (m, 2H), 7.45-7.41 (m, 3H), 7.39-7.32 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.62 (s, 1H), 4.59-4.52 (m, 2H), 4.51-4.47 (m, 1H), 4.36 (d, J=15.5 Hz, 1H), 3.93-3.87 (m, 1H), 3.80 (dd, J=11.0, 3.9 Hz, 1H), 3.36-3.32 (m, 2H), 3.17-2.91 (m, 8H), 2.68-2.60 (m, 1H), 2.50 (s, 3H), 2.31-2.18 (m, 3H), 2.16-2.01 (m, 2H), 1.63-1.52 (m, 4H), 1.40-1.25 (m, 12H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C56H71N8O9S+ 1031.5059, found 1031.5056.
LQ126-77 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (4.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-77 was obtained as white solid (4.6 mg, 58%). 1H NMR (600 MHz, Methanol-d4) δ 7.73 (d, J=8.0 Hz, 1H), 7.71-7.64 (m, 2H), 7.44-7.29 (m, 6H), 7.10 (d, J=9.6 Hz, 1H), 5.71-5.64 (m, 1H), 5.02 (dd, J=12.9, 5.4 Hz, 1H), 4.79-4.72 (m, 2H), 3.63-3.50 (m, 4H), 3.20-2.93 (m, 8H), 2.86-2.76 (m, 1H), 2.71-2.54 (m, 3H), 2.18-2.03 (m, 2H). HRMS m/z [M+H]+ calcd for C40H38N7O11+ 792.2624, found 792.2614.
LQ126-78 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(3-aminopropyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-78 was obtained as white solid (5.1 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 7.82-7.75 (m, 2H), 7.74-7.68 (m, 1H), 7.54-7.49 (m, 1H), 7.45-7.39 (m, 2H), 7.37-7.29 (m, 3H), 7.17 (s, 1H), 5.68-5.62 (m, 1H), 5.14-5.07 (m, 1H), 4.77-4.74 (m, 2H), 3.51-3.39 (m, 4H), 3.19-2.92 (m, 8H), 2.89-2.80 (m, 1H), 2.78-2.60 (m, 3H), 2.17-2.08 (m, 2H), 1.92-1.84 (m, 2H). HRMS m/z [M+H]+ calcd for C41H40N7O11+ 806.2780, found 806.2762.
LQ126-79 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-79 was obtained as white solid (5.8 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 7.80-7.69 (m, 3H), 7.49 (t, J=7.2 Hz, 1H), 7.44-7.30 (m, 5H), 7.15 (d, J=8.5 Hz, 1H), 5.70 (t, J=8.0 Hz, 1H), 5.09 (dd, J=12.7, 5.4 Hz, 1H), 4.76-4.68 (m, 2H), 3.50-3.35 (m, 4H), 3.18-2.94 (m, 8H), 2.85-2.62 (m, 4H), 2.18-2.04 (m, 2H), 1.76-1.59 (m, 4H). HRMS m/z [M+H]+ calcd for C42H42N7O11+ 820.2937, found 820.2929.
LQ126-80 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(5-aminopentyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-80 was obtained as white solid (5 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 7.81-7.73 (m, 2H), 7.68 (ddd, J=17.9, 7.9, 1.7 Hz, 1H), 7.49 (dd, J=11.0, 7.3 Hz, 1H), 7.44-7.39 (m, 2H), 7.38-7.33 (m, 2H), 7.29 (dd, J=14.8, 7.9 Hz, 1H), 7.16 (s, 1H), 5.69-5.63 (m, 1H), 5.16-5.07 (m, 1H), 4.75-4.70 (m, 2H), 3.43-3.34 (m, 3H), 3.19-2.93 (m, 8H), 2.91-2.82 (m, 1H), 2.78-2.60 (m, 3H), 2.18-2.09 (m, 2H), 1.69-1.61 (m, 4H), 1.51-1.44 (m, 2H). HRMS m/z [M+H]+ calcd for C43H44N7O11+ 834.3093, found 834.3091.
LQ126-81 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(6-aminohexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-81 was obtained as white solid (5.5 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 7.82-7.75 (m, 2H), 7.75-7.69 (m, 1H), 7.50 (dd, J=7.3, 4.4 Hz, 1H), 7.44-7.32 (m, 5H), 7.16 (s, 1H), 5.70 (t, J=7.9 Hz, 1H), 5.15-5.06 (m, 1H), 4.76-4.71 (m, 2H), 3.38-3.34 (m, 3H), 3.17-2.94 (m, 8H), 2.90-2.81 (m, 1H), 2.76-2.62 (m, 3H), 2.16-2.10 (m, 2H), 1.65-1.57 (m, 4H), 1.47-1.39 (m, 4H). HRMS m/z [M+H]+ calcd for C44H46N7O11+ 848.3250, found 848.3160.
LQ126-82 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(7-aminoheptyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-82 was obtained as white solid (6.2 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 7.81-7.76 (m, 2H), 7.73-7.68 (m, 1H), 7.48 (dd, J=8.9, 7.3 Hz, 1H), 7.44-7.38 (m, 2H), 7.37-7.32 (m, 3H), 7.16 (d, J=4.3 Hz, 1H), 5.69 (t, J=8.0 Hz, 1H), 5.16-5.11 (m, 1H), 4.76-4.71 (m, 2H), 3.32-3.28 (m, 3H), 3.18-2.94 (m, 8H), 2.91-2.82 (m, 1H), 2.79-2.63 (m, 3H), 2.19-2.10 (m, 2H), 1.63-1.54 (m, 4H), 1.43-1.34 (m, 6H). HRMS m/z [M+H]+ calcd for C45H48N7O11+ 862.3406, found 862.3405.
LQ126-83 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(2-(2-aminoethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-83 was obtained as white solid (5 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 7.80-7.75 (m, 2H), 7.70-7.67 (m, 1H), 7.48 (d, J=7.3 Hz, 1H), 7.40 (dd, J=7.9, 1.6 Hz, 1H), 7.37-7.31 (m, 3H), 7.21 (d, J=7.9 Hz, 1H), 7.13 (s, 1H), 5.58 (t, J=7.9 Hz, 1H), 5.04 (dd, J=12.6, 5.5 Hz, 1H), 4.73-4.61 (m, 2H), 3.73-3.59 (m, 5H), 3.57-3.49 (m, 3H), 3.20-2.96 (m, 8H), 2.92-2.85 (m, 1H), 2.80-2.56 (m, 4H), 2.16-2.04 (m, 1H). HRMS m/z [M+H]+ calcd for C42H42N7O12+ 836.2886, found 836.2879.
LQ126-84 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-84 was obtained as white solid (5.6 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 7.80 (d, J=7.3 Hz, 1H), 7.78-7.74 (m, 1H), 7.74-7.69 (m, 1H), 7.45 (t, J=7.5 Hz, 1H), 7.42-7.39 (m, 1H), 7.38-7.31 (m, 4H), 7.14 (d, J=5.1 Hz, 1H), 5.68 (t, J=8.0 Hz, 1H), 5.11 (dd, J=12.8, 5.5 Hz, 1H), 4.70-4.67 (m, 2H), 3.66-3.57 (m, 8H), 3.55-3.51 (m, 2H), 3.49-3.39 (m, 2H), 3.18-2.93 (m, 8H), 2.90-2.82 (m, 1H), 2.79-2.70 (m, 3H), 2.69-2.62 (m, 1H), 2.19-2.09 (m, 1H). HRMS m/z [M+H]+ calcd for C44H46N7O13+ 880.3148, found 880.3122.
LQ126-85 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (6.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-85 was obtained as white solid (6.2 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 7.79 (d, J=3.9 Hz, 1H), 7.77-7.74 (m, 1H), 7.74-7.70 (m, 1H), 7.46 (dd, J=7.3, 3.3 Hz, 1H), 7.41-7.36 (m, 2H), 7.36-7.30 (m, 3H), 7.13 (d, J=4.6 Hz, 1H), 5.67 (t, J=8.0 Hz, 1H), 5.13-5.07 (m, 1H), 4.71 (s, 2H), 3.63-3.50 (m, 14H), 3.44-3.39 (m, 2H), 3.15-2.93 (m, 8H), 2.89-2.81 (m, 1H), 2.77-2.60 (m, 3H), 2.16-2.07 (m, 2H). HRMS m/z [M+H]+ calcd for C46H50N7O14+ 924.3410, found 924.3384.
LQ126-86 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(14-amino-3,6,9,12-tetraoxatetradecyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (6.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-86 was obtained as white solid (6.7 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 7.82 (d, J=4.6 Hz, 1H), 7.78 (dd, J=8.4, 7.3 Hz, 1H), 7.77-7.73 (m, 1H), 7.50 (dd, J=7.3, 1.9 Hz, 1H), 7.44-7.39 (m, 2H), 7.38-7.33 (m, 3H), 7.16 (d, J=2.3 Hz, 1H), 5.70 (t, J=7.9 Hz, 1H), 5.13 (dd, J=12.8, 5.5 Hz, 1H), 4.76-4.73 (m, 2H), 3.66-3.53 (m, 18H), 3.47 (t, J=5.3 Hz, 2H), 3.17-2.95 (m, 8H), 2.92-2.84 (m, 1H), 2.79-2.62 (m, 3H), 2.18-2.09 (m, 2H). HRMS m/z [M+H]+ calcd for C48H54N7O15+ 968.3672, found 968.3661.
LQ126-87 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-87 was obtained as white solid (6.1 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 7.82 (d, J=3.8 Hz, 1H), 7.78 (dd, J=8.5, 7.3 Hz, 1H), 7.77-7.74 (m, 1H), 7.50 (dd, J=7.3, 2.3 Hz, 1H), 7.44-7.39 (m, 2H), 7.38-7.33 (m, 3H), 7.16 (d, J=1.8 Hz, 1H), 5.70 (t, J=8.0 Hz, 1H), 5.16-5.11 (m, 1H), 4.76-4.73 (m, 2H), 3.69-3.51 (m, 22H), 3.49 (t, J=5.3 Hz, 2H), 3.18-2.95 (m, 8H), 2.92-2.84 (m, 1H), 2.80-2.71 (m, 2H), 2.70-2.62 (m, 1H), 2.19-2.09 (m, 2H). HRMS m/z [M+H]+ calcd for C50H58N7O16+ 1012.3935, found 1012.3928.
Intermediate 41 was synthesized according to the procedures for the preparation of intermediate 3 as a white solid in 52% yield. MS (ESI): m/z 371.4 [M+H]+.
Intermediate 42 was synthesized according to the procedures for the preparation of intermediate 36 as a yellow oil in 64% yield. MS (ESI): m/z 527.5 [M+H]+.
Intermediate 43 was synthesized according to the procedures for the preparation of intermediate 37 as a yellow solid in 16% yield. MS (ESI): m/z 457.3 [M+H]+.
Intermediate 44 was synthesized according to the procedures for the preparation of intermediate 38 as a white solid in 62% yield. MS (ESI): m/z 454.4 [M+H]+.
Intermediate 45 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 17% yield. MS (ESI): m/z 440.6 [M+H]+.
Intermediate 46 was synthesized according to the procedures for the preparation of intermediate 3 as a white solid in 44% yield. MS (ESI): m/z 421.5 [M+H]+.
To a solution of Intermediate 46 (4 mg, 0.01 mmol) in DMSO (1 mL) were added 3-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)propanoic acid (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-70% acetonitrile/0.1% TFA in H2O) to afford LQ126-89 as white solid (8.7 mg, 78%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.48-7.39 (m, 5H), 7.38-7.31 (m, 3H), 7.29 (d, J=7.4 Hz, 1H), 7.27-7.20 (m, 2H), 7.15 (s, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.70-4.58 (m, 2H), 4.57-4.48 (m, 2H), 4.36 (t, J=18.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.82 (dd, J=10.9, 3.8 Hz, 1H), 3.79-3.59 (m, 8H), 3.56-3.41 (m, 2H), 3.16-3.07 (m, 3H), 3.03-2.91 (m, 4H), 2.65-2.57 (m, 1H), 2.49 (s, 3H), 2.44-2.37 (m, 1H), 2.28-2.22 (m, 1H), 2.13-2.03 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C51H61N8O10S+ 977.4226, found 977.4237.
LQ126-90 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(3-(4-(4-methylthiazol-5-yl)phenyl)propanoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)acetic acid (5.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-90 was obtained as white solid (7.4 mg, 75%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 7.46-7.38 (m, 5H), 7.35-7.18 (m, 6H), 7.12 (s, 1H), 5.63 (t, J=7.5 Hz, 1H), 4.76-4.68 (m, 1H), 4.64-4.56 (m, 1H), 4.51-4.48 (m, 2H), 4.36 (d, J=15.4 Hz, 1H), 4.12-3.98 (m, 4H), 3.92-3.68 (m, 6H), 3.58-3.52 (m, 1H), 3.44-3.35 (m, 1H), 3.15-3.05 (m, 3H), 3.02-2.88 (m, 4H), 2.63-2.56 (m, 1H), 2.47 (s, 3H), 2.28-2.21 (m, 1H), 2.10-2.01 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C51H61N8O11S+ 993.4175, found 993.4178.
LQ126-91 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)propanoic acid (6.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-91 was obtained as white solid (7.8 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 7.49-7.40 (m, 5H), 7.39-7.35 (m, 2H), 7.34-7.20 (m, 4H), 7.15 (s, 1H), 5.65 (t, J=7.8 Hz, 1H), 4.67 (s, 1H), 4.61 (t, J=8.3 Hz, 1H), 4.54 (d, J=15.4 Hz, 1H), 4.52-4.49 (m, 1H), 4.36 (d, J=15.5 Hz, 1H), 3.91 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.78-3.48 (m, 10H), 3.44-3.36 (m, 2H), 3.17-3.07 (m, 3H), 3.03-2.91 (m, 4H), 2.64-2.52 (m, 2H), 2.49 (s, 3H), 2.42-2.33 (m, 1H), 2.27-2.21 (m, 1H), 2.13-2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O11S+ 1021.4488, found 1021.4485.
LQ126-92 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecanoic acid (6.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-92 was obtained as white solid (6.9 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 7.49-7.41 (m, 5H), 7.39-7.31 (m, 3H), 7.29 (d, J=7.4 Hz, 1H), 7.27-7.20 (m, 2H), 7.15 (s, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.74-4.70 (m, 1H), 4.62-4.48 (m, 3H), 4.35 (d, J=15.4 Hz, 1H), 4.11-3.97 (m, 4H), 3.87 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.8 Hz, 1H), 3.78-3.64 (m, 8H), 3.48-3.38 (m, 2H), 3.18-3.07 (m, 3H), 3.02-2.91 (m, 4H), 2.65-2.57 (m, 1H), 2.49 (s, 3H), 2.24 (dd, J=13.2, 7.7 Hz, 1H), 2.13-2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O12S+ 1037.4437, found 1037.4430.
LQ126-93 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoic acid (6.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-93 was obtained as white solid (6.7 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.50-7.47 (m, 2H), 7.45-7.41 (m, 3H), 7.39-7.35 (m, 2H), 7.32 (d, J=7.3 Hz, 1H), 7.29 (d, J=7.3 Hz, 1H), 7.27-7.20 (m, 2H), 7.16 (s, 1H), 5.66 (t, J=7.8 Hz, 1H), 4.66 (s, 1H), 4.60 (t, J=8.3 Hz, 1H), 4.55 (d, J=15.5 Hz, 1H), 4.52-4.49 (m, 1H), 4.37 (d, J=15.5 Hz, 1H), 3.91 (d, J=10.9 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.78-3.66 (m, 6H), 3.64-3.50 (m, 10H), 3.18-3.06 (m, 3H), 3.04-2.91 (m, 4H), 2.64-2.54 (m, 2H), 2.50 (s, 3H), 2.49-2.45 (m, 2H), 2.44-2.37 (m, 1H), 2.26-2.21 (m, 1H), 2.12-2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H69N8O12S+ 1065.4750, found 1065.4745.
LQ126-94 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-18-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-19,19-dimethyl-16-oxo-4,7,10,13-tetraoxa-17-azaicosanoic acid (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-94 was obtained as white solid (7.3 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 7.49 (d, J=8.0 Hz, 2H), 7.46-7.41 (m, 3H), 7.39-7.36 (m, 2H), 7.32 (d, J=7.3 Hz, 1H), 7.29 (d, J=7.3 Hz, 1H), 7.27-7.20 (m, 2H), 7.16 (s, 1H), 5.66 (t, J=7.8 Hz, 1H), 4.66 (s, 1H), 4.62-4.57 (m, 1H), 4.55 (d, J=15.5 Hz, 1H), 4.52-4.49 (m, 1H), 4.37 (d, J=15.5 Hz, 1H), 3.91 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.78-3.50 (m, 18H), 3.18-3.07 (m, 3H), 3.04-2.91 (m, 4H), 2.65-2.54 (m, 2H), 2.50 (s, 3H), 2.49-2.45 (m, 2H), 2.43-2.34 (m, 1H), 2.27-2.20 (m, 1H), 2.13-2.03 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C57H73N8O13S+ 1109.5012, found 1109.5016.
LQ126-95 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosanoic acid (7.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-95 was obtained as white solid (7 mg, 62%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.50-7.45 (m, 2H), 7.46-7.42 (m, 3H), 7.39-7.34 (m, 2H), 7.32 (d, J=7.3 Hz, 1H), 7.29 (d, J=7.4 Hz, 1H), 7.27-7.20 (m, 2H), 7.16 (s, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.73-4.69 (m, 1H), 4.63-4.50 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 4.08-4.00 (m, 4H), 3.96-3.87 (m, 2H), 3.81 (dd, J=11.0, 3.8 Hz, 1H), 3.78-3.54 (m, 16H), 3.47-3.37 (m, 1H), 3.19-3.06 (m, 3H), 3.05-2.91 (m, 4H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.27-2.22 (m, 1H), 2.13-2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C57H73N8O13S+ 1125.4961, found 1125.4967.
LQ126-96 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-21-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-22,22-dimethyl-19-oxo-4,7,10,13,16-pentaoxa-20-azatricosanoic acid (7.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-96 was obtained as white solid (6.5 mg, 56%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 7.49 (d, J=8.0 Hz, 2H), 7.46-7.42 (m, 3H), 7.39-7.36 (m, 2H), 7.32 (d, J=7.3 Hz, 1H), 7.29 (d, J=7.4 Hz, 1H), 7.27-7.20 (m, 2H), 7.16 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 4.69-4.65 (m, 1H), 4.61-4.58 (m, 1H), 4.56 (d, J=15.5 Hz, 1H), 4.52-4.49 (m, 1H), 4.37 (d, J=15.5 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.78-3.51 (m, 22H), 3.18-3.07 (m, 3H), 3.03-2.91 (m, 4H), 2.64-2.55 (m, 2H), 2.51 (s, 3H), 2.50-2.46 (m, 2H), 2.43-2.37 (m, 1H), 2.26-2.21 (m, 1H), 2.12-2.04 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C59H77N8O14S+ 1153.5274, found 1153.5270.
LQ126-97 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid (5.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-97 was obtained as white solid (5.8 mg, 62%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 7.49-7.20 (m, 11H), 7.18-7.10 (m, 1H), 5.68-5.61 (m, 1H), 4.64-4.49 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.93 (d, J=11.0 Hz, 1H), 3.84-3.79 (m, 1H), 3.77-3.54 (m, 1H), 3.51-3.37 (m, 1H), 3.18-3.06 (m, 3H), 3.03-2.90 (m, 4H), 2.65-2.41 (m, 8H), 2.28-2.21 (m, 1H), 2.12-2.03 (m, 2H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C49H57N8O9S+ 933.3964, found 933.3966.
LQ126-98 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (5.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-98 was obtained as white solid (6.4 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 7.49-7.40 (m, 6H), 7.38-7.31 (m, 2H), 7.31-7.20 (m, 3H), 7.15 (s, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.66-4.60 (m, 2H), 4.56-4.51 (m, 2H), 4.36 (d, J=15.5 Hz, 1H), 3.97 (d, J=11.0 Hz, 1H), 3.83 (dd, J=10.9, 3.9 Hz, 1H), 3.79-3.39 (m, 2H), 3.18-3.06 (m, 3H), 3.05-2.90 (m, 4H), 2.65-2.58 (m, 1H), 2.49 (s, 3H), 2.39-2.06 (m, 7H), 2.00-1.79 (m, 2H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C50H59N8O9S+ 947.4120, found 947.4149.
LQ126-99 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (5.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-99 was obtained as white solid (6.7 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.50-7.41 (m, 5H), 7.39-7.20 (m, 6H), 7.15 (s, 1H), 5.65 (t, J=7.8 Hz, 1H), 4.67-4.49 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.93 (d, J=11.1 Hz, 1H), 3.82 (dd, J=11.0, 3.9 Hz, 1H), 3.56-3.50 (m, 1H), 3.45-3.37 (m, 1H), 3.18-3.05 (m, 3H), 3.03-2.91 (m, 4H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.37-2.03 (m, 8H), 1.72-1.52 (m, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C51H61N8O9S+ 961.4277, found 961.4277.
LQ126-100 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoic acid (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-100 was obtained as white solid (6.3 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 8.92 (s, 1H), 7.49-7.39 (m, 6H), 7.40-7.31 (m, 2H), 7.30-7.20 (m, 3H), 7.16 (s, 1H), 5.65 (t, J=7.6 Hz, 1H), 4.65 (s, 1H), 4.60 (t, J=8.6 Hz, 1H), 4.57-4.49 (m, 2H), 4.36 (d, J=15.5 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.82 (dd, J=10.9, 3.9 Hz, 1H), 3.74-3.68 (m, 1H), 3.56-3.49 (m, 1H), 3.18-3.06 (m, 3H), 3.03-2.91 (m, 4H), 2.65-2.57 (m, 1H), 2.48 (s, 3H), 2.32-2.19 (m, 4H), 2.17-2.04 (m, 2H), 1.71-1.50 (m, 4H), 1.43-1.28 (m, 3H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C52H63N8O9S+ 975.4433, found 975.4413.
LQ126-101 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctanoic acid (5.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-101 was obtained as white solid (7.2 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 8.93 (s, 1H), 7.48-7.39 (m, 5H), 7.37-7.18 (m, 6H), 7.14 (s, 1H), 5.64 (t, J=7.8 Hz, 1H), 4.63 (s, 1H), 4.60-4.47 (m, 3H), 4.34 (d, J=15.4 Hz, 1H), 3.90 (d, J=11.1 Hz, 1H), 3.80 (dd, J=11.0, 3.9 Hz, 1H), 3.71-3.59 (m, 1H), 3.52-3.35 (m, 1H), 3.17-3.04 (m, 3H), 3.02-2.88 (m, 4H), 2.62-2.56 (m, 1H), 2.47 (s, 3H), 2.32-2.02 (m, 4H), 1.66-1.46 (m, 5H), 1.43-1.26 (m, 6H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O9S+ 989.4590, found 989.4602.
LQ126-102 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoic acid (6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-102 was obtained as white solid (7.1 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 8.98 (s, 1H), 7.50-7.46 (m, 2H), 7.46-7.41 (m, 3H), 7.39-7.31 (m, 3H), 7.30-7.20 (m, 3H), 7.16 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 4.65 (s, 1H), 4.62-4.48 (m, 3H), 4.37 (d, J=15.4 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.74-3.67 (m, 1H), 3.54-3.48 (m, 1H), 3.16-3.06 (m, 3H), 3.03-2.90 (m, 4H), 2.64-2.57 (m, 1H), 2.49 (s, 3H), 2.34-2.20 (m, 3H), 2.16-2.03 (m, 2H), 1.68-1.50 (m, 4H), 1.40-1.24 (m, 8H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C54H67N8O9S+ 1003.4746, found 1003.4739.
LQ126-103 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 10-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecanoic acid (6.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-103 was obtained as white solid (6.5 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.51-7.42 (m, 5H), 7.39-7.31 (m, 3H), 7.30-7.20 (m, 3H), 7.16 (s, 1H), 5.66 (t, J=7.8 Hz, 1H), 4.65 (s, 1H), 4.61-4.48 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.74-3.64 (m, 1H), 3.56-3.49 (m, 1H), 3.17-3.07 (m, 3H), 3.04-2.90 (m, 4H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.33-2.20 (m, 4H), 2.16-2.05 (m, 2H), 1.68-1.48 (m, 4H), 1.40-1.26 (m, 9H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H69N8O9S+ 1017.4903, found 1017.4902.
LQ126-104 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 11-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoic acid (6.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-104 was obtained as white solid (7 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 7.50-7.42 (m, 5H), 7.40-7.31 (m, 3H), 7.30-7.20 (m, 3H), 7.16 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 4.65 (s, 1H), 4.62-4.48 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.82 (dd, J=11.0, 4.0 Hz, 1H), 3.73-3.68 (m, 1H), 3.56-3.50 (m, 1H), 3.17-3.06 (m, 3H), 3.04-2.90 (m, 4H), 2.65-2.57 (m, 1H), 2.49 (s, 3H), 2.33-2.19 (m, 4H), 2.14-2.03 (m, 2H), 1.68-1.50 (m, 5H), 1.39-1.25 (m, 10H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C56H71NO9S+ 1031.5059, found 1031.5083.
LQ126-105 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetyl)glycine (3.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-105 was obtained as white solid (4.7 mg, 59%). 1H NMR (600 MHz, Methanol-d4) δ 7.79-7.72 (m, 1H), 7.53-7.18 (m, 9H), 7.12-7.03 (m, 1H), 5.68-5.58 (m, 1H), 5.12 (dd, J=12.8, 5.4 Hz, 1H), 4.03-3.87 (m, 2H), 3.75-3.66 (m, 1H), 3.58-3.43 (m, 2H), 3.40-3.33 (m, 2H), 3.15-3.04 (m, 3H), 3.01-2.80 (m, 4H), 2.78-2.67 (m, 2H), 2.63-2.56 (m, 1H), 2.17-2.02 (m, 2H). HRMS m/z [M+H]+ calcd for C40H38N7O11+ 792.2624, found 792.2635.
LQ126-106 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)propanoic acid (4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-106 was obtained as white solid (5.1 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 7.83-7.74 (m, 1H), 7.56-7.46 (m, 1H), 7.44-7.21 (m, 8H), 7.12 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 5.14 (ddd, J=12.5, 5.6, 3.7 Hz, 1H), 4.74 (s, 2H), 3.76-3.36 (m, 7H), 3.16-3.06 (m, 2H), 3.03-2.83 (m, 3H), 2.80-2.71 (m, 2H), 2.66-2.58 (m, 1H), 2.55-2.39 (m, 2H), 2.18-2.13 (m, 1H), 2.13-2.04 (m, 1H). HRMS m/z [M+H]+ calcd for C41H40N7O11+ 806.2780, found 806.2771.
LQ126-107 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butanoic acid (4.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-107 was obtained as white solid (5.1 mg, 62%). 1H NMR (600 MHz, Methanol-d4) δ 7.80 (t, J=7.9 Hz, 1H), 7.55-7.51 (m, 1H), 7.47-7.39 (m, 2H), 7.36-7.20 (m, 6H), 7.13 (s, 1H), 5.68-5.62 (m, 1H), 5.17-5.10 (m, 1H), 4.78 (s, 2H), 3.76-3.63 (m, 1H), 3.57-3.50 (m, 1H), 3.45-3.36 (m, 5H), 3.16-3.07 (m, 2H), 3.05-2.83 (m, 3H), 2.80-2.69 (m, 2H), 2.64-2.56 (m, 1H), 2.35-2.03 (m, 4H), 1.94-1.75 (m, 2H). HRMS m/z [M+H]+ calcd for C42H42N7O11+ 820.2937, found 820.2938.
LQ126-108 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 5-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)pentanoic acid (4.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-108 was obtained as white solid (5 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 7.79 (t, J=7.9 Hz, 1H), 7.55-7.49 (m, 1H), 7.44-7.37 (m, 2H), 7.36-7.21 (m, 6H), 7.16-7.10 (m, 1H), 5.66 (t, J=7.3 Hz, 1H), 5.14 (ddd, J=12.8, 5.5, 3.0 Hz, 1H), 4.75 (s, 2H), 3.74-3.65 (m, 1H), 3.56-3.48 (m, 1H), 3.45-3.38 (m, 2H), 3.19-3.07 (m, 3H), 3.03-2.83 (m, 5H), 2.81-2.69 (m, 2H), 2.66-2.58 (m, 1H), 2.30-2.23 (m, 1H), 2.20-2.04 (m, 3H), 1.76-1.53 (m, 4H). HRMS m/z [M+H]+ calcd for C43H44N7O11+ 834.3093, found 834.3083.
LQ126-109 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexanoic acid (4.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-109 was obtained as white solid (5.6 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 7.79 (t, J=7.9 Hz, 1H), 7.52 (d, J=7.3 Hz, 1H), 7.43-7.39 (m, 2H), 7.35-7.18 (m, 6H), 7.12 (s, 1H), 5.66-5.60 (m, 1H), 5.15-5.11 (m, 1H), 4.74 (s, 2H), 3.73-3.65 (m, 1H), 3.54-3.46 (m, 1H), 3.44-3.36 (m, 2H), 3.15-3.06 (m, 3H), 3.02-2.82 (m, 5H), 2.80-2.68 (m, 2H), 2.63-2.55 (m, 1H), 2.26-2.02 (m, 4H), 1.71-1.49 (m, 4H), 1.43-1.27 (m, 2H). HRMS m/z [M+H]+ calcd for C44H46N7O11+ 848.3250, found 848.3245.
LQ126-110 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 7-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)heptanoic acid (4.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-110 was obtained as white solid (6 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 7.79 (t, J=7.9 Hz, 1H), 7.52 (d, J=7.3 Hz, 1H), 7.43-7.38 (m, 2H), 7.36-7.18 (m, 6H), 7.12 (s, 1H), 5.63 (t, J=7.6 Hz, 1H), 5.17-5.08 (m, 1H), 4.73 (s, 2H), 3.73-3.65 (m, 1H), 3.57-3.47 (m, 1H), 3.44-3.36 (m, 2H), 3.15-3.05 (m, 3H), 3.03-2.82 (m, 4H), 2.79-2.67 (m, 2H), 2.63-2.55 (m, 1H), 2.24-2.01 (m, 5H), 1.71-1.49 (m, 4H), 1.44-1.25 (m, 4H). HRMS m/z [M+H]+ calcd for C45H48N7O11+ 862.3406, found 862.3403.
LQ126-112 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 9-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)nonanoic acid (4.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-112 was obtained as white solid (6.5 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 7.81 (t, J=7.9 Hz, 1H), 7.54 (d, J=7.3 Hz, 1H), 7.45-7.40 (m, 2H), 7.39-7.31 (m, 3H), 7.30-7.19 (m, 3H), 7.15 (s, 1H), 5.65 (t, J=7.7 Hz, 1H), 5.14 (dd, J=12.6, 5.5 Hz, 1H), 4.75 (s, 2H), 3.75-3.66 (m, 1H), 3.59-3.50 (m, 1H), 3.47-3.38 (m, 2H), 3.17-3.06 (m, 3H), 3.04-2.84 (m, 4H), 2.81-2.70 (m, 2H), 2.64-2.57 (m, 1H), 2.25-2.03 (m, 5H), 1.69-1.48 (m, 4H), 1.41-1.24 (m, 8H). HRMS m/z [M+H]+ calcd for C47H52N7O11+ 890.3719, found 890.3695.
LQ126-113 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)propanoic acid (4.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-113 was obtained as white solid (5.2 mg, 62%). 1H NMR (600 MHz, Methanol-d4) δ 7.77 (t, J=7.9 Hz, 1H), 7.50 (d, J=7.3 Hz, 1H), 7.40-7.35 (m, 2H), 7.35-7.19 (m, 6H), 7.10 (s, 1H), 5.67-5.61 (m, 1H), 5.17-5.08 (m, 1H), 4.73 (s, 2H), 3.80-3.71 (m, 1H), 3.70-3.63 (m, 2H), 3.62-3.45 (m, 5H), 3.40-3.33 (m, 2H), 3.13-3.05 (m, 3H), 3.00-2.82 (m, 4H), 2.78-2.66 (m, 2H), 2.64-2.56 (m, 1H), 2.52-2.35 (m, 2H), 2.18-2.10 (m, 1H), 2.10-2.01 (m, 1H). HRMS m/z [M+H]+ calcd for C43H44N7O12+ 850.3042, found 850.3037.
LQ126-114 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)ethoxy)propanoic acid (4.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-114 was obtained as white solid (5.1 mg, 57%). 1H NMR (600 MHz, Methanol-d4) δ 7.80 (t, J=7.9 Hz, 1H), 7.53 (d, J=7.3 Hz, 1H), 7.44-7.40 (m, 2H), 7.38-7.31 (m, 3H), 7.31-7.20 (m, 3H), 7.13 (s, 1H), 5.65 (t, J=7.6 Hz, 1H), 5.18-5.10 (m, 1H), 4.77 (s, 2H), 3.79-3.57 (m, 8H), 3.55-3.45 (m, 3H), 3.44-3.38 (m, 2H), 3.15-3.07 (m, 3H), 3.04-2.85 (m, 4H), 2.81-2.69 (m, 2H), 2.65-2.57 (m, 1H), 2.50-2.35 (m, 2H), 2.20-2.12 (m, 1H), 2.12-2.04 (m, 1H). HRMS m/z [M+H]+ calcd for C45H48N7O13+ 894.3305, found 894.3297.
LQ126-115 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oic acid (5.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-115 was obtained as white solid (5.6 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 7.81 (t, J=7.9 Hz, 1H), 7.54 (d, J=7.3 Hz, 1H), 7.45-7.41 (m, 2H), 7.39-7.32 (m, 3H), 7.31-7.20 (m, 3H), 7.14 (s, 1H), 5.65 (t, J=7.6 Hz, 1H), 5.16-5.11 (m, 1H), 4.77 (s, 2H), 3.78-3.48 (m, 14H), 3.44-3.36 (m, 3H), 3.17-3.07 (m, 3H), 3.04-2.85 (m, 4H), 2.80-2.67 (m, 2H), 2.65-2.57 (m, 1H), 2.51-2.36 (m, 2H), 2.19-2.13 (m, 1H), 2.12-2.03 (m, 1H). HRMS m/z [M+H]+ calcd for C47H52N7O14+ 938.3567, found 938.3570.
LQ126-116 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (5.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-116 was obtained as white solid (6.5 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 7.79 (t, J=7.9 Hz, 1H), 7.52 (d, J=7.3 Hz, 1H), 7.43-7.39 (m, 2H), 7.36-7.29 (m, 3H), 7.28-7.18 (m, 3H), 7.13 (s, 1H), 5.63 (t, J=7.7 Hz, 1H), 5.15-5.09 (m, 1H), 4.75 (s, 2H), 3.75-3.45 (m, 18H), 3.44-3.34 (m, 3H), 3.16-3.05 (m, 3H), 3.02-2.82 (m, 4H), 2.78-2.68 (m, 2H), 2.62-2.56 (m, 1H), 2.49-2.34 (m, 2H), 2.17-2.12 (m, 1H), 2.09-2.02 (m, 1H). HRMS m/z [M+H]+ calcd for C49H56N7O15+ 982.3829, found 982.3830.
LQ126-117 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oic acid (6.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-117 was obtained as white solid (6.4 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 7.81 (t, J=7.9 Hz, 1H), 7.54 (d, J=7.3 Hz, 1H), 7.46-7.41 (m, 2H), 7.39-7.32 (m, 3H), 7.30-7.20 (m, 3H), 7.15 (s, 1H), 5.65 (t, J=7.8 Hz, 1H), 5.14 (ddd, J=12.9, 5.6, 2.1 Hz, 1H), 4.78 (s, 2H), 3.79-3.48 (m, 22H), 3.44-3.36 (m, 3H), 3.17-3.07 (m, 3H), 3.04-2.85 (m, 4H), 2.81-2.70 (m, 2H), 2.64-2.57 (m, 1H), 2.52-2.36 (m, 2H), 2.19-2.13 (m, 1H), 2.12-2.03 (m, 1H). HRMS m/z [M+H]+ calcd for C51H60N7O16+ 1026.4091, found 1026.4097.
LQ126-118 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), ((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanoyl)glycine (6.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-118 was obtained as white solid (6.3 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.51-7.20 (m, 11H), 7.13 (s, 1H), 5.66 (t, J=7.9 Hz, 1H), 5.45-5.37 (m, 1H), 4.76 (d, J=9.1 Hz, 1H), 4.68-4.56 (m, 1H), 4.47 (s, 1H), 3.89-3.73 (m, 2H), 3.60-3.51 (m, 1H), 3.44-3.37 (m, 1H), 3.18-3.06 (m, 2H), 3.03-2.83 (m, 7H), 2.65-2.57 (m, 1H), 2.48 (s, 3H), 2.23 (dd, J=13.2, 7.6 Hz, 1H), 2.11-2.04 (m, 2H), 2.02-1.97 (m, 2H), 1.41-1.24 (m, 4H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C53H61FN9O10S+ 1034.4241, found 1034.4245.
LQ126-120 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 4-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)butanoic acid (6.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-120 was obtained as white solid (5.8 mg, 55%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 7.52-7.39 (m, 5H), 7.36-7.18 (m, 6H), 7.13 (s, 1H), 5.64 (t, J=7.8 Hz, 1H), 5.35-5.29 (m, 1H), 4.73 (d, J=9.2 Hz, 1H), 4.62-4.55 (m, 1H), 4.44 (s, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.70-3.62 (m, 1H), 3.50-3.43 (m, 1H), 3.18-3.04 (m, 6H), 3.01-2.90 (m, 4H), 2.84 (dd, J=14.2, 6.4 Hz, 1H), 2.74 (dd, J=14.2, 8.1 Hz, 1H), 2.62-2.56 (m, 1H), 2.48 (s, 3H), 2.19 (dd, J=13.4, 7.7 Hz, 1H), 2.12-1.91 (m, 3H), 1.76-1.53 (m, 2H), 1.39-1.24 (m, 4H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C55H65FN9O10S+ 1062.4554, found 1062.4547.
LQ126-121 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 5-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)pentanoic acid (6.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-121 was obtained as white solid (6.2 mg, 58%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.51-7.41 (m, 5H), 7.40-7.28 (m, 4H), 7.28-7.20 (m, 2H), 7.15 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 5.32 (dd, J=8.1, 6.2 Hz, 1H), 4.75 (d, J=9.2 Hz, 1H), 4.61 (t, J=8.8 Hz, 1H), 4.46 (s, 1H), 3.84 (d, J=11.1 Hz, 1H), 3.78 (dd, J=11.1, 3.8 Hz, 1H), 3.72-3.63 (m, 1H), 3.55-3.45 (m, 1H), 3.18-3.05 (m, 6H), 3.03-2.91 (m, 4H), 2.87-2.81 (m, 1H), 2.75 (dd, J=14.2, 8.2 Hz, 1H), 2.66-2.57 (m, 1H), 2.50 (s, 3H), 2.23-2.14 (m, 1H), 2.11-2.03 (m, 2H), 2.00-1.93 (m, 1H), 1.60-1.50 (m, 2H), 1.49-1.28 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O10S+ 1076.4710, found 1076.4713.
LQ126-122 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 6-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)hexanoic acid (6.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-122 was obtained as white solid (6.6 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.51-7.39 (m, 5H), 7.38-7.26 (m, 4H), 7.25-7.18 (m, 2H), 7.13 (s, 1H), 5.64 (t, J=7.7 Hz, 1H), 5.30 (dd, J=8.1, 6.2 Hz, 1H), 4.73 (d, J=9.2 Hz, 1H), 4.58 (t, J=8.5 Hz, 1H), 4.44 (s, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.73-3.65 (m, 1H), 3.50-3.47 (m, 1H), 3.15-3.03 (m, 6H), 3.00-2.89 (m, 4H), 2.83 (dd, J=14.1, 6.2 Hz, 1H), 2.73 (dd, J=14.3, 8.2 Hz, 1H), 2.64-2.55 (m, 1H), 2.48 (s, 3H), 2.22-2.14 (m, 1H), 2.10-2.01 (m, 2H), 1.98-1.91 (m, 1H), 1.62-1.43 (m, 2H), 1.42-1.14 (m, 8H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C57H69FN9O10S+ 1090.4867, found 1090.4872.
LQ126-123 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 7-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)heptanoic acid (7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-123 was obtained as white solid (7.2 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.50-7.38 (m, 4H), 7.37-7.18 (m, 7H), 7.13 (s, 1H), 5.64 (t, J=7.8 Hz, 1H), 5.30 (dd, J=8.3, 6.0 Hz, 1H), 4.73 (d, J=9.2 Hz, 1H), 4.58 (t, J=8.5 Hz, 1H), 4.44 (s, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.72-3.65 (m, 1H), 3.52-3.46 (m, 1H), 3.15-2.90 (m, 10H), 2.83 (dd, J=14.1, 6.0 Hz, 1H), 2.73 (dd, J=14.0, 8.4 Hz, 1H), 2.64-2.55 (m, 1H), 2.49 (s, 3H), 2.21-2.12 (m, 1H), 2.09-2.02 (m, 2H), 1.98-1.91 (m, 1H), 1.60-1.51 (m, 1H), 1.49-1.40 (m, 1H), 1.38-1.13 (m, 10H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O10S+ 1104.5023, found 1104.5034.
LQ126-124 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 8-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)octanoic acid (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-124 was obtained as white solid (5.9 mg, 53%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 7.49-7.38 (m, 4H), 7.38-7.17 (m, 6H), 7.13 (s, 1H), 5.64 (t, J=7.7 Hz, 1H), 5.30 (dd, J=8.3, 6.0 Hz, 1H), 4.73 (d, J=9.3 Hz, 1H), 4.58 (dd, J=9.2, 7.7 Hz, 1H), 4.44 (s, 1H), 3.82 (d, J=11.1 Hz, 1H), 3.76 (dd, J=11.1, 3.8 Hz, 1H), 3.71-3.56 (m, 4H), 3.50 (d, J=5.8 Hz, 2H), 3.23-3.18 (m, 1H), 3.17-2.88 (m, 7H), 2.83 (dd, J=14.1, 5.9 Hz, 1H), 2.73 (dd, J=14.1, 8.4 Hz, 1H), 2.62-2.56 (m, 1H), 2.48 (s, 3H), 2.21-2.12 (m, 2H), 2.09-2.02 (m, 1H), 1.98-1.92 (m, 1H), 1.63-1.50 (m, 1H), 1.48-1.42 (m, 3H), 1.38-1.11 (m, 8H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C59H73FN9O10S+ 1118.5180, found 1118.5198.
LQ126-125 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-((S)-3-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-(4-(4-methylthiazol-5-yl)phenyl)propanamido)ethoxy)propanoic acid (6.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-125 was obtained as white solid (6.2 mg, 57%). 1H NMR (600 MHz, Methanol-d4) δ 8.96 (s, 1H), 7.51 (dd, J=9.3, 3.4 Hz, 1H), 7.47-7.35 (m, 6H), 7.34-7.20 (m, 4H), 7.14 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 5.33 (t, J=7.1 Hz, 1H), 4.75 (d, J=9.3 Hz, 1H), 4.63-4.57 (m, 1H), 4.45 (s, 1H), 3.83 (d, J=11.1 Hz, 1H), 3.77 (dd, J=11.1, 3.8 Hz, 1H), 3.72-3.57 (m, 3H), 3.56-3.51 (m, 1H), 3.49-3.36 (m, 3H), 3.18-3.07 (m, 3H), 3.03-2.91 (m, 4H), 2.85 (dd, J=14.2, 6.8 Hz, 1H), 2.76 (dd, J=14.2, 7.7 Hz, 1H), 2.65-2.58 (m, 1H), 2.48 (s, 3H), 2.47-2.33 (m, 2H), 2.24-2.16 (m, 1H), 2.12-2.04 (m, 1H), 2.00-1.93 (m, 1H), 1.42-1.26 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O11S+ 1092.4659, found 1092.4672.
LQ126-126 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5-dioxo-9,12-dioxa-2,6-diazapentadecan-15-oic acid (7.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-126 was obtained as white solid (7 mg, 62%).
1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 7.50 (dd, J=9.3, 3.4 Hz, 1H), 7.48-7.41 (m, 4H), 7.39-7.20 (m, 6H), 7.15 (s, 1H), 5.66 (t, J=7.8 Hz, 1H), 5.33 (t, J=7.2 Hz, 1H), 4.75 (d, J=9.0 Hz, 1H), 4.63-4.57 (m, 1H), 4.47-4.44 (m, 1H), 3.84 (d, J=11.1 Hz, 1H), 3.80-3.38 (m, 12H), 3.18-3.07 (m, 3H), 3.02-2.91 (m, 4H), 2.90-2.83 (m, 1H), 2.81-2.72 (m, 1H), 2.66-2.57 (m, 1H), 2.49 (s, 3H), 2.41-2.38 (m, 1H), 2.21 (dd, J=13.3, 7.8 Hz, 1H), 2.12-2.03 (m, 1H), 2.00-1.92 (m, 1H), 1.42-1.27 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O12S+ 1136.4921, found 1136.4917.
LQ126-127 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5-dioxo-9,12,15-trioxa-2,6-diazaoctadecan-18-oic acid (7.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-127 was obtained as white solid (8 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.53-7.42 (m, 5H), 7.39-7.35 (m, 2H), 7.34-7.20 (m, 4H), 7.15 (s, 1H), 5.66 (t, J=7.8 Hz, 1H), 5.34 (t, J=7.1 Hz, 1H), 4.75 (d, J=9.1 Hz, 1H), 4.63-4.58 (m, 1H), 4.46 (s, 1H), 3.84 (d, J=11.1 Hz, 1H), 3.80-3.65 (m, 4H), 3.62-3.38 (m, 12H), 3.18-3.07 (m, 3H), 3.04-2.91 (m, 4H), 2.86 (dd, J=14.2, 6.2 Hz, 1H), 2.77 (dd, J=14.1, 8.1 Hz, 1H), 2.65-2.58 (m, 1H), 2.50 (s, 3H), 2.43-2.38 (m, 1H), 2.21 (dd, J=13.0, 7.7 Hz, 1H), 2.11-2.04 (m, 1H), 2.00-1.93 (m, 1H), 1.42-1.25 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O13S+ 1180.5184, found 1180.5181.
LQ126-128 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (S)-1-((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidin-2-yl)-3-(4-(4-methylthiazol-5-yl)phenyl)-1,5-dioxo-9,12,15,18-tetraoxa-2,6-diazahenicosan-21-oic acid (8.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-128 was obtained as white solid (7.5 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.53-7.41 (m, 5H), 7.39-7.36 (m, 2H), 7.34-7.20 (m, 4H), 7.16 (s, 1H), 5.66 (t, J=7.7 Hz, 1H), 5.34 (t, J=7.1 Hz, 1H), 4.75 (d, J=9.2 Hz, 1H), 4.60 (t, J=8.5 Hz, 1H), 4.46 (s, 1H), 3.84 (d, J=11.1 Hz, 1H), 3.80-3.40 (m, 19H), 3.19-3.07 (m, 3H), 3.02-2.90 (m, 4H), 2.86 (dd, J=14.2, 6.1 Hz, 1H), 2.78 (dd, J=14.1, 8.1 Hz, 1H), 2.66-2.57 (m, 1H), 2.51 (s, 3H), 2.49-2.45 (m, 1H), 2.41-2.38 (m, 1H), 2.22 (dd, J=13.0, 7.8 Hz, 1H), 2.13-2.03 (m, 1H), 2.01-1.93 (m, 1H), 1.42-1.27 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C62H79FN9O14S+ 1224.5446, found 1224.5433.
LQ126-130 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), (2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetyl)glycine (6.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-130 was obtained as white solid (6 mg, 57%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.54-7.43 (m, 2H), 7.42-7.20 (m, 5H), 7.14-7.00 (m, 4H), 5.70-5.61 (m, 1H), 4.78-4.55 (m, 5H), 4.50-4.42 (m, 2H), 4.06-3.89 (m, 2H), 3.87-3.67 (m, 3H), 3.61-3.39 (m, 1H), 3.17-3.07 (m, 3H), 3.02-2.90 (m, 4H), 2.65-2.57 (m, 1H), 2.49 (s, 3H), 2.21 (dd, J=13.3, 7.6 Hz, 1H), 2.14-2.03 (m, 2H), 1.41-1.23 (m, 4H), 1.00 (s, 9H). HRMS m/z [M+H]+ calcd for C53H61FN9O11S+ 1050.4190, found 1050.4214.
LQ126-168 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)propanoic acid (6.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-168 was obtained as white solid (6.9 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 8.89 (s, 1H), 7.42-7.36 (m, 2H), 7.32-7.07 (m, 5H), 7.03-6.93 (m, 3H), 6.84 (s, 1H), 5.56-5.50 (m, 1H), 4.62 (d, J=9.2 Hz, 1H), 4.53-4.32 (m, 4H), 3.76-3.66 (m, 2H), 3.63-3.56 (m, 2H), 3.53-3.35 (m, 5H), 3.04-2.94 (m, 3H), 2.89-2.79 (m, 4H), 2.53-2.46 (m, 2H), 2.38 (s, 3H), 2.15-2.06 (m, 1H), 2.00-1.91 (m, 2H), 1.29-1.11 (m, 4H), 0.90 (s, 9H). HRMS m/z [M+H]+ calcd for C54H63FN9O11S+ 1064.4346, found 1064.4349.
LQ126-170 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 5-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)pentanoic acid (6.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-170 was obtained as white solid (7.6 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 7.49 (d, J=7.7 Hz, 2H), 7.42 (s, 1H), 7.37-7.20 (m, 5H), 7.17-7.06 (m, 2H), 6.96 (s, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.74 (d, J=9.2 Hz, 1H), 4.65-4.55 (m, 4H), 4.53-4.45 (m, 2H), 3.85 (d, J=11.1 Hz, 1H), 3.80 (dd, J=11.1, 3.8 Hz, 1H), 3.74-3.60 (m, 2H), 3.56-3.38 (m, 2H), 3.19-3.06 (m, 3H), 3.03-2.89 (m, 4H), 2.65-2.58 (m, 1H), 2.50 (s, 3H), 2.29-2.02 (m, 4H), 1.72-1.50 (m, 5H), 1.43-1.25 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O11S+ 1092.4659, found 1092.4687.
LQ126-171 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 6-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)hexanoic acid (7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-171 was obtained as white solid (6.9 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 7.52-7.46 (m, 2H), 7.43 (dd, J=7.9, 1.6 Hz, 1H), 7.40-7.20 (m, 5H), 7.15 (s, 1H), 7.10 (dd, J=7.7, 1.6 Hz, 1H), 6.99-6.96 (m, 1H), 5.67-5.62 (m, 1H), 4.74 (d, J=9.3 Hz, 1H), 4.66-4.57 (m, 4H), 4.52-4.45 (m, 2H), 3.85 (d, J=11.1 Hz, 1H), 3.79 (dd, J=11.0, 3.9 Hz, 1H), 3.74-3.67 (m, 1H), 3.55-3.38 (m, 3H), 3.18-3.06 (m, 3H), 3.04-2.91 (m, 4H), 2.69-2.56 (m, 1H), 2.50 (s, 3H), 2.26-2.17 (m, 2H), 2.14-2.03 (m, 2H), 1.69-1.51 (m, 5H), 1.42-1.24 (m, 6H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C57H69FN9O11S+ 1106.4816, found 1106.4817.
LQ126-172 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)ethoxy)propanoic acid (7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-172 was obtained as white solid (7.8 mg, 71%). 1H NMR (600 MHz, Methanol-d4) δ 8.94 (s, 1H), 7.49 (d, J=7.9 Hz, 2H), 7.42-7.20 (m, 6H), 7.12 (s, 1H), 7.09 (dd, J=7.7, 1.6 Hz, 1H), 6.97 (d, J=1.6 Hz, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.75 (d, J=9.2 Hz, 1H), 4.66-4.59 (m, 2H), 4.56 (d, J=15.2 Hz, 1H), 4.52-4.46 (m, 2H), 3.86 (d, J=11.1 Hz, 1H), 3.80 (dd, J=11.2, 3.7 Hz, 1H), 3.76-3.63 (m, 2H), 3.61-3.43 (m, 6H), 3.16-3.07 (m, 3H), 3.00-2.91 (m, 4H), 2.64-2.58 (m, 1H), 2.49 (s, 3H), 2.47-2.33 (m, 2H), 2.26-2.19 (m, 1H), 2.13-2.03 (m, 1H), 1.41-1.25 (m, 6H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O12S+ 1108.4608, found 1108.4611.
LQ126-173 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 3-(2-(2-(2-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetamido)ethoxy)ethoxy)propanoic acid (7.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-173 was obtained as white solid (7.8 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 7.53-7.49 (m, 1H), 7.42 (dd, J=7.9, 1.5 Hz, 1H), 7.37-7.34 (m, 2H), 7.32 (d, J=7.3 Hz, 1H), 7.29 (d, J=7.3 Hz, 1H), 7.27-7.20 (m, 2H), 7.14 (s, 1H), 7.09 (dd, J=7.7, 1.6 Hz, 1H), 6.98 (d, J=1.6 Hz, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.75 (d, J=9.1 Hz, 1H), 4.67-4.58 (m, 3H), 4.56-4.47 (m, 2H), 3.85 (d, J=11.1 Hz, 1H), 3.81 (dd, J=11.1, 3.7 Hz, 1H), 3.75-3.63 (m, 2H), 3.62-3.38 (m, 9H), 3.15-3.06 (m, 3H), 3.02-2.90 (m, 4H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.49-2.42 (m, 1H), 2.41-2.34 (m, 1H), 2.26-2.20 (m, 1H), 2.13-2.03 (m, 2H), 1.40-1.24 (m, 6H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O13S+ 1152.4871, found 1152.4870.
LQ126-174 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oic acid (7.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-174 was obtained as white solid (8.2 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.43 (dd, J=7.8, 1.5 Hz, 1H), 7.38-7.34 (m, 2H), 7.32 (d, J=7.3 Hz, 1H), 7.29 (d, J=7.3 Hz, 1H), 7.27-7.20 (m, 2H), 7.15 (s, 1H), 7.10 (dd, J=7.7, 1.6 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 5.65 (t, J=7.8 Hz, 1H), 4.75 (d, J=9.1 Hz, 1H), 4.67-4.59 (m, 3H), 4.56 (d, J=15.2 Hz, 1H), 4.53-4.48 (m, 1H), 3.85 (d, J=11.1 Hz, 1H), 3.81 (dd, J=11.0, 3.8 Hz, 1H), 3.75-3.64 (m, 2H), 3.62-3.52 (m, 11H), 3.49 (t, J=5.6 Hz, 2H), 3.17-3.06 (m, 3H), 3.03-2.91 (m, 4H), 2.64-2.57 (m, 1H), 2.51 (s, 3H), 2.49-2.37 (m, 2H), 2.23 (dd, J=13.1, 7.8 Hz, 1H), 2.13-2.03 (m, 2H), 1.41-1.26 (m, 6H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O14S+ 1196.5133, found 1196.5130.
LQ126-175 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (8.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-175 was obtained as white solid (7.9 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 8.97 (s, 1H), 7.53-7.49 (m, 2H), 7.45-7.42 (m, 1H), 7.39-7.34 (m, 1H), 7.32 (d, J=7.3 Hz, 1H), 7.30-7.20 (m, 3H), 7.15 (s, 1H), 7.10 (d, J=7.7 Hz, 2H), 6.99 (d, J=1.5 Hz, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.75 (d, J=9.2 Hz, 1H), 4.67-4.54 (m, 4H), 4.50 (d, J=15.0 Hz, 1H), 3.85 (d, J=11.1 Hz, 1H), 3.81 (dd, J=11.0, 3.8 Hz, 1H), 3.75-3.64 (m, 2H), 3.63-3.52 (m, 15H), 3.51-3.46 (m, 2H), 3.22-3.20 (m, 1H), 3.17-3.06 (m, 3H), 3.03-2.91 (m, 4H), 2.64-2.57 (m, 1H), 2.50 (s, 3H), 2.49-2.36 (m, 2H), 2.25-2.20 (m, 1H), 2.13-2.03 (m, 2H), 1.41-1.26 (m, 6H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C62H79FN9O15S+ 1240.5395, found 1240.5398.
LQ126-176 was synthesized following the standard procedure for preparing LQ126-89 from intermediate 46 (4 mg, 0.01 mmol), 1-(2-(((2S,4R)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-2-oxo-6,9,12,15,18-pentaoxa-3-azahenicosan-21-oic acid (8.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-176 was obtained as white solid (7.7 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 8.94 (s, 1H), 7.52-7.49 (m, 2H), 7.45-7.42 (m, 1H), 7.38-7.35 (m, 1H), 7.32 (d, J=7.2 Hz, 1H), 7.29 (d, J=7.4 Hz, 1H), 7.27-7.20 (m, 2H), 7.16 (s, 1H), 7.11-7.08 (m, 1H), 6.99 (d, J=1.6 Hz, 1H), 5.65 (t, J=7.7 Hz, 1H), 4.75 (d, J=9.4 Hz, 1H), 4.66-4.64 (m, 2H), 4.63-4.55 (m, 2H), 4.53-4.48 (m, 1H), 3.85 (d, J=11.2 Hz, 1H), 3.81 (dd, J=11.0, 3.8 Hz, 1H), 3.63-3.55 (m, 20H), 3.52-3.48 (m, 2H), 3.22-3.20 (m, 1H), 3.17-3.07 (m, 3H), 3.03-2.91 (m, 4H), 2.64-2.58 (m, 1H), 2.50 (s, 3H), 2.49-2.37 (m, 2H), 2.26-2.19 (m, 1H), 2.12-2.04 (m, 2H), 1.42-1.25 (m, 6H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C64H83FN9O16S+ 1284.5657, found 1284.5653.
Intermediate 47 was synthesized according to the procedures for the preparation of intermediate 4 as a white solid in 58% yield. 1H NMR (600 MHz, Methanol-d4) δ 7.44 (dd, J=7.9, 1.5 Hz, 1H), 7.37 (d, J=1.5 Hz, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.19 (d, J=8.3 Hz, 1H), 7.15 (s, 1H), 6.91 (d, J=2.5 Hz, 1H), 6.86 (dd, J=8.3, 2.5 Hz, 1H), 5.62 (t, J=7.7 Hz, 1H), 4.64 (s, 2H), 3.20-2.93 (m, 7H), 2.91-2.83 (m, 1H), 2.66-2.58 (m, 1H), 2.12-2.03 (m, 1H). MS (ESI): m/z 466.5 [M+H]+.
To a solution of Intermediate 47 (5 mg, 0.01 mmol) in DMSO (1 mL) were added (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv). After being stirred overnight at room temperature, the resulting mixture was purified by preparative HPLC (5%-70% acetonitrile/0.1% TFA in H2O) to afford LQ126-177 as white solid (7.2 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 7.58-7.29 (m, 7H), 7.15-7.11 (m, 2H), 6.94-6.84 (m, 2H), 5.54 (t, J=7.8 Hz, 1H), 4.72-4.67 (m, 1H), 4.62 (t, J=8.4 Hz, 1H), 4.58-4.47 (m, 5H), 4.02 (dd, J=15.2, 1.4 Hz, 1H), 3.92-3.84 (m, 2H), 3.79 (dd, J=11.0, 3.7 Hz, 1H), 3.69-3.54 (m, 2H), 3.53-3.45 (m, 2H), 3.18-2.90 (m, 7H), 2.84-2.74 (m, 1H), 2.59-2.50 (m, 1H), 2.46 (s, 3H), 2.28-2.21 (m, 1H), 2.13-2.00 (m, 2H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C50H59N8O11S+ 979.4019, found 979.4016.
LQ126-178 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(3-(2-aminoethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-178 was obtained as white solid (6.1 mg, 62%). 1H NMR (600 MHz, Methanol-d4) δ 9.19 (s, 1H), 7.50-7.29 (m, 7H), 7.23-7.13 (m, 2H), 6.98-6.84 (m, 2H), 5.56 (t, J=8.0 Hz, 1H), 4.68-4.60 (m, 2H), 4.54-4.41 (m, 4H), 4.33 (d, J=15.5 Hz, 1H), 3.86 (d, J=11.1 Hz, 1H), 3.73 (dd, J=11.0, 3.9 Hz, 1H), 3.69-3.62 (m, 1H), 3.60-3.51 (m, 2H), 3.50-3.39 (m, 3H), 3.21-2.89 (m, 7H), 2.87-2.79 (m, 1H), 2.61-2.53 (m, 1H), 2.49 (s, 3H), 2.44-2.33 (m, 1H), 2.27-2.20 (m, 1H), 2.11-2.01 (m, 3H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C51H61N8O11S+ 993.4175, found 993.4179.
LQ126-180 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-180 was obtained as white solid (7.1 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.19 (s, 1H), 7.50-7.45 (m, 2H), 7.43-7.40 (m, 3H), 7.37-7.32 (m, 2H), 7.20-7.15 (m, 2H), 6.93 (d, J=2.5 Hz, 1H), 6.87 (dd, J=8.2, 2.5 Hz, 1H), 5.59 (t, J=7.8 Hz, 1H), 4.65 (s, 1H), 4.61-4.51 (m, 2H), 4.51-4.42 (m, 3H), 4.34 (d, J=15.5 Hz, 1H), 3.86 (d, J=11.0 Hz, 1H), 3.76 (dd, J=10.9, 3.9 Hz, 1H), 3.74-3.62 (m, 2H), 3.60-3.50 (m, 6H), 3.42 (t, J=5.5 Hz, 2H), 3.18-2.92 (m, 7H), 2.89-2.80 (m, 1H), 2.62-2.55 (m, 1H), 2.52-2.39 (m, 5H), 2.24-2.17 (m, 1H), 2.12-2.02 (m, 2H), 1.01 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O12S+ 1037.4437, found 1037.4443.
LQ126-181 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-181 was obtained as white solid (7 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 7.56-7.32 (m, 7H), 7.25-7.14 (m, 2H), 6.98-6.85 (m, 2H), 5.64-5.56 (m, 1H), 4.72 (s, 1H), 4.64-4.29 (m, 6H), 4.05-3.71 (m, 4H), 3.71-3.50 (m, 10H), 3.47-3.41 (m, 2H), 3.24-2.82 (m, 8H), 2.64-2.56 (m, 1H), 2.49 (s, 3H), 2.27-2.21 (m, 1H), 2.17-2.02 (m, 2H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C54H67N8O13S+ 1067.4543, found 1067.4537.
LQ126-182 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-1-amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-182 was obtained as white solid (6.5 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 9.14 (s, 1H), 7.57-7.29 (m, 7H), 7.25-7.13 (m, 2H), 7.03-6.84 (m, 2H), 5.69-5.56 (m, 1H), 4.68-4.45 (m, 6H), 4.36 (d, J=15.0 Hz, 1H), 3.89 (d, J=10.9 Hz, 1H), 3.83-3.77 (m, 1H), 3.75-3.51 (m, 12H), 3.47-3.42 (m, 2H), 3.19-2.83 (m, 8H), 2.64-2.41 (m, 6H), 2.26-2.19 (m, 1H), 2.12-2.03 (m, 2H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C55H69N8O13S+ 1081.4699, found 1081.4670.
LQ126-183 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan-18-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-183 was obtained as white solid (6.4 mg, 57%). 1H NMR (600 MHz, Methanol-d4) δ 9.27 (s, 1H), 7.59-7.32 (m, 7H), 7.24-7.13 (m, 2H), 7.05-6.88 (m, 2H), 5.75-5.54 (m, 1H), 4.72-4.45 (m, 6H), 4.37 (d, J=15.3 Hz, 1H), 3.90 (d, J=11.1 Hz, 1H), 3.83-3.78 (m, 1H), 3.73-3.49 (m, 16H), 3.49-3.41 (m, 2H), 3.20-2.81 (m, 8H), 2.67-2.42 (m, 6H), 2.29-2.20 (m, 1H), 2.14-2.04 (m, 2H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C57H73N8O14S+ 1125.4961, found 1125.4937.
LQ126-184 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-1-amino-20-(tert-butyl)-18-oxo-3,6,9,12,15-pentaoxa-19-azahenicosan-21-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (8.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-184 was obtained as white solid (7.6 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.30 (s, 1H), 7.57-7.41 (m, 5H), 7.39-7.33 (m, 2H), 7.24-7.15 (m, 2H), 6.99-6.87 (m, 2H), 5.62 (t, J=7.8 Hz, 1H), 4.66 (s, 1H), 4.61-4.47 (m, 5H), 4.37 (d, J=15.6 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.8 Hz, 1H), 3.75-3.68 (m, 2H), 3.67-3.52 (m, 20H), 3.49-3.42 (m, 2H), 3.19-2.92 (m, 7H), 2.92-2.83 (m, 1H), 2.66-2.44 (m, 4H), 2.27-2.20 (m, 1H), 2.14-2.06 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C59H77N8O15S+ 1169.5224, found 1169.5227.
LQ126-185 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(2-aminoacetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-185 was obtained as white solid (5.9 mg, 63%). 1H NMR (400 MHz, Methanol-d4) δ 9.16 (s, 1H), 7.52-7.33 (m, 7H), 7.25-7.12 (m, 2H), 7.03-6.91 (m, 2H), 5.68-5.58 (m, 1H), 4.65 (d, J=5.7 Hz, 1H), 4.62-4.48 (m, 5H), 4.45-4.33 (m, 1H), 4.01 (s, 2H), 3.94-3.76 (m, 2H), 3.22-2.95 (m, 7H), 2.94-2.82 (m, 1H), 2.67-2.55 (m, 1H), 2.51 (s, 3H), 2.28-2.18 (m, 1H), 2.16-2.03 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C48H55N8O10S+ 935.3756, found 935.3755.
LQ126-186 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ126-186 was obtained as white solid (6.7 mg, 71%). 1H NMR (400 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.54-7.29 (m, 7H), 7.25-7.12 (m, 2H), 7.06-6.87 (m, 2H), 5.68-5.55 (m, 1H), 4.64-4.45 (m, 6H), 4.42-4.32 (m, 1H), 3.93 (d, J=10.7 Hz, 1H), 3.79 (d, J=11.5 Hz, 1H), 3.60-3.50 (m, 2H), 3.49-3.37 (m, 1H), 3.24-2.92 (m, 7H), 2.91-2.81 (m, 1H), 2.64-2.55 (m, 1H), 2.51 (s, 3H), 2.29-2.20 (m, 1H), 2.15-2.03 (m, 3H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C49H57N8O10S+ 949.3913, found 949.3919.
LQ141-1 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(4-aminobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-1 was obtained as white solid (7 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.16 (s, 1H), 7.51-7.47 (m, 2H), 7.45-7.40 (m, 3H), 7.37 (d, J=1.6 Hz, 1H), 7.34 (d, J=7.9 Hz, 1H), 7.21 (d, J=8.3 Hz, 1H), 7.18 (s, 1H), 6.97-6.95 (m, 1H), 6.93-6.90 (m, 1H), 5.63 (t, J=7.9 Hz, 1H), 4.64-4.53 (m, 3H), 4.52-4.46 (m, 3H), 4.37 (d, J=15.6 Hz, 1H), 3.94-3.89 (m, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.31-3.26 (m, 2H), 3.19-2.93 (m, 7H), 2.91-2.83 (m, 1H), 2.65-2.56 (m, 1H), 2.50 (s, 3H), 2.31-2.19 (m, 3H), 2.13-2.06 (m, 2H), 1.84-1.75 (m, 2H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C50H59N8O10S+ 963.4069, found 963.4061.
LQ141-2 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(5-aminopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-2 was obtained as white solid (6.8 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.21-9.10 (m, 1H), 7.60-7.29 (m, 7H), 7.26-7.11 (m, 2H), 7.01-6.82 (m, 2H), 5.66-5.56 (m, 1H), 4.65-4.32 (m, 7H), 3.91 (d, J=11.0 Hz, 1H), 3.83-3.74 (m, 1H), 3.30-3.21 (m, 2H), 3.18-2.80 (m, 8H), 2.67-2.55 (m, 1H), 2.51 (s, 3H), 2.37-2.17 (m, 3H), 2.16-2.02 (m, 2H), 1.71-1.43 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C51H61N8O10S+ 977.4226, found 977.4189.
LQ141-3 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(6-aminohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-3 was obtained as white solid (6.6 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 9.06 (s, 1H), 7.58-7.33 (m, 7H), 7.23-7.18 (m, 2H), 6.95-6.89 (m, 2H), 5.66-5.59 (m, 1H), 4.68-4.33 (m, 7H), 3.94-3.75 (m, 2H), 3.29-3.21 (m, 2H), 3.17-2.79 (m, 7H), 2.49 (s, 3H), 2.33-2.20 (m, 3H), 2.14-2.05 (m, 2H), 1.75-1.44 (m, 4H), 1.40-1.27 (m, 2H), 1.17-1.11 (m, 2H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C52H63N8O10S+ 991.4382, found 991.4363.
LQ141-4 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(7-aminoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-4 was obtained as white solid (7.6 mg, 76%). 1H NMR (600 MHz, Methanol-d4) δ 9.16 (s, 1H), 7.50 (d, J=8.0 Hz, 2H), 7.46-7.42 (m, 3H), 7.37 (d, J=1.5 Hz, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.20 (d, J=8.2 Hz, 1H), 7.17 (s, 1H), 6.94 (d, J=2.5 Hz, 1H), 6.89 (dd, J=8.2, 2.5 Hz, 1H), 5.61 (t, J=7.8 Hz, 1H), 4.64 (s, 1H), 4.62-4.53 (m, 2H), 4.52-4.49 (m, 1H), 4.47 (s, 2H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.24 (t, J=7.1 Hz, 2H), 3.17-2.92 (m, 7H), 2.91-2.83 (m, 1H), 2.65-2.56 (m, 1H), 2.51 (s, 3H), 2.32-2.20 (m, 3H), 2.14-2.04 (m, 2H), 1.62-1.54 (m, 2H), 1.54-1.46 (m, 2H), 1.34-1.25 (m, 4H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C53H65N8O10S+ 1005.4539, found 1005.4530.
LQ141-5 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(8-aminooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-5 was obtained as white solid (7.1 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.51-7.46 (m, 2H), 7.46-7.42 (m, 3H), 7.39-7.34 (m, 2H), 7.22-7.15 (m, 2H), 6.94 (d, J=2.5 Hz, 1H), 6.91-6.88 (m, 1H), 5.62 (t, J=7.8 Hz, 1H), 4.65 (s, 1H), 4.62-4.44 (m, 5H), 4.37 (d, J=15.5 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.81 (dd, J=10.9, 3.9 Hz, 1H), 3.28-3.21 (m, 2H), 3.16-2.95 (m, 7H), 2.91-2.83 (m, 1H), 2.64-2.58 (m, 1H), 2.49 (s, 3H), 2.31-2.19 (m, 3H), 2.14-2.04 (m, 2H), 1.62-1.55 (m, 2H), 1.52-1.46 (m, 2H), 1.34-1.23 (m, 6H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C54H67N8O10S+ 1019.4695, found 1019.4702.
LQ141-6 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(9-aminononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-6 was obtained as white solid (7.2 mg, 77%). 1H NMR (600 MHz, Methanol-d4) δ 9.21 (s, 1H), 7.50 (d, J=8.0 Hz, 2H), 7.47-7.41 (m, 3H), 7.39-7.33 (m, 2H), 7.20 (d, J=8.3 Hz, 1H), 7.17 (s, 1H), 6.94 (d, J=2.5 Hz, 1H), 6.90 (dd, J=8.3, 2.5 Hz, 1H), 5.61 (t, J=7.8 Hz, 1H), 4.65 (s, 1H), 4.62-4.45 (m, 5H), 4.38 (d, J=15.5 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.81 (dd, J=11.0, 3.9 Hz, 1H), 3.24 (t, J=7.1 Hz, 2H), 3.17-2.94 (m, 7H), 2.91-2.82 (m, 1H), 2.65-2.57 (m, 1H), 2.51 (s, 3H), 2.34-2.20 (m, 3H), 2.13-2.06 (m, 2H), 1.64-1.56 (m, 2H), 1.53-1.44 (m, 2H), 1.38-1.22 (m, 8H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C55H69N8O10S+ 1033.4852, found 1033.4809.
LQ141-7 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(10-aminodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-7 was obtained as white solid (7.6 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 7.56-7.30 (m, 7H), 7.25-7.12 (m, 2H), 6.99-6.85 (m, 2H), 5.61 (t, J=7.8 Hz, 1H), 4.70-4.43 (m, 6H), 4.37 (d, J=15.7 Hz, 1H), 3.92 (d, J=10.9 Hz, 1H), 3.81 (d, J=10.5 Hz, 1H), 3.28-2.94 (m, 9H), 2.91-2.81 (m, 1H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.36-2.20 (m, 3H), 2.09 (t, J=10.7 Hz, 2H), 1.71-1.46 (m, 5H), 1.40-1.20 (m, 9H), 1.05 (s, 9H). HRMS m/z [M+H]+ calcd for C56H71N8O10S+ 1047.5008, found 1047.5000.
LQ141-8 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (6.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-8 was obtained as white solid (7 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.15 (s, 1H), 7.62-7.30 (m, 7H), 7.26-7.11 (m, 2H), 7.02-6.84 (m, 2H), 5.67-5.56 (m, 1H), 4.72-4.31 (m, 7H), 4.02-3.76 (m, 2H), 3.28-3.19 (m, 2H), 3.18-2.80 (m, 9H), 2.68-2.56 (m, 2H), 2.51 (s, 3H), 2.40-2.01 (m, 5H), 1.70-1.42 (m, 5H), 1.40-1.20 (m, 11H), 1.04 (s, 9H). HRMS m/z [M+H]+ calcd for C57H73N8O10S+ 1061.5165, found 1061.5157.
LQ141-9 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (4.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-9 was obtained as white solid (6.5 mg, 79%). 1H NMR (600 MHz, Methanol-d4) δ 7.76 (dd, J=8.4, 7.3 Hz, 1H), 7.48 (d, J=7.3 Hz, 1H), 7.42-7.36 (m, 2H), 7.35-7.32 (m, 2H), 7.16 (d, J=8.3 Hz, 1H), 7.12 (s, 1H), 6.95 (d, J=2.4 Hz, 1H), 6.86 (dd, J=8.3, 2.5 Hz, 1H), 5.59 (t, J=7.9 Hz, 1H), 5.01 (dd, J=12.2, 5.3 Hz, 1H), 4.69 (d, J=6.2 Hz, 2H), 4.47 (d, J=4.1 Hz, 2H), 3.52-3.43 (m, 4H), 3.17-2.93 (m, 7H), 2.89-2.81 (m, 1H), 2.72-2.57 (m, 4H), 2.13-2.04 (m, 2H). HRMS m/z [M+H]+ calcd for C41H40N7O12+ 822.2729, found 822.2716.
LQ141-10 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(3-aminopropyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-10 was obtained as white solid (6.2 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 7.78-7.73 (m, 1H), 7.47 (dd, J=7.3, 5.9 Hz, 1H), 7.41-7.36 (m, 2H), 7.35-7.30 (m, 2H), 7.16 (dd, J=8.4, 3.9 Hz, 1H), 7.12 (d, J=3.4 Hz, 1H), 6.95 (t, J=2.7 Hz, 1H), 6.89-6.85 (m, 1H), 5.59 (t, J=7.9 Hz, 1H), 5.12 (ddd, J=12.6, 7.6, 5.5 Hz, 1H), 4.77-4.69 (m, 2H), 4.47 (s, 2H), 3.38-3.33 (m, 1H), 3.32-3.25 (m, 2H), 3.18-2.95 (m, 8H), 2.89-2.79 (m, 2H), 2.77-2.68 (m, 2H), 2.63-2.56 (m, 1H), 2.18-2.05 (m, 2H), 1.80-1.73 (m, 2H). HRMS m/z [M+H]+ calcd for C42H42N7O12+ 836.2886, found 836.2856.
LQ141-11 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-11 was obtained as white solid (5.9 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 7.82-7.74 (m, 1H), 7.55-7.29 (m, 5H), 7.24-7.06 (m, 2H), 7.02-6.83 (m, 2H), 5.64-5.54 (m, 1H), 5.14-5.07 (m, 1H), 4.75-4.70 (m, 2H), 4.51-4.45 (m, 2H), 3.33-3.23 (m, 4H), 3.18-2.93 (m, 8H), 2.89-2.65 (m, 3H), 2.62-2.56 (m, 1H), 2.17-2.02 (m, 2H), 1.64-1.49 (m, 4H). HRMS m/z [M+H]+ calcd for C43H44N7O12+ 850.3042, found 850.3041.
LQ141-12 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(5-aminopentyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-12 was obtained as white solid (6.4 mg, 74%). 1H NMR (600 MHz, Methanol-d4) δ 7.83-7.70 (m, 1H), 7.55-7.23 (m, 5H), 7.22-7.08 (m, 2H), 7.02-6.75 (m, 2H), 5.71-5.50 (m, 1H), 5.28-5.10 (m, 1H), 4.72 (s, 2H), 4.50-4.40 (m, 2H), 3.31-2.54 (m, 16H), 2.23-2.01 (m, 2H), 1.69-1.26 (m, 6H). HRMS m/z [M+H]+ calcd for C44H46N7O12+ 864.3199, found 864.3194.
LQ141-13 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(6-aminohexyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-13 was obtained as white solid (5.9 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 7.84-7.74 (m, 1H), 7.55-7.29 (m, 5H), 7.23-7.09 (m, 2H), 7.00-6.84 (m, 2H), 5.60 (t, J=7.9 Hz, 1H), 5.17-5.05 (m, 1H), 4.73 (s, 2H), 4.50-4.42 (m, 2H), 3.30-3.19 (m, 3H), 3.18-2.53 (m, 13H), 2.23-2.00 (m, 2H), 1.61-1.20 (m, 8H). HRMS m/z [M+H]+ calcd for C45H48N7O12+ 878.3355, found 878.3357.
LQ141-14 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(7-aminoheptyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-14 was obtained as white solid (5.6 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 7.82-7.77 (m, 1H), 7.51 (d, J=7.3 Hz, 1H), 7.44-7.39 (m, 2H), 7.37-7.32 (m, 2H), 7.19 (d, J=8.3 Hz, 1H), 7.14 (d, J=2.2 Hz, 1H), 6.96 (d, J=2.4 Hz, 1H), 6.89 (dd, J=8.3, 2.5 Hz, 1H), 5.64-5.58 (m, 1H), 5.14 (dd, J=12.5, 5.5 Hz, 1H), 4.75 (s, 2H), 4.47 (d, J=2.4 Hz, 2H), 3.30 (t, J=6.7 Hz, 2H), 3.23 (t, J=7.1 Hz, 2H), 3.16-2.94 (m, 8H), 2.91-2.83 (m, 2H), 2.78-2.70 (m, 2H), 2.64-2.58 (m, 1H), 2.18-2.11 (m, 1H), 2.11-2.04 (m, 1H), 1.58-1.52 (m, 2H), 1.51-1.45 (m, 2H), 1.37-1.23 (m, 5H). HRMS m/z [M+H]+ calcd for C46H50N7O12+ 892.3512, found 892.3510.
LQ141-15 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(2-(2-aminoethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-15 was obtained as white solid (5.9 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 7.79-7.68 (m, 1H), 7.52-7.26 (m, 4H), 7.19-7.03 (m, 2H), 6.98-6.86 (m, 2H), 6.85-6.70 (m, 1H), 5.60-5.48 (m, 1H), 5.15-5.08 (m, 1H), 4.74-4.63 (m, 2H), 4.43 (s, 2H), 3.70-3.38 (m, 7H), 3.23-2.91 (m, 8H), 2.88-2.53 (m, 5H), 2.22-2.01 (m, 2H). HRMS m/z [M+H]+ calcd for C43H44N7O13+ 866.2992, found 866.2989.
LQ141-16 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-16 was obtained as white solid (6.5 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 7.82-7.70 (m, 1H), 7.51-7.28 (m, 4H), 7.24-7.10 (m, 3H), 6.99-6.80 (m, 2H), 5.64-5.55 (m, 1H), 5.17-5.08 (m, 1H), 4.70 (s, 2H), 4.45 (s, 2H), 3.71-3.38 (m, 11H), 3.20-2.94 (m, 8H), 2.91-2.80 (m, 2H), 2.73 (t, J=15.2 Hz, 2H), 2.64-2.54 (m, 1H), 2.21-2.02 (m, 2H). HRMS m/z [M+H]+ calcd for C45H48N7O14+ 910.3254, found 910.3217.
LQ141-17 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (6.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-17 was obtained as white solid (6.3 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 7.81-7.75 (m, 1H), 7.49 (dd, J=7.3, 1.7 Hz, 1H), 7.42-7.37 (m, 2H), 7.36-7.31 (m, 2H), 7.20-7.16 (m, 1H), 7.14 (d, J=2.6 Hz, 1H), 6.96 (t, J=3.1 Hz, 1H), 6.90-6.85 (m, 1H), 5.60 (t, J=7.9 Hz, 1H), 5.11 (ddd, J=12.7, 5.5, 2.1 Hz, 1H), 4.73 (s, 2H), 4.48 (s, 2H), 3.63-3.52 (m, 11H), 3.49-3.45 (m, 2H), 3.43 (t, J=5.4 Hz, 2H), 3.16-2.95 (m, 8H), 2.90-2.82 (m, 2H), 2.79-2.68 (m, 2H), 2.63-2.57 (m, 1H), 2.18-2.12 (m, 1H), 2.12-2.05 (m, 1H). HRMS m/z [M+H]+ calcd for C47H52N7O15+ 954.3516, found 954.3493.
LQ141-18 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(14-amino-3,6,9,12-tetraoxatetradecyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (6.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-18 was obtained as white solid (6.9 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 7.81-7.75 (m, 1H), 7.50 (dd, J=7.3, 2.8 Hz, 1H), 7.44-7.30 (m, 4H), 7.25-7.10 (m, 2H), 6.99-6.84 (m, 2H), 5.60 (t, J=7.8 Hz, 1H), 5.11 (dd, J=13.1, 5.4 Hz, 1H), 4.74 (s, 2H), 4.48 (s, 2H), 3.73-3.39 (m, 21H), 3.20-2.95 (m, 8H), 2.91-2.83 (m, 2H), 2.79-2.67 (m, 2H), 2.65-2.54 (m, 1H), 2.22-2.04 (m, 2H). HRMS m/z [M+H]+ calcd for C49H56N7O16+ 998.3778, found 998.3761.
LQ141-19 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (7.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-19 was obtained as white solid (7.6 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 7.82-7.76 (m, 1H), 7.50 (d, J=7.3 Hz, 1H), 7.44-7.39 (m, 2H), 7.37-7.32 (m, 2H), 7.20 (d, J=8.3 Hz, 1H), 7.18-7.14 (m, 1H), 6.97-6.94 (m, 1H), 6.91-6.85 (m, 1H), 5.61 (t, J=7.9 Hz, 1H), 5.12 (dd, J=12.9, 5.5 Hz, 1H), 4.75 (s, 2H), 4.55-4.44 (m, 2H), 3.72-3.41 (m, 23H), 3.20-2.95 (m, 8H), 2.92-2.83 (m, 2H), 2.81-2.67 (m, 2H), 2.65-2.57 (m, 1H), 2.20-2.13 (m, 1H), 2.12-2.05 (m, 1H). HRMS m/z [M+H]+ calcd for C51H60N7O17+ 1042.4040, found 1042.4023.
LQ141-20 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((2-aminoethyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-20 was obtained as white solid (7 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.14 (s, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.45 (dd, J=9.4, 3.3 Hz, 1H), 7.42-7.39 (m, 1H), 7.37-7.31 (m, 2H), 7.18-7.15 (m, 2H), 7.08 (dd, J=7.7, 1.6 Hz, 1H), 6.97 (d, J=1.7 Hz, 1H), 6.92 (d, J=2.5 Hz, 1H), 6.86 (dd, J=8.2, 2.5 Hz, 1H), 5.59 (t, J=8.0 Hz, 1H), 4.72 (d, J=8.6 Hz, 1H), 4.62-4.56 (m, 2H), 4.54-4.44 (m, 3H), 4.35 (d, J=1.9 Hz, 2H), 3.84 (d, J=11.1 Hz, 1H), 3.79 (dd, J=11.1, 3.8 Hz, 1H), 3.52-3.41 (m, 4H), 3.17-2.94 (m, 8H), 2.87-2.79 (m, 1H), 2.63-2.55 (m, 1H), 2.49 (s, 3H), 2.21-2.14 (m, 1H), 2.11-2.02 (m, 2H), 1.40-1.22 (m, 4H), 1.00 (s, 9H). HRMS m/z [M+H]+ calcd for C54H63FN9O12S+ 1080.4295, found 1080.4245.
LQ141-21 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((3-aminopropyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-21 was obtained as white solid (7.5 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.09 (s, 1H), 7.58-7.27 (m, 5H), 7.25-7.04 (m, 3H), 7.01-6.85 (m, 3H), 5.59 (t, J=8.1 Hz, 1H), 4.77-4.39 (m, 8H), 3.87-3.71 (m, 2H), 3.40-3.22 (m, 4H), 3.20-2.81 (m, 8H), 2.63-2.54 (m, 1H), 2.48 (s, 3H), 2.24-2.00 (m, 3H), 1.80-1.67 (m, 2H), 1.41-1.16 (m, 6H), 0.99 (s, 9H). HRMS m/z [M+H]+ calcd for C55H65FN9O12S+ 1094.4452, found 1094.4426.
LQ141-22 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((4-aminobutyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-22 was obtained as white solid (7.1 mg, 64%). 1H NMR (600 MHz, Methanol-d4) δ 9.11 (s, 1H), 7.53-7.45 (m, 2H), 7.44-7.40 (m, 1H), 7.38-7.32 (m, 2H), 7.21 (d, J=8.3 Hz, 1H), 7.15 (d, J=1.5 Hz, 1H), 7.10 (dd, J=10.0, 3.8 Hz, 1H), 7.02-6.86 (m, 3H), 5.62 (t, J=7.9 Hz, 1H), 4.73 (d, J=9.3 Hz, 1H), 4.66-4.44 (m, 7H), 3.84 (d, J=11.0 Hz, 1H), 3.78 (dd, J=11.4, 4.0 Hz, 1H), 3.31-3.21 (m, 4H), 3.18-2.93 (m, 8H), 2.91-2.83 (m, 1H), 2.64-2.57 (m, 1H), 2.50 (s, 3H), 2.23-2.17 (m, 1H), 2.13-2.04 (m, 2H), 1.64-1.46 (m, 4H), 1.42-1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O12S+ 1108.4608, found 1108.4599.
LQ141-24 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((6-aminohexyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-24 was obtained as white solid (6.8 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 7.54-7.31 (m, 5H), 7.21 (d, J=8.3 Hz, 1H), 7.17-7.13 (m, 1H), 7.12-7.08 (m, 1H), 7.01-6.96 (m, 2H), 6.90 (dd, J=8.3, 2.6 Hz, 1H), 5.62 (t, J=7.8 Hz, 1H), 4.74 (d, J=9.2 Hz, 1H), 4.67-4.55 (m, 3H), 4.51-4.45 (m, 4H), 3.85 (d, J=10.9 Hz, 1H), 3.78 (dd, J=11.1, 3.8 Hz, 1H), 3.29-3.25 (m, 2H), 3.21 (t, J=7.1 Hz, 2H), 3.17-2.95 (m, 8H), 2.91-2.83 (m, 1H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.25-2.18 (m, 1H), 2.14-2.04 (m, 2H), 1.60-1.42 (m, 4H), 1.40-1.21 (m, 8H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O12S+ 1136.4921, found 1136.4898.
LQ141-26 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((8-aminooctyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-26 was obtained as white solid (8.1 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.00 (s, 1H), 7.55-7.31 (m, 5H), 7.21 (d, J=8.3 Hz, 1H), 7.17-7.08 (m, 2H), 7.01-6.94 (m, 1H), 6.90 (dd, J=8.4, 2.4 Hz, 1H), 5.62 (t, J=7.8 Hz, 1H), 4.75 (d, J=9.3 Hz, 1H), 4.66-4.56 (m, 3H), 4.51-4.45 (m, 4H), 3.86 (d, J=11.1 Hz, 1H), 3.79 (dd, J=11.1, 3.8 Hz, 1H), 3.28 (t, J=7.0 Hz, 2H), 3.23 (t, J=7.2 Hz, 2H), 3.18-2.94 (m, 8H), 2.91-2.83 (m, 1H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.25-2.19 (m, 1H), 2.14-2.04 (m, 2H), 1.58-1.44 (m, 6H), 1.42-1.19 (m, 10H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O12S+ 1164.5234, found 1164.5180.
LQ141-27 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((2-(2-aminoethoxy)ethyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-27 was obtained as white solid (8.1 mg, 72%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.55-7.29 (m, 5H), 7.22-7.05 (m, 3H), 7.00-6.82 (m, 3H), 5.63-5.54 (m, 1H), 4.78-4.42 (m, 8H), 3.90-3.73 (m, 2H), 3.69-3.36 (m, 8H), 3.20-2.94 (m, 8H), 2.89-2.80 (m, 1H), 2.66-2.56 (m, 1H), 2.48 (s, 3H), 2.25-2.16 (m, 1H), 2.15-2.04 (m, 2H), 1.42-1.19 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O13S+ 1124.4558, found 1124.4572.
LQ141-28 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-(2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-oxoethoxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-28 was obtained as white solid (7.9 mg, 68%). 1H NMR (600 MHz, Methanol-d4) δ 9.05 (s, 1H), 7.52-7.47 (m, 2H), 7.42 (dd, J=7.9, 1.6 Hz, 1H), 7.37-7.32 (m, 2H), 7.19 (d, J=8.3 Hz, 1H), 7.15 (s, 1H), 7.09 (dd, J=7.7, 1.6 Hz, 1H), 6.96 (dd, J=14.8, 2.0 Hz, 2H), 6.89 (dd, J=8.3, 2.5 Hz, 1H), 5.60 (t, J=7.8 Hz, 1H), 4.74 (d, J=9.3 Hz, 1H), 4.63-4.46 (m, 7H), 3.85 (d, J=11.1 Hz, 1H), 3.79 (dd, J=11.1, 3.8 Hz, 1H), 3.61-3.52 (m, 8H), 3.49-3.41 (m, 4H), 3.18-2.95 (m, 8H), 2.90-2.81 (m, 1H), 2.64-2.56 (m, 1H), 2.50 (s, 3H), 2.22 (dd, J=13.3, 7.7 Hz, 1H), 2.13-2.03 (m, 2H), 1.41-1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O14S+ 1168.4820, found 1168.4813.
LQ141-29 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N-(2-((14-amino-2-oxo-6,9,12-trioxa-3-azatetradecyl)oxy)-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-29 was obtained as white solid (7.4 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 9.10 (s, 1H), 7.53-7.48 (m, 2H), 7.42 (dd, J=7.9, 1.6 Hz, 1H), 7.38-7.32 (m, 2H), 7.20 (d, J=8.3 Hz, 1H), 7.15 (s, 1H), 7.10 (dd, J=7.8, 1.6 Hz, 1H), 6.99 (d, J=1.6 Hz, 1H), 6.95 (d, J=2.4 Hz, 1H), 6.89 (dd, J=8.2, 2.5 Hz, 1H), 5.61 (t, J=7.8 Hz, 1H), 4.75 (d, J=9.2 Hz, 1H), 4.64-4.58 (m, 3H), 4.54-4.46 (m, 4H), 3.85 (d, J=11.0 Hz, 1H), 3.80 (dd, J=11.0, 3.8 Hz, 1H), 3.60-3.51 (m, 12H), 3.49-3.41 (m, 4H), 3.17-2.94 (m, 8H), 2.90-2.82 (m, 1H), 2.64-2.56 (m, 1H), 2.51 (s, 3H), 2.22 (dd, J=13.3, 7.7 Hz, 1H), 2.13-2.04 (m, 2H), 1.41-1.23 (m, 4H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O15S+ 1212.5082, found 1212.5037.
LQ141-33 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((3-aminopropyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-33 was obtained as white solid (7.2 mg, 67%). 1H NMR (600 MHz, Methanol-d4) δ 9.14 (s, 1H), 7.51-7.41 (m, 5H), 7.38-7.32 (m, 2H), 7.21 (d, J=8.3 Hz, 1H), 7.16 (s, 1H), 6.96 (d, J=2.4 Hz, 1H), 6.91 (dd, J=8.3, 2.5 Hz, 1H), 5.62 (t, J=7.9 Hz, 1H), 5.33 (dd, J=8.1, 6.2 Hz, 1H), 4.75 (d, J=8.6 Hz, 1H), 4.64-4.58 (m, 1H), 4.47-4.43 (m, 3H), 3.84 (d, J=11.1 Hz, 1H), 3.77 (dd, J=11.1, 3.8 Hz, 1H), 3.19-2.93 (m, 12H), 2.91-2.82 (m, 2H), 2.75 (dd, J=14.2, 8.2 Hz, 1H), 2.65-2.57 (m, 1H), 2.49 (s, 3H), 2.24-2.17 (m, 1H), 2.15-2.06 (m, 1H), 1.99-1.92 (m, 1H), 1.57-1.51 (m, 2H), 1.41-1.24 (m, 3H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C55H65FN9O11S+ 1078.4503, found 1078.4519.
LQ141-36 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((6-aminohexyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-36 was obtained as white solid (6.7 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 9.04 (s, 1H), 7.51-7.42 (m, 6H), 7.38-7.34 (m, 2H), 7.20 (d, J=8.3 Hz, 1H), 7.15 (s, 1H), 6.95 (d, J=2.4 Hz, 1H), 6.90 (dd, J=8.3, 2.5 Hz, 1H), 5.62 (t, J=7.9 Hz, 1H), 5.32 (dd, J=8.3, 6.0 Hz, 1H), 4.77-4.73 (m, 1H), 4.60 (dd, J=9.3, 7.6 Hz, 1H), 4.48-4.43 (m, 3H), 3.84 (d, J=10.9 Hz, 1H), 3.78 (dd, J=11.1, 3.8 Hz, 1H), 3.21-2.95 (m, 12H), 2.91-2.82 (m, 2H), 2.76 (dd, J=14.1, 8.3 Hz, 1H), 2.64-2.57 (m, 1H), 2.49 (s, 3H), 2.23-2.18 (m, 1H), 2.13-2.06 (m, 1H), 2.01-1.93 (m, 1H), 1.45-1.26 (m, 7H), 1.23-1.11 (m, 4H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O11S+ 1120.4972, found 1120.4978.
LQ141-37 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((7-aminoheptyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-37 was obtained as white solid (7.9 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.03 (s, 1H), 7.52-7.41 (m, 5H), 7.39-7.33 (m, 2H), 7.21 (d, J=8.4 Hz, 1H), 7.15 (s, 1H), 6.96-6.94 (m, 1H), 6.92-6.88 (m, 1H), 5.62 (t, J=7.8 Hz, 1H), 5.32 (dd, J=8.4, 5.9 Hz, 1H), 4.75 (d, J=9.1 Hz, 1H), 4.62-4.58 (m, 1H), 4.49-4.43 (m, 3H), 3.84 (d, J=11.1 Hz, 1H), 3.80-3.75 (m, 1H), 3.21-2.94 (m, 12H), 2.92-2.82 (m, 2H), 2.75 (dd, J=14.0, 8.4 Hz, 1H), 2.65-2.57 (m, 1H), 2.50 (s, 3H), 2.24-2.17 (m, 1H), 2.14-2.05 (m, 1H), 2.00-1.94 (m, 1H), 1.46-1.26 (m, 7H), 1.23-1.15 (m, 4H), 1.14-1.09 (m, 2H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C59H73FN9O11S+ 1134.5129, found 1134.5123.
LQ141-38 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((8-aminooctyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.1 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-38 was obtained as white solid (8.6 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.59-7.31 (m, 7H), 7.27-7.13 (m, 2H), 7.02-6.88 (m, 2H), 5.62 (t, J=7.9 Hz, 1H), 5.35-5.30 (m, 1H), 4.75 (d, J=8.9 Hz, 1H), 4.63-4.57 (m, 1H), 4.54-4.43 (m, 3H), 3.84 (d, J=11.1 Hz, 1H), 3.78 (dd, J=11.1, 3.7 Hz, 1H), 3.26-2.94 (m, 12H), 2.93-2.84 (m, 2H), 2.75 (dd, J=13.9, 8.6 Hz, 1H), 2.65-2.58 (m, 1H), 2.50 (s, 3H), 2.24-2.18 (m, 1H), 2.14-2.05 (m, 1H), 2.01-1.94 (m, 1H), 1.47-1.26 (m, 9H), 1.23-1.14 (m, 6H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O11S+ 1148.5285, found 1148.5293.
LQ141-39 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((9-aminononyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-39 was obtained as white solid (8.1 mg, 70%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.57-7.32 (m, 7H), 7.25-7.13 (m, 2H), 7.00-6.88 (m, 2H), 5.62 (t, J=8.0 Hz, 1H), 5.35-5.30 (m, 1H), 4.75 (d, J=8.9 Hz, 1H), 4.63-4.57 (m, 1H), 4.53-4.45 (m, 3H), 3.85 (d, J=11.1 Hz, 1H), 3.78 (dd, J=11.1, 3.6 Hz, 1H), 3.22 (t, J=7.2 Hz, 2H), 3.19-2.93 (m, 10H), 2.92-2.82 (m, 2H), 2.79-2.72 (m, 1H), 2.64-2.59 (m, 1H), 2.50 (s, 3H), 2.24-2.18 (m, 1H), 2.13-2.05 (m, 1H), 2.01-1.94 (m, 1H), 1.55-1.26 (m, 10H), 1.24-1.11 (m, 7H), 1.08 (s, 9H). HRMS m/z [M+H]+ calcd for C61H77FN9O11S+ 1162.5442, found 1162.5441.
LQ141-42 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((2-(2-aminoethoxy)ethyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (7.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-42 was obtained as white solid (7.6 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 9.07 (s, 1H), 7.57-7.30 (m, 7H), 7.22-7.14 (m, 2H), 7.00-6.83 (m, 2H), 5.61 (t, J=7.9 Hz, 1H), 5.36-5.30 (m, 1H), 4.75 (d, J=8.9 Hz, 1H), 4.64-4.57 (m, 1H), 4.53-4.41 (m, 3H), 3.84 (d, J=11.1 Hz, 1H), 3.77 (dd, J=11.1, 3.9 Hz, 1H), 3.52-3.35 (m, 5H), 3.29-3.22 (m, 2H), 3.19-2.92 (m, 8H), 2.90-2.82 (m, 2H), 2.75 (dd, J=14.2, 8.0 Hz, 1H), 2.64-2.56 (m, 1H), 2.49 (s, 3H), 2.24-2.18 (m, 1H), 2.14-2.04 (m, 1H), 2.02-1.90 (m, 1H), 1.45-1.22 (m, 4H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C56H67FN9O12S+ 1108.4608, found 1108.4601.
LQ141-43 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-3-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.2 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-43 was obtained as white solid (7.5 mg, 65%). 1H NMR (600 MHz, Methanol-d4) δ 9.14 (s, 1H), 7.52-7.41 (m, 5H), 7.40-7.33 (m, 2H), 7.25-7.15 (m, 2H), 7.01-6.85 (m, 2H), 5.67-5.59 (m, 1H), 5.35-5.30 (m, 1H), 4.75 (d, J=8.5 Hz, 1H), 4.63-4.58 (m, 1H), 4.53-4.42 (m, 3H), 3.84 (d, J=10.7 Hz, 1H), 3.76 (dd, J=11.2, 3.9 Hz, 1H), 3.63-3.36 (m, 9H), 3.31-3.26 (m, 2H), 3.21-2.96 (m, 8H), 2.92-2.83 (m, 2H), 2.79-2.71 (m, 1H), 2.66-2.58 (m, 1H), 2.50 (s, 3H), 2.27-2.19 (m, 1H), 2.16-2.05 (m, 1H), 2.03-1.93 (m, 1H), 1.44-1.26 (m, 4H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C58H71FN9O13S+ 1152.4871, found 1152.4874.
LQ141-44 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-1-amino-15-(4-(4-methylthiazol-5-yl)phenyl)-13-oxo-3,6,9-trioxa-12-azapentadecan-15-yl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (8.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-44 was obtained as white solid (7.2 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 8.95 (s, 1H), 7.57-7.31 (m, 7H), 7.25-7.14 (m, 2H), 6.99-6.86 (m, 2H), 5.62 (t, J=7.8 Hz, 1H), 5.36-5.30 (m, 1H), 4.78-4.72 (m, 1H), 4.60 (t, J=8.6 Hz, 1H), 4.53-4.43 (m, 3H), 3.84 (d, J=11.1 Hz, 1H), 3.77 (dd, J=11.2, 3.7 Hz, 1H), 3.68-3.39 (m, 13H), 3.31-3.23 (m, 2H), 3.19-2.96 (m, 8H), 2.91-2.82 (m, 2H), 2.80-2.73 (m, 1H), 2.65-2.57 (m, 1H), 2.48 (s, 3H), 2.25-2.18 (m, 1H), 2.13-2.06 (m, 1H), 2.01-1.93 (m, 1H), 1.45-1.23 (m, 4H), 1.07 (s, 9H). HRMS m/z [M+H]+ calcd for C60H75FN9O14S+ 1196.5133, found 1196.5125.
LQ141-45 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-1-amino-18-(4-(4-methylthiazol-5-yl)phenyl)-16-oxo-3,6,9,12-tetraoxa-15-azaoctadecan-18-yl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (9 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-45 was obtained as white solid (9 mg, 73%). 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.60-7.30 (m, 7H), 7.25-7.13 (m, 2H), 6.99-6.86 (m, 2H), 5.62 (t, J=7.8 Hz, 1H), 5.37-5.31 (m, 1H), 4.75 (d, J=9.0 Hz, 1H), 4.60 (t, J=8.6 Hz, 1H), 4.54-4.42 (m, 3H), 3.84 (d, J=11.2 Hz, 1H), 3.77 (dd, J=11.1, 3.8 Hz, 1H), 3.71-3.34 (m, 17H), 3.31-3.22 (m, 2H), 3.18-2.93 (m, 8H), 2.91-2.82 (m, 2H), 2.81-2.74 (m, 1H), 2.64-2.57 (m, 1H), 2.49 (s, 3H), 2.25-2.18 (m, 1H), 2.14-2.05 (m, 1H), 2.01-1.93 (m, 1H), 1.43-1.24 (m, 4H), 1.08 (s, 9H). HRMS m/z [M+H]+ calcd for C62H79FN9O15S+ 1240.5395, found 1240.5403.
LQ141-46 was synthesized following the standard procedure for preparing LQ126-177 from intermediate 47 (5 mg, 0.01 mmol), (2S,4R)—N—((S)-1-amino-21-(4-(4-methylthiazol-5-yl)phenyl)-19-oxo-3,6,9,12,15-pentaoxa-18-azahenicosan-21-yl)-1-((S)-2-(1-fluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxypyrrolidine-2-carboxamide (9.5 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-46 was obtained as white solid (8.5 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.08 (s, 1H), 7.63-7.30 (m, 7H), 7.26-7.12 (m, 2H), 6.98-6.85 (m, 2H), 5.63 (t, J=8.5 Hz, 1H), 5.44-5.31 (m, 1H), 4.75 (d, J=9.0 Hz, 1H), 4.63-4.57 (m, 1H), 4.56-4.42 (m, 3H), 3.84 (d, J=11.3 Hz, 1H), 3.80-3.75 (m, 1H), 3.70-3.41 (m, 21H), 3.34-3.22 (m, 2H), 3.20-2.94 (m, 8H), 2.90-2.83 (m, 2H), 2.81-2.74 (m, 1H), 2.65-2.56 (m, 1H), 2.50 (s, 3H), 2.25-2.20 (m, 1H), 2.13-2.04 (m, 1H), 2.03-1.92 (m, 1H), 1.49-1.23 (m, 4H), 1.06 (s, 9H). HRMS m/z [M+H]+ calcd for C64H83FN9O16S+ 1284.5657, found 1284.5608.
LQ141-47 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(10-aminodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (7.3 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-47 was obtained as white solid (6.5 mg, 63%). 1H NMR (600 MHz, Methanol-d4) δ 9.18 (s, 1H), 7.79-7.70 (m, 2H), 7.53-7.40 (m, 5H), 7.40-7.32 (m, 3H), 7.15 (d, J=3.2 Hz, 1H), 5.69 (t, J=7.7 Hz, 1H), 4.99 (d, J=7.0 Hz, 1H), 4.65-4.60 (m, 1H), 4.57 (t, J=8.2 Hz, 1H), 4.46-4.39 (m, 1H), 3.98 (d, J=2.8 Hz, 1H), 3.87 (d, J=10.8 Hz, 1H), 3.79-3.69 (m, 1H), 3.37-3.29 (m, 6H), 3.19-3.05 (m, 4H), 3.05-2.91 (m, 4H), 2.69-2.60 (m, 1H), 2.51 (s, 3H), 2.34-2.07 (m, 5H), 1.95 (dd, J=8.8, 4.4 Hz, 1H), 1.66-1.54 (m, 5H), 1.52-1.48 (m, 2H), 1.41-1.26 (m, 10H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C56H71N8O9S+ 1031.5059, found 1031.5058.
LQ141-48 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(11-aminoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (7.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-48 was obtained as white solid (6.1 mg, 58%). 1H NMR (600 MHz, Methanol-d4) δ 9.22 (s, 1H), 7.75 (d, J=5.0 Hz, 1H), 7.72 (dd, J=7.9, 1.7 Hz, 1H), 7.50-7.30 (m, 8H), 7.14 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.99 (q, J=7.0 Hz, 1H), 4.60 (s, 1H), 4.56 (t, J=8.3 Hz, 1H), 4.41 (dt, J=4.3, 2.2 Hz, 1H), 3.97 (s, 1H), 3.86 (dt, J=11.2, 1.8 Hz, 1H), 3.73 (dd, J=11.0, 4.0 Hz, 1H), 3.35-3.30 (m, 6H), 3.14-3.02 (m, 4H), 2.96 (dt, J=16.5, 8.4 Hz, 4H), 2.64 (m, 1H), 2.50 (s, 3H), 2.31-2.05 (m, 5H), 1.93 (m, 1H), 1.63-1.52 (m, 4H), 1.49 (m, 3H), 1.31 (m, 12H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C57H73N8O9S+ 1045.5216, found 1045.5211.
LQ141-49 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(12-aminododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N—((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (7.6 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-49 was obtained as white solid (7 mg, 66%). 1H NMR (600 MHz, Methanol-d4) δ 9.21 (s, 1H), 7.85-7.66 (m, 2H), 7.60-7.25 (m, 8H), 7.14 (p, J=5.0 Hz, 1H), 5.78-5.62 (m, 1H), 5.08-5.00 (m, 1H), 4.69-4.51 (m, 2H), 4.42 (s, 1H), 3.97 (t, J=3.2 Hz, 1H), 3.87 (d, J=11.0 Hz, 1H), 3.78-3.68 (m, 1H), 3.47-3.23 (m, 6H), 3.21-2.87 (m, 8H), 2.75-2.43 (m, 5H), 2.39-1.87 (m, 6H), 1.73-1.45 (m, 7H), 1.30 (m, 13H), 1.02 (s, 9H). HRMS m/z [M+H]+ calcd for C58H75N8O9S+ 1059.5372, found 1059.5377.
LQ141-52 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), (2S,4R)-1-((S)-2-(12-aminododecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7.4 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-52 was obtained as white solid (6.3 mg, 60%). 1H NMR (600 MHz, Methanol-d4) δ 9.15 (s, 1H), 7.76 (s, 1H), 7.72 (dd, J=7.9, 1.7 Hz, 1H), 7.49 (d, J=8.0 Hz, 2H), 7.46-7.39 (m, 3H), 7.39-7.30 (m, 3H), 7.15 (s, 1H), 5.68 (t, J=7.9 Hz, 1H), 4.63 (s, 1H), 4.58 (d, J=8.5 Hz, 1H), 4.55 (d, J=15.2 Hz, 1H), 4.49 (m, 1H), 4.36 (d, J=15.5 Hz, 1H), 3.98 (s, 2H), 3.90 (d, J=11.0 Hz, 1H), 3.80 (dd, J=10.9, 3.9 Hz, 1H), 3.33 (m, 6H), 3.12 (m, 4H), 2.97 (m, 4H), 2.68-2.61 (m, 1H), 2.50 (s, 3H), 2.32-2.17 (m, 3H), 2.17-2.05 (m, 2H), 1.59 (m, 4H), 1.47-1.20 (m, 14H), 1.03 (s, 9H). HRMS m/z [M+H]+ calcd for C57H73N8O9S+ 1045.5216, found 1284.5206.
LQ141-57 was synthesized following the standard procedure for preparing LQ108-58 from intermediate 40 (5 mg, 0.01 mmol), N-(8-aminooctyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (5.7 mg, 0.01 mmol, 1.0 equiv), EDCI (2.9 mg, 0.015 mmol, 1.5 equiv), HOAt (2.1 mg, 0.015 mmol, 1.5 equiv), and NMM (3.1 mg, 0.03 mmol, 3.0 equiv) in DMSO (1 mL). LQ141-57 was obtained as white solid (5.3 mg, 61%). 1H NMR (600 MHz, Methanol-d4) δ 7.87-7.65 (m, 3H), 7.51 (d, J=7.0 Hz, 1H), 7.38 (m, 5H), 7.20-7.12 (m, 1H), 5.69 (t, J=7.9 Hz, 1H), 5.21-5.10 (m, 1H), 4.79-4.64 (m, 2H), 3.43-3.28 (m, 6H), 3.19-2.93 (m, 8H), 2.93-2.55 (m, 5H), 2.26-2.08 (m, 3H), 1.57 (m, 4H), 1.32 (m, 9H). HRMS m/z [M+H]+ calcd for C46H50N7O11+ 876.3563, found 876.3553.
Certain compounds disclosed herein have the structures shown in Table 1.
Compounds corresponding to Examples 1-326 have been synthesized and are provided with a Compound Code in Table 1.
As used herein, in case of discrepancy between the structure and chemical name provided for a particular compound, the given structure shall control.
Inhibitory effect of precursors was tested at 1 μM in AlphaScreen assay (
ENL-dependent MV4; 11 cells were seeded at 2×105 cells/mL density and treated with DMSO or the indicated compounds at 0.4, 2, 10 and 50 μM for 72 h. SGC-iMLLT was used as a control. Cell viability was measured using CellTiter-Glo reagent (Promega) and relative cell viability was calculated by normalization to DMSO samples.
ENL-dependent MV4; 11 and ENL-independent Jurkat cells were seeded at 2×105 cells/mL density and treated with DMSO or indicated compounds at 0.4, 2, 10 and 50 μM for 72 h. SGC-iMLLT was used as a control. Cell viability was measured using CellTiter-Glo reagent (Promega) and relative cell viability was calculated by normalization to DMSO samples.
MV4; 11 cells were treated with DMSO or the indicated compounds (the same panel of ENL degraders as shown in
MV4; 11 cells were treated with LQ076-122, LQ081-108 or LQ081-109 at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 8 μM for 24 h. Treatment with 8 μM of negative control compounds LQ081-107 (negative control of LQ076-122), LQ081-106 (negative control of LQ081-108), LQ081-158 (negative control of LQ081-109) or SGC-iMLLT were included as negative controls. The Western blot results show that LQ076-122, LQ081-108 and LQ081-109 reduced ENL protein levels in a concentration-dependent manner in MV4; 11 cells.
MOLM13 cells were treated with LQ076-122 or LQ081-108 at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 8 μM for 24 h. Treatment with 8 μM of negative control compounds LQ081-107 (negative control of LQ076-122), LQ081-106 (negative control of LQ081-108), or SGC-iMLLT were included as negative controls. The Western blot results show that LQ076-122 and LQ081-108 reduced ENL protein levels in a concentration-dependent manner in MOLM13 cells.
MV4; 11 cells were treated with LQ081-106, LQ081-108, LQ081-107, LQ076-122, or SGC-iMLLT at 0.3, 1, 3, and 10 μM for 12 and 24 h. DMSO treated cells were used as control. The Western blot results show that LQ076-122 and LQ081-108 reduced ENL protein levels in a concentration- and time-dependent manner in MV4; 11 cells. Negative control compounds and SGC-iMLLT did not affect ENL protein levels.
MV4; 11 cells were treated with DMSO or 4 μM of LQ076-122 for 12, 16, 20, 24 and 36 h. The Western blot results show that LQ076-122 reduced ENL protein levels in a time-dependent manner.
MOLM13 cells were treated with DMSO or 8 μM of LQ076-122 for 12, 16, 20, 24 and 36 h. The Western blot results show that LQ076-122 reduced ENL protein levels in a time-dependent manner.
MV4; 11 cells were treated with LQ076-122, or LQ081-107 at 0.3, 1, 3, 10, and 30 μM for 24 h. DMSO treated cells were used as control. Cells were lysed and expression of ENL and GAS41 was assessed by Western analysis. The Western blot results show that LQ076-122 selectively reduced the ENL protein level, but not the level of another YEATS domain-containing protein GAS41.
MV4; 11 cells were seeded at 2×105 cells/mL density and treated with DMSO or the indicated compounds at 0.5, 1, 2 and 4 μM for 72 h. SGC-iMLLT was used as a control. Cell viability was measured using CellTiter-Glo reagent (Promega) and relative cell viability was calculated by normalization to DMSO samples.
MV4; 11 (
MOLM13 cells were treated with LQ076-122 (
MV4; 11 cells were treated with DMSO, or LQ076-122 at 1, 2, and 4 μM for 6, 12, 18 and 24 h. RT-qPCR analysis was performed to detect the mRNA levels of selected ENL target genes. Results showed that LQ076-122 reduced ENL target gene expression in a concentration- and time-dependent manner.
MV4; 11 (
Three C57BL/6 mice at 6-8 weeks of age were used in PK study for each time point. After a single dose intraperitoneal (IP) injection of ENL degrader LQ076-122 (50 mg/kg), plasma concentrations of degrader were measured at 6 time points (0.5, 1, 2, 4, 8 and 12 h) from each test animal. The concentrations of LQ076-122 in plasma were maintained above 2 μM for 6 h with the maximum plasma concentration of about 6 μM.
Immuno-deficient NSG mice were irradiated and transplanted with 5×105 MV4; 11-Luc cells through tail-vein injections. Ten days after transplantation, mice (n=5) were treated with 100 mg/kg LQ076-122 or vehicle twice daily through IP injection in cycles. Each cycle contains 4 treatment days followed by 2 resting days. Day 0 is the time that the treatment started. Leukemia progression was monitored by bioluminescence imaging at different time points upon LQ076-122 or vehicle treatment (
MV4; 11 cells stably expressing 3Flag-HA-tagged ENL were treated with DMSO or the indicated compounds at 1 μM and 10 μM for 24 h. Cells were lysed and expression of 3Flag-HA-ENL was assessed by Western blot analysis. A panel of compounds significantly reduced ENL protein levels.
MV4; 11 cells stably expressing 3Flag-HA-tagged ENL were treated with DMSO or the indicated compounds at 1 μM and 10 μM for 6 h. Cells were lysed and expression of 3Flag-HA-ENL was assessed by Western blot analysis. A panel of compounds significantly reduced ENL protein levels.
MV4; 11 cells were treated with DMSO or the indicated compounds at 1 μM and 10 μM for 6 h. Cells were lysed and expression of endogenous ENL was assessed by Western blot analysis. Several compounds significantly reduced ENL protein levels.
MV4; 11, MOLM13 and Jurkat cells were treated with LQ108-69, LQ108-71, LQ108-72, LQ126-62 and LQ126-63 at 0, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM doses for 6 h. DMSO was used as negative control. The Western blot results show that LQ108-69, LQ108-71, LQ108-72, LQ126-62 and LQ126-63 reduced ENL protein levels in a concentration-dependent manner in all three tested cell lines.
MV4; 11, MOLM13 and Jurkat cells were treated with LQ108-69, LQ108-70, LQ108-71, LQ108-72, LQ126-62 and LQ126-63 at 1 μM for 48 and 72 h. DMSO treated cells were used as control. The Western blot results show that LQ108-69, LQ108-70, LQ108-71, LQ108-72, LQ126-62 and LQ126-63 maintained the ENL protein at low levels after 48 and 72 h treatment.
MG132 treatment partially blocks the ENL degradation induced by degraders LQ108-63, LQ108-69, LQ108-70, LQ126-62 and LQ126-63 in MV4; 11 cells. Cells were treated with 1 μM of ENL degrader with or without 1 μM proteasome inhibitor MG132 for 6 h.
ENL-dependent MV4; 11 cells were seeded at 2×105 cells/mL density and treated with DMSO or the indicated compounds at 0, 1.25, 2.5, 5 and 10 μM for 72 h. Cell viability was measured using CellTiter-Glo reagent (Promega) and relative cell viability was calculated by normalization to DMSO samples.
ENL-dependent MV4; 11 and ENL-independent Jurkat cells were seeded at 2×105 cells/mL density and treated with DMSO or indicated compounds at 10 nM, 100 nM, 1 μM and 10 μM for 3 days (A) or 6 days (B). Cell viability was measured using CellTiter-Glo reagent (Promega) and relative cell viability was calculated by normalization to DMSO samples.
Materials And Methods:
General Chemistry Methods
For the synthesis of intermediates and examples, HPLC spectra for all compounds were acquired using an Agilent 1200 Series system with DAD detector. Chromatography was performed on a 2.1×150 mm Zorbax 300SB-C18 5 μm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 ml/min. The gradient program was as follows: 1% B (0-1 min), 1-99% B (1-4 min), and 99% B (4-8 min). High-resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Nuclear Magnetic Resonance (NMR) spectra were acquired on a Bruker DRX-600 spectrometer with 600 MHz for proton (1H NMR) and 150 MHz for carbon (13C NMR); chemical shifts are reported in (6). Preparative HPLC was performed on Agilent Prep 1200 series with UV detector set to 254 nm. Samples were injected onto a Phenomenex Luna 250×30 mm, 5 μm, C18 column at room temperature. The flow rate was 40 ml/min. A linear gradient was used with 10% (or 50%) of MeOH (A) in H2O (with 0.1% TFA) (B) to 100% of MeOH (A). HPLC was used to establish the purity of target compounds. All final compounds had >95% purity using the HPLC methods described above.
AlphaScreen Assay
IC50 of ENL degrader precursor in inhibition of ENL YEATS-H3K9ac interaction was measured by AlphaScreen assay using AlphaScreen Histidine (Nickel Chelate) Detection Kit (PerkinElmer). Assays were set up in 30 μL volume with 100 nM His tagged-ENL YEATS protein, 30 nM biotinylated-H3K9ac peptide, indicated concentrations of ENL degrader precursor, 10 μg/mL of streptavidin-coated donor beads and 10 μg/mL of chelate nickle-coated acceptor beads in Alpha assay buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1.0 mg/mL BSA, and 0.05% CHAPS). Alpha signals were detected by an EnVision microplate reader equipped with an Alpha laser (PerkinElmer).
Cell Lines
All cell lines were purchased from ATCC. MV4; 11, MOLM13, and Jurkat were cultured in RPMI1640 supplemented with 10% FBS and 1% Penicillin/Streptomycin.
Compound Treatment
ENL degraders were dissolved in DMSO. DMSO with no degraders was used as the control. 1×106 leukemia cells were seeded in 5 mL medium. For prescreening of compounds, each test compound was added to the medium at 1 μM and 10 μM. Cells were collected after 24 h treatment. For the concentration-dependent treatment, candidate compounds were added to the medium at a series of concentration as indicated in figures. Cells were collected after 24 h treatment. For the time-course treatment, candidate compounds were added to the medium at a final concentration of 4 μM (MV4; 11 cells) or 8 μM (MOLM13 cells). Cells were collected at the indicated timepoints (in hours: 12, 16, 20, 24 and 36 h).
Immunoblotting
After ENL degrader treatment, cells were collected, lysed, and total cell lysates were used for Western blot. The following primary antibodies were used: ENL (Cell Signaling Technology), GAS41 (Santa Cruz), GAPDH (Santa Cruz), β-actin (Sigma). Blots were detected using HRP-conjugated secondary antibodies.
Cell Viability Assay
MV4; 11 or MOLM13 cells were seeded at 0.2×106 cells/mL density. Cells were treated with DMSO or ENL degraders at indicated concentrations. Each treatment was done in triplicates. After 72 h treatment, 100 μL of cell suspension from each treatment was mixed with 25 μL of CellTiter-Glo reagent (Promega) and incubated for 10 min before the luminescence signals were detected on a plate reader.
Apoptosis Assay
MV4; 11 or MOLM13 cells were seeded at 0.2×106 cells/mL density. Cells were treated with DMSO or ENL degraders at indicated concentrations. Each treatment was done in triplicates. After 24 h treatment, cells were collected and washed with ice-cold PBS once and resuspended in 250 μL of 1× binding buffer containing 5 μL of FITC-Annexin V and PI (BD Biosciences). After 15 min incubation at room temperature in the dark, 250 μL of 1× binding buffer was added and flow cytometry analysis was performed.
RNA Extraction and RT-qPCR
Total RNA was extracted using the RNeasy Plus kit (Qiagen) and reverse-transcribed using the iScript cDNA Synthesis kit (Bio-Rad). RT-qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on the CFX96 Real-Time PCR system (Bio-Rad). Gene expressions were calculated following normalization to 18s rRNA amounts using the comparative cycle threshold (Ct) method.
In Vivo Pharmacokinetics (PK) Study
The standard mouse PK study was conducted by Charles River Laboratories. Three C57BL/6 mice at 6-8 weeks of age were used for each time point. After a single dose intraperitoneal (IP) injection of ENL degrader (50 mg/kg), plasma concentrations of degrader were measured at 4 time points (in hours: 0.5, 1, 2, 4, 8 and 12 h) from each test animal.
Tumor Xenograft Study
Immunodeficient NOD.Cg-PrkdcscidIl2rgtm1wil/SzJ (NSG) mice at 6-8 weeks of age were produced at the Van Andel Institute Vivarium and Transgenic Core using breeders purchased from the Jackson laboratory. Mice were pretreated with acidified water and antibiotics for a week before a sublethal dose of total body irradiated (2 Gy). Then mice were transplanted with 0.5×106 MV4; 11-Luc cells through tail-vein injection. ENL degrader treatment was started ten days after transplantation with the successful engraftment confirmed by bioluminescence imaging. Mice were randomly assigned to two groups (n=5) and treated with IP injections of either ENL degrader LQ076-122 (100 mg/kg, twice daily) or vehicle. The treatment lasted for 4 consecutive days followed by a 2-day rest, and was repeated in three cycles. Leukemia progression in each animal was monitored by bioluminescence imaging after each treatment cycle. For whole-body bioluminescent imaging, mice were IP injected with 150 mg/kg D-luciferin 10 min prior to imaging using an AMI-1000 imaging system (Spectral Instruments Imaging). Mice were euthanized when they reached moribund stage according to the approved IACUC protocol. All procedures and studies with mice were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Van Andel Institute.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/055574 | 10/19/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63094771 | Oct 2020 | US |
