Patent Application
20250152585
The present invention relates to PARP1 inhibitor compounds, and in particular to PARP1 inhibitor compounds for use in medicine. The inhibitors of the invention may be used in pharmaceutical compositions, and in particular pharmaceutical compositions for treating a cancer. The invention also relates to methods of manufacture of such inhibitors, and methods of treatment using such inhibitors.
The family of poly(ADP-ribose) polymerases (PARPs) consists of 17 PARP proteins that catalyse the transfer of ADP-ribose to target proteins, a posttranslational process termed PARylation. Target protein modification by PARylation causes significant changes to function and as such PARPs play an important role in many cellular processes such as chromatin remodelling, transcription, replication, recombination, cell cycle progression and DNA damage repair (Kamaletdinova, T. et al. Cell. 2019; 8: 1625). PARP1 and 2 are the most widely studied PARP enzymes, primarily due to their role in DNA damage repair, in particular in the base excision repair (BER) process of DNA single-strand breaks (Ngoi, Y L. et al. Cancer J. 2021; 27: 521-528). PARP1 is activated by DNA damage breaks, and the subsequent PARylation of target proteins leads to recruitment of additional factors that initiate repair of DNA lesions. Auto-PARylation of PARP triggers the release of bound PARP from the DNA allowing other DNA repair proteins access to complete lesion repair. This highlights the critical role PARP plays in enabling a cancer cell to repair DNA damage caused by exogenous agents such as radiation therapy and chemotherapeutic agents. Hence, inhibition of PARP enzymes has been utilised as a strategy to selectively kill cancer cells that harbour genetic defects in complementary DNA damage repair pathways (Fanner, H. et al. Nature. 2005; 434: 917-921). This synthetic lethality approach has been demonstrated successfully in tumours with epigenetic modifications or deleterious mutations in BRCA1 and BRCA2, two functionally redundant tumour suppressor proteins involved in DNA double-strand break (DSB) repair by homologous recombination (HR) (Lord, C J. and Ashworth, A. Science. 2017; 355: 1152-1158). Such tumours with HR deficiency (HRD) are dependent on PARP function for survival—following PARP inhibition in these tumours, DSB breaks will be processed by alternative error-prone repair pathways leading to genomic instability and cancer cell death.
The inhibition of PARP can trap the inactivated PARP at the sites of DNA damage. This leads to replication fork stalling and subsequent collapse in S-phase when the fork reaches the site of the trapped PARP, resulting in the generation of genotoxic DNA double-strand breaks. It is believed that this PARP1-DNA trapping can lead to the selective death of cancer cells harbouring HRD (Farmer, H. et al. Nature. 2005; 434: 917-921.
This strategy has led to the successful approval of several PARP inhibitors for the treatment of cancers with HRD, such as in BRCA1/2-mutated breast, ovarian and prostate cancer, as well as in ovarian and prostate cancer harbouring genomic consequences of HRD, and ovarian cancer in the maintenance setting where platinum sensitivity acts as a surrogate for HRD (Fong, P C. et al. N. Engl. J. Med. 2009; 361:123-134).
It has recently been shown that genomic instability, in the form of unrepaired DNA double-strand breaks or micronuclei disruption can trigger innate immune system activation via the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), leading to generation of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and induction of dimerization of Stimulator of interferon genes (STING). STING subsequently translocates from the endoplasmic reticulum to the Golgi where it recruits and activates TANK-binding kinase 1 (TBK1). TBK1 phosphorylates interferon regulatory transcription factor 3 (IRF3) which drives the production of type I interferons and supports the induction of an adaptive immune response (Zhu, Y. et al. Mol. Cancer. 2019, 18: 152).
For example, PARP inhibitor-induced STING pathway activation and anti-tumour immune responses have been demonstrated in multiple tumour models, providing rationale for exploiting combinations of PARP inhibitors with immunotherapies for improved therapeutic efficacy (Sen, T. et al. Cancer Discov. 2019; 9: 646-661). For example, the PARP inhibitor Olaparib was also recently shown to induce synthetic lethal effects in combination with a synthetic cyclic dinucleotide STING agonist in DNA damage repair deficient cancer cells and a BRCA-deficient breast cancer model (Pantelidou, C. et al. 2021: bioRxiv).
Overall, modulation of nucleic acid sensing pathways via multiple mechanisms has been shown to promote anti-tumour efficacy in a variety of cell and animal models thus demonstrating therapeutic potential for augmenting efficacy of immunotherapies and overcoming resistance to immune checkpoint blockade through use of PARP inhibitors. There are numerous clinical trials ongoing combining PARP inhibitors with immunotherapies (reviewed in Chabanon, R M, et al. Nat. Rev. Cancer. 2021; 21: 701-717).
Recently, PARP1 has also been shown to bind the Epstein Barr Virus (EBV) genome and that PARP1 inhibition can alter EBV chromatin structure and latent gene expression (Morgan, S M. et al. Nat. Commun. 2022; 13: 187). Hence, PARP1 inhibitors may play a role in cancers where EBV plays a contributing role such as Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal and gastrointestinal cancers. Interestingly, EBV has also been shown to be a causative factor in multiple sclerosis (MS) whereby EBV infection greatly increases the risk of subsequent MS (Bjornevik, K. et al. Science (2021); 375: 296-301).
First-generation PARP inhibitors generally demonstrate non-selective activity at PARP1 and 2. Haematological toxicities such as anaemia, neutropenia and thrombocytopenia are associated with clinical use of these molecules which restricts their use in combination with cytotoxic chemotherapies and other targeted agents due to dose-limiting cytopenias (LaFargue, CJ. et al. Lancet Oncol. 2019, 20, e15-e28). Evidence from pre-clinical mouse studies strongly suggests that PARP2 inhibition is a major driver of these haematological toxicities, with PARP2 being particularly linked to erythrogenesis in mice (Farrés, J. et al. Blood. 2013; 122: 44-54). In addition, PARP2 function has been shown to be dispensable for anti-tumour activity in HRD mouse cancer models (Ronson, G E. et al. Nat. Commun. 2018, 9: 746). Taken together, these data suggest an unmet medical need for the development of inhibitors with improved selectivity for PARP1 over PARP2 and other PARPs, thus providing expanded therapeutic utility (1) as single agents and (2) in combination with other anti-cancer agents.
To date, two PARP1-selective inhibitors, AZD5305 and AZD9574, have entered clinical development. AZD5305 was described as a potent PARP1 inhibitor and trapper with 500-fold selectivity over PARP2 and less off-target activity against secondary pharmacology targets than first-generation PARP inhibitors (Johannes, J W. et al. J. Med. Chem. 2021; 64: 14498-14512). Importantly, significantly less haematotoxicity was observed for AZD5305 in rodent models than with first-generation PARP inhibitors, confirming the reported pathogenic role of PARP2 in haematologic toxicity (Illuzzi, G. et al. Clin. Cancer Res. 2022; CCR-22-0301).
Having regard to the above, it is an aim of the present invention to provide PARP1 inhibitors, and in particular PARP1 inhibitors for use in medicine. It is a further aim to provide pharmaceutical compositions comprising such inhibitors, and in particular to provide compounds and pharmaceutical compositions for treating a cancer. It is also an aim to provide methods of synthesis of the compounds.
Accordingly, the present invention provides a PARP1 inhibitor compound for use in medicine, which compound comprises the following structure:
In the context of the present invention, the Z1, Z2, X1, X2, X3, X4, X5 and X6 atoms maintain their normal valency and the number of substituents attached to these will depend on their nature and the number of other bonds they possess. Maintaining the valency means ensuring that an atom has its normal (typically most common) valency in organic compounds (i.e. 2 for oxygen and divalent sulphur, 3 for nitrogen and 4 for carbon). Nitrogen atoms may, in some instances, have 4 bonds, but in such cases they are typically positively charged such that the compound may have a counter-ion. Such compounds are also considered to be part of the invention, and in these cases, due to the positive charge, it will be clear that the nitrogen atom still maintains its normal valency of 3. For the avoidance of doubt, where the number of R groups may vary according to the choice of X group, it may vary as follows.
R6 is absent when Zi is N. R2 is absent when Z2 is N. Each R41 may be the same or different, provided that for each X1: R41 is absent when X1 is O or divalent S; R41 is absent when X1 is N and is double bonded to an adjacent atom; one R41 is present when X1 is N and is not double bonded to an adjacent atom; one R41 is present when X1 is C and is double bonded to an adjacent atom; and two R41 are present when X1 is C and is not double bonded to an adjacent atom. Each R43 may be the same or different, provided that for each X3: R43 is absent when X3 is O or divalent S; R43 is absent when X3 is N and is double bonded to an adjacent atom; one R43 is present when X3 is N and is not double bonded to an adjacent atom; one R43 is present when X3 is C and is double bonded to an adjacent atom; and two R43 are present when X3 is C and is not double bonded to an adjacent atom. For each X6: R43 is absent when X6 is N or is C and is double bonded to an adjacent atom; and one R43 is present when X6 is C and not double bonded to an adjacent atom. For the X3 with which it is associated: R44 is absent when X3 is O or divalent S; R4 is absent when X3 is N and is double bonded to an adjacent atom; one R4 is present when X3 is N and is not double bonded to an adjacent atom; one R44 is present when X3 is C and is double bonded to an adjacent atom; and one R4 is present when X3 is C and the C is attached to R43 and not double bonded to an adjacent atom. Each R42 may be the same or different, provided that for each X2: R42 is absent when X2 is O or divalent S; R42 is absent when X2 is N and is double bonded to an adjacent atom; one R42 is present when X2 is N and is not double bonded to an adjacent atom; one R42 is present when X2 is C and is double bonded to an adjacent atom; and two R42 are present when X2 is C and is not double bonded to an adjacent atom. For X5: R42 is absent when X5 is N or is C and is double bonded to an adjacent atom; and one R42 is present when X5 is C and not double bonded to an adjacent atom. Each R11 may be the same or different, provided that for each X4: R11 is absent when X4 is O or divalent S; R11 is absent when X4 is N and is double bonded to an adjacent atom; one R11 is present when X4 is N and is not double bonded to an adjacent atom; one R11 is present when X4 is C and is double bonded to an adjacent atom; and two R11 are present when X4 is C and is not double bonded to an adjacent atom. Each R14 bonded to N may be the same or different, provided that R14 is absent when the N is double bonded to an adjacent atom.
In these compounds, and elsewhere herein, in some embodiments any R group may form a ring with any other R group on an adjacent and/or proximal atom, although in most embodiments this is not preferred, except where explicitly stated. Thus, in some embodiments the following substituents may together form a ring: R5 with R4; R11 with another R11; R13 with another R13; R41 with another R41; R42 with another R42; R43 with another R43; R45 with another R45. In the context of the present invention, an adjacent and/or proximal atom may mean another atom directly bonded to an atom (adjacent) or may be two atoms with only a single atom in between (proximal), or may mean two atoms close enough sterically to be capable of forming a ring (proximal). Preferably R groups attached to the same atom do not together form a ring, although this is not excluded.
In the present context the invention includes compounds in which a single R group on an atom, or two R groups on the same atom, form a group which is double bonded to that atom. Accordingly, an R group, or two R groups attached to the same atom, may together form a ═O group, or a ═C(R′)2 group (wherein each R′ group is the same or different and is H or an organic group, preferably H or a straight or branched C1-C6 alkyl group). This is more typical in cases where the R groups are attached to a C atom, such that together they form a C═O group or a C═C(R′)2 group. Thus is some cases a C ring atom in a ring may comprise a ═O group, as may an X, and/or one or more of R11, R41, R42, R43, R44, and R45.
In the present context a part of any structure present in brackets may be repeated the number of times given by the numbers next to the brackets (whether regular brackets or square brackets). For example, in the case of (C(R))0,1,2 or [C(R)]0,1,2 the C—R group may be absent, present once i.e. —C(R)—; or present twice i.e. —C(R)—C(R)—.
In the context of the present invention, a compound is considered to be a PARP1 inhibitor if its presence is capable of preventing or reducing the ability of immobilised PARP1 to undergo auto-poly-ADP ribosylation (AutoPARylation) following incubation with biotinylated-NAD+ as compared to the same process in its absence. Typically, the compound is considered to be a PARP1 inhibitor if it has an IC50< 10 μM in a suitable assay. A suitable assay may be conducted using 2 nM PARP1, 2 μM biotin-NAD+ assay solution in 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.1% BSA (w/v), 0.02% Tween (v/v) assay buffer. PARylation may take place for 2 h at room temperature and may be detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. A particularly suitable assay is described in the Examples below. Preferably, the compound has an IC50<1 μM, more preferably <100 nM and most preferably <10 nM in the PARP1 inhibitor assay.
A compound is also considered to be a selective PARP1 inhibitor if its presence is capable of displacing or reducing the ability of a high affinity Cy5 fluorescent dye-labelled chemical probe to bind to PARP1 whilst displacing the same chemical probe at PARP2 with at least 10-fold weaker activity. Typically, the compound is considered to be a selective PARP1 inhibitor if it has an IC50<10 μM in this assay at PARP1 with at least 10-fold selectivity preference over PARP2. A suitable such assay may be conducted for 1 h at room temperature using 10 nM PARP1 or PARP2, Tb-cryptate antibody and PARP1/2 binding probe in 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.1% BSA (w/v), 0.02% Tween (v/v) assay buffer. Probe binding displacement may be detected using homogeneous time-resolved fluorescence. A particularly suitable assay is described in the Examples below. Preferably the selectivity preference of PARP1 over PARP2 is at least 50-fold, more preferably at least 100-fold.
The compound is also considered to be a selective PARP1 inhibitor if it has an IC50<10 μM at PARP1 with at least 10-fold selectivity preference over PARP2 in NanoBRET assays demonstrating cellular target engagement. These assays are based on bioluminescence resonance energy transfer (BRET) between a Nano-luc-tagged protein (eg PARP1 or PARP2) and a fluorescent group on a high affinity NAD+ competitive binding probe. Such cellular probe displacement assays can be utilised to measure inhibitor affinities and selectivity ratios at PARP1 and 2. A particularly suitable assay is described in the Examples below. Preferably the selectivity preference of PARP1 over PARP2 is at least 50-fold, more preferably at least 100-fold.
In all of the embodiments of this invention (both above and below herein), the substituents (each of the R groups) are not especially limited, provided that they do not prevent the PARP1 inhibitory function from occurring. In all of the embodiments mentioned in connection with this invention, both above and in the following, the substituents are selected from H and an organic group. Thus, both above and in the following, the terms ‘substituent’ and ‘organic group’ are not especially limited and may be any functional group or any atom, especially any functional group or atom common in organic chemistry. Thus, ‘substituent’ and ‘organic group’ may have any of the following meanings.
The organic group may comprise any one or more atoms from any of groups IIIA, IVA, VA, VIA or VIIA of the Periodic Table, such as a B, Si, N, P, O, or S atom (e.g. OH, OR, NH2, NHR, NR2, SH, SR, SO2R, SO3H, PO4H2) or a halogen atom (e.g. F, Cl, Br or I) where R is a linear or branched lower hydrocarbon (1-6 C atoms) or a linear or branched higher hydrocarbon (7 C atoms or more, e.g. 7-40 C atoms).
The organic group preferably comprises a hydrocarbon group. The hydrocarbon group may comprise a straight chain, a branched chain or a cyclic group. Independently, the hydrocarbon group may comprise an aliphatic or an aromatic group. Also independently, the hydrocarbon group may comprise a saturated or unsaturated group.
The number of carbon atoms in the hydrocarbon group is not especially limited, but preferably the hydrocarbon group comprises from 1-40 C atoms. The hydrocarbon group may thus be a lower hydrocarbon (1-6 C atoms) or a higher hydrocarbon (7 C atoms or more, e.g. 7-40 C atoms). The lower hydrocarbon group may be a methyl, ethyl, propyl, butyl, pentyl or hexyl group or regioisomers of these, such as isopropyl, isobutyl, tert-butyl, etc. The number of atoms in the ring of the cyclic group is not especially limited, but preferably the ring of the cyclic group comprises from 3-10 atoms, such as 3, 4, 5, 6, 7, 8, 9 or 10 atoms.
The groups comprising heteroatoms described above, as well as any of the other groups defined above, may comprise one or more heteroatoms from any of groups IIIA, IVA, VA, VIA or VIIA of the Periodic Table, such as a B, Si, N, P, O, or S atom or a halogen atom (e.g. F, Cl, Br or I). Thus, the substituent may comprise one or more of any of the common functional groups in organic chemistry, such as hydroxy groups, carboxylic acid groups, ester groups, ether groups, aldehyde groups, ketone groups, amine groups, amide groups, imine groups, thiol groups, thioether groups, sulphate groups, sulphonic acid groups, sulphonyl groups, and phosphate groups etc. The substituent may also comprise derivatives of these groups, such as carboxylic acid anhydrides and carboxylic acid halides.
In addition, any substituent may comprise a combination of two or more of the substituents and/or functional groups defined above.
The rings A, B (when present) and C of the compounds of the present invention form a bicyclic or tricyclic ring structure (which may comprise further fused rings when the substituents on either ring themselves form a ring). Each of rings A, B, C and D are not necessarily limited, provided that they do not prevent the PARP1 inhibitory function from occurring. Rings A, B, C and D may independently be comprised of an aromatic ring, a non-aromatic ring, an aliphatic ring, and/or a heterocyclic group. The rings may be fully saturated, partially saturated, or fully unsaturated. Each ring may thus independently comprise a benzene, naphthalene, anthracene, phenanthrene, phenalene, biphenylene, pentalene, indene, as-indacene, s-indacene, acenaphthylene, fluorene, fluoranthene, acephenanthrylene, azulene, heptalene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, pyrrolidine, oxetane, furan, tetrahydrofuran, 2-aza-tetrahydrofuran, 3-aza-tetrahydrofuran, oxazole, isoxazole, furazan, 1,2,4-oxadiazol, 1,3,4-oxadiazole, thiophene, isothiazole, thiazole, thiolane, pyridine, pyridazine, pyrimidine, pyrazine, piperidine, 2-azapiperidine, 3-azapiperidine, piperazine, pyran, tetrahydropyran, 2-azapyran, 3-azapyran, 4-azapyran, 2-aza-tetrahydropyran, 3-aza-tetrahydropyran, morpholine, thiopyran, 2-azathiopyran, 3-azathiopyran, 4-azathiopyran, thiane, indole, indazole, benzimidazole, 4-azaindole, 5-azaindole, 6-azaindole, 7-azaindole, isoindole, 4-azaisoindole, 5-azaisoindole, 6-azaisoindole, 7-azaisoindole, indolizine, 1-azaindolizine, 2-azaindolizine, 3-azaindolizine, 5-azaindolizine, 6-azaindolizine, 7-azaindolizine, 8-azaindolizine, 9-azaindolizine, purine, carbazole, carboline, benzofuran, isobenzofuran, benzothiophene, isobenzothiophene, quinoline, cinnoline, quinazoline, quinoxaline, 5-azaquinoline, 6-azaquinoline, 7-azaquinoline, isoquinoline, phthalazine, 6-azaisoquinoline, 7-azaisoquinoline, pteridine, chromene, isochromene, acridine, phenanthridine, perimidine, phenanthroline, phenoxazine, xanthene, phenoxanthiin, and/or thianthrene, as well as regioisomers of the above groups. These rings may generally be substituted at any point in the group, and also may be substituted at a hetero-atom or at a carbon atom. All tautomeric ring forms are included in these definitions. For example, pyrrole is intended to include 1H-pyrrole, 2H-pyrrole and 3H-pyrrole.
In typical embodiments, the invention provides a compound as defined above, wherein Q3 is absent or is a group independently selected from the following structures:
such as a —CH2— group.
In typical embodiments, the invention provides a compound as defined above, wherein Q2 is present and each Q1 is independently absent or is a group independently selected from the following structures:
such as a —CH2— group, particularly when Q2 is present.
The Q1 group bound to ring A is preferably
such as a —CH2— group.
In some embodiments, the invention provides a compound as defined above, wherein the group L comprises a group having the following structure:
In some embodiments, the group L comprises a group having the following structure:
In some embodiments, the group L comprises a group having the following structure:
In some embodiments, the group L comprises a group having the following structure:
In some embodiments, the group L comprises a group having the following structure:
In some embodiments, the group L comprises a group having the following structure:
In some embodiments, the group L comprises a group selected from the following structures:
In these structures for the L group it is preferred that m is selected from 1 or 2, n is selected from 2 or 3, p is selected from 1, 2 or 3 (more preferably for 2 or 3) and q is selected from 1 or 2.
In some embodiments, group L comprises a group having any the following structures:
Typically, R41, R42, R43, R44 and R45 are each independently selected from H and a group selected from the following groups:
In some arrangements, a pair of R41 groups attached to different atoms may together form a ring with ring A atoms, and/or a pair of R42 groups attached to different atoms may together form a ring with ring B atoms, optionally wherein each of the pair of R41 groups and/or pair of R42 groups independently comprises (X7)1 or 2, wherein each X7 may be the same or different and is independently selected from C, N, O and S; and wherein each X7 is independently unsubstituted or (i) independently substituted with H or an organic group selected from a C1-C6 alkyl group, a C1-C6 halogenated alkyl group, a halogen such as F, or hydroxyl, when X7 is C; and (ii) independently substituted with H or an organic group selected from a C1-C6 alkyl group or a C1-C6 amide group, when X7 is N.
In some arrangements, groups R5 and R44 may together form a ring with atoms X6 and X3 of ring C to which they are attached, optionally wherein the R5 and R44 groups together comprise (X8)3, 4 or 5, wherein each X8 may be the same or different and is independently selected from C, N, O and S; and wherein each X8 is independently unsubstituted or (i) independently substituted with H or an organic group selected from a C1-C6 alkyl group, a C1-C6 halogenated alkyl group, a halogen such as F, or hydroxyl, when X8 is C; and (ii) independently substituted with H or an organic group selected from a C1-C6 alkyl group, a C1-C6 halogenated alkyl group, or a C1-C6 amide group, when X8 is N.
It is preferred that R41, R42, R43 and R44 are each independently selected from H, deuterium, a halogen (such as —F, —Cl, —Br, and —I, preferably F or Cl), a nitrile group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted linear or branched C1-C6 halogenated alkyl group, (preferably CF3 or CHF2), a cyclopropyl group, an —OH group or a substituted or unsubstituted linear or branched C1-C6 alcohol group, a substituted or unsubstituted linear or branched C1-C7 amino carbonyl group (such as —NH—CO-Me), an —NH2 group or a substituted or unsubstituted C1-C6 amino group and a substituted or unsubstituted C1-C6 alkoxy group; wherein, when a pair of R41 groups attached to different atoms together forms a ring with ring A atoms, and/or a pair of R42 groups attached to different atoms together forms a ring with ring B atoms, each of the pair of R41 groups and/or pair of R42 groups independently comprises —CH2— or —CH2CH2—; and wherein, when groups R5 and R44 together form a ring with atoms of ring C, R5 and R44 together comprise —CH═CH—CH═CH— or —NH—CO—NH—.
It is preferred that R45 is selected from H, a halogen (such as —F, —Cl, —Br, and —I, preferably —F), a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted linear or branched C1-C6 halogenated alkyl group (preferably CF3), an —NH2 group or a substituted or unsubstituted C1-C6 amino group, an —OH group or a substituted or unsubstituted linear or branched C1-C6 alcohol group and a substituted or unsubstituted C1-C6 alkoxy group.
Typically, R46 is selected from H and a group selected from the following groups:
It is preferred that R46 is selected from H, a substituted or unsubstituted C1-C6 alkyl group or a substituted or unsubstituted linear or branched C1-C6 halogenated alkyl group.
Ring A of the group L preferably comprises any of the following structures:
More preferably, ring A of the group L comprises
Still more preferably, ring A of the group L comprises any of the following structures:
Particularly preferably, ring A of the group L comprises.
In one arrangement, Q2 of the group L is present and comprises any of the following structures:
More preferably, Q2 of the group L comprises any of the following structures:
Alternatively, group Q2 of the group L is absent and only one Q1 group is present. In this arrangement the Q1 group typically separates ring A from ring C by 3 or 4 atoms in the linear direction and may comprise any of the following structures:
In this arrangement, the Q1 group preferably comprises any of the following structures:
Preferably, ring C of the group L comprises any of the following structures:
More preferably, ring C of the group L comprises any of the following structures:
In some arrangements, R5 is a substituted or unsubstituted organic group.
Preferably, the group R5 is not MeO, and is more preferably selected from H, —F, —Cl, —Br, —I, —CN, —CONR51R51, —NR51COR52, —SO2NR51R51, —NR51SO2R53, —O—CR52R52R52, —CR52R52NR51R51 and any of the following structures:
Still more preferably, the group R5 is selected from —F, —Cl, —CN, —CONH2, —CONHMe, —CONHEt, —CONMe2, —CONHCOMe, —CONHCH2—CH2OMe, —CONH—CH2—CH2F, —CONH—CH2—CF3, —CONH—CH2—CHF2, —OCHF2, —NHCOMe, —NHSO2Me, —SO2NHMe, —CONHSO2Me,
A particularly preferred group R5 is —CONHMe, especially where ring C of the group L comprises
preferably
In some embodiments, the group L of the compound according to the invention comprises a group having any the following structures:
In some embodiments, a compound is provided which comprises any of the following structures:
Preferably, the compound comprises any of the following structures:
More preferably, the compound comprises any of the following structures:
Still more preferably, the compound comprises any of the following structures:
Each R11 may independently be selected from H, a halogen, a nitrile group, a linear or branched C1-C3 alkyl group, a linear or branched C1-C3 halogenated alkyl group (preferably fluoroalkyl), an —OH group, a linear or branched C1-C3 alcohol group, a halogenated (preferably fluoro-) linear or branched C1-C3 alcohol group, —NH2, a linear or branched primary secondary or tertiary C1-C3 amine group, a halogenated (preferably fluoro-) linear or branched primary, secondary or tertiary C1-C3 amine group, a linear or branched C1-C3 alkoxy group, a linear or branched C1-C3 halogenated alkoxy group (preferably fluoroalkoxy), and/or a pair of R11 groups attached to the same atom forming ═O; and independently, and/or when a pair of R11 groups attached to different atoms together forms a ring with ring D atoms, the pair of R11 groups comprises —CH2CH2CH2—.
It is preferred that each R11 is independently selected from H, Cl, F, CHF2, CF3, CH3, OH, CH3O, and NH2, and/or a pair of R11 groups attached to the same atom forming ═O; and independently, when a pair of R11 groups attached to different atoms together forms a ring with ring D atoms, the pair of R11 groups comprises —CH2CH2CH2—.
In some embodiments, a compound is provided which comprises any of the following structures:
In some embodiments, a compound is provided which comprises any of the following structures:
At least one R12 is preferably selected from —CH3, —CH2CH3, —CH2CH2CH3, —CH2Cl, —CHCl2, —CCl3, —CH2F, —CHF2, —CF3, —C1, —F, —Cl, —CH2CF3, —CH2CH2F, —CH2CH2OH, methoxy, methoxymethyl, methoxyethyl, isopropyl, cyclopropyl or cyclopropylmethyl.
R13 is preferably selected from H, F, C1 to C3 alkyl or C1 to C3 fluoroalkyl.
R3 is preferably H.
R6 is preferably selected from H, halogen, C1 to C3 alkyl, C1 to C3 haloalkyl, C1 to C3 alcohol or C1 to C3 aminooalkyl.
In some embodiments, a compound is provided which comprises any of the following structures:
In some embodiments, the present invention provides a PARP1 inhibitor compound which comprises a formula selected from one of the following:
The compounds of the present invention have been described in detail above in terms of their structures. For the avoidance of doubt, any compounds for use in the invention may comprise compounds or compositions in accordance with their structure as follows:
For the above numbered compounds, compounds 1, 2, 5, 8, 12, 15, 17 to 22, 25, 27 to 30, 36, 40, 41, 45, 47, 48, 54, 59, 63, 75, 168, 173, 179, 192, 193, 227, 228, 238, 259, 260, 284, 295, 299, 300, 302-305, 310-312, 330, 361, 368, 369, 373, 375, 376, 379-381, 383, 384, 387-389, 394 and 411 are achiral. The remaining compounds represent more than one enantiomeric structure which may have PARP1 inhibitory activity as a racemic mixture and/or as a separated enantiomer(s). In the examples below, a compound with a suffix “a” (eg 10a) represents an enantiomer eluted as a first fraction when a racemic mixture of the two enantiomers is applied to a Daicel CHIRALPAK chiral chromatography column. In the examples below, a compound with a suffix “b” (eg 10b) represents an enantiomer eluted as a second fraction when a racemic mixture of the two enantiomers is applied to a Daicel CHIRALPAK chiral chromatography column. In the examples below, a compound with no suffix represents either an achiral compound or a racemic mixture of the enantiomers. In the examples below, a compound which bears a suffix “rac” represents a racemic mixture of the enantiomers.
The compounds described herein may be provided for use in medicine. In the context of the present invention, the medicinal use is not especially limited, provided that it is a use which is facilitated by the PARP1 inhibitory effect of the compound. Thus, the compounds of the invention may be for use in any disease, condition or disorder that may be prevented, ameliorated or treated using a PARP1 inhibitor. Typically, this comprises a disease condition and/or a disorder selected from: a cancer, it is not especially limited, provided that the cancer is one which may be treated, prevented or ameliorated by using a PARP1 inhibitor. Thus the cancer may be a cancer selected from: a solid or liquid tumour including cancer of the eye, brain (such as gliomas, glioblastomas, medullablastomas, craniopharyngioma, ependymoma, and astrocytoma), spinal cord, kidney, mouth, lip, throat, oral cavity, nasal cavity, small intestine, colon, parathyroid gland, gall bladder, head and neck, breast, bone, bile duct, cervix, heart, hypopharyngeal gland, lung, bronchus, liver, skin, ureter, urethra, testicles, vagina, anus, laryngeal gland, ovary, thyroid, oesophagus, nasopharyngeal gland, pituitary gland, salivary gland, prostate, pancreas, adrenal glands; an endometrial cancer, oral cancer, melanoma, neuroblastoma, gastric cancer, an angiomatosis, a hemangioblastoma, a pheochromocytoma, a pancreatic cyst, a renal cell carcinoma, Wilms' tumour, squamous cell carcinoma, sarcoma, osteosarcoma, Kaposi sarcoma, rhabdomyosarcoma, hepatocellular carcinoma, PTEN Hamartoma-Tumor Syndromes (PHTS) (such as Lhermitte-Duclos disease, Cowden syndrome, Proteus syndrome, and Proteus-like syndrome), leukaemias and lymphomas (such as acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, acute myelogenous leukaemia, chronic myelogenous leukaemia, hairy cell leukaemia, T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, juvenile myelomonocytic leukaemia, Hodgkin lymphoma, non-Hodgkin lymphoma, mantle lymphoma, follicular lymphoma, primary effusion lymphoma, AIDS-related lymphoma, Hodgkin lymphoma, diffuse B cell lymphoma, Burkitt lymphoma, and cutaneous T-cell lymphoma). In addition, the compounds described herein may be of use in cancers where EBV plays a contributing role such as Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal and gastrointestinal cancers.
The compounds described herein may be provided for use in for treating a cancer which is deficient in DNA damage response repair pathways, in particular in Homologous Recombination dependent DNA DSB DNA repair activity. Components of HR dependent DNA DSB repair pathways and other DNA damage response pathways include but are not limited to the following proteins: ATM, ATR, ERCC1, XRCC1, XRCC2, XRCC3, RAD51, RAD51L1, RAD51C, RAD51D, RAD51L3, DMC1, RAD52, RAD54L, RAD54B, RAD50, MRE11A, NBS1, BRCA1, BRCA2, FANCP (SLX4), FEN1, PALB2, PBRM1, SMARCA4, ARID1A, ARID1B, FANCD2, BLM. Other components involved in HR dependent DNA DSB repair include regulatory factors such as ESMY (Hughes-Davies, L. et al. Cell. 2003; 115: 523-535). A cancer which is deficient in HR-dependent DNA DSB repair typically becomes dependent on alternative DSB pathway repair mechanisms. Such cancers include but are not limited to cancers of the ovary, prostate, breast, lung, gastrointestine, blood and pancreas.
In some embodiments, the cancer cells may have a BRCA1 and/or BRCA2 deficient phenotype ie they may be deficient in BRCA1 and/or 2 expression and function by means of mutation, polymorphism or epigenetic silencing in the encoding nucleic acids or by means of amplification, polymorphism, mutation in a gene encoding a regulatory factor eg, the ESMY gene which encodes a BRCA2 regulatory factor (Hughes-Davies, L. et al. Cell. 2003; 115: 523-535). Amplification of the ESMY gene is associated with breast and ovarian cancer. Carriers of mutations in the tumour suppressor BRCA1 and/or BRCA2 genes are known to have an elevated risk of developing certain cancers including ovarian, prostate and breast. Wild-type alleles of BRCA1 and/or BRCA2 are frequently lost in tumours of heterozygous carriers (Jasin, M. et al. Oncogene. 2002; 21: 8981-93) and their detection, as a means of patient selection, is well known in the art (Radice, P J. et al. Exp. Clin. Cancer. Res. 2002; 21: 9-12; Chappnis, PO and Foulkes WO. Cancer Treat Res. 2002; 107: 29-59).
In some embodiments, the compounds described herein are selective PARP1 inhibitors, as defined above. Selective inhibition of PARP1 over PARP2 reduces PARP2 associated side effects including one or more haematological toxicities such as anaemia, neutropenia and thrombocytopenia. This enables treatment of cancer patients with reduced haematological side effects. This also enables higher doses of PARP1 inhibitors to be administered to patients and for such inhibitors to be administered in combination with chemotherapeutic agents.
The present invention also provides a pharmaceutical composition comprising a compound as defined above. Whilst the pharmaceutical composition is not especially limited, typically the composition further comprises a pharmaceutically acceptable additive and/or excipient. In the pharmaceutical composition, the compound as defined above may be present in the form described above, but may alternatively be in a form suitable for improving bioavailability, solubility, and/or activity, and/or may be in a form suitable for improving formulation. Thus, the compound may be in the form of a pharmaceutically acceptable salt, hydrate, acid, ester, or other alternative suitable form. Typically, the composition is for treating a disease, condition or disorder as defined above. In some instances, the compound may be present in the composition as a pharmaceutically acceptable salt, or other alternative form of the compound, in order to ameliorate pharmaceutical formulation.
In some embodiments the pharmaceutical composition is a composition for treating a cancer, further comprising a further agent for treating cancer. The further agent for treating cancer is not especially limited, provided that it affords some utility for cancer treatment. However, typically the further agent for treating cancer is selected from ionising radiation, chemotherapeutic agents such as anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, senolytic agents, hormones and hormone analogues, signal transduction pathway inhibitors, other DNA damage repair pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, antibody-drug conjugates, immunotherapeutic agents, hormone-deprivation therapies, proapoptotic agents and cell cycle signalling inhibitors. An immunotherapeutic agent may consist of but is not limited to an anti-tumour vaccine, an oncolytic virus, an immune stimulatory antibody such as anti-CTLA4, anti-PD1, anti-PDL-1, anti-OX40, anti-41BB, anti-CD27, anti-CD40, anti-LAG3, anti-TIM3, and anti-GITR, a pattern recognition receptor agonist such as a STING, TLR-9 or RIG-I Helicase agonist, an IDO or TDO inhibitor, a novel adjuvant, a peptide, a cytokine, a chimeric antigen receptor T cell therapy (CAR-T), a small molecule immune modulator, tumour microenvironment modulators, and anti-angiogenic agents.
In still further embodiments the invention provides a pharmaceutical kit for treating a cancer, which pharmaceutical kit comprises:
Further provided by the invention is a method of treating a disease and/or a condition and/or a disorder, which method comprises administering to a patient (or subject) a compound, or a composition, or a kit as defined above. The method is typically a method for treating any disease condition or disorder mentioned herein. In typical embodiments, the method is a method for treating a cancer. Preferably such a method comprises administering to a patient (or subject) a compound or a composition as defined above and a further agent for treating cancer as defined above. The compound or composition and the further agent may be administered simultaneously, sequentially or separately, depending upon the agents and patients involved, and the type of cancer indicated.
Typically, in all embodiments of the invention, both above and below, the patient (or subject) is an animal, typically a mammal, including canines, equines and felines, and more typically a human.
Further provided by the invention is a method of synthesis of a compound as defined above, which method comprises conducting a reaction between (i) a first reactant comprising ring E bearing a portion of substituent group L and (ii) a second reactant comprising the remainder of substituent group L, so as to form the PARP1 inhibitor compound.
Typically, in one method of synthesis, the first reactant comprises ring E and ring A, and the second reactant comprises a Q1 or Q2 precursor bearing a reactive group, which method comprises joining the N atom of ring A to the Q1 or Q2 precursor. In this method, the reactive group of the Q1 or Q2 precursor may comprise a carbonyl group, an alkyl halide or an alkyl sulfonate. Typically, the reaction comprises alkylation, reductive amination or amide formation so as to form group L.
Typically, in another method of synthesis, the first reactant comprises ring E, ring A, and at least one of Q1 and Q2, and the second reactant comprises a ring C derivative bearing a leaving group such as a halide or sulfonate. In this method, the reaction comprises a nucleophilic substitution reaction, such as a nucleophilic aromatic substitution reaction, so as to form group L.
The skilled person may select the reaction conditions in these methods with reference to known synthesis techniques depending on the appropriate starting materials. In some embodiments, the method comprises one or more additional steps. Exemplary synthesis methodology is shown in the Examples herein Typically, the above formulae (and all formulae herein) are shown in non-stereoisomeric form.
For the avoidance of doubt, throughout the present disclosure a single formula is intended to represent all possible stereoisomers of a particular structure, including all possible isolated enantiomers corresponding to the formula, all possible mixtures of enantiomers corresponding to the formula, all possible mixtures of diastereomers corresponding to the formula, all possible mixtures of epimers corresponding to the formula and all possible racemic mixtures corresponding to the formula. In addition to this, the above formulae (and all formulae herein) are intended to represent all tautomeric forms equivalent to the corresponding formula.
The term “comprises” as used throughout the description and claims herein means “includes or consists of”. The term denotes the inclusion of at least the features following the term and does not exclude the inclusion of other features which have not been explicitly mentioned. The term may also denote an entity which consists only of the features following the term.
The invention will now be described in more detail, by way of example only, with reference to the following specific embodiments.
The compounds of the invention may be synthesised using readily available starting materials and known reactions. Exemplary syntheses of three compounds are shown below:
To a solution of 5-ethyl-1,3-dihydropyrimidine-2,4-dione 1 (10.00 g, 0.07 mol) in POCl3 (55.00 g, 0.36 mol) at 0° C. and added DIEA (23.00 g, 0.18 mol). Then the mixture was stirred for 2 h at 120° C. The hot reaction mixture was poured into ice water and the aqueous layer was extracted with ethyl acetate (50 mL×3). The combined organic layers were dried over Na2SO4. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 92:8) to give 2,4-dichloro-5-ethylpyrimidine 2 (4.80 g, 38% yield) as a white solid.
LCMS (ESI) calcd for C6H6Cl2N2 [M+H]+ m/z 176.99, found 177.00
To a solution of (4-methoxyphenyl) methanol 3 (4.54 g, 32.90 mmol) in THF (20 mL) was added tBuOLi (2.30 g, 28.75 mmol) at 70° C. for 15 min. Then 2,4-dichloro-5-ethylpyrimidine 2 (4.80 g, 27.27 mmol) was added to the mixture at 0° C. The mixture was stirred for 3 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 93:7) to give 2-chloro-5-ethyl-4-(2-methoxy-5-methylphenoxy)pyrimidine INT-4 (4.00 g, 53% yield) as a white solid.
LCMS (ESI) calcd for C14H15ClN2O2 [M+H]+ m/z 279.08, found 279.15.
To a solution tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydropyrrole-1-carboxylate 1 (500 mg, 1.69 mmol) in 1,4-dioxane/H2O=4:1 (15 mL) was added 2-chloro-5-ethyl-4-(2-methoxy-5-methylphenoxy)pyrimidine INT-4 (377.72 mg, 1.76 mmol) and Na2CO3 (189.11 mg, 3.01 mmol), then Pd(dppf)Cl2 (247.89 mg, 0.34 mmol) was added at room temperature. The reaction mixture was refluxed under nitrogen at 100° C. and stirred for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with petroleum ether/ethyl acetate=100:0 to 85:15) to give tert-butyl 3-(5-ethyl-4-((4-methoxybenzyl)oxy)pyrimidin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 2 (250 mg, 31% yield) as a colorless oil.
LCMS (ESI) calcd for C23H29N3O4 [M+H]+ m/z 412.22, found 412.28.
To a solution 3-(5-ethyl-4-((4-methoxybenzyl)oxy)pyrimidin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 2 (250 mg, 0.61 mmol) in MeOH (15 mL) was added Pd/C (97 mg, 0.91 mmol) at room temperature. The reaction mixture was stirred for 2 h at 50° C. under hydrogen. After cooling to room temperature, the mixture was filtered through a Celite pad and the filtrate was concentrated to give tert-butyl 3-(5-ethyl-4-hydroxypyrimidin-2-yl)pyrrolidine-1-carboxylate 3 (150 mg, 80% yield) as a colourless oil.
LCMS (ESI) calcd for C15H23N3O3 [M+H]+ m/z 294.17, found 294.19.
To a solution 3-(5-ethyl-4-hydroxypyrimidin-2-yl)pyrrolidine-1-carboxylate 3 (150 mg, 0.51 mmol) was added HCl in dioxane (4 M, 10 mL) at room temperature. The reaction mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure to give 5-ethyl-2-(pyrrolidin-3-yl)-3H-pyrimidin-4-one 4 (100 mg, 94% yield) as a white solid.
LCMS (ESI) calcd for C10H15N3O [M+H]+ m/z 194.12, found 194.19.
Preparation of tert-butyl 4-[3-(5-ethyl-4-oxo-3H-pyrimidin-2-yl)pyrrolidin-1-yl]piperidine-1-carboxylate (6) To a solution 5-ethyl-2-(pyrrolidin-3-yl)-3H-pyrimidin-4-one 4 (100 mg, 0.52 mmol) in MeOH (15 mL) was added tert-butyl 4-oxopiperidine-1-carboxylate 5 (124.48 mg, 0.61 mmol) and NaBH3CN (145 mg, 3.11 mmol). The reaction mixture was refluxed under nitrogen at 50° C. and stirred for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 95:5) to give tert-butyl 4-[3-(5-ethyl-4-oxo-3H-pyrimidin-2-yl)pyrrolidin-1-yl]piperidine-1-carboxylate 6 (120 mg, 55% yield) as a white solid.
LCMS (ESI) calcd for C20H32N4O3 [M+H]+ m/z 377.25, found 377.30.
Tert-butyl 4-[3-(5-ethyl-4-oxo-3H-pyrimidin-2-yl)pyrrolidin-1-yl]piperidine-1-carboxylate 6 (120 mg, 0.32 mmol) was added to HCl in dioxane (4 M, 10 mL) at room temperature. The reaction mixture was stirred for 1 h at room temperature, then concentrated under reduced pressure to give 5-ethyl-2-[1-(piperidin-4-yl)pyrrolidin-3-yl]-3H-pyrimidin-4-one 7 (90 mg, 92% yield) as a white solid.
LCMS (ESI) calcd for C15H24N4O [M+H]+ m/z 277.20, found 277.24.
To a solution 5-ethyl-2-[1-(piperidin-4-yl)pyrrolidin-3-yl]-3H-pyrimidin-4-one 7 (90 mg, 0.33 mmol) in DMF (10 mL) was added 5-fluoro-N-methylpyridine-2-carboxamide 8 (104 mg, 0.68 mmol) and Cs2CO3 (884 mg, 2.71 mmol). The reaction mixture was irradiated in a microwave reactor at 150° C. for 3 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 95:5) to give 5-{4-[3-(5-ethyl-4-oxo-3H-pyrimidin-2-yl)pyrrolidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide Compound 42 (20 mg, 95% purity, 20% yield) as a white solid.
1H NMR (400 MHz, DMSO) δ 12.16 (s, 1H), 8.42-8.38 (m, 1H), 8.28 (d, J=2.4 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.73 (s, 1H), 7.42-7.39 (dd, J=8.8, 2.8 Hz, 1H), 3.87 (d, J=12.8 Hz, 2H), 3.29-3.25 (m, 2H), 3.17-3.13 (m, 1H), 2.94-2.74 (m, 8H), 2.34 (q, J=7.2 Hz, 2H), 2.18-1.94 (m, 4H), 1.56-1.47 (m, 2H), 1.08 (t, J=7.6 Hz, 3H).
LCMS (ESI) calcd for C22H30N6O2 [M+H]+ m/z 411.24, found 411.33.
To a solution of 5-fluoro-N-methylpyridine-2-carboxamide 1 (1.00 g, 6.50 mmol) in DMF (15 mL) was added 1,4-dioxa-8-azaspiro[4.5]decane 2 (1.40 g, 9.75 mmol), then Cs2CO3 (2.12 g, 6.50 mmol) was added at room temperature. The reaction mixture was stirred at 150° C. using microwave for 5 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 97:3) to give 5-{1,4-dioxa-8-azaspiro[4.5]decan-8-yl}-N-methylpyridine-2-carboxamide 3 (1.50 g, 76% yield) as a white solid.
LCMS (ESI) calcd for C14H19N3O3 [M+H]+ m/z 278.14, found 278.14.
To a solution of 5-{1,4-dioxa-8-azaspiro[4.5]decan-8-yl}-N-methylpyridine-2-carboxamide 3 (1.50 g, 5.40 mmol) in H2O (10 mL) was added HCl in 1,4-dioxane (4 M, 20 mL) at room temperature. The reaction mixture was stirred at 50° C. for 1 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was adjusted pH>7 using NaHCO3 solution, then extracted with EtOAc (50 mL×3), the organic phase dried by Na2SO4 and concentrated to give N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (1.50 g, 76% yield) as a yellow solid.
LCMS (ESI) calcd for C12H15N3O2 [M+H]+ m/z 234.12, found 234.18.
To a solution of 6-bromo-2-methoxypyridin-3-amine 1 (2.00 g, 9.90 mmol) in 1,4-dioxane/H2O=4:1 (45 mL) was added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-2H-pyridine-1-carboxylate 2 (3.98 g, 12.87 mmol), then Pd(dppf)Cl2 (0.72 g, 0.99 mmol) and Na2CO3 (3.12 g, 99.00 mmol) were added at room temperature. The reaction mixture was stirred under nitrogen at 80° C. for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 85:15) to give tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)-5,6-dihydro-2H-pyridine-1-carboxylate 3 (3.00 g, 89% yield) as a white solid.
LCMS (ESI) calcd for C16H23N3O3 [M+H]+ m/z 306.17, found 306.19.
To a solution of tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)-5,6-dihydro-2H-pyridine-1-carboxylate 3 (2.80 g, 9.20 mmol) in MeOH (50 mL) was added Pd/C (0.78 g, 7.36 mmol) at room temperature. The reaction mixture was stirred under hydrogen at 50° C. for 2 h. After cooling to room temperature, the reaction mixture was filtered, the solution was concentrated under reduced pressure to give product of tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)piperidine-1-carboxylate 4 (2.80 g, 89% yield) as a white solid.
LCMS (ESI) calcd for C16H25N3O3 [M+H]+ m/z 308.19, found 308.19.
To a solution of tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)piperidine-1-carboxylate 4 (2.80 g, 9.10 mmol) in ACN (50 mL) was added tert-Butyl nitrite (2.82 g, 27.30 mmol), stirred for 15 minutes, then CuBr (5.22 g, 36.40 mmol) was added at room temperature. The reaction mixture was stirred at 50° C. for 2 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 85:15) to give tert-butyl tert-butyl 3-(5-bromo-6-methoxypyridin-2-yl)piperidine-1-carboxylate 5 (1.10 g, 30% yield) as a white solid.
LCMS (ESI) calcd for C16H23BrN2O3 [M+H]+ m/z 371.09, found 371.15.
To a solution of tert-butyl 3-(5-bromo-6-methoxypyridin-2-yl)piperidine-1-carboxylate 5 (1.10 g, 2.96 mmol) in 1,4-dioxane (65 mL) was added Pd(dppf)Cl2 (219 mg, 0.30 mmol). Then Et2Zn (1M, 11.84 mL, 11.84 mml) was added at room temperature. The reaction mixture under nitrogen was stirred at 80° C. for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 85:15) to give tert-butyl 3-(5-ethyl-6-methoxypyridin-2-yl)piperidine-1-carboxylate 6 (700 mg, 67% yield) as a white solid.
LCMS (ESI) calcd for C18H28N2O3 [M+H]+ m/z 321.21, found 321.30.
To a solution of tert-butyl 3-(5-ethyl-6-methoxypyridin-2-yl)piperidine-1-carboxylate 6 (200 mg, 0.62 mmol) in HBr in water (48%, 6 mL). The reaction mixture was stirred at 100° C. for 6 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to give 3-ethyl-6-(piperidin-3-yl)-1H-pyridin-2-one 7 (80 mg, 56% yield) as a yellow solid.
LCMS (ESI) calcd for C12H18N2O [M+H]+ m/z 207.14, found 206.95.
To a solution of 3-ethyl-6-(piperidin-3-yl)-1H-pyridin-2-one 7 (80 mg, 0.38 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide INT (89 mg, 0.38 mmol). Then two drops of acetic acid and NaBH3CN (24 mg, 0.38 mml) was added at room temperature. The reaction mixture was stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 95:5) to give 5-{4-[3-(5-ethyl-6-oxo-1H-pyridin-2-yl)piperidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide 8 (48 mg, 31% yield) as a white solid.
LCMS (ESI) calcd for C24H33N5O2 [M+H]+ m/z 424.26, found 424.37.
5-{4-[3-(5-ethyl-6-oxo-1H-pyridin-2-yl)piperidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide 8 was separated by SFC (Column: Daicel CHIRALPAK AD-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=75/25) and concentrated under reduced pressure to afford the first fraction as Compound 53a (7.16 mg, 95% purity, ee %: 100, white solid) and the second fraction as Compound 53b (6.57 mg, 99% purity, ee %: 100, white solid) Compound 53a 1H NMR (400 MHz, DMSO) δ 11.48 (s, 1H), 8.39 (q, J=4.8 Hz, 1H), 8.26 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 7.17 (d, J=6.8 Hz, 1H), 5.98 (d, J=7.2 Hz, 1H), 3.97 (d, J=12.4 Hz, 2H), 2.91-2.77 (m, 7H), 2.67-2.54 (m, 2H), 2.36-2.28 (m, 4H), 1.83-1.81 (m, 3H), 1.68-1.65 (m, 1H), 1.59-1.40 (m, 4H), 1.05 (t, J=7.6 Hz, 3H).
1H NMR (400 MHz, DMSO) δ 11.48 (s, 1H), 8.39 (q, J=4.4 Hz, 1H), 8.26 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.40 (dd, J=9.2, 3.2 Hz, 1H), 7.17 (d, J=7.2 Hz, 1H), 5.98 (d, J=6.8 Hz, 1H), 3.97 (d, J=12.4 Hz, 2H), 2.91-2.77 (m, 7H), 2.66-2.54 (m, 2H), 2.36-2.25 (m, 4H), 1.83-1.80 (m, 3H), 1.67-1.64 (m, 1H), 1.59-1.36 (m, 4H), 1.05 (t, J=7.6 Hz, 3H).
To a solution of 5-hydroxypicolinic acid 1 (4.00 g, 28.72 mmol) in DMF (15 mL) was added HATU (12.55 g, 33.01 mmol) and DIEA (9.28 g, 71.80 mmol). Then methanamine (2M, 15 mL, 30.00 mmol) was added at room temperature. Then the mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 93:7) to give 5-hydroxy-N-methylpicolinamide 2 (2.00 g, 46% yield) as a white solid.
LCMS (ESI) calcd for C7H8N2O2 [M+H]+ m/z 153.06, found 153.10
To a solution of 5-hydroxy-N-methylpicolinamide 2 (1.00 g, 6.61 mmol) in DMF (10 mL) was added 3-bromopropan-1-ol (2.75 g, 19.58 mmol) and Cs2CO3 (3.23 g, 9.93 mmol) at room temperature. Then the mixture was stirred for 18 h at room temperature. The reaction mixture was filtered to give 5-(3-hydroxypropoxy)-N-methylpicolinamide 3 (1.00 g, 65% yield) as an oil.
LCMS (ESI) calcd for C10H14N2O3 [M+H]+ m/z 211.10, found 211.05.
To a solution of 5-(3-hydroxypropoxy)-N-methylpicolinamide 3 (250.00 mg, 1.19 mmol) in DCM (15 mL) was added CBr4 (788.74 mg, 2.38 mmol). Then PPh3 (623.83 mg, 2.38 mmol) was added at room temperature. Then the mixture was stirred for 2 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 95:5) to give 5-(3-bromopropoxy)-N-methylpicolinamide INT-4-1 (300 mg, 69% yield) as a white sold.
LCMS (ESI) calcd for C10H13BrN2O2 [M+H]+ m/z 273.02, found 274.85.
To a solution of 5-(3-bromopropoxy)-N-methylpicolinamide INT-4-1 (150.00 mg, 0.55 mmol) in ACN (10 mL) was added 2-(piperidin-4-yl)-3H-quinazolin-4-one INT-5 (151.10 mg, 0.66 mmol). Then DIEA (212.94 mg, 1.65 mmol) was added at room temperature. Then the mixture was stirred for 18 h at 67° C. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give N-methyl-5-(3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)propoxy)picolinamide Compound 2 (100 mg, 78% yield) as a white sold.
1H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 8.57-8.54 (m, 1H), 8.29 (d, J=2.8 Hz, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.98 (d, J=8.8 Hz, 1H), 7.78 (t, J=7.6 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.54 (dd, J=8.8, 2.8 Hz, 1H), 7.47 (t, J=7.6 Hz, 1H), 4.18 (t, J=5.6 Hz, 2H), 3.10-2.86 (m, 2H), 2.79 (d, J=4.8 Hz, 3H), 2.65-2.53 (m, 1H), 2.49-2.41 (m, 2H), 1.94 (s, 8H).
LCMS (ESI) calcd for C23H27N5O3 [M+H]+ m/z 422.21, found 422.30.
To a solution of 2-aminobenzamide 1 (6.00 g, 44.10 mmol) and 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid 2 (10.11 g, 44.10 mmol) in pyridine (50 mL) was added EDCI (8.45 g, 44.10 mmol). then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers was washed by 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give product of tert-butyl 3-((2-carbamoylphenyl)carbamoyl)piperidine-1-carboxylate 3 (12.00 g, 79% yield) as a white solid
LCMS (ESI) calcd for C18H25N3O4 [M−H]− m/z 346.18, found 346.20.
To the solution of tert-butyl 3-((2-carbamoylphenyl)carbamoyl)piperidine-1-carboxylate 3 (12.00 g, 34.50 mmol) in Diglyme (80 mL) was added KOH (2.13 g, 37.95 mmol), heated to 140° C. stirred for 2 h, cooled to 0° C., ice-water (100 mL) added then adjusted pH<7 using 1M HCl solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of tert-butyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate 4 (11.00 g, 97% yield) as a white solid.
LCMS (ESI) calcd for C18H23N3O3 [M+H]+ m/z 330.17, found 330.20.
To the solution of tert-butyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate 4 (3.30 g, 10.00 mmol) in DCM (10 mL) were added TFA (10 mL), stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give crude product, ice-water (20 mL) added then adjusted pH>7 using ammonium hydroxide solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of 2-(piperidin-3-yl)quinazolin-4(3H)-one INT-5-1 (1.50 g, 66% yield) as a white solid.
LCMS (ESI) calcd for C13H15N3O [M+H]+ m/z 230.12, found 230.00.
To a solution of 2-(piperidin-3-yl)quinazolin-4(3H)-one INT-5-1 (460 mg, 2.01 mmol) in MeOH (15 mL) was added tert-butyl 4-oxopiperidine-1-carboxylate 6 (8.45 g, 44.10 mmol), then two drops of acetic acid and NaBH3CN (189.11 mg, 3.01 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 93:7) to give tert-butyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-[1,4′-bipiperidine]-1′-carboxylate 7 (300 mg, 36% yield) as a white solid.
1H NMR (400 MHz, DMSO) δ 12.40 (s, 1H), 8.11 (d, J=7.2 Hz, 1H), 7.81 (s, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.51 (s, 1H), 4.10-3.99 (m, 2H), 3.53-3.44 (m, 1H), 3.16-3.04 (m, 1H), 2.94-2.62 (m, 4H), 1.99 (d, J=2.4 Hz, 4H), 1.91 (d, J=2.4 Hz, 2H), 1.82-1.53 (m, 4H), 1.41 (s, 9H).
Tert-butyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-[1,4′-bipiperidine]-1′-carboxylate 7 (300 mg, 0.65 mmol) was added to HCl in dioxane (4 M, 10 mL), stirred for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to give 2-([1,4′-bipiperidin]-3-yl)quinazolin-4(3H)-one 8 (200 mg, 88% yield) as a white solid.
LCMS (ESI) calcd for C18H24N4O [M+H]+ m/z 313.20, found 313.15.
To a solution 2-([1,4′-bipiperidin]-3-yl)quinazolin-4(3H)-one 8 (200.00 mg, 0.64 mmol) in DMF (10 mL) was added Cs2CO3 (1042.94 mg, 3.20 mmol) and 5-fluoro-N-methylpicolinamide (148.02 mg, 0.96 mmol). The mixture was stirred for 6 h under 150° C. using microwave. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to N-methyl-5-(3-(4-oxo-3,4-dihydroquinazolin-2-yl)-[1,4′-bipiperidin]-1′-yl)picolinamide 9 (100 mg, 95% purity, 33% yield) as a yellow solid.
LCMS (ESI) calcd for C25H30N6O2 [M+H]+ m/z 447.24, found 447.20
Compound 9 was separated by SFC (Column: Daicel CHIRALPAK OJ-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=70/30) and concentrated under reduced pressure to afford the first fraction as Compound 6a (40.6 mg, 100% purity, ee %: 100, white solid) and the second fraction as Compound 6b (39.5 mg, 99% purity, ee %: 100, white solid)
1H NMR (400 MHz, DMSO) δ 12.25 (s, 1H), 8.40 (q, J=4.8 Hz, 1H), 8.29 (d, J=2.8 Hz, 1H), 8.10 (dd, J=7.6, 1.6 Hz, 1H), 7.85-7.78 (m, 2H), 7.63 (d, J=8.0 Hz, 1H), 7.49 (t, J=7.4 Hz, 1H), 7.42 (dd, J=8.8, 2.8 Hz, 1H), 3.99 (d, J=12.8 Hz, 2H), 3.08 (d, J=8.8 Hz, 1H), 2.87-2.81 (m, 4H), 2.80 (d, J=4.8 Hz, 3H), 2.61-2.55 (m, 2H), 2.32 (t, J=9.8 Hz, 1H), 1.98-1.96 (m, 1H), 1.87 (d, J=12.4 Hz, 2H), 1.78-1.75 (m, 1H), 1.69-1.55 (m, 4H).
LCMS (ESI) calcd for C25H30N6O2 [M+H]+ m/z 447.24, found 447.
1H NMR (400 MHz, DMSO) δ 12.25 (s, 1H), 8.39 (q, J=4.8 Hz, 1H), 8.29 (d, J=2.8 Hz, 1H), 8.10 (dd, J=7.6, 1.6 Hz, 1H), 7.84-7.78 (m, 2H), 7.63 (d, J=8.0 Hz, 1H), 7.50-7.47 (m, 1H), 7.42 (dd, J=8.8, 2.8 Hz, 1H), 3.99 (d, J=12.8 Hz, 2H), 3.08 (d, J=8.8 Hz, 1H), 2.87-2.81 (m, 4H), 2.80 (d, J=4.8 Hz, 3H), 2.61-2.55 (m, 2H), 2.31 (t, J=9.6 Hz, 1H), 1.98-1.96 (m, 1H), 1.87 (d, J=12.4 Hz, 2H), 1.78-1.75 (m, 1H), 1.63-1.55 (m, 4H).
LCMS (ESI) calcd for C25H30N6O2 [M+H]+ m/z 447.24, found 447.
To a solution of 2-aminobenzamide 1 (6.00 g, 44.10 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid 2 (10.11 g, 44.10 mmol) in pyridine (50 mL) was added EDCI (8.45 g, 44.10 mmol). then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers was washed by 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give product of tert-butyl 4-((2-carbamoylphenyl)carbamoyl)piperidine-1-carboxylate 3 (12.00 g, 79% yield) as a white solid.
LCMS (ESI) calcd for C18H25N3O4 [M+Na]+ m/z 370.42, found 370.15.
To the solution of tert-butyl 4-((2-carbamoylphenyl)carbamoyl)piperidine-1-carboxylate 3 (12.00 g, 34.50 mmol) in Diglyme (80 mL) was added KOH (2.13 g, 37.95 mmol), heated to 140° C. stirred for 2 h, cooled to 0° C., ice-water (100 mL) added then adjusted pH<7 using 1M HCl solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of tert-butyl 4-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate 4 (11.00 g, 97% yield) as a white solid.
LCMS (ESI) calcd for C18H23N3O3 [M+H]+ m/z 330.17, found 330.20.
To the solution of tert-butyl 4-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate 4 (3.30 g, 10.00 mmol) in DCM (10 mL) were added TFA (10 mL), stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give crude product, ice-water (20 mL) added then adjusted pH>7 using ammonium hydroxide solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of 2-(piperidin-4-yl)quinazolin-4(3H)-one INT-5 (1.50 g, 66% yield) as a white solid.
LCMS (ESI) calcd for C13H15N3O [M+H]+ m/z 230.12, found 230.20.
To a solution of tert-butyl 4-formylpiperidine-1-carboxylate 1 (837.00 mg, 3.85 mmol) in MeOH (50 mL) was added 2-(piperidin-4-yl) quinazolin-4(3H)-one INT-5 (300.00 mg, 1.31 mmol). Then two drops of acetic acid and NaBH3CN (400.00 mg, 6.37 mmol) were added at room temperature. Then the mixture was stirred for 18 h at 65° C. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give tert-butyl 4-((4-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate 2 (500.00 mg, 90% yield) as a white solid.
LCMS (ESI) calcd for C24H34N4O3 [M+H]+ m/z 427.26, found 427.30.
To a solution of tert-butyl 4-((4-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate 2 (500.00 mg, 1.17 mmol) in MeOH (5 mL) was added 4M HCl-Dioxane (10 mL) at room temperature. The reaction mixture stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give 2-(1-(piperidin-4-ylmethyl) piperidin-4-yl) quinazolin-4(3H)-one 3 (350.00 mg, 87% yield) as a white solid.
LCMS (ESI) calcd for C19H26N4O [M+H]+ m/z 327.21, found 327.15.
To a solution of 2-(1-(piperidin-4-ylmethyl)piperidin-4-yl)quinazolin-4(3H)-one 3 (300.00 mg, 0.92 mmol) in DMF (5 mL) was added 5-fluoro-N-methylpicolinamide 4 (354.14 mg, 2.30 mmol). Then Cs2CO3 (2994.20 mg, 9.19 mmol) was added at room temperature. The reaction mixture was irradiated in a microwave reactor at 150° C. for 5 h. the reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (columns: Gemini 5 um C18 150×21.2 mm, mobile phase: ACN-H2O (0.1% FA), gradient: 15-25) to give N-methyl-5-(4-((4-(4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidin-1-yl)picolinamide Compound 8 (100 mg, 98% purity, 24% yield) as a white sold.
LCMS (ESI) calcd for C26H32N6O2 [M+H]+ m/z 461.26, found 461.20.
1H NMR (400 MHz, DMSO) δ 12.13 (s, 1H), 8.37 (d, J=4.8 Hz, 1H), 8.26 (d, J=2.8 Hz, 1H), 8.08 (d, J=7.6 Hz, 1H), 7.82-7.76 (m, 2H), 7.60 (d, J=8.0 Hz, 1H), 7.50-7.35 (m, 2H), 3.91 (d, J=12.4 Hz, 2H), 2.95 (d, J=10.8 Hz, 2H), 2.89-2.75 (m, 5H), 2.62-2.53 (m, 1H), 2.18 (d, J=6.4 Hz, 2H), 1.98-1.73 (m, 9H), 1.23-1.17 (m, 2H).
To a solution of 2-aminobenzamide 1 (6.00 g, 44.10 mmol) and 1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid 2 (9.49 g, 44.10 mmol) in pyridine (50 mL) was added EDCI (8.45 g, 44.10 mmol). then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water and the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers was washed by 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give tert-butyl 3-((2-carbamoylphenyl)carbamoyl)pyrrolidine-1-carboxylate 3 (12.00 g, 77% yield) as a white solid
LCMS (ESI) calcd for C17H23N3O4 [M+Na]+ m/z 356.17, found 356.00.
To the solution of tert-butyl 3-((2-carbamoylphenyl)carbamoyl)pyrrolidine-1-carboxylate 3 (12.00 g, 36.04 mmol) in Diglyme (80 mL) was added KOH (2.42 g, 43.25 mmol), heated to 140° C. stirred for 0.5 h, cooled to 0° C., ice-water (100 mL) added then adjusted pH<7 using 1M HCl solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of tert-butyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate 4 (10.00 g, 83% yield) as a white solid.
LCMS (ESI) calcd for C17H21N3O3 [M+H]+ m/z 316.16, found 316.05.
To the solution of tert-butyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate 4 (10.00 g, 31.75 mmol) in DCM (20 mL) was added TFA (20 mL), stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give crude product, ice-water (20 mL) added then adjusted pH>7 using ammonium hydroxide solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of 2-(pyrrolidin-3-yl)quinazolin-4(3H)-one INT-5-2 (8.00 g, 80% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6, ppm) δ 8.14-8.07 (m, 1H), 7.87-7.76 (m, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.57-7.44 (m, 1H), 3.76-3.61 (m, 1H), 3.61-3.49 (m, 2H), 3.43-3.33 (m, 1H), 3.29 (dt, J=11.4, 5.6 Hz, 1H), 2.37 (td, J=14.0, 7.1 Hz, 1H), 2.21 (td, J=13.5, 7.2 Hz, 1H).
LCMS (ESI) calcd for C12H13N3O [M−H]− m/z 216.11, found 215.95.
To a solution of 2-(pyrrolidin-3-yl)quinazolin-4(3H)-one INT-5-2 (600.00 mg, 2.79 mmol) in MeOH (30 mL) was added tert-butyl 4-oxopiperidine-1-carboxylate 6 (1.67 g, 8.36 mmol), then two drops of acetic acid and NaBH3CN (1.05 g, 16.72 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 93:7) to give tert-butyl 4-(3-(4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidin-1-yl)piperidine-1-carboxylate 7 (400 mg, 34% yield) as a white solid.
LCMS (ESI) calcd for C22H30N4O3 [M+H]+ m/z 399.23, found 399.30.
Tert-butyl 4-(3-(4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidin-1-yl)piperidine-1-carboxylate 7 (400 mg, 0.81 mmol) was added to HCl in dioxane (4 M, 10 mL), stirred for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to give 2-(1-(piperidin-4-yl)pyrrolidin-3-yl)quinazolin-4(3H)-one 8 (200.00 mg, 79% yield) as a white solid.
LCMS (ESI) calcd for C17H22N4O [M+H]+ m/z 299.18, found 299.25.
To a solution 2-(1-(piperidin-4-yl)pyrrolidin-3-yl)quinazolin-4(3H)-one 8 (200.00 mg, 0.67 mmol) in DMF (10 mL) was added Cs2CO3 (2.18 g, 6.70 mmol) and 5-fluoro-N-methylpicolinamide 9 (432.44 mg, 2.01 mmol). The mixture was stirred for 6 h under 150° C. using microwave. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to N-methyl-5-(4-(3-(4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidin-1-yl)piperidin-1-yl)picolinamide 9 (100 mg, 95% purity, 33% yield) as a yellow solid.
LCMS (ESI) calcd for C24H28N6O2 [M+H]+ m/z 433.23, found 433.25.
Compound 9 was separated by SFC (Column: Daicel CHIRALPAK OJ-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=70/30) and concentrated under reduced pressure to afford the first fraction as Compound 10a (16.4. mg, 100% purity, ee %: 100, off-white solid) and the second fraction as Compound 10b (12.3 mg, 100% purity, ee %: 100, off-white solid)
1H NMR (400 MHz, DMSO-d6, ppm) δ 12.16 (s, 1H), 8.40-8.37 (m, 1H), 8.27 (d, J=2.8 Hz, 1H), 8.07 (dd, J=7.6, 1.6 Hz, 1H), 7.82-7.75 (m, 2H), 7.61 (d, J=8.0 Hz, 1H), 7.48-7.44 (m, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 3.82 (d, J=12.8 Hz, 2H), 3.07 (t, J=8.6 Hz, 2H), 2.95 (t, J=10.8 Hz, 2H), 2.86-2.79 (m, 2H), 2.78-2.77 (m, 3H), 2.69-2.63 (m, 1H), 2.33-2.32 (m, 1H), 2.20-2.13 (m, 2H), 1.96-1.93 (m, 2H), 1.58-1.47 (m, 2H).
LCMS (ESI) calcd for C24H28N6O2 [M+H]+ m/z 433.23, found 433.20.
1H NMR (400 MHz, DMSO-d6, ppm) δ 8.39-8.38 (m, 1H), 8.28 (d, J=3.2 Hz, 1H), 8.08 (dd, J=7.6, 1.6 Hz, 1H), 7.83-7.76 (m, 2H), 7.61 (d, J=8.0 Hz, 1H), 7.47 (t, J=7.6 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 3.83 (d, J=12.8 Hz, 2H), 3.09 (d, J=8.4 Hz, 2H), 2.95 (t, J=12.4 Hz, 2H), 2.88-2.79 (m, 2H), 2.79 (d, J=4.8 Hz, 3H), 2.68-2.66 (m, 1H), 2.39-2.31 (m, 1H), 2.21-2.14 (m, 2H), 1.97-1.94 (m, 2H), 1.59-1.47 (m, 2H).
LCMS (ESI) calcd for C24H28N6O2 [M+H]+ m/z 433.23, found 433.20.
To a solution of 2-aminobenzamide 1 (2.80 g, 20.60 mmol) and 2-(1-(tert-butoxycarbonyl) piperidin-3-yl)acetic acid 2 (5.00 g, 20.60 mmol) in pyridine (50 mL) was added EDCI (3.95 g, 20.60 mmol). then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers was washed with 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give product of tert-butyl 3-(2-((2-carbamoylphenyl)amino)-2-oxoethyl)piperidine-1-carboxylate 3 (7.00 g, 94% yield) as a white solid.
LCMS (ESI) calcd for C19H27N3O4 [M+H]+ m/z 362.20, found 362.25.
To the solution of tert-butyl 3-(2-((2-carbamoylphenyl)amino)-2-oxoethyl)piperidine-1-carboxylate 3 (7.60 g, 21.00 mmol) in Diglyme (80 mL) was added KOH (1.18 g, 21.00 mmol), heated to 140° C. stirred for 2 h, cooled to 0° C., ice-water (100 mL) added then adjusted pH<7 using 1M HCl solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of tert-butyl 3-((4-oxo-3,4-dihydroquinazolin-2-yl)methyl)piperidine-1-carboxylate 4 (6.80 g, 94% yield) as a white solid.
LCMS (ESI) calcd for C19H25N3O3 [M+H]+ m/z 344.19, found 344.15.
To a solution of tert-butyl 3-((4-oxo-3,4-dihydroquinazolin-2-yl)methyl)piperidine-1-carboxylate 4 (2.00 g, 5.80 mmol) in DCM (10 mL) were added TFA (10 mL), stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give crude product, ice-water (20 mL) added then adjusted pH>7 using ammonium hydroxide solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of 2-(piperidin-3-ylmethyl)quinazolin-4(3H)-one INT-5-3 (1.20 g, 85% yield) as a white solid.
LCMS (ESI) calcd for C14H17N3O [M+H]+ m/z 244.14, found 244.20.
To a solution of 5-hydroxy-N-methylpicolinamide 1 (500.00 mg, 3.29 mmol) and Cs2CO3 (3.22 g, 9.87 mmol) in DMF (15 mL) was added 1-bromo-2-chloroethane 2 (943.64 mg, 6.58 mmol). then the mixture was stirred for 3 h at 50° C. The reaction mixture was quenched with water, aqueous layer was extracted with EtOAc (50 mL×3), the combined organic layers was concentrated under reduced pressure. the residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 80:20) to give tert-butyl 4-((2-carbamoylphenyl)carbamoyl)piperidine-1-carboxylate 2 (500.00 mg, 71% yield) as a colorless oil.
LCMS (ESI) calcd for C9H11ClN2O2 [M+H]+ m/z 215.05, found 215.00.
To a solution of tert-butyl 4-((2-carbamoylphenyl)carbamoyl)piperidine-1-carboxylate 2 (500.00 mg, 2.33 mmol) and Cs2CO3 (2.28 g, 6.99 mmol) in DMF (15 mL) were added 2-(piperidin-3-ylmethyl)quinazolin-4(3H)-one INT5-3 (566.91 mg, 2.33 mmol) and KI (1.16 g, 6.99 mmol). then the mixture was stirred for 3 h at 80° C. The reaction mixture was quenched with water, aqueous layer was extracted with EtOAc (50 mL×3), the combined organic layers was concentrated under reduced pressure. the residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give N-methyl-5-(2-(3-((4-oxo-3,4-dihydroquinazolin-2-yl)methyl)piperidin-1-yl)ethoxy)picolinamide 3 (200.00 mg, 20% yield) as a yellow solid.
LCMS (ESI) calcd for C23H27N5O3 [M+H]+ m/z 422.21, found 422.10.
Compound 3 was separated by SFC (Column: Daicel CHIRALPAK OJ-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (FA)]=70/30) and concentrated under reduced pressure to afford the first fraction as Compound 14a (31.6 mg, 100% purity, ee %: 100, white solid) and the second fraction as Compound 14b (28.1 mg, 99% purity, ee %: 99, white solid) Compound 14a 1H NMR (400 MHz, DMSO-d6, ppm) δ 12.15 (s, 1H), 8.53 (q, J=9.6, 4.8 Hz, 1H), 8.23 (d, J=2.8 Hz, 1H), 8.07 (dd, J=8.0, 1.2 Hz, 1H), 7.92 (d, J=8.8 Hz, 1H), 7.79-7.73 (m, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.49-7.42 (m, 2H), 4.18 (t, J=5.6 Hz, 2H), 2.90-2.81 (m, 2H), 2.79 (d, J=4.8 Hz, 3H), 2.73-2.65 (m, 2H), 2.55-2.51 (m, 2H), 2.17-2.00 (m, 2H), 1.93-1.85 (m, 1H), 1.70-1.59 (m, 2H), 1.51-1.38 (m, 1H), 1.02-0.99 (m, 1H).
LCMS (ESI) Calcd for C23H27N5O3 [M+H]+ m/z 422.21, Found 422.25.
1H NMR (400 MHz, DMSO-d6, ppm) δ 12.15 (s, 1H), 8.53 (q, J=9.6, 4.8 Hz, 1H), 8.23 (d, J=2.8 Hz, 1H), 8.07 (dd, J=8.0, 1.2 Hz, 1H), 7.92 (d, J=8.8 Hz, 1H), 7.79-7.73 (m, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.49-7.42 (m, 2H), 4.18 (t, J=5.6 Hz, 2H), 2.90-2.81 (m, 2H), 2.79 (d, J=4.8 Hz, 3H), 2.73-2.65 (m, 2H), 2.55-2.51 (m, 2H), 2.17-2.00 (m, 2H), 1.93-1.85 (m, 1H), 1.70-1.59 (m, 2H), 1.51-1.38 (m, 1H), 1.02-0.99 (m, 1H).
LCMS (ESI) calcd for C23H27N5O3 [M+H]+ m/z 422.21, found 422.25.
To a solution of ethyl 1H-pyrazole-4-carboxylate 1 (2.00 g, 14.30 mmol) and tert-butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate 2 (4.99 g, 17.87 mmol) in DMF (60 mL) was added Cs2CO3 (10.02 g, 30.74 mmol). Then the mixture was stirred for 18 h at 120° C. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (200 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 95:5) to give to give product of tert-butyl 4-(4-(ethoxycarbonyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 3 (2.00 g, 41% yield) as a white solid.
LCMS (ESI) calcd for C16H25N3O4 [M+H]+ m/z 324.18, found 324.20.
To a solution of tert-butyl 4-(4-(ethoxycarbonyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 3 (1.50 g, 4.80 mmol) in MeOH/H2O=1:1 (40 mL) was added LiOH (460.00 mg, 19.20 mmol).
The mixture was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure at 40° C. to obtain residue. To this was added water and acidified with 1 M aqueous HCl to adjust pH=4-5 at 0° C., the aqueous layer was extracted with EtOAc (200 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 95:5) to give 1-(1-(tert-butoxycarbonyl)piperidin-4-yl)-1H-pyrazole-4-carboxylic acid 4 (1.00 g, 67% yield) as a white solid.
LCMS (ESI) calcd for C14H21N3O4 [M−56+H]+ m/z 240.15, found 240.20.
1H NMR (400 MHz, DMSO-d6, ppm) δ 12.29 (s, 1H), 8.30 (s, 1H), 7.81 (s, 1H), 4.52-4.30 (m, 1H), 4.03 (d, J=11.2 Hz, 2H), 2.88 (s, 2H), 1.99 (d, J=12.4 Hz, 2H), 1.78 (dt, J=12.4, 8.0 Hz, 2H), 1.41 (s, 9H).
To a solution of 2-aminobenzamide 5 (299.66 mg, 2.20 mmol) and 1-(1-(tert-butoxycarbonyl)piperidin-4-yl)-1H-pyrazole-4-carboxylic acid 4 (650.00 mg, 2.20 mmol) in pyridine (25 mL) was added EDCI (421.91 mg, 2.20 mmol). then the mixture was stirred for 12 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers was washed by 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give product of tert-butyl 4-(4-((2-carbamoylphenyl)carbamoyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 6 (720.00 mg, 71% yield) as a white solid.
LCMS (ESI) calcd for C21H27N5O4 [M+H]+ m/z 414.21, found 414.25.
To the solution of tert-butyl 4-(4-((2-carbamoylphenyl)carbamoyl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 6 (700.00 mg, 1.69 mmol) in Diglyme (15 mL) was added KOH (113.99 mg, 2.03 mmol), heated to 140° C. stirred for 2 h, cooled to 0° C., ice-water (10 mL) added then adjusted pH<7 using 1M HCl solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of tert-butyl 4-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 7 (600 mg, 67% yield) as a white solid.
LCMS (ESI) calcd for C21H25N5O3 [M+H]+ m/z 396.20, found 396.25.
To a solution of tert-butyl 4-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate 7 (350.00 mg, 0.88 mmol) at room temperature was added HCl-Dioxane (4 M, 40 mL). The reaction mixture was stirred at room temperature for 1 h. After reaction completed, the mixture was concentrated under reduced pressure to obtain crude 2-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)quinazolin-4(3H)-one 8 (200 mg, 73% yield) as a white solid.
LCMS (ESI) calcd for C16H17N5O [M+H]+ m/z 296.14, found 296.25.
To a solution 2-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)quinazolin-4(3H)-one 8 (200.00 mg, 0.68 mmol) in DMF (10 mL) was added Cs2CO3 (2.21 g, 6.77 mmol) and 5-fluoro-N-methylpicolinamide INT-9-1 (260.96 mg, 1.69 mmol). The mixture was stirred for 6 h under 150° C. using microwave. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give crude product which was purified by prep-HPLC (Gemini 5 um C18 150×21.2 mm, mobile phase: ACN-H2O (0.1% FA), gradient: 25-75) to give N-methyl-5-(4-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-1-yl)piperidin-1-yl)picolinamide Compound 17 (30 mg, 98% purity, 9% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6, ppm) δ 12.30 (s, 1H), 8.67 (s, 1H), 8.43-8.40 (m, 1H), 8.35 (d, J=2.8 Hz, 1H), 8.26 (s, 1H), 8.09 (d, J=8.0 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.80-7.76 (m, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.50-7.42 (m, 2H), 4.56-4.53 (m, 1H), 4.08 (d, J=12.8 Hz, 2H), 3.10 (t, J=11.6, 10.8 Hz, 2H), 2.79 (d, J=4.8 Hz, 3H), 2.19-2.17 (m, 2H), 2.06-1.98 (m, 2H).
LCMS (ESI) calcd for C23H23N7O2 [M+H]+ m/z 430.19, found 430.25.
To a solution of 2-amino-3-chlorobenzoic acid 1 (10.00 g, 58.30 mmol) in HOAC (100 mL) was added KOCN (14.19 g, 174.90 mmol) slowly at room temperature. The mixture was heated to 100° C. stirred for 15 hours, cooled to 0° C., ice-water (30 mL) added. The precipitate was collected by filtration, washed with petroleum ether:EtOAc=5:1 (100 mL), filtered to provide 8-chloroquinazoline-2,4(1H,3H)-dione 2 (5.00 g, 40% yield) as a white solid which was used directly in next step without further purification.
LCMS (ESI) calcd for C8H5ClN2O2[M+H]+ m/z 197.00, found 196.95.
To a solution of 8-chloroquinazoline-2,4(1H,3H)-dione 2 (5.00 g, 25.40 mmol) in POCl3 (45 mL) was added DMF (3 mL) slowly at room temperature. The mixture was heated to 100° C. for 12 hours. The resulting mixture was diluted with ice water (200 mL) and extracted with EtOAc (400 mL×3). The combine organic phases were washed with brine, dried over sodium sulfate, concentrated and purified by silica gel column chromatography (eluting with EtOAc/petroleum ether, 100:0 to 50:50) to give 2,4,8-trichloroquinazoline 3 (2.00 g, 32% yield) as a white solid.
LCMS (ESI) calcd for C8H3Cl3N2[M+H]+ m/z 232.94, found 232.90.
To a solution of 2,4,8-trichloroquinazoline 3 (2.00 g, 8.60 mmol) in THF/H2O=1:1 (200 mL) was added NaOH (690 mg, 17.20 mmol). The mixture was stirred at room temperature for 2 h.
The reaction mixture was concentrated under reduced pressure at 40° C. to obtain residue. To this was added water and acidified with 1 M aqueous HCl to adjust pH 4-5 at 0° C. The mixture was extracted with EtOAc (200 mL×3). The combined organic phases were washed with brine (100 mL×3), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by Flash chromatography (eluent: DCM/MeOH=100:0 to 95:5) to afford 2,8-dichloroquinazolin-4(3H)-one 4 (1.60 g, 82% yield) as a white solid.
LCMS (ESI) calcd for C8H4Cl2N2O [M+H]+ m/z 214.97, found 214.95.
To a solution of 2,8-dichloroquinazolin-4(3H)-one 4 (400 mg, 1.86 mmol) in Dioxane/H2O=10:1 (40 mL) were added tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (690 mg, 2.23 mmol), Pd(dppf)Cl2 (50 mg, 0.07 mmol) and Na2CO3 (591 mg, 5.58 mmol) successively at room temperature. The reaction mixture was stirred at 80° C. for 18 h under N2. The mixture was diluted with water (50 mL) and extracted with EtOAc (100 mL×3). The combined organic layer was washed with brine (100 mL×2), dried over Na2SO4, filtered and concentrated in vacuo to get crude product, which was purified by flash column chromatography (PE/EtOAc=100:0 to 20:80) to afford tert-butyl 4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate 5 (550 mg, 77% yield) as a yellow solid.
LCMS (ESI) calcd for C18H20ClN3O3[M+H]+ m/z 362.12, found 362.10.
To a solution of tert-butyl 4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate 5 (550 mg, 1.52 mmol) in EtOAc (200 mL) was added PtO2 (110 mg). The mixture was evacuated and backfilled with hydrogen three times and then charged with hydrogen. The resulting mixture was stirred at room temperature for 1 hour. Then the mixture was filtered through celite and concentrated under vacuum to give crude product, which was purified by flash column chromatography (PE/EtOAc=100:0 to 50:50) to afford tert-butyl 4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate 6 (400 mg, 65% yield) as a yellow solid.
LCMS (ESI) calcd for C18H22ClN3O3[M+H]+ m/z 364.13, found 364.10.
To a solution of tert-butyl 4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate 6 (400 mg, 1.21 mmol) in DCM (5 mL) was added TFA (2 mL), stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give crude product, ice-water (2 mL) added then adjusted pH>7 using ammonium hydroxide solution, precipitate formed, filtered, the filter cake washed with ice-water (5 mL*3), then dried to give product of 8-chloro-2-(piperidin-4-yl)quinazolin-4(3H)-one 7 (300 mg, 89% yield) as a white solid.
LCMS (ESI) calcd for C13H14ClN3O [M+H]+ m/z 264.08, found 264.10.
To a solution of 8-chloro-2-(piperidin-4-yl)quinazolin-4(3H)-one 7 (300 mg, 1.14 mmol) in MeOH (60 mL) was added tert-butyl 4-oxopiperidine-1-carboxylate (727 mg, 3.41 mmol), then two drops of acetic acid and NaBH3CN (428 mg, 6.82 mmol) were added at room temperature.
The reaction mixture stirred at 50° C. for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give tert-butyl 4-((4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate 8 (300 mg, 51% yield) as a white solid.
LCMS (ESI) calcd for C24H33ClN4O3[M+H]+ m/z 361.17, found 361.05.
Tert-butyl 4-((4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate 8 (300 mg, 0.65 mmol) was added to HCl in dioxane (4 M, 30 mL), stirred for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to give 8-chloro-2-(1-(piperidin-4-ylmethyl)piperidin-4-yl)quinazolin-4(3H)-one 9 (200 mg, 81% yield) as a white solid.
LCMS (ESI) calcd for C19H25ClN4O [M+H]+ m/z 361.17, found 361.05.
To a solution 8-chloro-2-(1-(piperidin-4-ylmethyl)piperidin-4-yl)quinazolin-4(3H)-one 9 (150 mg, 0.42 mmol) in DMF (10 mL) were added Cs2CO3 (1.35 g, 4.16 mmol) and 5-fluoro-N-methylpicolinamide INT-9-1 (160 mg, 1.03 mmol). The mixture was stirred for 6 h under 150° C. using microwave. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give crude 5-(4-((4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidin-1-yl)-N-methylpicolinamide Compound 40 as a yellow solid. The crude product was purified by prep-HPLC (Gemini 5 um C18 150×21.2 mm, mobile phase: ACN—H2O (0.1% FA), gradient: 20-80) to give 5-(4-((4-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidin-1-yl)methyl)piperidin-1-yl)-N-methylpicolinamide Compound 40 (24.2 mg, 96% purity, 11% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6, ppm) δ 12.39 (s, 1H), 8.40-8.37 (m, 1H), 8.26 (d, J=2.8 Hz, 1H), 8.04 (dd, J=7.6, 1.2 Hz, 1H), 7.94 (dd, J=7.8, 1.2 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.46-7.36 (m, 2H), 3.91 (d, J=12.8 Hz, 2H), 2.97-2.94 (m, 2H), 2.86-2.81 (m, 2H), 2.77 (d, J=4.8 Hz, 3H), 2.65-2.53 (m, 1H), 2.18 (d, J=6.8 Hz, 2H), 1.97-1.80 (m, 9H), 1.23-1.17 (m, 2H).
LCMS (ESI) calcd for C26H31ClN6O2[M+H]+ m/z 495.22, found 495.15.
To a solution of 2,8-dichloroquinazolin-4(3H)-one 1 (500 mg, 2.35 mmol) in 1,4-dioxane/H2O=5:1 (60 mL) was added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 2 (900 mg, 3.00 mmol), Pd(dppf)Cl2 (170 mg, 0.23 mmol) and Na2CO3 (740 mg, 6.98 mmol) under N2. The reaction mixture was stirred at 80° C. for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 91:9) to give tert-butyl 3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 3 (400 mg, 40% yield) as a white solid.
LCMS (ESI) calcd for C17H18ClN3O3 [M+H]+ m/z 348.10, found 348.17.
To a solution of tert-butyl 3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 3 (400 mg, 1.15 mmol) in EtOAc (50 mL) was added PtO2 (80 mg, 0.35 mmol) under H2. The reaction mixture stirred for 2 h at room temperature. The mixture was filtered through a Celite pad, and the filtrate was concentrated to give crude product of tert-butyl 3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate 4 (200 mg, 50% yield) as a white solid.
LCMS (ESI) calcd for C17H20ClN3O3 [M+H]+ m/z 350.12, found 350.18.
Tert-butyl 3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate 4 (200 mg, 0.57 mmol) was added to HCl in dioxane (4 M, 5 mL), stirred for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to give product of 8-chloro-2-(pyrrolidin-3-yl)quinazolin-4(3H)-one 5 (130 mg, 82% yield) as a white solid.
LCMS (ESI) calcd for C12H12ClN3O [M+H]+ m/z 250.07, found 250.12.
To a solution of 8-chloro-2-(pyrrolidin-3-yl)quinazolin-4(3H)-one 5 (80 mg, 0.32 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide 6 (50 mg, 0.21 mmol), then three drops of acetic acid and NaBH3CN (15 mg, 0.24 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give 5-(4-(3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 7 (40 mg, 40% yield) as a white solid.
LCMS (ESI) calcd for C24H27ClN6O2 [M+H]+ m/z 467.19, found 467.28.
5-(4-(3-(8-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 7 was separated by SFC (Column: DAICEL AS-H 4.6 mm I.D.*250 mmL 5 m; Mobile phase: CO2/IPA[0.1% NH3(7M Solution in MeOH)]=60/40) and concentrated under reduced pressure to afford the first fraction as Compound 52a (14.4 mg, 97% purity, ee %: 100, white solid) and the second fraction as Compound 52b (15.4 mg, 99% purity, ee %: 100, white solid)
1H NMR (400 MHz, DMSO) δ 8.42 (d, J=4.8 Hz, 1H), 8.27 (d, J=2.8 Hz, 1H), 8.05 (d, J=8.0 Hz, 1H), 7.94 (d, J=8.0 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.48-7.38 (m, 2H), 3.83 (d, J=12.8 Hz, 2H), 3.40-3.32 (m, 1H), 3.06 (t, J=9.2 Hz, 1H), 2.95 (dd, J=22.0, 12.0 Hz, 3H), 2.80-2.68 (m, 5H), 2.38 (t, J=10.4 Hz, 1H), 2.24-2.15 (m, 2H), 1.96 (d, J=11.2 Hz, 2H), 1.53 (dd, J=22.8, 10.8 Hz, 2H).
LCMS (ESI) calcd for C24H27ClN6O2 [M+H]+ m/z 467.19, found 467.28.
1H NMR (400 MHz, DMSO) δ 8.49 (d, J=4.8 Hz, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.11 (d, J=7.6 Hz, 1H), 8.01 (d, J=7.6 Hz, 1H), 7.89 (d, J=8.8 Hz, 1H), 7.55-7.45 (m, 2H), 3.90 (d, J=12.4 Hz, 2H), 3.45-3.39 (m, 1H), 3.13 (t, J=8.8 Hz, 1H), 3.01 (dd, J=22.8, 11.6 Hz, 3H), 2.87-2.77 (m, 5H), 2.43 (d, J=10.0 Hz, 1H), 2.26 (d, J=6.4 Hz, 2H), 2.02 (d, J=11.2 Hz, 2H), 1.59 (d, J=12.0 Hz, 2H).
LCMS (ESI) calcd for C24H27ClN6O2 [M+H]+ m/z 467.19, found 467.28.
To a solution of 2-aminobenzamide 1 (2.00 g, 14.70 mmol) and {1-[(tert-butoxy)carbonyl]piperidin-4-yl}acetic acid 2 (3.58 g, 14.70 mmol) in pyridine (50 mL) was added EDCI (2.82 g, 14.70 mmol). then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers was washed by 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give tert-butyl 4-{[(2-carbamoylphenyl)carbamoyl]methyl}piperidine-1-carboxylate 3 (5.76 g, 92% yield) as a yellow oil.
LCMS (ESI) calcd for C19H27N3O4 [M+H]+ m/z 362.20, found 262.30.
To the solution of tert-butyl 4-{[(2-carbamoylphenyl)carbamoyl]methyl}piperidine-1-carboxylate 3 (5.76 g, 15.90 mmol) in Diglyme (40 mL) was added KOH (1.16 g, 20.67 mmol), heated to 140° C. stirred for 2 h, cooled to 0° C., ice-water (50 mL) added then adjusted pH<7 using 1M HCl solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of tert-butyl 4-[(4-oxo-3H-quinazolin-2-yl)methyl]piperidine-1-carboxylate 4 (4.20 g, 73% yield) as a white solid.
LCMS (ESI) calcd for C19H25N3O3 [M+H]+ m/z 344.19, found 344.25.
To a solution of tert-butyl 4-[(4-oxo-3H-quinazolin-2-yl)methyl]piperidine-1-carboxylate 4 (4.20 g, 10.00 mmol) in DCM (13 mL) was added TFA (13 mL), stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give crude product, ice-water (20 mL) added then adjusted pH>7 using ammonium hydroxide solution, precipitate formed, filtered, the filter cake washed with ice-water (50 mL*3), then dried to give product of 2-(piperidin-4-ylmethyl)-3H-quinazolin-4-one 5 (2.32 g, 90% yield) as a white solid.
LCMS (ESI) calcd for C14H17N3O [M+H]+ m/z 244.14, found 244.25.
To a solution of 2-(piperidin-4-ylmethyl)-3H-quinazolin-4-one 5 (500 mg, 2.06 mmol) in MeOH (50 mL) was added tert-butyl 4-oxopiperidine-1-carboxylate (532 mg, 2.67 mmol), then two drops of acetic acid and NaBH3CN (504 mg, 8.01 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 18 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 91:9) to give tert-butyl 4-{4-[(4-oxo-3H-quinazolin-2-yl)methyl]piperidin-1-yl}piperidine-1-carboxylate 6 (244 mg, 25% yield) as a white solid.
LCMS (ESI) calcd for C24H34N4O3 [M+H]+ m/z 427.26, found 427.40.
Tert-butyl 4-{4-[(4-oxo-3H-quinazolin-2-yl)methyl]piperidin-1-yl}piperidine-1-carboxylate 6 (244 mg, 0.57 mmol) was added to HCl in dioxane (4 M, 8 mL), stirred for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to give 2-{1[1-(piperidin-4-yl)piperidin-4-yl]methyl}-3H-quinazolin-4-one 7 (187 mg, 90% yield) as a white solid.
LCMS (ESI) calcd for C19H26N4O [M+H]+ m/z 327.21, found 327.44.
To a solution of 2-{[1-(piperidin-4-yl)piperidin-4-yl]methyl}-3H-quinazolin-4-one 7 (200 mg, 0.61 mmol) in DMF (16 mL) were added Cs2CO3 (1.20 g, 3.68 mmol) and 5-fluoro-N-methylpyridine-2-carboxamide 5 (208 mg, 1.35 mmol). The mixture was stirred for 5 h under 150° C. using microwave. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 91:9) to give N-methyl-5-(4-{4-[(4-oxo-3H-quinazolin-2-yl)methyl]piperidin-1-yl}piperidin-1-yl)pyridine-2-carboxamide Compound 54 (20.11 mg, 99% purity, 7% yield) as a white solid.
1H NMR (400 MHz, DMSO) δ 12.16 (s, 1H), 8.37 (d, J=4.8 Hz, 1H), 8.25 (d, J=2.8 Hz, 1H), 8.07 (d, J=8.0 Hz, 1H), 7.83-7.74 (m, 2H), 7.59 (d, J=8.0 Hz, 1H), 7.45 (t, J=7.6 Hz, 1H), 7.37 (dd, J=8.8, 2.8 Hz, 1H), 3.93 (d, J=12.4 Hz, 2H), 2.90-2.74 (m, 8H), 2.14 (t, J=10.8 Hz, 2H), 1.81 (d, J=11.6 Hz, 3H), 1.64 (d, J=10.8 Hz, 2H), 1.49 (d, J=8.8 Hz, 2H), 1.23 (d, J=9.2 Hz, 3H).
LCMS (ESI) calcd for C24H28N6O2 [M+H]+ m/z 461.26, found 461.40.
To a solution of 2-fluoro-6-methylbenzoic acid 1 (1.00 g, 6.50 mmol) and diethylamine 2 (0.57 g, 7.80 mmol) in DCM (50 mL) was added tritolylphosphine (50% in EtOAc) (3.10 g, 9.75 mmol) and DIEA (4.20 g, 32.50 mmol) at room temperature. The mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAC=100:0 to 90:10) to give N,N-diethyl-2-fluoro-6-methylbenzamide 3 (0.90 g, 63% yield) as a white solid.
LCMS (ESI) calcd for C12H16FNO [M+H]+ m/z 210.12, found 210.09.
A dry three-necked round-bottomed flask with an addition funnel at −78° C. under inert atmosphere was charged with anhydrous THF (30 ml). A solution of LDA (2.5 M in THF, 0.9 ml) was added dropwise. The internal temperature was maintained below −70° C. during the entire addition process. After 0.5 h at −78° C. a solution of N,N-diethyl-2-fluoro-6-methylbenzamide 3 (300 mg, 1.43 mmol) in anhydrous THF (10 ml) was added dropwise and the mixture was stirred for 1 h, then tert-butyl 3-cyanopyrrolidine-1-carboxylate 4 (366 mg, 1.86 mmol) was added at −78° C. The mixture was stirred for 18 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAC=100:0 to 80:20) to give 3-hydroxy-1-(1-methylphenyl)pyrrolidine-3-carboxylic acid 5 (160 mg, 32% yield) as a white solid.
LCMS (ESI) calcd for C18H21FN2O3 [M+H]+ m/z 333.15, found 333.25.
3-Hydroxy-1-(1-methylphenyl)pyrrolidine-3-carboxylic acid 5 (160 mg, 0.48 mmol) was added to HCl-dioxane (4M, 10 mL). The mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure to give 8-fluoro-3-(pyrrolidin-3-yl)-2H-isoquinolin-1-one 6 (120 mg, 97% yield) as a white solid.
LCMS (ESI) calcd for C13H13FN2O [M+H]+ m/z 233.10, found 233.12.
To a solution of 8-fluoro-3-(pyrrolidin-3-yl)-2H-isoquinolin-1-one 6 (120 mg, 0.52 mmol) in MeOH (15 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide INT (100 mg, 0.55 mmol), then two drops of acetic acid and NaBH3CN (30 mg, 0.46 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give 5-{4-[3-(8-fluoro-1-oxo-2H-isoquinolin-3-yl)pyrrolidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide Compound 136rac (65 mg, 25% yield, 99% purity) as a yellow solid.
1H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.41-8.38 (m, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.62-7.58 (m, 1H), 7.45-7.36 (m, 2H), 7.10 (dd, J=11.6, 8.0 Hz, 1H), 6.46 (d, J 1.6 Hz, 1H), 3.84-3.80 (m, 2H), 3.23-3.16 (m, 1H), 3.00-2.92 (m, 3H), 2.84-2.65 (m, 6H), 2.38-2.33 (m, 1H), 2.24-2.18 (m, 1H), 1.97-1.97 (m, 2H), 1.84-1.76 (m, 1H), 1.60-1.49 (m, 2H).
LCMS (ESI) calcd for C25H28FN5O2 [M+H]+ m/z 450.22, found 450.30.
5-{4-[3-(8-fluoro-1-oxo-2H-isoquinolin-3-yl)pyrrolidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide 136rac was separated by SFC (Column: Daicel CHIRALPAK OJ-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=70/30) and concentrated under reduced pressure to afford the first fraction as Compound 136a (12.8 mg, 99% purity, ee %: 100, white solid) and the second fraction as Compound 136b (13.8 mg, 99% purity, ee %: 97, white solid)
1H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.41 (q, J=4.4 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.62-7.58 (m, 1H), 7.42-7.36 (m, 2H), 7.10 (dd, J=12.0, 8.0 Hz, 1H), 6.46 (s, 1H), 3.84-3.80 (m, 2H), 3.22-3.15 (m, 1H), 3.00-2.90 (m, 3H), 2.81-2.63 (m, 6H), 2.36-2.31 (m, 1H), 2.23-2.18 (m, 1H), 1.97-1.93 (m, 2H), 1.87-1.79 (m, 1H), 1.56-1.52 (m, 2H).
LCMS (ESI) calcd for C25H28FN5O2 [M+H]+ m/z 450.22, found 450.20.
1H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.40 (q, J=4.8 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.61-7.58 (m, 1H), 7.42-7.36 (m, 2H), 7.10 (dd, J=11.6, 8.0 Hz, 1H), 6.46 (s, 1H), 3.84-3.80 (m, 2H), 3.22-3.15 (m, 1H), 3.00-2.90 (m, 3H), 2.81-2.63 (m, 6H), 2.36-2.31 (m, 1H), 2.25-2.18 (m, 1H), 1.97-1.94 (m, 2H), 1.87-1.79 (m, 1H), 1.56-1.52 (m, 2H).
LCMS (ESI) calcd for C25H28FN5O2 [M+H]+ m/z 450.22, found 450.25.
To a solution of 6-bromo-2-methoxypyridin-3-amine 1 (1.38 g, 6.80 mmol) in 1,4-dioxane/H2O=4:1 (20 mL) was added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydropyrrole-1-carboxylate 2 (2.01 g, 6.80 mmol), then Pd(dppf)Cl2 (0.50 g, 0.68 mmol) and Na2CO3 (2.23 g, 21.08 mmol) were added at room temperature. The reaction mixture was stirred under nitrogen at 80° C. for 3 h under N2. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 85:15) to give tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)-2,5-dihydropyrrole-1-carboxylate 3 (1.50 g, 72% yield) as a white solid.
LCMS (ESI) calcd for C15H21N3O3 [M+H]+ m/z 292.16, found 292.19.
To a solution of tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)-2,5-dihydropyrrole-1-carboxylate 3 (1.50 g, 5.10 mmol) in MeOH (20 mL) was added Pd/C (0.54 g, 5.10 mmol) at room temperature. The reaction mixture was stirred under hydrogen at 50° C. for 2 h. After cooling to room temperature, the reaction mixture was filtered, the solution was concentrated under reduced pressure to give product of tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)pyrrolidine-1-carboxylate 4 (1.20 g, 76% yield) as a white solid.
LCMS (ESI) calcd for C15H23N3O3 [M+H]+ m/z 294.17, found 294.18.
To a solution of tert-butyl 3-(5-amino-6-methoxypyridin-2-yl)pyrrolidine-1-carboxylate 4 (1.40 g, 4.80 mmol) in ACN (20 mL) was added tert-Butyl nitrite (1.14 g, 11.04 mmol). Then CuBr (1.38 g, 9.60 mmol) was added at room temperature. The reaction mixture was stirred at 50° C. for 2 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 85:15) to give tert-butyl 3-(5-bromo-6-methoxypyridin-2-yl)pyrrolidine-1-carboxylate 5 (0.65 g, 35% yield) as a white solid.
LCMS (ESI) calcd for C15H21BrN2O3 [M+H]+ m/z 357.07, found 357.11.
To a solution of tert-butyl 3-(5-bromo-6-methoxypyridin-2-yl)pyrrolidine-1-carboxylate 5 (300 mg, 0.84 mmol) in 1,4-dioxane (15 mL) was added Pd(dppf)Cl2 (61 mg, 0.08 mmol). Then Et2Zn (5 mL, 0.14 mml) was added at room temperature. The reaction mixture under nitrogen was stirred at 80° C. for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EA=100:0 to 85:15) to give tert-butyl 3-(5-ethyl-6-methoxypyridin-2-yl)pyrrolidine-1-carboxylate 6 (120 mg, 44% yield) as a white solid.
LCMS (ESI) calcd for C17H26N2O3 [M+H]+ m/z 307.19, found 307.25.
Tert-butyl 3-(5-ethyl-6-methoxypyridin-2-yl)pyrrolidine-1-carboxylate 6 (120 mg, 0.39 mmol) was added to HBr in water (48%, 15 mL). The reaction mixture was stirred at 100° C. for 6 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to give 3-ethyl-6-(pyrrolidin-3-yl)-1H-pyridin-2-one 7 (70 mg, 88% yield) as a white solid.
LCMS (ESI) calcd for C11H16N2O [M+H]+ m/z 193.13, found 193.14.
To a solution of 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT-2 (100 mg, 0.40 mmol) in MeOH (40 mL) was added 3-ethyl-6-(pyrrolidin-3-yl)-1H-pyridin-2-one 7 (115 mg, 0.60 mmol), then two drops of acetic acid and NaBH3CN (38 mg, 0.61 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 12 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to afford 5-(4-(3-(5-ethyl-6-oxo-1,6-dihydropyridin-2-yl)pyrrolidin-1-yl)piperidin-1-yl)-6-fluoro-N-methylpicolinamide 151rac (40 mg, 95% purity, 22.34% yield) as a white solid.
LCMS (ESI) calcd for C23H30FN5O2[M+H]+ m/z 428.24, found 428.10.
Compound 151 was separated by SFC (Column: Daicel Chiralpak AD-H SFC 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=80/20) and concentrated under reduced pressure to afford the first fraction as Compound 151a (15.8 mg, 92% purity, ee %: 100, white solid) and the second fraction as Compound 151b (15.9 mg, 95% purity, ee %: 100, white solid)
1H NMR (400 MHz, DMSO-d6, ppm) δ: 11.28 (s, 1H), 8.44-8.37 (m, 1H), 7.83 (dd, J=8.0, 1.2 Hz, 1H), 7.61-7.52 (m, 1H), 7.17 (d, J=7.2 Hz, 1H), 6.03 (d, J=6.4 Hz, 1H), 3.56-3.43 (m, 2H), 3.17-3.09 (m, 1H), 2.90-2.74 (m, 7H), 2.68-2.60 (m, 2H), 2.35-2.17 (m, 4H), 1.96 (d, J=12.0 Hz, 2H), 1.79-1.70 (m, 1H), 1.63-1.52 (m, 2H), 1.06 (t, J=7.2 Hz, 3H).
LCMS (ESI) calcd for C23H30FN5O2[M+H]+ m/z 428.24, found 428.10.
1H NMR (400 MHz, DMSO-d6, ppm) δ: 11.28 (s, 1H), 8.45-8.35 (m, 1H), 7.83 (dd, J=8.0, 1.2 Hz, 1H), 7.64-7.51 (m, 1H), 7.17 (d, J=6.8 Hz, 1H), 6.03 (d, J=6.8 Hz, 1H), 3.58-3.41 (m, 2H), 3.17-3.09 (m, 1H), 2.93-2.74 (m, 7H), 2.68-2.60 (m, 2H), 2.35-2.16 (m, 4H), 2.01-1.92 (m, 2H), 1.79-1.70 (m, 1H), 1.64-1.52 (m, 2H), 1.06 (t, J=7.2 Hz, 3H).
LCMS (ESI) calcd for C23H30FN5O2[M+H]+ m/z 428.24, found 428.20.
To a solution of 6-chloro-5-fluoropicolinic acid 1 (2.00 g, 11.40 mmol) in DMF (50 mL) was added methanamine (420 mg, 13.68 mmol), DIEA (4.42 g, 34.20 mmol), HATU (6.50 g, 17.10 mmol) at room temperature successively. The mixture was kept stirring at room temperature for 1 h. The resulting mixture was diluted with water and extracted with EtOAc (200 mL×3). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 70:30) to afford 6-chloro-5-fluoro-N-methylpicolinamide 2 (2.00 g, 88% yield) as a white solid.
LCMS (ESI) calcd for C7H6ClFN2O [M+H]+ m/z 189.02, found 188.90.
To a solution 6-chloro-5-fluoro-N-methylpicolinamide 2 (2.00 g, 10.60 mmol) in DMF (20 mL) was added Cs2CO3 (6.91 g, 21.20 mmol) and 1,4-dioxa-8-azaspiro[4.5]decane 3 (3.04 g, 21.20 mmol). The mixture was stirred for 4 h under 120° C. using a sealed tube. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAC=100:0 to 50:50) to afford 6-chloro-N-methyl-5-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)picolinamide 4 (1.60 g, 42% yield) as a white solid.
LCMS (ESI) calcd for C14H18ClN3O3[M+H]+ m/z 312.10, found 311.95.
To a solution 6-chloro-N-methyl-5-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)picolinamide 4 (300 mg, 0.96 mmol) in DMF (20 mL) was added CsF (293 mg, 1.93 mmol). The mixture was stirred for 20 h under 150° C. using microwave. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAC=100:0 to 30:70) to 6-fluoro-N-methyl-5-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)picolinamide 5 (200 mg, 69% yield) as a yellow solid.
LCMS (ESI) calcd for C14H18FN3O3[M+H]+ m/z 296.13, found 295.95.
To the solution of 6-fluoro-N-methyl-5-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)picolinamide 5 (200 mg, 0.68 mmol) in H2O (3 mL) was added formic acid (2 mL), stirred at 50° C. for 1 h, The aqueous solution was adjusted to pH 7-8 with aqueous NaHCO3 solution. The mixture was diluted with water (50 mL) and extracted with EtOAc (100 mL×3). The combined organic layer was washed with brine (100 mL×2), dried over Na2SO4, concentrated to give crude product of 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT-2 (100 mg, 47% yield) as a yellow solid.
LCMS (ESI) calcd for C12H14FN3O2[M+H]+ m/z 252.11, found 251.90.
To a solution of 1,3-dichloro-7-fluoroisoquinoline 1 (500 mg, 2.32 mmol) in MeOH (50 mL), was added MeONa (150 mg, 2.78 mmol) at room temperature. The mixture was stirred for 72 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAC=100:0 to 98:2) to give 3-chloro-7-fluoro-1-methoxyisoquinoline 2 (160 mg, 33% yield) as a white solid.
LCMS (ESI) calcd for C10H7ClFNO [M+H]+ m/z 212.02, found 211.90.
To a solution of 3-chloro-7-fluoro-1-methoxyisoquinoline 2 (160 mg, 0.76 mmol) in dioxane:H2O=5:1 (20 mL) was added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 3 (270 mg, 0.92 mmol), Pd(dppf)Cl2 (50 mg, 0.07 mmol) and K2CO3 (250 mg, 1.81 mmol) at room temperature. The reaction mixture was stirred under N2 at 90° C. for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 95:5) to afford tert-butyl 3-(7-fluoro-1-methoxyisoquinolin-3-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 4 (160 mg, 61% yield) as a white solid.
LCMS (ESI) calcd for C19H21FN2O3 [M+H]+ m/z 345.15, found 345.11.
To a solution of tert-butyl 3-(7-fluoro-1-methoxyisoquinolin-3-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 4 (160 g, 0.46 mmol) in MeOH (20 mL) was added Pd/C (35 mg, 0.29 mmol) under H2. The reaction mixture was stirred at room temperature for 5 h. The mixture was filtered through a Celite pad and the filtrate was concentrated to give tert-butyl 3-(7-fluoro-1-methoxyisoquinolin-3-yl)pyrrolidine-1-carboxylate 5 (140 mg, 88% yield) as a white solid.
LCMS (ESI) calcd for C19H23FN2O3 [M+H]+ m/z 347.17, found 347.18.
Tert-butyl 3-(7-fluoro-1-methoxyisoquinolin-3-yl)pyrrolidine-1-carboxylate 5 (140 mg, 0.40 mmol) was added to HBr in water (48%, 3 mL). The mixture was stirred at 100° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give 7-fluoro-3-(pyrrolidin-3-yl)isoquinolin-1(2H)-one 6 (90 mg, 97% yield) as a white solid.
LCMS (ESI) calcd for C13H13FN2O [M+H]+ m/z 233.10, found 232.95.
To a solution of 7-fluoro-3-(pyrrolidin-3-yl)isoquinolin-1(2H)-one 6 (90 mg, 0.39 mmol) in MeOH (15 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide (INT, 100 mg, 0.43 mmol), then two drops of acetic acid and NaBH3CN (25 mg, 0.40 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the residue was purified by prep-HPLC (columns: Gemini 5 um C18 150×21.2 mm, mobile phase: ACN-H2O (0.1% FA), gradient: 10-25) to give 5-(4-(3-(7-fluoro-1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 154rac (35 mg, 99% purity, 20% yield) as a white solid.
1H NMR (400 MHz, DMSO) δ 11.35 (s, 1H), 8.39 (q, J=4.8 Hz, 1H), 8.30 (d, J=2.8 Hz, 1H), 7.87-7.76 (m, 2H), 7.70 (dd, J=8.8, 5.2 Hz, 1H), 7.59 (td, J=8.8, 2.8 Hz, 1H), 7.43 (dd, J=8.8, 2.8 Hz, 1H), 6.57 (s, 1H), 4.02-3.85 (m, 2H), 3.31-2.71 (m, 11H), 2.40-2.25 (m, 1H), 2.13-1.88 (m, 3H), 1.68-1.52 (m, 2H).
LCMS (ESI) calcd for C25H28FN5O2 [M+H]+ m/z 450.22, found 450.23.
5-(4-(3-(7-fluoro-1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 154rac was separated by SFC (Column: DAICEL OJ-H 4.6 mm I.D.*250 mmL 5 m; Mobile phase: CO2/MeOH[0.1% NH3(7M Solution in MeOH)]=75/25) and concentrated under reduced pressure to afford the first fraction as 154a (10 mg, 99% purity, ee %: 100, white solid) and the second fraction as 154b (10 mg, 97% purity, ee %: 100, white solid)
1H NMR (400 MHz, DMSO) δ 11.25 (s, 1H), 8.39 (d, J=4.8 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.77 (dd, J=9.2, 2.8 Hz, 1H), 7.68 (dd, J=8.8, 5.6 Hz, 1H), 7.56 (td, J=8.8, 2.8 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 6.51 (s, 1H), 3.87-3.77 (m, 2H), 3.25-3.17 (m, 1H), 3.03-2.91 (m, 3H), 2.85-2.75 (m, 4H), 2.74-2.66 (m, 2H), 2.35-2.32 (m, 1H), 2.28-2.17 (m, 1H), 2.01-1.90 (m, 2H), 1.88-1.78 (m, 1H), 1.60-1.47 (m, 2H).
LCMS (ESI) calcd for C25H28FN5O2 [M+H]+ m/z 450.22, found 450.20.
1H NMR (400 MHz, DMSO) δ 11.25 (s, 1H), 8.39 (d, J=4.8 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.77 (dd, J=9.2, 2.8 Hz, 1H), 7.68 (dd, J=8.8, 5.2 Hz, 1H), 7.56 (td, J=8.8, 2.8 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 6.51 (s, 1H), 3.88-3.78 (m, 2H), 3.26-3.16 (m, 1H), 3.03-2.91 (m, 3H), 2.83-2.78 (m, 4H), 2.74-2.66 (m, 2H), 2.35-2.31 (m, 1H), 2.28-2.17 (m, 1H), 2.00-1.92 (m, 2H), 1.89-1.79 (m, 1H), 1.61-1.47 (m, 2H).
LCMS (ESI) calcd for C25H28FN5O2 [M+H]+ m/z 450.22, found 450.20.
To a solution of 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine 1 (5 g, 26.50 mmol) in ACN/DMF=1:1 (100 mL) was added NIS (8.9 g, 39.75 mmol). The resulting mixture was stirred at 90° C. for 3 h. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 72:28) to give 4,6-dichloro-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 2 (2.9 g, 35% yield) as white solid.
LCMS (ESI) calcd for C5HCl2IN4 [M+H]+ m/z 314.86, found 314.85.
To a solution of 4,6-dichloro-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 2 (2.9 g, 9.24 mmol) in THF (40 mL) was added NaH (290 mg, 11.96 mmol) at 0° C., stirred 10 min, then added SEMCl (2.3 g, 13.80 mmol). The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give 4,6-dichloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine 3 (3.6 g, 88% yield) as white solid.
LCMS (ESI) calcd for C11H15Cl2IN4OSi [M+H]+ m/z 444.94, found 444.85.
To a solution of 4,6-dichloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine 3 (1.5 g, 3.39 mmol) in MeOH (45 mL) was added MeONa (183 mg, 3.39 mmol). The mixture was stirred at room temperature for 1 h. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 95:5) to give 6-chloro-3-iodo-4-methoxy-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine 4 (282 mg, 19% yield) as a white solid.
LCMS (ESI) calcd for C12H18ClIN4O2Si [M+H]+ m/z 440.99 found 441.01.
To a solution of 6-chloro-3-iodo-4-methoxy-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine 4 (282 mg, 0.64 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate 5 (491 mg, 2.56 mmol) in DMF (15 mL) was added CuCl2 (131 mg, 0.98 mmol) under N2. The reaction mixture was stirred at 100° C. for 2 h. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 96:4) to 6-chloro-4-methoxy-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine 6 (220 mg, 90% yield) as a white solid.
LCMS (ESI) calcd for C13H18ClF3N4O2Si [M+H]+ m/z 383.08, found 383.08.
To a solution of tert-butyl 3-(4-methoxy-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 6 (200 mg, 0.52 mmol), tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 7 (154 mg, 0.52 mmol) and Pd(dppf)Cl2 (23 mg, 0.031 mmol) in 1,4-dioxane/H2O=10:1 (11 mL) was added Na2CO3 (165 mg, 1.56 mmol) under N2. The reaction mixture was stirred at 100° C. for 2 h. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 93:7) to give tert-butyl 3-(4-methoxy-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 8 (280 mg, 94% yield) as an orange oil.
LCMS (ESI) calcd for C27H32F3N5O4Si [M+H]+ m/z 516.22, found 516.23.
To a solution of tert-butyl 3-(4-methoxy-5-(trifluoromethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)pyrrolidine-1-carboxylate 8 (280 mg, 0.54 mmol) in MeOH (15 mL) was added Pd/C (114 mg, 1.08 mmol) under H2. The reaction mixture was stirred at room temperature for 2 h. Then the mixture was filtered through celite and concentrated under vacuum to give tert-butyl 3-(4-methoxy-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyrrolidine-1-carboxylate 9 (158 mg, 56% yield) as a white solid.
LCMS (ESI) calcd for C22H34F3N5O4Si [M+H]+ m/z 518.23, found 518.25.
Tert-butyl 3-(4-methoxy-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyrrolidine-1-carboxylate 9 (158 mg, 0.31 mmol) was added to HBr water (5 mL). The reaction mixture was stirred at 100° C. for 2 h. Then the mixture was concentrated under vacuum to give 6-(pyrrolidin-3-yl)-3-(trifluoromethyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 10 (80 mg, 96% yield) as a white solid.
LCMS (ESI) calcd for C10H10F3N5O [M+H]+ m/z 274.08, found 274.00.
To a solution of 6-(pyrrolidin-3-yl)-3-(trifluoromethyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 10 (80 mg, 0.29 mmol), N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (90 mg, 0.29 mmol) and sodium triacetoxyborohydride (92 mg, 0.44 mmol) in MeOH (10 mL) was added two drops of HOAc and the solution was stirred for 0.5 h at 50° C. Then was added NaBH3CN (22 mg, 0.35 mmol). The mixture was stirred for 18 h at 50° C. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give product of N-methyl-5-(4-(3-(4-oxo-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)pyrrolidin-1-yl)piperidin-1-yl)picolinamide 162rac (69 mg, 48% yield) as white solid.
LCMS (ESI) calcd for C22H25F3N8O2 [M+H]+ m/z 491.21, found 491.25.
Compound 162rac was separated by SFC (Column: DAICEL OJ-H 4.6 mm I.D.*250 mmL, 5 μm; Mobile phase: CO2/MeOH[0.1% NH3 (7M Solution in MeOH)]=70/30) and concentrated under reduced pressure to afford the first fraction as 162a (39 mg, 93% purity, ee %: 100, white solid) and the second fraction as 162b (31 mg, 99% purity, ee %: 100, white solid) Compound 162a 1H NMR (400 MHz, DMSO) δ 14.37 (s, 1H), 12.27 (s, 1H), 8.39 (q, J=4.4 Hz, 1H), 8.27 (d, J=2.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.40 (dd, J=8.4, 2.8 Hz, 1H), 3.83 (d, J=12.8 Hz, 2H), 3.38-3.35 (m, 1H), 3.06 (t, J=8.4 Hz, 1H), 2.98-2.88 (m, 2H), 2.83-2.73 (m, 5H), 2.68-2.61 (m, 1H), 2.39-2.29 (m, 1H), 2.21-2.09 (m, 2H), 1.93 (d, J=11.6 Hz, 2H), 1.55-1.43 (m, 2H).
LCMS (ESI) calcd for C22H25F3N8O2 [M+H]+ m/z 491.21, found 491.26.
1H NMR (400 MHz,) δ 14.41 (s, 1H), 12.41 (s, 1H), 8.39 (q, J=4.8 Hz, 1H), 8.28 (d, J=2.0 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.41 (dd, J=8.8, 2.0 Hz, 1H), 3.86 (s, 1H), 3.30-3.27 (m, 1H), 3.16-3.03 (m, 1H), 2.96-2.58 (m, 8H), 2.34-2.10 (m, 3H), 2.03-1.88 (m, 2H), 1.63-1.44 (m, 2H).
LCMS (ESI) calcd for C22H25F3N8O2 [M+H]+ m/z 491.21, found 491.25.
To a solution of 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide INT-1 (92 mg, 0.40 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide 2 (intermediate 6 in synthesis of compound 136) (100 mg, 0.40 mmol). Then two drops of acetic acid and NaBH3CN (25 mg, 0.26 mmol) was added at room temperature. The reaction mixture was stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give product of 6-fluoro-5-{4-[3-(8-fluoro-1-oxo-2H-isoquinolin-3-yl)pyrrolidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide 184rac (60 mg, 27% yield) as a colorless oil.
LCMS (ESI) calcd for C25H27F2N5O2 [M+H]+ m/z 468.21, found 468.26.
6-chloro-5-{4-[3-(8-fluoro-1-oxo-2H-isoquinolin-3-yl)pyrrolidin-1-yl]piperidin-1-yl}-N-methylpyridine-2-carboxamide 184rac was separated by SFC (Column: Daicel CHIRALPAK OJ-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=80/20) and concentrated under reduced pressure to afford the first fraction as 184a (24.5 mg, 98% purity, ee %: 100, white solid) and the second fraction as 184b (24.4 mg, 99% purity, ee %: 97, white solid) Compound 184a
1H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.41 (q, J=5.2 Hz, 1H), 7.85 (d, J=7.2 Hz, 1H), 7.64-7.56 (m, 2H), 7.39 (d, J=7.6 Hz, 1H), 7.10 (dd, J=12.0, 8.0 Hz, 1H), 6.47 (s, 1H), 3.52-3.51 (m, 2H), 3.22-3.16 (m, 1H), 2.91-2.73 (m, 8H), 2.67-2.63 (m, 1H), 2.30-2.22 (m, 2H), 2.00-1.97 (m, 2H), 1.86-1.79 (m, 1H), 1.68-1.53 (m, 2H).
LCMS (ESI) calcd for C25H27F2N5O2 [M+H]+ m/z 468.21, found 468.22.
1H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.41 (q, J=4.8 Hz, 1H), 7.85 (dd, J=8.0, 1.2 Hz, 1H), 7.64-7.56 (m, 2H), 7.39 (d, J=7.6 Hz, 1H), 7.11 (dd, J=11.6, 8.0 Hz, 1H), 6.46 (s, 1H), 3.53-3.49 (m, 2H), 3.21-2.87 (m, 1H), 2.93-2.71 (m, 8H), 2.68-2.63 (m, 1H), 2.28-2.19 (m, 2H), 2.00-1.97 (m, 2H), 1.88-1.79 (m, 1H), 1.64-1.54 (m, 2H).
LCMS (ESI) calcd for C25H27F2N5O2 [M+H]+ m/z 468.21, found 468.18.
To a solution of 2-aminobenzamide 1 (360 mg, 2.64 mmol) and 2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxylic acid 2 (500 mg, 2.20 mmol) in pyridine (30 mL) was added EDCI (422 mg, 2.20 mmol). Then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (200 mL×3). The combined organic layers was washed by 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give product of tert-butyl 4-((2-carbamoylphenyl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (600 mg, 90% yield) as a white solid.
LCMS (ESI) calcd for C18H23N3O4 [M+Na]+ m/z 368.17, found 368.05.
To the solution of tert-butyl 4-((2-carbamoylphenyl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (600 mg, 1.74 mmol) in DME (60 mL) was added KOH (195 mg, 3.47 mmol), heated to 60° C. stirred for 2 h, cooled to 25° C., the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give tert-butyl 4-(4-oxo-3,4-dihydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 4 (400 mg, 63% yield) as a white solid.
LCMS (ESI) calcd for C18H21N3O3 [M+H]+ m/z 328.16, found 328.05.
Tert-butyl 4-(4-oxo-3,4-dihydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 4 (400 mg, 0.56 mmol) was added to HCl in dioxane (4M, 10 mL). The mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure to give product of 2-(2-azabicyclo[2.1.1]hexan-4-yl)quinazolin-4(3H)-one 5 (320 mg, 93% yield) as a white solid.
LCMS (ESI) calcd for C13H13N30 [M+H]+ m/z 228.11, found 228.02.
To a solution of 2-(2-azabicyclo[2.1.1]hexan-4-yl)quinazolin-4(3H)-one 5 (80 mg, 0.35 mmol) in MeOH (40 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide (INT, 98 mg, 0.42 mmol), then two drops of acetic acid and NaBH(OAC)3 (149 mg, 0.70 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 1 h. Then was added NaBH3CN (11.06 mg, 0.176 mmol). The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by C18 column (mobile phase: ACN-H2O (0.1% FA), gradient: 10-95) to give N-methyl-5-(4-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexan-2-yl)piperidin-1-yl)picolinamide 192 (25.6 mg, 99% purity, 16% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6, ppm) δ:12.17 (s, 1H), 8.43-8.36 (m, 1H), 8.29 (d, J=2.8 Hz, 1H), 8.17 (s, 1H), 8.10 (dd, J=8.0, 1.2 Hz, 1H), 7.86-7.76 (m, 2H), 7.62 (d, J=7.8 Hz, 1H), 7.52-7.47 (m, 1H), 7.42 (dd, J=8.8, 2.9 Hz, 1H), 3.93-3.86 (m, 2H), 3.76 (s, 1H), 3.12 (s, 2H), 2.97-2.90 (m, 2H), 2.78 (d, J=4.8 Hz, 3H), 2.69-2.62 (m, 1H), 2.19-2.14 (m, 2H), 2.02-1.90 (m, 4H), 1.54-1.44 (m, 2H).
LCMS (ESI) calcd for C25H28N6O2 [M+H]+ m/z 445.23, found 445.15.
To a solution of 6-chloro-1H-pyrazolo[4,3-c]pyridine 1 (10.00 g, 0.06 mol) in DMF (200 mL) was added NIS (8.44 g, 0.08 mol). The mixture was stirred for 3 h at 60° C. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layers were concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 60:40) to give product of 6-chloro-3-iodo-1H-pyrazolo[4,3-c]pyridine 2 (10.5 g, 64% yield) as a yellow solid.
LCMS (ESI) calcd for C6H3ClIN3 [M+H]+ m/z 279.91, found 280.00.
A suspension of 6-chloro-3-iodo-1H-pyrazolo[4,3-c]pyridine 2 (10.50 g, 0.04 mol) and NaH (2.70 g, 0.11 mol) in DMF (200 mL) was stirred for 30 min at 0° C., then SEMCl (12.30 g, 0.08 mol) was added. The mixture was stirred for 3 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 70:30) to give product of 6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridine 3 (9.90 g, 61% yield) as a yellow solid.
LCMS (ESI) calcd for C12H17ClIN3OSi [M+H]+ m/z 409.99, found 410.05.
To a solution of 6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridine 3 (9.90 g, 0.02 mol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate 4 (23.04 g, 0.12 mol) in DMF (80 mL) were added CuI (9.15 g, 0.04 mmol) and HMPA (21.35 g, 0.12 mmol) at room temperature. The mixture was stirred for 2 h at 110° C. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (50 mL×3). The combined organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 80:20) to give product of 6-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridine 5 (3.30 g, 38% yield) as a yellow solid.
LCMS (ESI) calcd for for C13H17ClF3N3OSi [M+H]+ m/z 352.08, found 352.20.
To a solution of 6-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridine 5 (3.30 g, 0.01 mmol) in HOAc (20 mL) was added H2O2(20 mL). The reaction mixture stirred at 70° C. for 5 h. After cooling to room temperature, the reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (30 mL×3). The combined organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 55:45) to give product of 6-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridine 5-oxide 6 (1.60 g, 48% yield) as an yellow oil.
LCMS (ESI) calcd for C13H17ClF3N3O2Si [M+H]+ m/z 368.07, found 367.97.
To a solution of 6-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-c]pyridine 5-oxide 6 (800 mg, 2.16 mmol) in H2O (15 mL) and MsCl (1.00 g, 8.62 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (20 mL×3). The combined organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 85:15) to give product of 6-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydro-4H-pyrazolo[4,3-c]pyridin-4-one 7 (400 mg, 48% yield) as an yellow oil.
LCMS (ESI) calcd for C13H17ClF3N3O2Si [M+H]+ m/z 368.07, found 368.01.
To a solution of 6-chloro-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydro-4H-pyrazolo[4,3-c]pyridin-4-one 7 (400 mg, 1.07 mmol) and Cs2CO3 (1.25 g, 3.85 mmol) in DMF (15 mL) was added PMBCl (0.84 g, 5.35 mmol) at room temperature. The reaction mixture was stirred at 50° C. for 2 h. After cooling to room temperature, the combined organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 85:15) to give product of 6-chloro-5-(4-methoxybenzyl)-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydro-4H-pyrazolo[4,3-c]pyridin-4-one 8 (200 mg, 32% yield) as a yellow oil.
LCMS (ESI) calcd for C21H25ClF3N3O3Si [M+H]+ m/z 488.13, found 488.15.
To a solution of 6-chloro-5-(4-methoxybenzyl)-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydro-4H-pyrazolo[4,3-c]pyridin-4-one 8 (200 mg, 0.48 mmol) and tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydropyrrole-1-carboxylate 9 (150 mg, 0.50 mmol) in Dioxan:H2O=5:1 (15 mL) was added Pd(dppf)Cl2 (50 mg, 0.07 mmol) and Na2CO3 (70 mg, 0.06 mmol) at room temperature. The reaction mixture stirred under nitrogen at 80° C. for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure and purified by flash chromatography (eluting with PE/EtOAc=100:0 to 80:20) to give product of tert-butyl 3-(5-(4-methoxybenzyl)-4-oxo-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5-dihydro-1H-pyrazolo[4,3-c]pyridin-6-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 10 (140 mg, 60% yield) as a yellow oil.
LCMS (ESI) calcd for C30H39F3N4O5Si [M+H]+ m/z 621.26, found 621.30.
To a solution of tert-butyl 3-(5-(4-methoxybenzyl)-4-oxo-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5-dihydro-1H-pyrazolo[4,3-c]pyridin-6-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 10 (140 mg, 0.30 mmol) in MeOH (15 mL) was added Pd/C (60 mg, 0.57 mmol). The reaction mixture stirred under hydrogen at room temperature for 2 h. The reaction mixture was filtered, the solution was concentrated under reduced pressure to give product of tert-butyl 3-(5-(4-methoxybenzyl)-4-oxo-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5-dihydro-1H-pyrazolo[4,3-c]pyridin-6-yl)pyrrolidine-1-carboxylate 11 (120 mg, 80% yield) as a colourless oil.
LCMS (ESI) calcd for C30H41F3N4O5Si [M+H]+ m/z 623.28, found 623.30.
To a solution of tert-butyl 3-(5-(4-methoxybenzyl)-4-oxo-3-(trifluoromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5-dihydro-1H-pyrazolo[4,3-c]pyridin-6-yl)pyrrolidine-1-carboxylate 11 (120 mg, 0.19 mmol) in TFA (5 mL) was added two drops of TfOH. The reaction mixture was stirred at 50° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give product of 6-(pyrrolidin-3-yl)-3-(trifluoromethyl)-1,5-dihydro-4H-pyrazolo[4,3-c]pyridin-4-one 12 (50 mg, 90% yield) as a yellow oil.
LCMS (ESI) calcd for C11H11F3N4O [M+H]+ m/z 273.09, found 273.15.
To a solution of 6-(pyrrolidin-3-yl)-3-(trifluoromethyl)-1,5-dihydro-4H-pyrazolo[4,3-c]pyridin-4-one 12 (50 mg, 0.18 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide INT (43 mg, 0.18 mmol). Then two drops of acetic acid and NaBH3CN (12 mg, 0.18 mmol) were added at room temperature. The reaction mixture was stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 92:8) to give N-methyl-5-(4-(3-(4-oxo-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazolo[4,3-c]pyridin-6-yl)pyrrolidin-1-yl)piperidin-1-yl)picolinamide a racemic mixture of compounds 194a and 194b (30 mg, 32% yield) as a white solid.
LCMS (ESI) calcd for C23H26F3N7O2 [M+H]+ m/z 490.21, found 490.17.
A racemic mixture of N-methyl-5-(4-(3-(4-oxo-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazolo[4,3-c]pyridin-6-yl)pyrrolidin-1-yl)piperidin-1-yl)picolinamide (194rac) was separated by SFC (Column: Daicel CHIRALPAK OJ-H 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH[0.1% (NH3)]=70/30) and concentrated under reduced pressure to afford the first fraction as 194a (9.9 mg, 98% purity, ee %: 100, white solid) and the second fraction as 194b (9.4 mg, 100% purity, ee %: 98, white solid) Compound 194a
1H NMR (400 MHz, DMSO) δ 13.82 (s, 1H), 11.10 (s, 1H), 8.39 (q, J=4.4 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.41 (dd, J=8.8, 2.8 Hz, 1H), 6.39 (s, 1H), 3.84-3.81 (m, 2H), 3.29-3.25 (m, 1H), 2.98 (t, J=11.2 Hz, 2H), 2.89-2.85 (m, 1H), 2.81-2.78 (m, 5H), 2.60-2.56 (m, 1H), 2.39-2.30 (m, 1H), 2.30-2.17 (m, 1H), 1.97 (d, J=12.0 Hz, 2H), 1.83-1.77 (m, 1H), 1.56-1.51 (m, 2H).
LCMS (ESI) calcd for C23H26F3N7O2 [M+H]+ m/z 490.21, found 490.20.
1H NMR (400 MHz, DMSO) δ 13.81 (s, 1H), 11.11 (s, 1H), 8.39 (q, J=4.8 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 6.40 (s, 1H), 3.91-3.81 (m, 2H), 3.27-3.23 (m, 1H), 2.98 (t, J=11.6 Hz, 2H), 2.90-2.86 (m, 1H), 2.80-2.77 (m, 5H), 2.60-2.54 (m, 1H), 2.36-2.33 (m, 1H), 2.25-2.19 (m, 1H), 1.97 (d, J=11.2 Hz, 2H), 1.86-1.75 (m, 1H), 1.58-1.48 (m, 2H).
LCMS (ESI) calcd for C23H26F3N7O2 [M+H]+ m/z 490.21, found 490.16.
To a solution of 2,4-dichloropyrido[2,3-d]pyrimidine 1 (1.5 g, 0.0075 mol) in EtOH (30 mL) at room temperature was added NaOH (0.66 g, 0.017 mol) in H2O (16.5 mL) at rt. The reaction mixture was stirred at rt for 1 h. The reaction solution was quenched with water, then adjusted pH to 4-5 with 1 M HCl. The solution was filtered, and the filter cake was collected and dried in vacuo pressure to give 2-chloropyrido[2,3-d]pyrimidin-4(3H)-one 2 (1.1 g, 90% purity, 73% yield) as yellow solid.
LCMS (ESI) calcd for C7H4ClN3O [M+H]+ m/z 182.00, found 181.90.
To a solution of 2-chloropyrido[2,3-d]pyrimidin-4(3H)-one 2 (1.1 g, 0.0061 mol) in THF/H2O=4:1 (50 mL) was added PtO2 (0.14 g). Then the mixture was stirred under hydrogen for 18 h at rt. The mixture was filtered through a celite pad, and the filtrate was concentrated under reduced pressure to give 2-chloro-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 3 (1.1 g, 95% purity, 91% yield) as a yellow solid.
LCMS (ESI) calcd for C7H8ClN3O [M+H]+ m/z 186.04, found 185.95.
To a solution of 2-chloro-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 3 (0.9 g, 4.85 mmol) in DMSO (150 mL) at room temperature were added Cs2CO3 (4.7 g, 14.5 mmol) and PMBCl (1.52 g, 9.7 mmol). The reaction mixture was stirred at 50° C. for 1 h. The reaction solution was quenched with water and extracted with EtOAc (100 mL×3). The combined organic phase was washed three times with brine and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 50:50) to give 2-chloro-3-(4-methoxybenzyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 4 (1.23 g, 90% purity, 74% yield) as white solid.
LCMS (ESI) calcd for C15H16ClN3O2[M+H]+ m/z 306.09, found 305.80.
To a solution of 2-chloro-3-(4-methoxybenzyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 4 (1.2 g, 0.004 mol) in DMF (50 mL) at 0° C. were added MeI (3.05 g, 0.02 mol) and NaH (60% in mineral oil, 0.32 g, 0.008 mol). The reaction mixture was stirred at rt for 2 h. The reaction solution was quenched with water and extracted with EtOAc (100 mL×3). The combined organic phase was washed three times with brine and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 50:50) to give 2-chloro-3-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 5 (450 mg, 90% purity, 33% yield) as yellow oil.
LCMS (ESI) calcd for C16H18ClN3O2[M+H]+ m/z 320.11, found 320.00.
To a solution of 2-chloro-3-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 5 (450 mg, 1.41 mmol) in dioxane/H2O=5:1 (30 mL) at room temperature were added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 6 (500 mg, 1.69 mmol), Pd(dppf)Cl2-DCM (115 mg, 0.14 mmol) and K2CO3 (587 mg, 4.22 mmol). The reaction mixture was stirred at 90° C. for 3 h. The reaction solution was quenched with water and extracted with EtOAc (50 mL×3). The combined organic phase was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 50:50) to give tert-butyl 3-(3-(4-methoxybenzyl)-8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 7 (550 mg, 90% purity, 77% yield) as white solid.
LCMS (ESI) calcd for C25H32N4O4 [M+H]+ m/z 453.24, found 453.20.
To a solution of tert-butyl 3-(3-(4-methoxybenzyl)-8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 7 (550 mg, 1.2 mmol) in MeOH (15 mL) at room temperature were added Pd/C (55 mg). The reaction mixture was stirred at rt for 18 h under a hydrogen atmosphere. The reaction solution was filtered, and the filter cake was washed with MeOH (5 mL×2). The filtrate was concentrated under reduced pressure to give tert-butyl 3-(8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)pyrrolidine-1-carboxylate 8 (400 mg, 90% purity, 88% yield) as yellow oil.
LCMS (ESI) calcd for C17H26N4O3 [M+H]+ m/z 335.20, found 335.30.
A solution of tert-butyl 3-(8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)pyrrolidine-1-carboxylate 8 (200 mg, 0.60 mmol) in HCl-dioxane (4 M, 10 mL) was stirred at room temperature for 2 h under N2 atmosphere. The precipitate formed was washed with DCM (5 mL×3), collected and dried under reduced pressure to obtain 8-methyl-2-(pyrrolidin-3-yl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one hydrochloride 9 (110 mg, 90% purity, 70% yield) as white solid.
LCMS (ESI) calcd for C12H18N4O [M+H]+ m/z 235.15, found 235.00.
To a solution of 8-methyl-2-(pyrrolidin-3-yl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one hydrochloride 9 (110 mg, 0.46 mmol) in MeOH (1 mL) was added TEA (1 mL) at rt and stirred for 5 mins, then the reaction was concentrated to dryness under reduced pressure. The residue was dissolved in MeOH (15 mL) and AcOH (0.01 mL) at room temperature. N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (131 mg, 0.56 mmol) and NaBH3CN (88 mg, 1.40 mmol) were added. The reaction mixture was stirred at 50° C. for 1 h. The reaction solution was concentrated under reduced pressure and the residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give a racemic mixture of N-methyl-5-(4-(3-(8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)pyrrolidin-1-yl)piperidin-1-yl)picolinamide 208rac (120 mg, 99% purity, 55% yield) as a white solid.
LCMS (ESI) calcd for C24H33N7O2 [M+H]+ m/z 452.27, found 452.20.
A racemic mixture of compounds 208a and 208b was separated by SFC (Column: Daicel Chiralpak IH SFC, 250 mm×20 mm I.D., 5 mm; Mobile phase: CO2/MeOH [0.1% NH3]=70/30) and concentrated under reduced pressure to afford the first fraction as 208a (52.3 mg, 99% purity, 100% ee, white solid) and the second fraction as 208b (54.2 mg, 100% purity, 98% ee, white solid).
1H NMR (400 MHz, DMSO-d6, ppm) δ:11.37 (s, 1H), 8.43-8.34 (m, 1H), 8.26 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.39 (dd, J=8.8, 3.2 Hz, 1H), 3.82-3.77 (m, 2H), 3.23-3.20 (m, 2H), 3.13-3.09 (m, 1H), 3.03 (s, 3H), 2.99-2.92 (m, 3H), 2.78 (d, J=4.8 Hz, 3H), 2.68-2.66 (m, 1H), 2.59-2.57 (m, 1H), 2.35-2.26 (m, 4H), 2.07-2.00 (m, 2H), 1.94-1.88 (m, 2H), 1.78-1.72 (m, 2H), 1.53-1.44 (m, 2H).
LCMS (ESI) calcd for C24H33N7O2 [M+H]+ m/z 452.27, found 452.30.
1H NMR (400 MHz, DMSO-d6, ppm) δ:11.35 (s, 1H), 8.41-8.33 (m, 1H), 8.26 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.39 (dd, J=8.8, 2.8 Hz, 1H), 3.85-3.75 (m, 2H), 3.24-3.19 (m, 2H), 3.14-3.07 (m, 1H), 3.03 (s, 3H), 3.00-2.88 (m, 3H), 2.77 (d, J=4.8 Hz, 3H), 2.67-2.62 (m, 1H), 2.61-2.56 (m, 1H), 2.35-2.21 (m, 4H), 2.09-1.99 (m, 2H), 1.96-1.87 (m, 2H), 1.79-1.70 (m, 2H), 1.55-1.43 (m, 2H).
LCMS (ESI) calcd for C24H33N7O2 [M+H]+ m/z 452.27, found 452.30.
To a solution of tert-butyl 3-(1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidine-1-carboxylate 1 (500 mg, 1.59 mmol) in DMAC (20 mL) was added Selectfluor (563 mg, 1.59 mmol) at rt. The mixture was stirred at 150° C. for 10 min. After cooling to rt, the reaction mixture was added into cold water and then extracted with EtOAc (100 mL×3). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash silica chromatography (eluting with PE/EtOAc=100:0 to 50:50) to give tert-butyl 3-(4-fluoro-1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidine-1-carboxylate INT-3 (100 mg, 60% purity, 11% yield) as yellow oil.
LCMS (ESI) calcd for C18H21FN2O3[M+H]+ m/z 333.15, found 332.90.
To a solution of 2-amino-5-fluorobenzamide 1 (30 mg, 2.21 mmol) and 2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxylic acid 2 (520 mg, 2.20 mmol) in pyridine (20 mL) was added EDCI (423 mg, 2.21 mmol), then the mixture was stirred for 18 h at room temperature. The reaction mixture was quenched with water, the aqueous layer was extracted with EtOAc (150 mL×3). The combined organic layers were washed with 1M HCl solution and brine, dried over Na2SO4, concentrated under reduced pressure to give product of tert-butyl 4-((2-carbamoyl-4-fluorophenyl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (500 mg, 56% yield) as a white solid.
LCMS (ESI) calcd for C18H22FN3O4[M+Na]+ m/z 386.16, found 385.95.
To the solution of tert-butyl 4-((2-carbamoyl-4-fluorophenyl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (500 mg, 1.38 mmol) in DME (50 mL) was added KOH (154 mg, 2.75 mmol). The solution was stirred at 60° C. for 2 h, cooled to room temperature, then the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give tert-butyl 4-(6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 4 (400 mg, 76% yield) as a white solid.
LCMS (ESI) calcd for C18H20FN3O3[M+H]+ m/z 346.15, found 345.85.
To a solution of tert-butyl 4-(6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 4 (400 mg, 1.16 mmol) in DCM (10 mL) was added HCl in dioxane (4M, 20 mL). The mixture was stirred for 1 h at room temperature. The reaction mixture was concentrated under reduced pressure to give product of 2-(2-azabicyclo[2.1.1]hexan-4-yl)-6-fluoroquinazolin-4(3H)-one 5 (200 mg, 63% yield) as a white solid.
LCMS (ESI) calcd for C15H17N3O [M+H]+ m/z 246.10, found 245.95.
To a solution of 2-(2-azabicyclo[2.1.1]hexan-4-yl)-6-fluoroquinazolin-4(3H)-one 5 (60 mg, 0.24 mmol) in MeOH (20 mL) was added 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT-2 (61 mg, 0.24 mmol), then two drops of acetic acid and NaBH(OAC)3 (104 mg, 0.49 mmol) were added at room temperature. After 1 h, NaBH3CN (8 mg, 0.13 mmol) was added. The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by Cis column (mobile phase: ACN-H2O (0.1% FA), gradient: 10-95) to give 6-fluoro-5-(4-(4-(6-fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexan-2-yl)piperidin-1-yl)-N-methylpicolinamide 259 (45.5 mg, 99% purity, 15% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6, ppm) δ:12.29 (s, 1H), 8.40 (q, 1H), 7.84 (dd, J=8.0, 1.2 Hz, 1H), 7.77 (dd, J=8.4, 2.8 Hz, 1H), 7.72-7.65 (m, 2H), 7.62-7.56 (m, 1H), 3.70 (s, 1H), 3.59-3.53 (m, 2H), 3.05 (s, 2H), 2.86 (t, J=11.2 Hz, 2H), 2.77 (d, J=4.8 Hz, 3H), 2.57-2.52 (m, 1H), 2.15-2.09 (m, 2H), 1.99 (d, J=10.8 Hz, 2H), 1.92-1.86 (m, 2H), 1.58-1.47 (m, 2H).
LCMS (ESI) calcd for C25H26F2N6O2[M+H]+ m/z 481.21, found 481.05.
To a solution of 2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxylic acid 1 (500 mg, 2.19 mmol) in DCM (20 mL) were added NH4Cl (234 mg, 4.38 mmol), DIPEA (1415 mg, 10.95 mmol) and HATU (1249 mg, 3.28 mmol) at room temperature successively. The mixture was kept stirring at room temperature for 1 h. The resulting mixture was diluted with water and extracted with DCM (50 mL×3). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with MeOH/DCM, 3% to 5%) to give tert-butyl 4-carbamoyl-2-azabicyclo[2.1.1]hexane-2-carboxylate 2 (400 mg, 90% purity, 72% yield) as a white solid.
LCMS (ESI) calcd for C11H18N2O3 [M−56+H]+ m/z 171.13, found 171.10.
Tert-butyl 4-carbamoyl-2-azabicyclo[2.1.1]hexane-2-carboxylate 2 (400 mg, 1.76 mmol) was added to a suspension of Me3O+BF4− (312 mg, 2.1 mmol) in anhydrous DCM (10 mL). The mixture was stirred under argon atmosphere at rt for 2 h. Solvent was then removed to provide crude imidate salt. This crude was dissolved in NH3/MeOH (7 M, 10 mL) and stirred at rt for 16 h. Then Boc2O (1152 mg, 5.28 mmol) was added. The mixture was stirred at rt for 10 min and then concentrated to give crude tert-butyl 4-carbamimidoyl-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (500 mg, 50% purity, 62% yield) as a white solid.
LCMS (ESI) calcd for C11H19N3O2 [M+H]+ m/z 226.15, found 226.05.
To a solution of tert-butyl 4-carbamimidoyl-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (500 mg, 2.21 mmol) in DMF (20 mL) were added K2CO3 (916 mg, 6.63 mmol) and methyl (E)-3-methoxyacrylate 4 (770 mg, 6.63 mmol) in one portion. The reaction mixture was heated with stirring at 120° C. for 2 h. The reaction mixture was concentrated and purified by silica gel column chromatography (eluting with EtOAc/PE, 50% to 100%) to obtain tert-butyl 4-(6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 5 (200 mg, 90% purity, 29% yield) as a white solid.
LCMS (ESI) calcd for C14H19N3O3 [M+H]+ m/z 278.15, found 277.95.
To a solution of tert-butyl 4-(6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 5 (215 mg, 0.77 mmol) in AcOH (5 mL) was added NIS (260 mg, 1.16 mmol) in one portion. The reaction mixture was heated with stirring at 50° C. for 2 h. The reaction mixture was concentrated and purified by silica gel column chromatography (eluting with EtOAc/PE, 50% to 70%) to obtain tert-butyl 4-(5-iodo-6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 6 (180 mg, 90% purity, 51% yield) as a white solid.
LCMS (ESI) calcd for C14H18IN3O3[M+H]+ m/z 404.04, found 403.85.
A solution of tert-butyl 4-(5-iodo-6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 6 (180 mg, 0.44 mmol), tributyl(vinyl)stannane 7 (282 mg, 0.89 mmol) and Pd(AMPHOS)Cl2 (32 mg, 0.04 mmol) in ACN (10 mL) was purged with N2 for 2 min and then stirred at 80° C. for 1 h. After cooling to rt, the reaction mixture was concentrated and purified by flash chromatography (eluting with EtOAc/PE, 50% to 70%) to give tert-butyl 4-(6-oxo-5-vinyl-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 8 (120 mg, 90% purity, 79% yield) as a white solid.
LCMS (ESI) calcd for C16H21N3O3 [M+H]+ m/z 304.16, found 304.20.
To a solution of give tert-butyl 4-(6-oxo-5-vinyl-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 8 (120 mg, 0.40 mmol) in MeOH (5 mL) was added 10% Pd/C (12 mg). The mixture was evacuated and backfilled with nitrogen three times and then charged with hydrogen. The resulting mixture was stirred at room temperature for 16 hours. Then the mixture was filtered through celite and concentrated under vacuum to give crude tert-butyl 4-(5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 9 (120 mg, 90% purity, 89% yield) as a white solid which was used directly in the next step without further purification.
LCMS (ESI) calcd for C16H23N3O3 [M+H]+ m/z 306.18, found 306.00.
To a solution of tert-butyl 4-(5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 9 (120 mg, 0.39 mmol) in DCM (4 mL) was added TFA (1 mL) dropwise at 0° C. The reaction solution was stirred at room temperature for 1 hour then concentrated under vacuum. The resulted mixture quenched with Et3N and concentrated under vacuum to afford 2-(2-azabicyclo[2.1.1]hexan-4-yl)-5-ethylpyrimidin-4(3H)-one 10 (120 mg, 50% purity, 74% yield).
LCMS (ESI) calcd for C11H15N3O [M+H]+ m/z 206.12, found 206.00.
To a solution of 2-(2-azabicyclo[2.1.1]hexan-4-yl)-5-ethylpyrimidin-4(3H)-one 10 (40 mg, 0.19 mmol) in MeOH (5 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (68 mg, 0.29 mmol), then two drops of acetic acid and NaBH(OAc)3 (82 mg, 0.39 mmol) were added at room temperature. After 1 h, NaBH3CN (12 mg, 0.19 mmol) was added. The reaction mixture stirred at 50° C. for 1 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (Gemini 5 um C18 150×21.2 mm, eluting with 10% to 40% ACN/H2O containing 0.1% NH3—H2O) and prep-TLC (MeOH/DCM, 1/20) to give 5-(4-(4-(5-ethyl-6-oxo-1,6-dihydropyrimidin-2-yl)-2-azabicyclo[2.1.1]hexan-2-yl)piperidin-1-yl)-N-methylpicolinamide 299 (3.2 mg, 99% purity, 3% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6, ppm) δ: 12.30 (s, 1H), 8.39 (q, J=4.8 Hz, 1H), 8.27 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.72 (s, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 3.94-3.81 (m, 2H), 3.64 (s, 1H), 3.00-2.85 (m, 4H), 2.78 (d, J=4.8 Hz, 3H), 2.57-2.52 (m, 1H), 2.40-2.27 (m, 2H), 2.07-2.01 (m, 2H), 1.96-1.91 (m, 2H), 1.87-1.72 (m, 2H), 1.50-1.36 (m, 2H), 1.07 (t, J=7.6 Hz, 3H).
LCMS (ESI) calcd for C23H30N6O2 [M+H]+ m/z 423.25, found 423.15
To a solution of 2,6-dichloropyridin-4-amine 1 (1.0 g, 6.1 mmol) in dioxane (15 mL) was added MeONa (3.3 g, 61.0 mmol) and TBAI (230 mg, 0.6 mmol) at rt. The reaction mixture was stirred at 150° C. for 15 h in a sealed tube. The reaction solution was cooled to rt and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with PE/EtOAc=100:0 to 95:5) to afford 2-chloro-6-methoxypyridin-4-amine 2 (880 mg, 90% purity, 81% yield) as a white solid.
LCMS (ESI) calcd for C6H7ClN2O [M+H]+ m/z 159.02, found 158.93.
To a solution of 2-chloro-6-methoxypyridin-4-amine 2 (880 mg, 5.5 mmol) in DMF (40 mL) was added NBS (1.2 g, 6.7 mmol) at rt. The reaction mixture was stirred at rt for 1 h. The reaction mixture was poured into water. The aqueous layer was extracted with EtOAc (100 mL×3). The combined organic layers were washed with brine (100 mL×3), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with PE/EtOAc=100:0 to 70:30) to afford 3-bromo-6-chloro-2-methoxypyridin-4-amine 3 (950 mg, 90% purity, 64% yield) as a white solid.
LCMS (ESI) calcd for C6H6BrClN2O [M+H]+ m/z 236.94, found 236.87.
To a solution of 3-bromo-6-chloro-2-methoxypyridin-4-amine 3 (950 mg, 4.0 mmol) in DCM (30 mL) was added Boc2O (1.31 g, 6.0 mmol), Et3N (810 mg, 8.0 mmol) and DMAP (244 mg, 2.0 mmol) successively at rt. The reaction mixture was stirred at rt for 1 h. The reaction solution was concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with PE/EtOAc=100:0 to 80:20) to afford a mixture of tert-butyl (3-bromo-6-chloro-2-methoxypyridin-4-yl)carbamate 4 and tert-butyl (3-bromo-6-chloro-2-methoxypyridin-4-yl)(tert-butoxycarbonyl)carbamate 4a (750 mg, 90% purity, 49% yield) as a white solid.
LCMS (ESI) calcd for C11H14BrClN2O3[M+H]+ m/z 336.99, found 336.92.
To a solution of the mixture of tert-butyl (3-bromo-6-chloro-2-methoxypyridin-4-yl)carbamate 4 and tert-butyl (3-bromo-6-chloro-2-methoxypyridin-4-yl)(tert-butoxycarbonyl)carbamate 4a (750 mg, 2.2 mmol) in dioxane/H2O (25 mL, 10:1) was added ethyl (E)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate 5 (751 mg, 3.3 mmol), Pd(dppf)Cl2DCM (181 mg, 0.22 mmol) and K3PO4—H2O (1.5 g, 6.6 mmol) successively at rt. The reaction mixture was stirred at 100° C. for 6 h under N2. The reaction solution was cooled to rt and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with PE/EtOAc=100:0 to 70:30) to afford a mixture of ethyl (E)-3-(4-((tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)acrylate 6 and ethyl (E)-3-(4-(bis(tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)acrylate 6a (410 mg, 90% purity, 46% yield) as a white solid.
LCMS (ESI) calcd for C16H21ClN2O5[M+H]+ m/z 357.11, found 357.00.
A solution of the mixture of ethyl (E)-3-(4-((tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)acrylate 6 and ethyl (E)-3-(4-(bis(tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)acrylate 6a (410 mg, 0.9 mmol), and PtO2 (50 mg) in EtOAc (10 mL) was stirred at rt for 1 h under H2 atmosphere. The resulting solution was filtered through diatomaceous earth and the filter cake was washed with DCM (20 mL). The filtrate was concentrated under reduced pressure to give a mixture of ethyl 3-(4-((tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)propanoate 7 and ethyl 3-(4-(bis(tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)propanoate 7a (210 mg, 90% purity, 45% yield) as a colorless oil.
LCMS (ESI) calcd for C16H23ClN2O5[M+H]+ m/z 359.13, found 359.08.
A solution of the mixture of ethyl 3-(4-((tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)propanoate 7 and ethyl 3-(4-(bis(tert-butoxycarbonyl)amino)-6-chloro-2-methoxypyridin-3-yl)propanoate 7a (210 mg, 0.6 mmol) in HCl-dioxane (10 mL, 4 M) was stirred at rt for 2 h. The reaction mixture was concentrated under reduced pressure to give 7-chloro-5-methoxy-3,4-dihydro-1,6-naphthyridin-2(1H)-one 8 (110 mg, 90% purity, 79% yield) as a white solid.
LCMS (ESI) calcd for C9H9ClN2O2[M+H]+ m/z 213.04, found 212.92.
To a solution of 7-chloro-5-methoxy-3,4-dihydro-1,6-naphthyridin-2(1H)-one 8 (110 mg, 0.5 mmol) in dioxane/H2O (10 mL, 10:1) was added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 9 (184 mg, 0.6 mmol), Pd(dppf)Cl2 (42 mg, 0.06 mmol) and K3PO4·H2O (357 mg, 1.6 mmol) successively at rt. The reaction mixture was stirred at 100° C. for 6 h under N2 atmosphere. The reaction solution was cooled to rt and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with PE/EtOAc=100:0 to 20:80) to afford tert-butyl 3-(5-methoxy-2-oxo-1,2,3,4-tetrahydro-1,6-naphthyridin-7-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 10 (150 mg, 90% purity, 75% yield) as a white solid.
LCMS (ESI) calcd for C18H23N3O4 [M+H]+ m/z 346.17, found 346.05.
A solution of tert-butyl 3-(5-methoxy-2-oxo-1,2,3,4-tetrahydro-1,6-naphthyridin-7-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 10 (150 mg, 0.4 mmol) and Pd/C (20 mg) in MeOH (10 mL) was stirred at rt for 2 h under H2 atmosphere. The resulting solution was filtered through diatomaceous earth and the filter cake was washed with DCM (20 mL×3). The filtrate was concentrated under reduced pressure to give tert-butyl 3-(5-methoxy-2-oxo-1,2,3,4-tetrahydro-1,6-naphthyridin-7-yl)pyrrolidine-1-carboxylate 11 (140 mg, 90% purity, 83% yield) as a colorless oil.
LCMS (ESI) calcd for C18H25N3O4 [M+H]+ m/z 348.18, found 347.95.
A solution of tert-butyl 3-(5-methoxy-2-oxo-1,2,3,4-tetrahydro-1,6-naphthyridin-7-yl)pyrrolidine-1-carboxylate 11 (120 mg, 0.34 mmol) in HBr (48% in H2O, 5 mL) was stirred at 110° C. for 2 h. The reaction solution was cooled to rt and concentrated under reduced pressure. The residue was dissolved in MeOH (5 mL) and Et3N (1 mL) was added dropwise. The reaction mixture was stirred at rt for 5 min. The reaction solution was concentrated under reduced pressure to give 7-(pyrrolidin-3-yl)-4,6-dihydro-1,6-naphthyridine-2,5(1H,3H)-dione 12 (65 mg, 90% purity, 72% yield) as a brown solid.
LCMS (ESI) calcd for C12H15N3O2 [M+H]+ m/z 234.12, found 234.06.
To a solution of 7-(pyrrolidin-3-yl)-4,6-dihydro-1,6-naphthyridine-2,5(1H,3H)-dione 12 (65 mg, 0.3 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (78 mg, 0.3 mmol), AcOH (2 drops) and NaBH3CN (35 mg, 0.6 mmol) and at rt. The reaction mixture was stirred at 50° C. for 1 h. The reaction mixture was quenched with water (2 mL) and the reaction solution was concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with DCM/MeOH=100:0 to 90:10) to afford 5-(4-(3-(2,5-dioxo-1,2,3,4,5,6-hexahydro-1,6-naphthyridin-7-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide a racemic mixture of compounds 309a and 309b (35 mg, 98% purity, 27% yield) as a white solid.
A racemic mixture of compounds 309a and 309b was separated by SFC (Column: Daicel Chiralpak IH SFC; 20 mm I.D.×250 mm, 5 mm; Mobile phase: CO2/MeOH [0.1% NH3 (7 M Solution in MeOH)]=60/40) and concentrated under reduced pressure to afford the first fraction as 309a (4.8 mg, 95% purity, 100% ee, white solid) and the second fraction as 309b (8.7 mg, 95% purity, 100% ee, white solid).
1H NMR (400 MHz, DMSO-d6, ppm) δ:11.08 (s, 1H), 10.04 (s, 1H), 8.43-8.34 (m, 1H), 8.27 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.48-7.36 (m, 1H), 5.83 (s, 1H), 3.89-3.76 (m, 2H), 3.14-3.07 (m, 1H), 3.01-2.91 (m, 2H), 2.84-2.72 (m, 5H), 2.60-2.56 (m, 3H), 2.43-2.41 (m, 3H), 2.24-2.10 (m, 2H), 1.96-1.88 (m, 2H), 1.72-1.62 (m, 1H), 1.55-1.46 (m, 2H).
LCMS (ESI) calcd for C24H30N6O3 [M+H]+ m/z 451.24, found 451.40.
1H NMR (400 MHz, DMSO-d6, ppm) δ:11.07 (s, 1H), 10.04 (s, 1H), 8.46-8.32 (m, 1H), 8.27 (d, J=2.8 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 5.83 (s, 1H), 3.87-3.76 (m, 2H), 3.14-3.04 (m, 1H), 3.02-2.91 (m, 2H), 2.84-2.75 (m, 5H), 2.58-2.54 (m, 3H), 2.44-2.37 (m, 3H), 2.30-2.24 (m, 1H), 2.23-2.12 (m, 1H), 1.97-1.90 (m, 2H), 1.75-1.63 (m, 1H), 1.59-1.44 (m, 2H).
LCMS (ESI) calcd for C24H30N6O3 [M+H]+ m/z 451.24, found 451.20.
To a solution of ethyl 3-oxobutanoate 2 (4.57 g, 0.0351 mol) in DME (250 mL) was added t-BuOK (5.25 g, 0.0468 mol). The mixture was stirred at room temperature for 1 hour. Cu(OAc)2 (1.70 g, 0.00936 mol) and 2-chloro-5-fluoropyridine-3-carboxylic acid 1 (4.1 g, 0.0234 mol) were added and the resulting mixture was stirred at 100° C. for 24 hours under nitrogen atmosphere. The mixture was diluted with water, acidified with 1 M HCl to pH=2 and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica (DCM/MeOH=100:0 to 90:10) to afford 2-(2-ethoxy-2-oxoethyl)-5-fluoropyridine-3-carboxylic acid 3 (2.4 g, 90% purity, 40% yield) as a white solid.
LCMS (ESI) calcd for C10H10FNO4 [M+H]+ m/z 228.06, found 228.00.
To a solution of 2-(2-ethoxy-2-oxoethyl)-5-fluoropyridine-3-carboxylic acid 3 (2400 mg, 10.56 mmol) and TEA (2137 mg, 21.12 mmol) in THF (40 mL) was added ethyl carbonochloridate (2281 mg, 21.12 mmol) dropwise at 0° C. under N2 atmosphere. The mixture was stirred at this temperature for 1 hour before NH3—H2O (28%, 1285 mg) was added dropwise and the resulting mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by Biotage Isolera One (C18 column, eluting with 5% to 90% MeCN/H2O containing 0.1% FA) to afford 3-fluoro-6,8-dihydro-1,6-naphthyridine-5,7-dione 4 (505 mg, 90% purity, 23% yield) as a white solid.
LCMS (ESI) calcd for C8H5FN2O2[M+H]+ m/z 181.03, found 180.90.
A solution of 3-fluoro-6,8-dihydro-1,6-naphthyridine-5,7-dione 4 (300 mg, 1.66 mmol) in PhPOCl2 (3 mL) was stirred at 110° C. for 12 hours under nitrogen atmosphere. The mixture was cooled and adjusted pH to 9-10 with aqueous KOH (5% wt) and diluted with ethyl acetate. The mixture was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE/EtOAc=100:0 to 50:50) to afford 5,7-dichloro-3-fluoro-1,6-naphthyridine 5 (125 mg, 90% purity, 31% yield) as a white solid.
LCMS (ESI) calcd for C8H3C2FN2 [M+H]+ m/z 216.97, found 216.90.
To a solution of 5,7-dichloro-3-fluoro-1,6-naphthyridine 5 (195 mg, 0.8985 mmol) in MeOH (20 mL) was added MeONa (48 mg, 0.8985 mmol), then stirred for 2 h at room temperature. The reaction mixture was separated with EtOAc (20 mL×3) and brine. The combined organic phase was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 95:5) to give 7-chloro-3-fluoro-5-methoxy-1,6-naphthyridine 6 (135 mg, 90% purity, 63% yield) as white solid.
LCMS (ESI) calcd for C9H6ClFN2O [M+H]+ m/z 213.02, found 212.90.
A mixture of 7-chloro-3-fluoro-5-methoxy-1,6-naphthyridine 6 (100 mg, 0.470 mmol), tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 7 (167 mg, 0.564 mmol), Pd(dppf)Cl2 (38 mg, 0.047 mmol) and Na2CO3 (306 mg, 0.941 mmol) in dioxane and H2O (3:1, 2 mL) was degassed with N2 and heated at 80° C. for 3 h under N2 atmosphere. The reaction mixture was concentrated and purified by flash silica chromatography to obtain tert-butyl 3-(3-fluoro-5-methoxy-1,6-naphthyridin-7-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 8 (100 mg, 90% purity, 24% yield) as a colorless oil.
LCMS (ESI) calcd for C18H20FN3O3[M+H]+ m/z 346.15, found 346.15.
A mixture of 3-(3-fluoro-5-methoxy-1,6-naphthyridin-7-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 8 (100 mg, 0.289 mmol) and Pd/C (31 mg) in MeOH (2 mL) was degassed with H2 and stirred at rt for 2 h under H2 atmosphere. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure to obtain tert-butyl 3-(3-fluoro-5-methoxy-1,6-naphthyridin-7-yl)pyrrolidine-1-carboxylate 9 (100 mg, 60% purity, 59% yield) as a yellow oil.
LCMS (ESI) calcd for C18H22FN3O3[M+H]+ m/z 348.16, found 348.05.
A solution of tert-butyl 3-(3-fluoro-5-methoxy-1,6-naphthyridin-7-yl)pyrrolidine-1-carboxylate 9 (100 mg, 0.287 mmol) in HBr-water (48%, 10 mL) was heated at 100° C. for 2 h. The mixture was concentrated under reduced pressure to obtain 3-fluoro-7-(pyrrolidin-3-yl)-1,6-naphthyridin-5(6H)-one hydrobromide 10 (80 mg, 90% purity, 79% yield) as a white solid.
LCMS (ESI) calcd for C12H12FN3O [M+H]+ m/z 234.10, found 233.95.
To a solution of 3-fluoro-7-(pyrrolidin-3-yl)-6H-1,6-naphthyridin-5-one hydrobromide 10 (40 mg, 0.127 mmol) in MeOH (10 mL) was added 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide INT-2 (32 mg, 0.127 mmol), then two drops of acetic acid and NaBH3CN (40 mg, 0.636 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography to give 6-fluoro-5-(4-(3-(3-fluoro-5-oxo-5,6-dihydro-1,6-naphthyridin-7-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide racemic 324rac (13.2 mg, 95% purity, 21% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6 ppm) δ 11.56 (s, 1H), 8.92 (d, J=2.8 Hz, 1H), 8.42-8.35 (m, 1H), 8.23-8.17 (m, 1H), 8.16 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.62-7.54 (m, 1H), 6.61 (s, 1H), 3.54-3.49 (m, 2H), 3.31-3.27 (m, 1H), 2.97-2.84 (m, 4H), 2.83-2.79 (m, 1H), 2.77 (d, J=4.8 Hz, 3H), 2.70-2.64 (m, 1H), 2.38-2.31 (m, 1H), 2.30-2.23 (m, 1H), 1.99 (d, J=12.4 Hz, 2H), 1.92-1.81 (m, 1H), 1.67-1.55 (m, 2H).
LCMS (ESI) calcd for C24H26F2N6O2[M+H]+ m/z 469.21, found 469.10.
2H-isoquinolin-1-one (8 g, 0.05 mol) and PtO2 (0.9 g, 0.003 mol) were added to TFA (80 mL), stirred under H2 (1 Mpa) at 100° C. for 12 h. The precipitate was collected by filtration, extracted with EtOAc (400 mL×3). The combine organic phases were washed with brine, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (eluent: PE/EtOAC=100:0 to 80:20) to give 5,6,7,8-tetrahydroisoquinolin-1(2H)-one 2 (7.00 g, 77% yield) as a black oil.
LCMS (ESI) calcd for C9H11NO [M+H]+ m/z 150.08, found 150.00
A solution of sodium 5,6,7,8-tetrahydro-2H-isoquinolin-1-one 2 (7 g, 0.047 mmol) in POCl3 (100 mL) was stirred at 110° C. for 3 h. After completion of the reaction, the mixture was cooled to room temperature, taken into ice-water, neutralized with NH3·H2O under cooling, extracted with EtOAc (300 mL×3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (eluting with PE/EtOAc=100:0 to 80:20) to afford 1-chloro-5,6,7,8-tetrahydroisoquinoline 3 (5 g, 90% purity, 57% yield) as a colorless oil.
LCMS (ESI) calcd for C9H10ClN [M+H]+ m/z 168.05, found 168.00.
1-chloro-5,6,7,8-tetrahydroisoquinoline 3 (5 g, 0.029 mol) was added to AcOH/H2O2 (100 mL). The reaction mixture was stirred at 70° C. for 2 h. The precipitate was collected by filtration, extracted with EtOAc (200 mL×3). The combine organic phases were washed with brine, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (eluent: DCM/MeOH=100:0 to 90:10) to give 1-chloro-5,6,7,8-tetrahydroisoquinoline 2-oxide 4 (2.00 g, 73% yield) as a yellow oil.
LCMS (ESI) calcd for C9H10ClNO [M+H]+ m/z 184.05, found 184.00.
1-chloro-5,6,7,8-tetrahydroisoquinoline 2-oxide 4 (2.00 g, 0.019 mmol) was added to POCl3 (50 mL) slowly at room temperature. The mixture was heated to 110° C. stirred for 3 h. The resulting mixture was diluted with ice water (200 mL) and extracted with EtOAc (400 mL×3). The combined organic phases were washed with brine, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (eluent: PE/EtOAc=100:0 to 70:30) to give 1,3-dichloro-5,6,7,8-tetrahydroisoquinoline 5 (370 mg, 15% yield) as a yellow oil.
LCMS (ESI) calcd for C9H9Cl2N [M+H]+ m/z 202.01, found 201.95.
To a solution of 1,3-dichloro-5,6,7,8-tetrahydroisoquinoline 5 (350 mg, 1.73 mmol) in MeOH (15 mL) was added MeONa (280 mg, 5.20 mmol), the mixture was heated to 60° C. with stirring for 3 h. The precipitate was collected by filtration, extracted with EtOAc (100 mL×3). The combined organic phases were washed with brine, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (eluent: PE/EtOAC=100:0 to 90:10) to afford 3-chloro-1-methoxy-5,6,7,8-tetrahydroisoquinoline 6 (250 mg, 66% yield) as a white solid.
LCMS (ESI) calcd for C10H12ClNO [M+H]+ m/z 198.06, found 197.95.
To a solution of 3-chloro-1-methoxy-5,6,7,8-tetrahydroisoquinoline 6 (240 mg, 1.21 mmol) in dioxane/H2O=10:1 (60 mL) were added tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 7 (394 mg, 1.33 mmol), Pd(dppf)Cl2 (88 mg, 0.12 mmol) and Na2CO3 (503 mg, 3.64 mmol) successively at room temperature. The reaction mixture was stirred at 90° C. for 3 h under N2. The mixture was diluted with water (50 mL) and extracted with EtOAc (100 mL×3). The combined organic layer was washed with brine (100 mL×2), dried over Na2SO4, filtered and concentrated in vacuo to obtain the crude product, which was purified by flash column chromatography (PE/EtOAc=100:0 to 85:15) to afford tert-butyl 3-(1-methoxy-5,6,7,8-tetrahydroisoquinolin-3-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 8 (140 mg, 31% yield) as a yellow solid.
LCMS (ESI) calcd for C19H26N2O3 [M+H]+ m/z 331.19, found 331.15.
To a solution of tert-butyl 3-(1-methoxy-5,6,7,8-tetrahydroisoquinolin-3-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate 8 (130 mg, 0.39 mmol) in MeOH (50 mL) was added Pd/C (30 mg). The mixture was evacuated and backfilled with hydrogen three times and then charged with hydrogen. The resulting mixture was stirred at room temperature for 5 h. Then the mixture was filtered through celite and concentrated under vacuum to give crude product, which was purified by flash column chromatography (PE/EtOAc=100:0 to 50:50) to afford tert-butyl 3-(1-methoxy-5,6,7,8-tetrahydroisoquinolin-3-yl)pyrrolidine-1-carboxylate 9 (130 mg, 89% yield) as a yellow solid.
LCMS (ESI) calcd for C19H28N2O3 [M+H]+ m/z 333.21, found 333.10.
Tert-butyl 3-(1-methoxy-5,6,7,8-tetrahydroisoquinolin-3-yl)pyrrolidine-1-carboxylate 9 (115 mg, 0.71 mmol) was added to HBr in water (10 mL). The mixture was stirred for 2 h at 100° C. The reaction mixture was concentrated under reduced pressure to give product of 3-(pyrrolidin-3-yl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-one 10 (80 mg, 95% yield) as a white solid.
LCMS (ESI) calcd for C13H18N2O [M+H]+ m/z 219.14, found 219.05.
To a solution of 3-(pyrrolidin-3-yl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-one 10 (60 mg, 0.27 mmol), 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)pyridine-2-carboxamide INT-2 (69 mg, 0.27 mmol) and AcOH (two drops) in MeOH (5 mL) was added sodium triacetoxyborohydride (146 mg, 0.69 mmol) and then stirred at 50° C. for 1 h, then NaBH3CN (21 mg, 0.33 mmol) was added and stirred for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give 6-fluoro-N-methyl-5-(4-(3-(1-oxo-1,2,5,6,7,8-hexahydroisoquinolin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)picolinamide, a racemic mixture of compounds 337a and 337b (22 mg, 18% yield) as a white solid.
LCMS (ESI) calcd for C25H32FN5O2[M+H]+ m/z 454.25, found 454.15.
A racemic mixture of compounds 337a and 337b was separated by SFC (Column: DAICEL OD-H 4.6 mm I.D.*250 mmL 5 μm; Mobile phase: CO2/MeOH [0.1% NH3(7M Solution in MeOH)]=70/30) and concentrated under reduced pressure to afford the first fraction as 337a (7 mg, 95% purity, ee %: 100, white solid) and the second fraction as 337b (5 mg, 92% purity, ee %: 100, white solid).
1H NMR (400 MHz, DMSO) δ 11.03 (s, 1H), 8.40 (d, J=4.8 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.57 (dd, J=10.6, 8.4 Hz, 1H), 5.83 (s, 1H), 3.55-3.44 (m, 2H), 3.14-3.04 (m, 1H), 2.86 (t, J=11.6 Hz, 3H), 2.75 (t, J=6.4 Hz, 4H), 2.63 (dd, J=14.8, 8.0 Hz, 2H), 2.44 (s, 2H), 2.27 (s, 3H), 2.21-2.10 (m, 1H), 1.95 (d, J=13.2 Hz, 2H), 1.73 (dd, J=12.8, 8.4 Hz, 1H), 1.58 (dd, J=22.4, 12.0 Hz, 6H).
LCMS (ESI) calcd for C25H32FN5O2[M+H]+ m/z 454.25, found 454.20.
1H NMR (400 MHz, DMSO) δ 11.03 (s, 1H), 8.40 (d, J=4.4 Hz, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.70-7.45 (m, 1H), 5.83 (s, 1H), 3.49 (d, J=11.2 Hz, 2H), 3.08 (d, J=6.4 Hz, 1H), 2.85 (t, J=11.2 Hz, 3H), 2.76 (d, J=4.4 Hz, 4H), 2.69-2.57 (m, 2H), 2.44 (s, 2H), 2.27 (s, 3H), 2.20-2.10 (m, 1H), 1.95 (d, J=12.4 Hz, 2H), 1.80-1.68 (m, 1H), 1.68-1.51 (m, 6H).
LCMS (ESI) calcd for C25H32FN5O2[M+H]+ m/z 454.25, found 454.15.
N-BuLi (2.4 M in hexanes, 10 mL, 23.9 mmol) was added to THF (100 mL) and cooled to −78° C. To this solution was added i-PrNH2 (2.54 g, 25.0 mmol) slowly followed by 4-chloro-3-fluoropyridine 1 (3 g, 22.8 mmol). After stirring for 2 h at −78° C., crushed dry ice was added in one portion. The reaction mixture was kept stirring until the reaction mixture warmed to room temperature. The reaction was quenched by addition of aqueous ammonium chloride solution and acidified to pH=2 using conc. HCl and extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford 4-chloro-5-fluoronicotinic acid 2 (2.2 g, 70% purity, 36% yield) as a yellow solid.
LCMS (ESI) calcd for C6H3ClFNO2 [M+H]+ m/z 175.99, found 175.80.
To a solution of 4-chloro-5-fluoronicotinic acid 2 (2.2 g, 11.6 mmol) in ACN (100 mL) were added DBU (5.29 g, 34.8 mmol) and CH3I (8.2 g, 58.0 mmol) dropwise at 0° C. The mixture was stirred at rt for 5 hours. The resulting mixture was diluted with water (300 mL) and extracted with EtOAc (100 mL×3). The combined organic phases were washed with brine, dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (eluting with EtOAc/PE, 20% to 50%) to give methyl 4-chloro-5-fluoronicotinate 3 (1.5 g, 90% purity, 63% yield) as a white solid.
LCMS (ESI) calcd for C7H5ClFNO2 [M+H]+ m/z 190.00, found 189.95.
A mixture of methyl 4-chloro-5-fluoronicotinate 3 (150 mg, 0.79 mmol), tert-butyl 3-ethynylpyrrolidine-1-carboxylate 4 (233 mg, 1.19 mmol), PdCl2(PPh3)2(55 mg, 0.079 mmol) and DIPEA (511 mg, 3.96 mmol) in ACN (15 mL) was heated at 85° C. for 5 hours under an atmosphere of N2. After cooling to ambient temperature, the mixture was filtered through celite, and the filtrate was concentrated under vacuum. The residue was diluted with water and extracted with EtOAc. The combined organic phases were washed with water and brine, dried over sodium sulfate, concentrated under reduced pressure, and purified by flash chromatography (eluting with EtOAc/PE, 20% to 40%) to obtain methyl 4-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)ethynyl)-5-fluoronicotinate 5 (150 mg, 90% purity, 48% yield) as a yellow solid.
LCMS (ESI) calcd for C18H21FN2O4[M+H]+ m/z 349.15, found 348.95.
To a solution of methyl 4-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)ethynyl)-5-fluoronicotinate 5 (150 mg, 0.43 mmol) in THF/H2O (3/1, 10 mL) was added LiOH (31 mg, 1.29 mmol). The mixture was stirred at room temperature for 2 h. Then the organic solvent was removed under reduced pressure. The aqueous solution was adjusted to pH 3-4 with 1 M HCl and extracted with DCM (50 mL×3). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give 4-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)ethynyl)-5-fluoronicotinic acid 6 (120 mg, 90% purity, 75% yield) as a yellow oil.
LCMS (ESI) calcd for C17H19FN2O4[M+H]+ m/z 335.14, found 335.00.
To a solution of 4-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)ethynyl)-5-fluoronicotinic acid 6 (120 mg, 0.36 mmol) in DCM (10 mL) was added TfOH (161 mg, 1.07 mmol) dropwise at 0° C. The mixture was stirred at rt for 16 hours. The reaction was quenched with DIPEA (231 mg, 1.79 mmol), then Boc2O (156 mg, 0.716 mmol) was added. After stirring at rt for 1 h, the resulting mixture were concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluting with EtOAc/PE, 20% to 40%) to give tert-butyl 3-(5-fluoro-1-oxo-1H-pyrano[3,4-c]pyridin-3-yl)pyrrolidine-1-carboxylate 7 (70 mg, 90% purity, 52% yield) as a white solid.
LCMS (ESI) calcd for C17H19FN2O4[M−t-Bu+H]+ m/z 279.14, found 278.85.
A solution of tert-butyl 3-(5-fluoro-1-oxo-1H-pyrano[3,4-c]pyridin-3-yl)pyrrolidine-1-carboxylate 7 (70 mg, 0.21 mmol) in NH3·MeOH (7 M, 5 mL) was stirred at 90° C. for 1 h in a sealed tube. Then the reaction was concentrated under reduced pressure to provide tert-butyl 3-(5-fluoro-1-oxo-1,2-dihydro-2,7-naphthyridin-3-yl)pyrrolidine-1-carboxylate 8 (70 mg, 90% purity, 90% yield) as a yellow solid.
LCMS (ESI) calcd for C17H20FN3O3[M+H]+ m/z 334.15, found 333.95.
A mixture of tert-butyl 3-(5-fluoro-1-oxo-1,2-dihydro-2,7-naphthyridin-3-yl)pyrrolidine-1-carboxylate 8 (70 mg, 0.44 mmol) in HCl dioxane solution (4 M, 5 mL) was stirred at room temperature for 1 hours. The mixture was concentrated under reduced pressure then quenched with Et3N. The resulting solution was concentrated under vacuum to give 5-fluoro-3-(pyrrolidin-3-yl)-2,7-naphthyridin-1(2H)-one 9 (50 mg, 90% purity, 92% yield) as a yellow oil.
LCMS (ESI) calcd for C12H12FN3O [M+H]+ m/z 234.10, found 234.05.
To a solution of 5-fluoro-3-(pyrrolidin-3-yl)-2,7-naphthyridin-1(2H)-one 9 (70 mg, 0.30 mmol) in MeOH (10 mL) was added 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT-2 (113 mg, 0.45 mmol), then two drops of acetic acid, NaBH3CN (19 mg, 0.30 mmol) and NaBH(OAc)3 (191 mg, 0.90 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 1 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give 6-fluoro-5-(4-(3-(5-fluoro-1-oxo-1,2-dihydro-2,7-naphthyridin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide, a racemic mixture of compounds 343a and 343b (50 mg, 95% purity, 33% yield) as a white solid.
A racemic mixture of compounds 343a and 343b was separated by SFC (Column: Daicel Chiralpak IH 20 mm I.D.×250 mm, 5 m; Mobile phase: CO2/MeOH [0.1% (NH3)]=70/30) and concentrated under reduced pressure to afford the first fraction as 343a (19.7 mg, 99% purity, 100% ee, white solid) and the second fraction as 343b (24.7 mg, 98% purity, 90% ee, white solid)
1H NMR (400 MHz, DMSO-d6, ppm) δ: 11.74 (s, 1H), 9.09 (s, 1H), 8.68 (d, J=2.0 Hz, 1H), 8.40 (d, J=4.8 Hz, 1H), 7.87-7.80 (m, 1H), 7.58 (dd, J=10.6, 8.2 Hz, 1H), 6.54 (s, 1H), 3.58-3.47 (m, 2H), 3.29-3.16 (m, 1H), 2.91-2.83 (m, 5H), 2.77 (d, J=4.8 Hz, 3H), 2.72-2.58 (m, 1H), 2.47-2.23 (m, 2H), 2.10-1.96 (m, 2H), 1.95-1.81 (m, 1H), 1.77-1.50 (m, 2H).
LCMS (ESI) calcd for C24H26F2N6O2[M+H]+ m/z 469.21, found 469.07.
1H NMR (400 MHz, DMSO-d6, ppm) δ: 11.77 (s, 1H), 9.09 (s, 1H), 8.68 (d, J=2.0 Hz, 1H), 8.45-8.31 (m, 1H), 7.89-7.76 (m, 1H), 7.58 (dd, J=10.6, 8.2 Hz, 1H), 6.55 (s, 1H), 3.54-3.46 (m, 2H), 3.29-3.23 (m, 1H), 2.94-2.82 (m, 5H), 2.77 (d, J=4.8 Hz, 3H), 2.69-2.58 (m, 1H), 2.36-2.25 (m, 2H), 2.02-1.94 (m, 2H), 1.91-1.78 (m, 1H), 1.67-1.56 (m, 2H).
LCMS (ESI) calcd for C24H26F2N6O2[M+H]+ m/z 469.21, found 469.10.
To a solution of 2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxylic acid 1 (110 mg, 0.48 mmol) in pyridine (10 mL) was added ethyl 2-aminocyclohex-1-ene-1-carboxylate 2 (160 mg, 0.95 mmol) at room temperature, then added 10 drops of POCl3. The reaction mixture stirred at room temperature for 3 h. After cooling to room temperature, the reaction mixture was poured into water, The aqueous was extracted with organic solvent (50 mL) 3 times. The combined organic layers were washed with brine (50 mL) and dried over Na2SO4, then concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 85:15) to give tert-butyl 4-((2-(ethoxycarbonyl)cyclohex-1-en-1-yl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (100 mg, 55% yield) as a white solid.
LCMS (ESI) calcd for C20H30N2O5 [M−56+H]+ m/z 323.22, found 323.05.
To a solution of tert-butyl 4-((2-(ethoxycarbonyl)cyclohex-1-en-1-yl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (100 mg, 0.26 mmol) in MeOH:H2O=1:1 (10 mL) was added LiOH (30 mg, 1.25 mmol). The reaction mixture stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give 2-(2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxamido)cyclohex-1-ene-1-carboxylic acid 4 (80 mg, 88% yield) as a white solid.
LCMS (ESI) calcd for C18H26N2O5 [M−56+H]+ m/z 295.18, found 295.05.
To a solution of 2-(2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxamido)cyclohex-1-ene-1-carboxylic acid 4 (80 mg, 0.23 mmol) in ACN (10 mL) was added N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate/TCFH (150 mg, 0.54 mmol) and N-methyl imidazole/NMI (50 mg, 0.61 mmol) at room temperature. After 10 mins, excess of NH3·MeOH (7 M, 5 mL) was added at 50° C. The mixture was stirred at 50° C. for 18 h. The residue was concentrated under reduced pressure, then purified by flash chromatography (eluting with DCM/MeOH=100:0 to 96:4) to give tert-butyl 4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 5 (60 mg, 79% yield) as a white solid.
LCMS (ESI) calcd for C18H25N3O3 [M+H]+ m/z 332.19, found 332.10.
Tert-butyl 4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 5 (60 mg, 0.18 mmol) was added to HCl in dioxane (4 M, 3 mL), stirred for 1 h at room temperature, the reaction mixture was concentrated under reduced pressure to give 2-(2-azabicyclo[2.1.1]hexan-4-yl)-5,6,7,8-tetrahydroquinazolin-4(3H)-one 6 (40 mg, 96% yield) as a white solid.
LCMS (ESI) calcd for C13H17N3O [M+H]+ m/z 232.14, found 232.05.
To a solution of 2-(2-azabicyclo[2.1.1]hexan-4-yl)-5,6,7,8-tetrahydroquinazolin-4(3H)-one 6 (40 mg, 0.17 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (40 mg, 0.17 mmol) and 3 drops of HOAc, then added NaBH3CN (10 mg, 0.16 mmol). The reaction mixture stirred at 50° C. for 4 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (columns: Gemini 5 um C18 150×21.2 mm, mobile phase: ACN—H2O (0.1% FA), gradient: 10-25) to give N-methyl-5-(4-(4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)-2-azabicyclo[2.1.1]hexan-2-yl)piperidin-1-yl)picolinamide 361 (30 mg, 97% purity, 38% yield) as a white solid.
1H NMR (400 MHz, DMSO) δ 12.11 (s, 1H), 8.38 (q, J=4.8 Hz, 1H), 8.28 (d, J=2.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 7.40 (dd, J=8.8, 2.8 Hz, 1H), 3.87 (d, J=12.8 Hz, 2H), 3.69 (s, 1H), 2.98 (s, 2H), 2.91 (t, J=11.2 Hz, 2H), 2.78 (d, J=4.8 Hz, 3H), 2.61-2.54 (m, 1H), 2.48-2.44 (m, 2H), 2.31 (t, J=6.0 Hz, 2H), 2.09-2.02 (m, 2H), 1.99-1.92 (m, 2H), 1.84-1.78 (m, 2H), 1.72-1.59 (m, 4H), 1.51-1.41 (m, 2H).
LCMS (ESI) calcd for C25H32N6O2 [M+H]+ m/z 449.26, found 449.20.
To a solution of 2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexane-4-carboxylic acid 2 (500 mg, 2.19 mmol) in DCM (15 mL) were added 2-aminonicotinamide 1 (330 mg, 2.41 mmol), T3P (50% wt in EtOAc, 4.18 g, 6.57 mmol) and DIPEA (850 mg, 6.57 mmol) at room temperature successively. The mixture was kept stirring at room temperature for 1 h. The reaction solution was concentrated under reduced pressure and the residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give tert-butyl 4-((3-carbamoylpyridin-2-yl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (330 mg, 90% purity, 39% yield) as white solid.
LCMS (ESI) calcd for C17H22N4O4 [M+H]+ m/z 347.16, found 346.85.
To a solution of tert-butyl 4-((3-carbamoylpyridin-2-yl)carbamoyl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 3 (330 mg, 0.95 mmol) in DME (20 mL) at rt was added KOH (160 mg, 2.85 mmol). The reaction mixture was stirred at 60° C. for 6 h. The reaction solution was filtered to remove KOH. The filtrate was diluted with water and extracted with EtOAc (10 mL×3). The water phase was purified by silica gel C18 column (eluting with 10% to 50% MeCN/H2O, containing 0.1% formic acid) to give tert-butyl 4-(4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 4 (220 mg, 80% purity, 56% yield) as white solid.
LCMS (ESI) calcd for C17H20N4O3[M+H]+ m/z 329.15, found 329.00.
To a solution of tert-butyl 4-(4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 4 (200 mg, 0.607 mmol) in THF/H2O=4:1 (20 mL) was added PtO2 (41 mg). Then the mixture was stirred under hydrogen for 18 h at rt. The mixture was filtered through a celite pad, and the filtrate was concentrated under reduced pressure to give tert-butyl 4-(4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 5 (200 mg, 90% purity, 88% yield) as a yellow solid.
LCMS (ESI) calcd for C17H24N4O3 [M+H]+ m/z 333.18, found 333.25.
To a solution of tert-butyl 4-(4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 5 (200 mg, 0.6 mmol) in DMSO (10 mL) at room temperature were added Cs2CO3 (586 mg, 1.8 mmol) and PMBCl (188 mg, 1.2 mmol). The reaction mixture was stirred at 50° C. for 1 h. The reaction solution was quenched with water and extracted with EtOAc (30 mL×3). The combined organic phase was washed three times with brine and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 50:50) to give tert-butyl 4-(3-(4-methoxybenzyl)-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 6 (150 mg, 90% purity, 49% yield) as white solid.
LCMS (ESI) calcd for C25H32N4O4 [M+H]+ m/z 453.24, found 453.05.
To a solution of methyl tert-butyl 4-(3-(4-methoxybenzyl)-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 6 (50 mg, 0.11 mmol) in DMF (10 mL) at 0° C. were added NaH (7 mg, 0.17 mmol, 60% wt) and MeI (24 mg, 0.17 mmol). The reaction mixture was stirred at rt for 1 h. The reaction solution was quenched with water and extracted with EtOAc (50 mL×3). The organic phase was concentrated under reduced pressure and the residue was purified by flash chromatography (eluting with PE/EtOAc=100:0 to 70:30) to give tert-butyl 4-(3-(4-methoxybenzyl)-8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 7 (30 mg, 90% purity, 52% yield) as white solid.
LCMS (ESI) calcd for C26H34N4O4 [M+H]+ m/z 467.26, found 467.30.
To a solution of tert-butyl 4-(3-(4-methoxybenzyl)-8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate 7 (30 mg, 0.064 mmol) in TFA (5 mL) was added TfOH (0.2 mL). The reaction was stirred at rt for 0.5 h. The reaction solution was adjusted to pH 8 with saturated aqueous NaHCO3 at 0° C. The basified solution was extracted with DCM (10 mL×3). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give 2-(2-azabicyclo[2.1.1]hexan-4-yl)-8-methyl-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 8 (25 mg, 60% purity, 94% yield) as a yellow oil.
LCMS (ESI) calcd for C13H18N4O [M+H]+ m/z 247.15, found 247.25.
To a solution of 2-(2-azabicyclo[2.1.1]hexan-4-yl)-8-methyl-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one 8 (25 mg, 0.10 mmol) in MeOH (15 mL) and AcOH (0.01 mL) at room temperature was added 6-fluoro-N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT-2 (31 mg, 0.12 mmol) and NaBH3CN (10 mg, 0.15 mmol). The reaction mixture was stirred at 50° C. for 1 h. The reaction solution was concentrated under reduced pressure and the residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) and prep-HPLC (Gemini 5 um C18 150×21.2 mm, mobile phase: ACN-H2O (0.05% NH3), gradient: 25-95) to give 6-fluoro-N-methyl-5-(4-(4-(8-methyl-4-oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-2-azabicyclo[2.1.1]hexan-2-yl)piperidin-1-yl)picolinamide 375 (5.0 mg, 97% purity, 10% yield) as white solid.
1H NMR (400 MHz, DMSO-d6, ppm) δ:11.32 (s, 1H), 8.43-8.35 (m, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.63-7.53 (m, 1H), 3.61 (s, 1H), 3.57-3.50 (m, 2H), 3.25-3.18 (m, 2H), 3.03 (s, 3H), 2.91 (s, 2H), 2.84 (t, J=11.2 Hz, 2H), 2.76 (d, J=4.8 Hz, 3H), 2.47-2.42 (m, 1H), 2.37-2.31 (m, 2H), 2.03-1.90 (m, 4H), 1.80-1.71 (m, 4H), 1.55-1.42 (m, 2H).
LCMS (ESI) calcd for C25H32FN7O2[M+H]+ m/z 482.26, found 482.15.
To a solution of 3-chloro-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 1 (2000 mg, 6.294 mmol) and Pd(AMPHOS)Cl2 (445 mg, 0.6294 mmol) in ACN (30 mL) was added tributyl(vinyl)stannane 2 (1995 mg, 6.294 mmol). The mixture was heated at 100° C. in sealed tube for 5 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with EtOAc/PE, 0 to 15%) to give 8-fluoro-2-(4-methoxybenzyl)-3-vinylisoquinolin-1(2H)-one 3 (1500 mg, 90% purity, 69% yield) as a white solid.
LCMS (ESI) calcd for C19H16FNO2 [M+H]+ m/z 310.12, found 309.90.
To a solution of 8-fluoro-2-(4-methoxybenzyl)-3-vinylisoquinolin-1(2H)-one 3 (1500 mg, 4.85 mmol) in MeOH/H2O (3:1, 80 mL) was added NaIO4 (4150 mg, 19.40 mmol) and K2OsO4·2H2O (178 mg, 0.48 mmol). The reaction stirred for 1 h at room temperature. The reaction mixture was diluted with water and extracted with EtOAc (200 mL×3). The combined organic layers were washed by brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (eluting with EtOAc/PE, 0 to 25%) to give 8-fluoro-2-(4-methoxybenzyl)-1-oxo-1,2-dihydroisoquinoline-3-carbaldehyde 4 (430 mg, 90% purity, 25% yield) as a yellow solid.
LCMS (ESI) calcd for C18H14FNO3 [M+H]+ m/z 312.10, found 311.90.
To a solution of 8-fluoro-2-(4-methoxybenzyl)-1-oxo-1,2-dihydroisoquinoline-3-carbaldehyde 4 (430 mg, 1.38 mmol), PPh3 (724 mg, 2.76 mmol) and LiI (369 mg, 2.76 mmol) in DMF/Dioxane (8%, 10 mL) was added TMSCF3 (491 mg, 3.45 mmol). The mixture was heated at 130° C. in a sealed tube for 3 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with EtOAc/PE, 0 to 15%) to give 3-(2,2-difluorovinyl)-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 5 (200 mg, 60% purity, 25% yield) as a white solid.
LCMS (ESI) calcd for C19H14F3NO2 [M+H]+ m/z 346.10, found 346.00.
To a solution of 3-(2,2-difluorovinyl)-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 5 (260 mg, 0.75 mmol) and LiF (39 mg, 1.51 mmol) in ACN (50 mL) was added N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine 6 (357 mg, 1.51 mmol). The mixture was kept stirring at 60° C. for 16 h. The resulting mixture was diluted with water and extracted with DCM (30 mL×3). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with EtOAc/PE, 0 to 15%) to give 3-(1-benzyl-4,4-difluoropyrrolidin-3-yl)-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 7 (25 mg, 90% purity, 6% yield) as a white solid.
LCMS (ESI) calcd for C28H25F3N2O2[M+H]+ m/z 479.19, found 479.20.
To a solution of 3-(1-benzyl-4,4-difluoropyrrolidin-3-yl)-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 7 (130 mg, 0.271 mmol) in MeOH (5 mL) was added one drop of conc. HCl, 10% Pd/C (20 mg) and 10% Pd(OH)2/C (20 mg). The mixture was evacuated and backfilled with hydrogen three times and then charged with hydrogen. The resulting mixture was stirred at room temperature for 16 hours. Then the mixture was filtered through celite and concentrated under vacuum to give crude 3-(4,4-difluoropyrrolidin-3-yl)-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 8 (100 mg, 50% purity, 47% yield) as a white solid which was used directly in next step without further purification.
LCMS (ESI) calcd for C21H19F3N2O2[M+H]+ m/z 389.14, found 389.00.
To a solution of 3-(4,4-difluoropyrrolidin-3-yl)-8-fluoro-2-(4-methoxybenzyl)isoquinolin-1(2H)-one 8 (100 mg, 0.257 mmol) in MeOH (10 mL) was added N-methyl-5-(4-oxopiperidin-1-yl)picolinamide INT (90 mg, 0.386 mmol), then two drops of acetic acid and NaBH3CN (24 mg, 0.386 mmol) were added at room temperature. The reaction mixture stirred at 50° C. for 1 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with DCM/MeOH=100:0 to 90:10) to give 5-(4-(3,3-difluoro-4-(8-fluoro-2-(4-methoxybenzyl)-1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 10 (80 mg, 50% purity, 25% yield) as a white solid.
LCMS (ESI) calcd for C33H34F3N5O3[M+H]+ m/z 606.26, found 606.20.
To a solution of 5-(4-(3,3-difluoro-4-(8-fluoro-2-(4-methoxybenzyl)-1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 10 (80 mg, 0.13 mmol) in TFA (5 mL) was added TfOH (1 mL) dropwise at rt. The reaction mixture was stirred at 100° C. for 5 min. The pH of the resulting mixture was adjusted to around 8.0 by progressively adding saturated NaHCO3 solution, and then extracted with DCM (50 mL×3). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by prep-HPLC (Gemini 5 um C18 150×21.2 mm, eluting with 35% to 40% ACN/H2O containing 0.1% FA) to give 5-(4-(3,3-difluoro-4-(8-fluoro-1-oxo-1,2-dihydroisoquinolin-3-yl)pyrrolidin-1-yl)piperidin-1-yl)-N-methylpicolinamide 393rac (6.2 mg, 93% purity, 9% yield) as a brown solid.
1H NMR (400 MHz, CD3OD_SPE, ppm) δ: 8.31 (d, J=2.8 Hz, 1H), 7.95 (d, J=8.8 Hz, 1H), 7.72-7.65 (m, 1H), 7.57-7.47 (m, 1H), 7.43 (d, J=8.0 Hz, 1H), 7.17 (dd, J=11.8, 8.2 Hz, 1H), 6.76-6.68 (m, 1H), 4.10-3.94 (m, 2H), 3.85-3.50 (m, 5H), 3.28-3.25 (m, 1H), 3.07 (t, J=12.4 Hz, 2H), 2.96-2.90 (m, 3H), 2.24-2.09 (m, 2H), 1.84-1.66 (m, 2H).
LCMS (ESI) calcd for C25H26F3N5O2[M+H]+ m/z 486.21, found 486.40.
Exemplary compounds of the invention were prepared and tested to determine their effect as PARP1 and PARP2 inhibitors. Typical assays are described below.
Optiplate HB 384-well plates were coated with anti-FLAG antibody, supplied as a 4 mg/ml solution, using a Na2CO3/HCO3 coating buffer at pH 9.6, overnight at 4° C., in order to achieve a final immobilisation per well of 0.3 μg. Wells were then washed 3×5 min in coating wash buffer (PBS/0.05% Tween (v/v)), and blocked with 2% BSA (w/v) in coating wash buffer overnight at 4° C. Prior to assay, wells were washed 3×5 min in coating wash buffer. For the assay 20 μl of 2.5 nM recombinant full length human N-terminally FLAG-tagged PARP1 was added to each well of the 384-well plate for 30 min at room temperature followed by addition of 50 nL of compound solution in DMSO using pintool technology. Following incubation for 30 min at room temperature, 5 μl of 10 μM biotin-NAD+ and 10 nM activation DNA (sequence shown below) in solution in 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.1% BSA (w/v), 0.02% Tween (v/v) assay buffer. Auto-PARylation proceeded for 2 h at room temperature prior to the addition of 5 μl of 12 mM NAD+ quenching solution. After 30 min at room temperature, assay solution was removed and following washing 5 times for 3 min, 100 μl of a 1:1000 dilution of DELFIA Eu—N1 Streptavidin reagent was added. Plates were then incubated for 30 min at room temperature. Reaction mixture was removed and plates washed 5 times for 3 min prior to the addition of 25 μl DELFIA enhancement solution. Following incubation for 30 min at room temperature, fluorescence was measured on a Pherastar FS (Ex337 nm, Em620 nm; integration start 60 μs; integration time 400 μs).
Typically compounds were tested from 20 μM at 3-fold dilution intervals in 12-point concentration-response curves to determine IC50 values. Data was analysed using ActivityBase software and replicate values for the low (without enzyme, 0.2% DMSO) and high (0.2% DMSO) % controls were averaged and the data obtained from the test compounds expressed as a % of 100% using the below formulae:
% value=100−(100*((high control−unknown)/(high control−low control))
% data was fitted to a non-linear regression equation (log inhibitor vs response-variable slope 4-parameters) to obtain IC50 values.
The IC50 values for a variety of test compounds are shown in Table 1.
10 nM full length N-terminally FLAG-tagged PARP1 was incubated with 2 nM Anti-FLAG Tb-cryptate antibody and PARP1/2 Cy5 fluorescent dye-labelled binding probe (10-fold probe Kd═270 nM) in 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.1% BSA (w/v), 0.02% Tween (v/v) assay buffer for 40 min at room temperature. A Cy5-labelled binding probe is shown below and described in Papeo, G. et al. J. Biomol. Screen. 2014; 19:1212-1219. 6 μl of this reaction mixture was then transferred to each well of a black non-binding surface 384-well plate and 35 nl of compound solution in DMSO was then added using pintool technology. Following incubation for 1 h at room temperature, fluorescence was measured on a Pherastar FS (Ex 337 nm, Em620 nm, em665 nm; integration start 60 μs; integration time 400 μs) using the HTRF module.
Typically compounds were tested from 58.5 μM at factor 3 dilution intervals in 12-point concentration-response curves to determine IC50 values. Data was analysed using ActivityBase software and replicate values for the low (without enzyme but with probe and Tb-cryptate antibody, 0.6% DMSO) and high (0.6% DMSO) % controls were averaged and the data obtained from the test compounds expressed as a % of 100% using the below formulae:
% activity=100*(value−low control)/(high control−low control)
% activity data was fitted to a non-linear regression equation to obtain IC50 values
Kd values were calculated using Cheng-Prussoff formula:
IC50=(1+([probe concentration]/[Kmprobe]))*Kd
Therefore Kd=IC50/(1+[[probe concentration]/[Kmprobe])); using probe at 10×Km, this equated to Kd=IC50/11
This assay was performed under identical conditions as for PARP1, except that N-terminally FLAG-tagged PARP2 (amino acids 1-583) was used instead of PARP1, and PARP1/2 binding probe was used at 10-fold probe Kd=540 nM. Data analysis was performed identical as for PARP1.
NanoBRET assays were employed to demonstrate cellular target engagement and selectivity at PARP1 and PARP2. These assays are based on bioluminescence resonance energy transfer (BRET) between a Nano-luc-tagged protein (eg PARP1 or PARP2) and a fluorescent group on a high affinity NAD+ competitive binding probe. Such cellular probe displacement assays can be utilised to measure inhibitor affinities and selectivity ratios at PARP1 and 2.
Frozen HEK293 cells transiently transfected with either PARP1-NanoLuc® fusion or PARP2-NanoLuc® fusion constructs (Promega) were thawed and dispensed as a suspension in 384-well microplates each at a density of 1750 cells per well. NanoBRET™ TE PARP Tracer-01 was then added to final concentrations of 11 and 2 nM for PARP1 and PARP2 assays, respectively. Compounds were added from 25 μM at factor 3 dilution intervals in 12-point concentration-response curves and plates were incubated for 2 hours at 37° C. BRET ratios were then measured using a NanoBRET module (LUM 610-LP 450-80) and PHERAstar FS or FSX reader following addition of NanoBRET™ Nano-Glo® Substrate and Extracellular NanoLuc® Inhibitor according to manufacturer's instructions. Kd values were calculated using Cheng-Prussoff formula:
IC50=(1+([tracer concentration]/[Kmtracer]))*Kd
Binned potency, affinity and selectivity data for a variety of test compounds are shown in Table 1 where DELFIA and Probe Displacement HTRF assays were used. Binned potency, affinity and selectivity data for a subset of test compounds where the NanoBRET assay was used are shown in Table 2.
The selectivity values relate to the selectivity preference of PARP1 over PARP2. They are calculated from the ratio of Kd values for PARP1 and PARP2 inhibition as Kd (PARP2)/Kd (PARP1).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2202070.5 | Feb 2022 | GB | national |
| 2212967.0 | Sep 2022 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/053638 | 2/14/2023 | WO |
