Patent Application
20250170266
The present disclosure relates to the field of therapeutics and, in particular, to purine analogues, conjugates comprising such purine-derived compounds, their use for modulating an immune response in a cell or a subject, as well as the use of such compounds and conjugates for treatment of a disease (e.g., cancer, inflammation, etc.).
Immunostimulatory compounds and immuno-conjugates (e.g., antibody-drug conjugates (ADCs) such as immune-stimulating antibody conjugates (ISACs)) comprising such compounds have previously been evaluated for the treatment of various diseases, including cancer. However, such compounds and conjugates remain clinically unvalidated, mostly due to dose-limiting cytotoxicity (e.g., cytokine-release syndrome (CRS)), which can restrict their use to doses that are unable to provide sufficient efficacy.
Furthermore, current immunotherapies lack efficacy specifically in solid tumors due to highly suppressive tumor microenvironments (TMEs) and associated lack of effector immune cells (e.g., T cells). Compounds that agonize Toll-like receptor 7 (TLR7) are immunostimulatory compounds capable of “reprogramming” a TME locally at a tumor site; however, significant tolerability issues remain unsolved which limits systemic administration of these compounds.
Although clinical data support the use of TLR7 agonists in oncology, such treatment requires local administration. For example, the TLR7 agonist imiquimod has been approved for topical use in dermal oncology applications, including basal cell carcinoma and actinic keratosis (see, Geisse et al. J Am Acad Dermatol. 2004; 50(5):722-33, and Korman et al. Arch Dermatol. 2005; 141(4):467-473). Other uses in invasive skin cancers (e.g., squamous cell carcinoma, Bowen's disease, melanoma, and/or lentigo maligna) are similarly efficacious when applied locally on surface lesions (see, Meyer et al. Expert Opin Investig Drugs. 2008; 17(7):1051-65, and Wolf et al. Arch Dermatol. 2003; 139(3):273-6).
Unfortunately, studies have reported that systemic administration of imiquimod and other TLR agonists leads to dose-limiting toxicity below efficacious doses (see, Dudek et al. Clin Cancer Res 2007; 13:7119-7125). Furthermore, overexpression and/or activation of some TLRs (including TLR7) can result in conflicting anti-tumor/pro-tumor activity, in some cases contributing to, rather than diminishing, inflammation, tumor growth, cell survival, metastasis, and the upregulation of pro-inflammatory cytokines (see, Kaczanowska S, et al. J Leukoc Biol. 2013; 93(6):847-863).
Disclosed herein are purine-derived compounds, e.g., compounds having a structure according to any one of Formulae (I)-(IV) herein, as well as conjugates comprising such compounds, e.g., conjugates according to Formula (X). Furthermore, the present disclosure discloses methods of producing the herein described compounds and conjugates and their use for, e.g., the treatment of a disease such as cancer.
As further described herein, in vitro agonism of TLR7 using the compounds and conjugates of the present disclosure has been demonstrated by incubating immune cells expressing TLR7 (e.g., PBMCs) with the compounds and conjugates of the present disclosure and measuring one or more downstream effects of TLR7 agonism, such as cytokine induction (see, e.g., EXAMPLE 3). Moreover, in vivo immunostimulatory antibody-drug conjugate (ISAC) therapy utilizing antibodies targeting tumor-associated antigens (e.g., Her2) conjugated to at least one of the disclosed compounds can generate a potent anti-tumor response, as evidenced by inhibition of tumor growth rate and/or reduction in tumor volume (see, e.g., EXAMPLE 6). Surprisingly, a significant reduction in off-target effects—and hence an increased tolerability—was observed during in vivo testing of certain ISACs comprising a TLR7-agonizing compound of the present disclosure, when compared to the use of conventional immunostimulatory compounds (see, e.g., EXAMPLE 7).
More specifically, in various embodiments, the present disclosure relates to a compound according to Formula (I):
In some embodiments, a compound of Formula (I) can have a structure according to Formula (II):
In various embodiments, the present disclosure relates to compounds of Formula (III):
In certain embodiments of the present disclosure, a compound according to Formula (III) can have a structure according to Formula (IV):
wherein:
In some embodiments, the present disclosure relates to methods of agonizing TLR7, the method comprising contacting a cell that expresses TLR7 with a compound of any one of Formulae (I)-(IV), thereby agonizing TLR7.
In some embodiments, the present disclosure relates to a method of inducing release of a cytokine from a cell expressing TLR7, the method comprising contacting the cell with a compound of any one of Formulae (I)-(IV), thereby inducing release of the cytokine from the cell.
In some embodiments, the present disclosure relates to a method of inhibiting the proliferation of cancer cells, the method comprising contacting a cell population comprising the cancer cells and immune cells expressing TLR7 with an effective amount of a compound of any one of Formulae (I)-(IV). Such method can further comprise agonizing the TLR7 with the compound of any one of Formulae (I)-(IV), thereby inhibiting the proliferation of cancer cells.
In some embodiments, this disclosure relates to a method of killing cancer cells, the method comprising contacting a cell population comprising the cancer cells and immune cells expressing TLR7 with an effective amount of a compound of any one of Formulae (I)-(IV). Such method can further comprise agonizing the TLR7 with the compound of any one of Formulae (I)-(IV), thereby killing the cancer cells.
Other embodiments of the present disclosure relate to a method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of any one of Formulae (I)-(IV). In some embodiments, the compound agonizes TLR7 in the subject, thereby stimulating the immune response in the subject.
Further embodiments of this disclosure relate to a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of any one of Formulae (I)-(IV). Such method can further comprise agonizing TLR7 in the subject, thereby treating the cancer in the subject.
In various embodiments, the present disclosure relates to conjugates comprising one or more compounds of any one of Formulae (I)-(IV). In various embodiments, such conjugate has a structure according to Formula (X):
T-[L-(C)p]r (X)
wherein:
In various embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof.
The present disclosure also relates to methods of agonizing TLR7, the method comprising contacting a cell that expresses TLR7 with a conjugate of Formula (X), thereby agonizing TLR7.
Further embodiments herein relate to a method of inducing release of a cytokine from a cell expressing TLR7, the method comprising contacting the cell with a conjugate of Formula (X), thereby inducing release of the cytokine from the cell.
Further embodiments herein relate to a method of inhibiting the proliferation of cancer cells, the method comprising contacting a cell population comprising the cancer cells and immune cells expressing TLR7 with an effective amount of a conjugate of Formula (X). Such method can further comprise agonizing the TLR7 with a compound of the conjugate of Formula (X), thereby inhibiting the proliferation of cancer cells.
Other embodiments of this disclosure relate to a method of killing cancer cells, the method comprising contacting a cell population comprising the cancer cells and immune cells expressing TLR7 with an effective amount of a conjugate of Formula (X). Such method can further comprise agonizing the TLR7 with a compound of the conjugate of Formula (X) (e.g., a compound of any one of Formulae (I)-(IV) that is comprised by the conjugate), thereby killing the cancer cells.
Further embodiments herein relate to a method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of Formula (X). In some embodiments, the conjugate agonizes TLR7, e.g., via a compound of Formulae (I)-(IV), in the subject, thereby stimulating the immune response in the subject.
Further embodiments of this disclosure relate to a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of Formula (X). In some embodiments, the conjugate agonizes TLR7, e.g., via a compound of Formulae (I)-(IV), in the subject, thereby treating the cancer in the subject.
The implementations disclosed herein are illustrated by way of example, and not by way of limitation, in the accompanying drawings. The description and drawings are only for the purpose of illustration and as an aid to understanding and are not intended as a definition of the limits of the compounds, conjugates, and methods of the present disclosure.
In various embodiments, the present disclosure relates to immunostimulatory purine-derived compounds having a structure according to any one of Formulae (I)-(IV) that are capable of agonizing TLR7. Also disclosed herein are compound-linker constructs of Formula (A) that comprise a compound of the present disclosure coupled to a linker moiety. Further disclosed herein are immunostimulatory conjugates of Formula (X) comprising one or more of the purine-derived compounds described herein (e.g., those of Formulae (I)-(IV)) coupled to a targeting moiety via a linker, e.g., by using a compound-linker construct of Formula (A) as described herein. Methods for making and using the compounds, compound-linker constructs, and conjugates, e.g., for the treatment of cancer, are also disclosed.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The term “about,” as used herein in the context of a numerical value or range, generally refers to ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the numerical value or range recited or claimed, unless otherwise specified. In various embodiments, the term “about” refers to an approximately ±10% variation from a given value. In other embodiments, the term “about” refers to an approximately ±5% variation from a given value. In yet other embodiments, the term “about” refers to an approximately ±1% variation from a given value. It is to be understood that such a variation is always included in any given value described herein, whether it is specifically referred to or not.
The use of the word “a” or “an” when used herein in conjunction with the term “comprising” can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”
As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term “consisting essentially of” when used herein in connection with a compound, composition, use or method, denotes that additional elements and/or method steps can be present, but that these additions do not materially affect the manner in which the recited compound, composition, method or use functions. The term “consisting of,” when used herein in connection with a compound, composition, use or method, excludes the presence of additional elements and/or method steps. A compound, composition, use, or method described herein as comprising certain elements and/or steps can also, in certain embodiments, consist essentially of those elements and/or steps, and, in other embodiments, consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
The terms “alkyloxycarbonyl” and “alkoxycarbonyl” can be used interchangeably herein and refer to the group —C(O)OR, wherein R is alkyl.
The term “alkyl,” as used herein, refers to a straight chain or branched saturated hydrocarbon group containing the specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, t-pentyl, neo-pentyl, 1-methylbutyl, 2-methylbutyl, n-hexyl, and the like.
The term “alkylcycloalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one cycloalkyl group as defined herein. Examples include (C1-C6 alkyl)-cycloalkyl, e.g., (C1-C2 alkyl)-cycloalkyl or (C1-C4 alkyl)-cycloalkyl. Accordingly, the terms “alkylheterocycloalkyl,” “alkylaryl” and “alkylheteroaryl, as used herein, refer to an alkyl group as defined herein substituted with one heterocycloalkyl group, aryl group, or heteroaryl group, respectively, and as further defined herein. Examples include (C1-C6 alkyl)-heterocycloalkyl, (C1-C6 alkyl)-aryl and (C1-C6 alkyl)-heteroaryl. In various embodiments, any cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can itself be substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, (C0-C2 alkyl)-heterocycloalkyl, (C0-C2 alkyl)-aryl, and (C0-C2 alkyl)-heteroaryl, as described herein.
The term “amido,” as used herein, refers to the group —C(O)NRR′, where R and R′ are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl.
The term “amino,” as used herein, refers to the group —NRR′, wherein R and R′ are independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl.
The term “carboxy,” as used herein, refers to the group —C(O)OR, wherein R is H, alkyl, aryl, heteroaryl, cycloalkyl or heterocycloalkyl.
The term “haloalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one or more halogen atoms.
The terms “halogen” and “halo,” as used herein, refer to fluorine (F), bromine (Br), chlorine (Cl) and iodine (I).
The term “aminoalkyl,” as used herein, refers to an alkyl group as defined herein substituted with one or more amino groups, for example, one, two or three amino groups.
The term “aminoaryl,” as used herein, refers to an aryl group as defined herein substituted with one amino group.
The term “aryl,” as used herein, and unless otherwise defined, refers to a 6- to 12-membered mono- or bicyclic hydrocarbon ring system in which at least one ring is aromatic.
Examples of aryl include, but are not limited to, phenyl, naphthalenyl, 1,2,3,4-tetrahydro-naphthalenyl, 5,6,7,8-tetrahydro-naphthalenyl, indanyl, and the like.
The term “cycloalkyl,” as used herein and unless otherwise defined, refers to a mono- or bicyclic saturated hydrocarbon containing the specified number of carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptane, bicyclo[1.1.1]pentane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, and the like.
The term “heteroaryl,” as used herein and unless otherwise defined, refers to a 5- to 12-membered mono- or bicyclic ring system containing the specified number of carbon atoms (e.g., C3-C7, incl., e.g., imidazole, thiazole, etc.) and in which at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of heteroatoms include, but are not limited to, O, S and N.
Examples of heteroaryl include, but are not limited to: pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, and the like.
The term “heterocycloalkyl,” as used herein, refers to a mono- or bicyclic non-aromatic ring system containing the specified number of carbon atoms (e.g., C2-C6, incl., e.g., aziridine, piperazine, etc.) and in which at least one ring atom is a heteroatom, for example, O, S or N. A heterocyclyl substituent can be attached via any of its available ring atoms, for example, a ring carbon, or a ring nitrogen. Examples of heterocycloalkyl include, but are not limited to, aziridinyl, azetidinyl, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and the like.
As used herein with reference to an alkyl or heteroalkyl ring system, the term “bicyclic” generally includes both fused and spiro ring systems, unless otherwise defined herein.
The terms “hydroxy” and “hydroxyl,” as used herein, refer to the group —OH.
The term “hydroxyalkyl,” as used herein, refers to a linear or branched alkyl group as defined herein substituted with one or more hydroxy groups. In some embodiments, such one or more hydroxy groups can be terminal hydroxy groups.
The term “alkylthio,” as used herein, refers to the group —SR, where R is a C1-C6 alkyl group. In some embodiments, the alkyl group can optionally be substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl and C1-C2 hydroxyalkyl.
The terms “thio” and “thiol,” as used herein, refer to the group —SH.
Unless specifically stated as being “unsubstituted,” any alkyl (e.g., any haloalkyl, hydroxyalkyl, aminoalkyl, etc.), cycloalkyl, heterocycloalkyl, spirocycloalkyl, heterospirocycloalkyl, aryl or heteroaryl group referred to herein is understood to be “optionally substituted,” i.e., each such reference includes both unsubstituted and substituted versions of these groups. For example, reference to a “—C1-C6 alkyl” group includes both unsubstituted —C1-C6 alkyl and —C1-C6 alkyl substituted with one or more substituents further described herein.
In various embodiments, a substituent of any one of Formulae (I)-(IV) in any of the embodiments herein can be optionally substituted with one or more groups selected from the following: —NH2, —CO2H, —OH, carbonyl, halogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 carboxyalkyl, (C0-C4 alkyl)-cycloalkyl, (C0-C4 alkyl)-heterocycloalkyl, (C0-C4 alkyl)-spirocycloalkyl, (C0-C4 alkyl)-heterospirocycloalkyl, (C0-C4 alkyl)-aryl, and (C0-C4 alkyl)-heteroaryl, wherein each of the alkyl, hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 carboxyalkyl, cycloalkyl, heterocycloalkyl, spirocycloalkyl, heterospirocycloalkyl, aryl or heteroaryl group can itself be substituted with one or more of: —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl, or unsubstituted C1-C4 carboxyalkyl.
In other embodiments, a substituent of any one of Formulae (I)-(IV) in any of the embodiments herein can be optionally substituted with one or more groups selected from the following: —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl, unsubstituted C1-C4 carboxyalkyl, unsubstituted (C0-C4 alkyl)-cycloalkyl, unsubstituted (C0-C2 alkyl)-heterocycloalkyl, unsubstituted (C0-C2 alkyl)-aryl, and unsubstituted (C0-C2 alkyl)-heteroaryl.
In yet other embodiments, a substituent of any one of Formulae (I)-(IV) in any of the embodiments herein can be optionally substituted with one or more groups selected from the following: —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl and unsubstituted C1-C4 carboxyalkyl.
Generally, a substituent of any one of Formulae (I)-(IV) described herein as “substituted,” can include one substituent or a plurality of substituents up to the full valence of substitution for that group. For example, a methyl group can include 1, 2, or 3 substituents, and a phenyl group can include 1, 2, 3, 4, or 5 substituents. When a group is substituted with more than one substituent, the substituents can be the same or they can be different.
Throughout the present disclosure, and unless otherwise specified, the following system for numbering ring atoms in the purine moiety of the compounds described herein was used ( indicates potential attachment points to substituent(s)):
The terms “subject” and “patient” can be used interchangeably herein and refer to an animal in need of treatment. An animal in need of treatment can be a human or a non-human animal, such as a mammal, bird, or fish. In certain embodiments, the subject or patient is a mammal. In some embodiments, the subject is a human. In other embodiments, the subject is a rodent or a non-human primate.
An “effective amount” of a compound or conjugate described herein in respect of a particular result to be achieved is an amount sufficient to achieve the desired result. For example, an “effective amount” of a compound when referred to in respect of the killing of cancer cells, refers to an amount of that compound sufficient to produce a killing effect.
It is further to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. In particular, where a list of options is presented for a given embodiment or claim, it is to be understood that one or more option can be deleted from the list and the shortened list can form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.
It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition disclosed herein, and vice versa.
In various embodiments, the present disclosure discloses purine-derived compounds capable of agonizing TLR7.
In some embodiments, the present disclosure relates to a compound having Formula (I):
wherein * is the point of attachment to R5 and #is the point of attachment to N;
In some embodiments, in compounds of Formula (I), R2 is halogen, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy. In some of these embodiments, R2 is halogen or optionally substituted C1-C6 alkoxy. In certain embodiments, R2 is halogen or unsubstituted C1-C6 alkoxy. In some embodiments, R2 can be fluorine or methoxy. In some embodiments, R2 is fluorine. In other embodiments, R2 is methoxy.
Disclosed herein are compounds of Formula (I) in which X is O. In other embodiments, in compounds of Formula (I), X is NH.
In some embodiments, in compounds of Formula (I), R1 is optionally substituted C2-C6 alkyl. In some embodiments, R1 is unsubstituted C2-C6 alkyl, such as unsubstituted C2-C4 alkyl. In these embodiments, X can be O.
In yet other embodiments, R1 is branched and optionally substituted C3-C8 hydroxyalkyl. In some embodiments, R1 can be branched and unsubstituted C3-C8 hydroxyalkyl. In some embodiments, R1 is branched and unsubstituted C4-C6 hydroxyalkyl.
In such embodiments, the compound of Formula (I) can be:
In some embodiments, in compounds of Formula (I), R3 and R4 are independently H or Q-R5. In some embodiments, R3 and R4 can both be H. In other embodiments, R3 and R4 are both Q-R5. In some embodiments in which R3 and R4 are both Q-R5, Q can be optionally substituted C1-C6-alkyl, and R5 can be H or OH. In some embodiments in which R3 and R4 are both Q-R5, Q is unsubstituted C1-C6-alkyl and R5 is H.
In other embodiments, in compounds of Formula (I), Q is a bond and R5 is unsubstituted C3-C6-carboxyalkyl.
In various other embodiments, in compounds of Formula (I), Q is a bond, optionally substituted C1-C6-alkyl, optionally substituted C2-C6-alkenyl or
wherein * is the point of attachment to R5 and #is the point of attachment to N.
In some embodiments in which both R3 and R4 are H, the compound of Formula (I) can be:
In some embodiments in which both R3 and R4 are Q-R5, the compound of Formula (I) can be:
In yet other embodiments, in compounds of Formula (I), R3 is H and R4 is Q-R5. In such embodiments, Q can be a bond and R5 can be optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In such embodiments, the compound of Formula (I) can be selected from the compounds shown in SUBTABLE 1C.
In some embodiments, in compounds of Formula (I), R3 is H and R4 is Q-R5, wherein Q is optionally substituted C1-C6-alkyl and R5 is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C1-C6-alkyl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, (C0-C2 alkyl)-heterocycloalkyl, (C0-C2 alkyl)-aryl, and (C0-C2 alkyl)-heteroaryl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl, and C1-C2 hydroxyalkyl. In some embodiments, the compound of Formula (I) can be selected from the compounds shown in SUBTABLE 1D.
In some embodiments, in compounds of Formula (I), R3 is H and R4 is Q-R5, wherein Q is optionally substituted C2-C6-alkenyl and R5 is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C2-C6-alkenyl.
In various embodiments, in compounds of Formula (I), in which one or more of R3 and R4 is Q-R5, Q is C1-C6-alkyl substituted with one or more of C3-C6-cycloalkyl, C2-C6-heterocycloalkyl, C3-C7-heterobicycloalkyl, C6-C10-heterospirocycloalkyl, aryl, or heteroaryl. In some of these embodiments, such ring substituent (referred to below as “W”) can be a bridging moiety so that Q is:
wherein:
In some of these embodiments, W is unsubstituted C3-C6-cycloalkyl, unsubstituted C2-C6-heterocycloalkyl, unsubstituted C3-C7-heterobicycloalkyl, unsubstituted C6-C10-heterospirocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In such embodiments, Q can be selected from the group consisting of:
wherein a and b are independently 1, 2, or 3.
In some embodiments, in compounds of Formula (I), R3 and R4 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring, or an optionally substituted heteroaryl ring. In some embodiments, R3 and R4 together with the N form a C3-C6-heterocycloalkyl ring, a C3-C6-heterobicycloalkyl ring, a C6-C10-heterospirocycloalkyl ring, or a heteroaryl ring, wherein the ring moiety is optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, and (C0-C2 alkyl)-heterocycloalkyl.
In some embodiments, R3 and R4 together with the N form a ring selected from the group consisting of:
wherein:
In some embodiments, in compounds of Formula (I), R3 and R4 together with the N form a ring selected from the group consisting of:
In some embodiments, in compounds of Formula (I), R3 and R4 together with the N form a ring moiety as described herein, and wherein the compound is selected from the compounds shown in SUBTABLE 1B.
In some embodiments, in compounds of Formula (I) in which at least one of R3 and R4 is Q-R5, R5 can be NR6R7. In such embodiments, R6 and R7 can be independently H, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxycarbonyl. In some embodiments, R6 and R7 are both H. In some embodiments, R6 and R7 are both optionally substituted C1-C6 alkyl, and wherein R6 is identical to R7. In such embodiments, R6 and R7 can both be C1-C6 alkyl, substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, and C1-C2 hydroxyalkyl. In some embodiments, R6 and R7 are C1-C2 alkyl substituted with one or more of —NH2, —CO2H, —OH, or halogen. In other embodiments, R6 is H and R7 is optionally substituted C1-C6 alkyl. In some embodiments, R6 is H and R7 can be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (I) in which at least one of R3 and R4 is Q-R5, R5 can be NR6R7, wherein R6 and R7 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring, or an optionally substituted heteroaryl ring. In some embodiments, such ring moiety can be optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C3 aminoalkyl and C1-C2 hydroxyalkyl. In some embodiments, R6 and R7 together with the N form a ring selected from the group consisting of:
In some embodiments, a compound of Formula (I) is selected from the compounds shown in SUBTABLE 1E.
In some embodiments, in compounds of Formula (I), n is 1.
In some embodiments, in compounds of Formula (I), m is an integer from 0 to 3 or from 1 to 3. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In yet other embodiments, m is 3.
In some embodiments, in compounds of Formula (I), R2 is H and (a) R1 is branched and optionally substituted C3-C8 hydroxyalkyl, and/or (b) X is NH and R1 is optionally substituted C5-C6 alkyl, and/or (c) m is 0 or 1 and R3 and R4 together with the N form an unsubstituted piperazinyl ring or an optionally substituted C6-C10-heterospirocycloalkyl ring. In some embodiments, R2 is H and R1 is branched and optionally substituted C3-C8 hydroxyalkyl. In such embodiments, R1 can be branched and unsubstituted C3-C8 hydroxyalkyl. In other embodiments, R2 is H, X is NH, and R1 is optionally substituted C5-C6 alkyl. In some of those embodiments, R1 is unsubstituted C5-C6 alkyl. In yet other embodiments, R2 can be H, m can be 0 or 1, and R3 and R4 together with the N can form an unsubstituted piperazinyl ring or an optionally substituted C6-C10-heterospirocycloalkyl ring. In some embodiments, R3 and R4 together with the N form an unsubstituted piperazinyl ring. In yet other embodiments, R3 and R4 together with the N form an optionally substituted C6-C10-heterospirocycloalkyl ring.
In some embodiments, a compound of Formula (I) is selected from TABLE 1 herein, which comprises SUBTABLES 1A-1F.
In certain embodiments of the present disclosure, a compound of Formula (I) has the structure of Formula (II):
wherein:
wherein * is the point of attachment to R5 and #is the point of attachment to N;
In some embodiments, in compounds of Formula (II), X is O and R1 is optionally substituted C2-C6 alkyl. In some of these embodiments, R1 is unsubstituted C2-C6 alkyl. In some specific embodiments, R1 is ethyl or n-butyl.
In some embodiments, in compounds of Formula (II), Y is CH.
In some embodiments, in compounds of Formula (II), m is 0 or 1, 1 or 2, or 1 or 3. In such embodiments, m can be 0. In other embodiments, m is 1, 2, or 3. In some embodiments, m is 1. In some embodiments, m is 2. In yet other embodiments, m is 3.
In some embodiments, in compounds of Formula (II), R3 and R4 are both H. In other embodiments, R3 and R4 are both Q-R5. In such embodiments, Q can be optionally substituted C1-C6-alkyl, and R5 can be H or OH. In some of these embodiments, Q is unsubstituted C1-C6-alkyl and R5 is H. In other embodiments, Q is a bond and R5 is unsubstituted C3-C6-carboxyalkyl.
In yet other embodiments, in compounds of Formula (II), R3 is H and R4 is Q-R5. In such embodiments, Q can be a bond and R5 can be optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (II), R3 is H and R4 is Q-R5, wherein Q is optionally substituted C1-C6-alkyl and R5 is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C1-C6-alkyl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, (C0-C2 alkyl)-heterocycloalkyl, (C0-C2 alkyl)-aryl, and (C0-C2 alkyl)-heteroaryl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl, and C1-C2 hydroxyalkyl.
In some embodiments, in compounds of Formula (II), R3 is H and R4 is Q-R5, wherein Q is optionally substituted C2-C6-alkenyl and R5 is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C2-C6-alkenyl.
In some of these embodiments, the compound is:
In various embodiments, in compounds of Formula (II), in which one or more of R3 and R4 is Q-R5, Q is C1-C6-alkyl substituted with one or more of C3-C6-cycloalkyl, C2-C6-heterocycloalkyl, C3-C7-heterobicycloalkyl, C6-C10-heterospirocycloalkyl, aryl, or heteroaryl. In some of these embodiments, such ring substituent (referred to below as “W”) can be a bridging moiety so that Q is:
wherein:
In such embodiments, W is an unsubstituted ring moiety such that Q can be selected from the group consisting of:
wherein a and b are independently 1, 2, or 3.
In some embodiments, in compounds of Formula (II), R3 and R4 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring. In some embodiments, R3 and R4 together with the N form a C2-C6-heterocycloalkyl ring, a C3-C7-heterobicycloalkyl ring, a C6-C10-heterospirocycloalkyl ring or a heteroaryl ring, wherein the ring moiety is optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, or (C0-C2 alkyl)-heterocycloalkyl. In some embodiments, R3 and R4 together with the N form a ring selected from the group consisting of:
wherein:
In some embodiments, in compounds of Formula (II), R3 and R4 together with the N form a ring selected from the group consisting of:
In some embodiments, in compounds of Formula (II) in which at least one of R3 and R4 is Q-R5, R5 can be NR6R7. In such embodiments, R6 and R7 can be independently H, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxycarbonyl. In some embodiments, R6 and R7 are both H. In some embodiments, R6 and R7 are both optionally substituted C1-C6 alkyl such that R6 is identical to R7. In such embodiments, R6 and R7 can both be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, and C1-C2 hydroxyalkyl. In some embodiments, R6 and R7 are C1-C2 alkyl substituted with one or more of —NH2, —CO2H, —OH, and halogen. In other embodiments, R6 is H and R7 is optionally substituted C1-C6 alkyl. In some embodiments, R6 is H and R7 can be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (II), R5 is NR6R7, wherein R6 and R7 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring. In some embodiments, such ring moiety can be optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C3 aminoalkyl and C1-C2 hydroxyalkyl. In some embodiments, R6 and R7 together with the N form a ring selected from the group consisting of:
In some embodiments, a compound of Formula (II) is selected from SUBTABLE 1A.
In some embodiments, the compound of Formula (I) or Formula (II) is:
In various embodiments, the present disclosure relates to compounds of Formula (III):
In some embodiments, in compounds of Formula (III), R1a is optionally substituted C2-C6 alkyl. In such embodiments, Ria can be unsubstituted C2-C6 alkyl. In some of these embodiments, R1a can be ethyl or n-butyl.
In some embodiments, in compounds of Formula (III), R2a is halogen or optionally substituted C1-C6 alkoxy. In such embodiments, R2a can be halogen or unsubstituted C1-C6 alkoxy. In certain embodiments, R2a is fluorine or methoxy.
In some embodiments, in compounds of Formula (III), R3a and R4a are both H. In other embodiments, R3a and R4a are both Q-R5a. In such embodiments, Q can be optionally substituted C1-C6-alkyl, and R5a can be H or OH. In some of these embodiments, Q is unsubstituted C1-C6-alkyl and R5a is H. In other embodiments, Q is a bond and R5a is unsubstituted C3-C6-carboxyalkyl.
In yet other embodiments, in compounds of Formula (III), R3a is H and R4a is Q-R5a. In such embodiments, Q can be a bond and Ra can be optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (III), R3a is H and R4a is Q-R5a, wherein Q is optionally substituted C1-C6-alkyl and R5a is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C1-C6-alkyl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, (C0-C2 alkyl)-heterocycloalkyl, (C0-C2 alkyl)-aryl, and (C0-C2 alkyl)-heteroaryl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl, and C1-C2 hydroxyalkyl.
In some embodiments, in compounds of Formula (III), R3a is H and R4a is Q-R5a, wherein Q is optionally substituted C2-C6-alkenyl and R5a is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C2-C6-alkenyl.
In various embodiments, in compounds of Formula (III), in which one or more of R3a and R4a is Q-R5a, Q is C1-C6-alkyl substituted with one or more of C3-C6-cycloalkyl, C2-C6-heterocycloalkyl, C3-C7-heterobicycloalkyl, C6-C10-heterospirocycloalkyl, aryl, or heteroaryl. In some of these embodiments, such ring substituent (referred to below as “W”) can be a bridging moiety so that Q is:
wherein:
In some of these embodiments, W is an unsubstituted ring moiety such that Q can be selected from the group consisting of:
wherein a and b are independently 1, 2, or 3.
In some embodiments, in compounds of Formula (III), R3a and R4a together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring. In some embodiments, R3a and R4a together with the N form a C2-C6-heterocycloalkyl ring, a C3-C7-heterobicycloalkyl ring, a C6-C10-heterospirocycloalkyl ring or a heteroaryl ring, wherein the ring moiety can be optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, and (C0-C2 alkyl)-heterocycloalkyl.
In some embodiments, R3a and R4a together with the N form a ring selected from the group consisting of:
wherein:
In some embodiments, in compounds of Formula (III), R3a and R4a together with the N form a ring selected from the group consisting of:
In some embodiments, in compounds of Formula (III), R5a is NR6aR7a. In such embodiments, R6a and R7a can be independently H, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxycarbonyl. In some embodiments, R6a and R7a are both H. In other embodiments, R6a and R7a are both optionally substituted C1-C6 alkyl, wherein R6a is identical to R7a. In such embodiments, R6a and R7a can both be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, and C1-C2 hydroxyalkyl. In some embodiments, R6a and R7a are C1-C2 alkyl substituted with one or more of —NH2, —CO2H, —OH, or halogen. In other embodiments, R6a is H and R7a is optionally substituted C1-C6 alkyl. In some embodiments, R6a is H and R7a can be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (III) in which at least one of R3a and R4a is Q-R5a, R5a can be NR6aR7a, wherein R6a and R7a together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring. In some embodiments, such ring moiety can be optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C3 aminoalkyl and C1-C2 hydroxyalkyl. In some embodiments, R6 and R7 together with the N form a ring selected from the group consisting of:
In various embodiments, in compounds of Formula (III), u is 1.
In various embodiments, in compounds of Formula (III), v is an integer from 0 to 4, from 1 to 4, 1 to 3, or from 1 to 2. In some embodiments, v is 0. In some embodiments, v is 1. In other embodiments, v is 2. In yet other embodiments, v is 3.
In some embodiments, a compound of Formula (III) is selected from SUBTABLE 1A.
In various embodiments of this disclosure, a compound herein of Formula (III) is a compound according to Formula (IV):
wherein:
In some embodiments, in compounds of Formula (IV), R1a is unsubstituted C2-C6 alkyl, such as unsubstituted C2-C4 alkyl. In other embodiments, R1a is branched and unsubstituted C3-C8 hydroxyalkyl.
In some embodiments, in compounds of Formula (IV), R3a and R4a are both H. In other embodiments, R3a and R4a are both Q-R5a. In such embodiments, Q can be optionally substituted C1-C6-alkyl, and R5a can be H or OH. In some of these embodiments, Q is unsubstituted C1-C6-alkyl and R5a is H. In other embodiments, Q is a bond and R5a is unsubstituted C3-C6-carboxyalkyl.
In yet other embodiments, in compounds of Formula (IV), R3a is H and R4a is Q-R5a. In such embodiments, Q can be a bond and R5a can be H, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (IV), R3a is H and R4a is Q-R5a, wherein Q is optionally substituted C1-C6-alkyl and R5a is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C1-C6-alkyl. In other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, (C0-C2 alkyl)-heterocycloalkyl, (C0-C2 alkyl)-aryl, and (C0-C2 alkyl)-heteroaryl. In yet other embodiments, Q is C1-C6-alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkyl, and C1-C2 hydroxyalkyl.
In some embodiments, in compounds of Formula (IV), R3a is H and R4a is Q-R5a, wherein Q is optionally substituted C2-C6-alkenyl and R5a is H, OH, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Q is unsubstituted C2-C6-alkenyl.
In various embodiments, in compounds of Formula (IV), in which one or more of R3a and R4a is Q-R5a, Q is C1-C6-alkyl substituted with one or more of C3-C6-cycloalkyl, C2-C6-heterocycloalkyl, C3-C7-heterobicycloalkyl, C6-C10-heterospirocycloalkyl, aryl, or heteroaryl. In some of these embodiments, such ring substituent (referred to below as “W”) can be a bridging moiety so that Q is:
wherein:
In such embodiments, W is an unsubstituted ring moiety such that Q can be selected from the group consisting of:
wherein a and b are independently 1, 2, or 3.
In some embodiments, in compounds of Formula (IV), R3a and R4a together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring. In some embodiments, R3a and R4a together with the N form a C2-C6-heterocycloalkyl ring, a C3-C7-heterobicycloalkyl ring, a C6-C10-heterospirocycloalkyl ring or a heteroaryl ring, wherein the ring moiety can be optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, (C0-C2 alkyl)-cycloalkyl, or (C0-C2 alkyl)-heterocycloalkyl. In some embodiments, R3a and R4a together with the N form a ring selected from the group consisting of:
wherein:
In some embodiments, in compounds of Formula (IV), R3a and R4a together with the N form a ring selected from the group consisting of:
In some embodiments, in compounds of Formula (IV) in which one or more of R3a and R4a is Q-R5a, R5a can be NR6aR7a. In such embodiments, R6a and R7a can be independently H, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxycarbonyl. In some embodiments, R6a and R7a are both H. In some embodiments, R6a and R7a are both optionally substituted C1-C6 alkyl, wherein R6a is identical to R7a. In such embodiments, R6a and R7a can be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, and C1-C2 hydroxyalkyl. In some embodiments, R6a and R7a are C1-C2 alkyl substituted with one or more of —NH2, —CO2H, —OH, or halogen. In other embodiments, R6a is H and R7a is optionally substituted C1-C6 alkyl. In some embodiments, R6a is H and R7a can be C1-C6 alkyl substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C2 hydroxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl.
In some embodiments, in compounds of Formula (IV) in which one or more of R3a and R4a is Q-R5a, R5a can be NR6aR7a, wherein R6a and R7a together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring. In some embodiments, such ring moiety can be optionally substituted with one or more of —NH2, —CO2H, —OH, carbonyl, halogen, C1-C2 alkylthio, C1-C2 alkyl, C1-C3 aminoalkyl and C1-C2 hydroxyalkyl. In some embodiments, R6 and R7 together with the N form a ring selected from the group consisting of:
In some embodiments, in compounds of Formula (IV), v is 0. In other embodiments, v is 1. In some embodiments, v is 2. In yet other embodiments, v is 3.
In some embodiments, a compound of Formula (IV) is selected from the compounds shown in SUBTABLE 1A.
In some embodiments, a compound of the present disclosure, e.g., a compound of any one of Formulae (I)-(IV) herein, is selected from TABLE 1, which comprises SUBTABLES 1A-1F.
It is to be understood that reference to compounds of Formula (I) throughout this disclosure, includes in various embodiments, compounds of Formulae (I)-(IV), to the same extent as if embodiments reciting each of these Formulae individually were specifically recited.
In certain embodiments, a compound having Formula (I), as described herein, can possess a sufficiently acidic group, a sufficiently basic group, or both functional groups, and accordingly react with a number of organic and inorganic bases, or organic and inorganic acids, to form pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt,” as used herein, refers to a salt of a compound having Formula (I), which is substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound having Formula (I), with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts.
Acids commonly employed to form acid addition salts are inorganic acids including, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, phosphoric acid, and organic acids including, but not limited to, p-toluenesulphonic acid, methanesulphonic acid, oxalic acid, p-bromophenylsulphonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, sulphates, pyrosulphates, bisulphates, sulphites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, hydrochlorides, dihydrochlorides, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulphonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, methanesulphonates, propanesulphonates, naphthalene-1-sulfonates, napththalene-2-sulfonates and mandelates. Pharmaceutically acceptable acid addition salts of particular interest are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulphonic acid.
Salts of amine groups can also comprise quaternary ammonium salts in which the amino nitrogen carries a suitable organic group such as an alkyl, lower alkenyl, lower alkynyl or aralkyl moiety.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing pharmaceutically acceptable salts include, but are not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide and calcium carbonate.
One skilled in the art will understand that the particular counterion forming a part of a pharmaceutically acceptable salt is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
As described herein, a compound of Formulae (I)-(IV) can comprise one or more free amino, hydroxy, carbonyl (for example, keto or aldehyde) and/or carboxylic acid groups. Also encompassed by the present disclosure are protected versions of the compounds of Formulae (I)-(IV) in which an otherwise free amino, hydroxy, carbonyl (for example, keto or aldehyde) and/or carboxylic acid group is protected with an appropriate protecting group. The term “protecting group” refers to a chemical group that, when attached to a potentially reactive functional group, masks, reduces or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed as desired during the course of a synthesis.
Protecting groups are well-known in the art and various examples are described, for example, in “Protective Groups in Organic Chemistry” (Greene, W. & Wuts, P. G. M., 2006, John Wiley & Sons). Examples of amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl (Bn), benzoyl (Bz), benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethylsilyl (TMS), 2-trimethylsilyl-ethanesulfonyl (TES), trityl, substituted trityl, tosyl, phthalimide, alloxycarbonyl (Alloc) and 9-fluorenylmethyloxycarbonyl (FMOC). Examples of hydroxy protecting groups include, but are not limited to, acetyl, benzyl (Bn), t-butyl, benzoyl (Bz), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl](MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl, trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS or TBS), tri-iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS). Examples of carbonyl protecting groups include, but are not limited to, acetals, hemi-acetals, and ketals. Examples of carboxylic acid protecting groups include, but are not limited to, methyl esters, benzyl esters, tert-butyl esters, silyl esters, orthoesters and oxazoline.
Certain embodiments relate to pharmaceutically acceptable solvates of a compound having Formula (I)-(IV). One skilled in the art will appreciate that certain compounds having Formula (I)-(IV), can combine with solvents such as water, methanol, ethanol, or acetonitrile to form pharmaceutically acceptable solvates such as the corresponding hydrate, methanolate, ethanolate or acetonitrilate. Other examples of solvents that can be used to prepare solvates include isopropanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, and acetone, as well as miscible formulations of solvate mixtures as would be known by the skilled artisan.
In various embodiments, a compound of the present disclosure, e.g., a compound having a structure according to any one of Formulae (I)-(IV), is an agonist of TLR7. In some embodiments, a compound of the present disclosure (e.g., one according to any one of Formulae (I)-(IV)) is capable of inducing production of one or more cytokine(s) in immune cells (e.g., PBMCs). In such embodiments, the compound can induce production of, e.g., IL6, IFN-α and/or TNF-α in immune cells (e.g., PBMCs) when such cells are contacted with the compound.
In some embodiments, a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), can have an EC50 of about 1 μM or less, 750 nM or less, 650 nM or less, 500 nM or less, 300 nM or less, 275 nM or less, 250 nM or less, 225 nM or less, 200 nM or less, 175 nM or less, 150 nM or less, 125 nM or less, 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, or about 10 nM or less, for agonism of TLR7, as determined, e.g., in a reporter gene assay. The TLR7 can be a human TLR7 or a murine TLR7, or both. In some embodiments, a compound of any one of Formulae (I)-(IV) has an EC50 value for agonizing human and/or murine TLR7 of about 1 μM or less. In some embodiments, a compound of any one of Formulae (I)-(IV) has an EC50 value for agonizing human and/or murine TLR7 of about 500 nM or less. In some embodiments, a compound of any one of Formulae (I)-(IV) has an EC50 value for agonizing human and/or murine TLR7 of about 300 nM or less. In some embodiments, a compound of any one of Formulae (I)-(IV) has an EC50 value for agonizing human and/or murine TLR7 of about 100 nM or less. In other embodiments, a compound of any one of Formulae (I)-(IV) has an EC50 value for agonizing human and/or murine TLR7 of about 50 nM or less. In yet other embodiments, a compound of any one of Formulae (I)-(IV) has an EC50 value for agonizing human and/or murine TLR7 of about 25 nM or less. As used herein, an EC50 value generally refers to the half maximal effective concentration of the respective compound, wherein the value of the EC50 indicates the concentration of the compound that induces a biological response (e.g., TLR7 agonism, stimulation of the immune system, production of cytokines by immune cells, and/or killing of tumor cells) halfway between the baseline and the maximum after a defined duration of exposure.
In some embodiments, the present disclosure relates to purine-derived compounds, e.g., those having a structure according to any one of Formulae (I)-(IV), that have an EC50 for agonizing TLR7 from about 1 μM to about 750 nM, from about 1 μM to about 500 nM, from about 1 μM to about 300 nM, from about 1 μM to about 200 nM, from about 1 μM to about 100 nM, from about 1 μM to about 50 nM, from about 500 nM to about 25 nM, from about 500 nM to about 15 nM, from about 500 nM to about 5 nM, from about 500 nM to about 1 nM, from about 500 nM to about 0.1 nM, from about 500 nM to about 0.01 nM, from about 300 nM to about 25 nM, from about 300 nM to about 15 nM, from about 300 nM to about 5 nM, from about 300 nM to about 1 nM, from about 300 nM to about 0.1 nM, from about 300 nM to about 0.01 nM, from about 200 nM to about 25 nM, from about 200 nM to about 15 nM, from about 200 nM to about 5 nM, from about 200 nM to about 1 nM, from about 200 nM to about 0.1 nM, from about 200 nM to about 0.01 nM, from about 100 nM to about 25 nM, from about 100 nM to about 15 nM, from about 100 nM to about 5 nM, from about 100 nM to about 1 nM, from about 100 nM to about 0.1 nM, from about 100 nM to about 0.01 nM, from about 50 nM to about 25 nM, from about 50 nM to about 15 nM, from about 50 nM to about 5 nM, from about 50 nM to about 1 nM, from about 50 nM to about 0.1 nM, or from about 50 nM to about 0.01 nM. In various embodiments, the TLR7 is a human TLR7.
An EC50 value for TLR7 agonism of a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), can be determined using various methods known in the art, e.g., in vitro using a reporter gene assay employing TLR7 reporter cells. In such embodiments, the EC50 value can be determined in vitro using a reporter gene assay employing HEK-Blue™ TLR7 reporter cells (e.g., available from Invivogen, San Diego, CA), and as described in, e.g., EXAMPLE 3 herein.
In some embodiments, and as further described herein, it was surprisingly found that particularly those purine-derived compounds of Formulae (I)-(IV) that contain a substituent at R2 (i.e., R2≠H) can provide potent immunomodulatory activity in vitro as well as in vivo, when used both as a free molecule and in form of an immunostimulatory antibody drug conjugate (ISAC). Such compounds can elicit their immunomodulatory activities in both murine and human settings targeting murine as well as human TLR7. In various embodiments, such substituent at the R2 position can be an alkoxy moiety or a halogen atom. In specific embodiments, the R2 substituent can be methoxy or fluorine. Such compounds herein can have an EC50 for agonizing TLR7 of equal to or less than about 500 nM, 100 nM, 50 nM, 30 nM, 20 nM, equal to or less than about 10 nM, or lower, as determined, e.g., using reporter gene assays (RGAs), as described herein.
In other embodiments, a compound of Formula (I) in which R2 is H and either (i) R1 is branched and optionally substituted C3-C8 hydroxyalkyl, (ii) X is NH and R1 is optionally substituted C5-C6 alkyl, or (iii) m is 0 or 1 and R3 and R4 together with the N form an unsubstituted piperazinyl, can possess strong immunomodulatory properties that can be useful for the treatment of diseases such as cancer.
In some embodiments, a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), can have an EC50 for inducing production of one or more human and/or murine cytokines from human or murine immune cells, respectively, of about 5 μM or less, 3 μM or less, 1 μM or less, 750 nM or less, about 500 nM or less, about 300 nM or less, about 275 nM or less, about 250 nM or less, about 225 nM or less, about 200 nM or less, about 175 nM or less, about 150 nM or less, about 125 nM, about 100 nM or less, about 50 nM or less, about 25 nM or less, or about 10 nM or less. In some embodiments, a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), can have an EC50 for inducing production of one or more human and/or murine cytokines from human or murine immune cells, respectively, from about 5 μM to about 5 nM, from about 1 μM to about 1 nM, from about 750 nM to about 1 nM, from about 500 nM to about 1 nM, from about 300 nM to about 1 nM or from about 200 nM to about 1 nM. In various embodiments, the one or more human and/or murine cytokines comprise IL6. In some embodiments, the human immune cells comprise PBMCs, and the murine immune cells comprise murine splenocytes. In various embodiments, the EC50 values for inducing production of human or murine IL6 from PBMCs or mouse splenocytes are determined using a human PBMC assay or a mouse splenocyte assay, respectively, e.g., as described in EXAMPLE 3 herein.
In some embodiments, a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), can have an EC50 for inducing production of human interferon-α (hIFN-α) of about 10 nM or less, 5 nM or less, 1 nM or less, or 0.1 nM or less. In some embodiments, a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), can have an EC50 for inducing production of hIFN-α of from about 10 nM to about 1 nM, from about 1 nM to about 0.1 nM, or lower than 0.1 nM. In various embodiments, the EC50 values for inducing production of hIFN-α from PBMCs are determined using a human PBMC assay, e.g., as described in EXAMPLE 3 herein.
In certain embodiments, the EC50 for inducing production of a cytokine from PBMCs by a compound of the present disclosure, e.g., one having a structure according to any one of Formulae (I)-(IV), is determined in vitro by treating PBMCs isolated from peripheral blood with titrating concentrations of the tested compound followed by assaying for cytokines, e.g., by homogeneous time resolved fluorescence (HTRF). An exemplary method for determining EC50 values for inducing production of a cytokine from PBMCs is provided in EXAMPLE 3 herein.
The purine-derived compounds of the present disclosure, e.g., those of Formulae (I)-(IV), can be prepared by standard synthetic organic chemistry methods from commercially available starting materials and reagents. Representative examples of suitable synthetic routes are described in detail in EXAMPLE 1 provided herein (see also,
A purine-derived compound of the present disclosure can be coupled to a linker moiety. In certain embodiments, the present disclosure relates to compound-linker constructs comprising one or more compounds (C) of the present disclosure, e.g., those having a structure according to any one of Formulae (I)-(IV), e.g., any of the compounds listed in TABLE 1, coupled to a linker moiety (L).
In various embodiments, the present disclosure relates to a compound-linker construct having Formula (A):
L-(C)p (A)
wherein:
In various embodiments of a compound-linker construct, p can be an integer from 1 to 4. In other embodiments, p is an integer from 1 to 3. In yet other embodiments, p is an integer from 1 to 2. In various embodiments, p is 1. In other embodiments, p is 2. In yet other embodiments, p is 3.
A linker (L) herein can be a bifunctional or multifunctional moiety capable of linking one or more immunomodulatory purine-derived compounds, C, of the present disclosure to another molecule, such as a targeting moiety, T, as further described elsewhere herein.
A bifunctional (or monovalent) linker, L, links a single compound C to a single site (e.g., functional group) on a targeting moiety, T. In such embodiments, p is 1. A multifunctional (or polyvalent) linker, L, links more than one compound, C, to a single site (e.g., functional group) on a targeting moiety, T. A linker that links one compound, C, to more than one site on targeting moiety, T, can also be considered to be multifunctional. However, in embodiments where the linker (L) is polyvalent and couples more than one compound (C) to another moiety, such as a targeting moiety (T), p can be >1, such as an integer from 2 to 5.
A linker, L, of a compound-linker construct herein can comprise a functional group capable of reacting with a target group or groups on a targeting moiety, T, and at least one functional group capable of reacting with a target group on a compound of the present disclosure (e.g., a compound according to any one of Formulae (I)-(IV)), C. Suitable functional groups are known in the art and include those described herein, for example, in Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). Functional groups on targeting moiety, T, and the purine-derived compound of the present disclosure, C, that can serve as target groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.
In some embodiments, a linker herein comprises or consists of a substituted or unsubstituted alkyl or a substituted or unsubstituted heteroalkyl chain comprising a first and a second terminal functional group. In some embodiments, the first terminal functional group of the linker forms a first linkage or bond with a first reactive functional group on a first conjugation partner (e.g., a targeting moiety, T), and the second terminal functional group of the linker forms a second linkage or bond with a second reactive functional group of a second conjugation partner (e.g., a compound C of Formulae (I)-(IV)). In some embodiments, the linker comprises a substituted or unsubstituted hydrocarbon backbone. In some embodiments, the substituted or unsubstituted hydrocarbon backbone is interrupted by one or more heteroatoms (e.g., O, N, S, P), thereby forming, e.g., a heteroalkyl linker.
In some embodiments, a linker (L) is a cleavable linker. In other embodiments, the linker (L) is a non-cleavable linker. A cleavable linker herein is a linker that is susceptible to cleavage under specific conditions, for example, intracellular conditions (such as in an endosome or lysosome) or within the vicinity of a target cell (such as in the tumor microenvironment). Examples include linkers that are protease-sensitive, acid-sensitive, or reduction-sensitive. Non-cleavable linkers by contrast, can rely on the degradation of a targeting moiety (e.g., antibody)—to which it is attached—in the cell, which typically results in the release of an amino acid-linker-compound moiety. In some embodiments, the linker includes an alkylene oxide, e.g., a poly (alkylene oxide), or a poly (ethylene glycol) (PEG) moiety.
In some embodiments, the linker (L) of a compound-linker construct herein comprises a polyethylene glycol (PEG) moiety. Such PEG moiety can have a molecular weight from about 500 Da to about 5 kDa, from about 500 Da to about 3 kDa, from about 500 Da to about 1 kDa, from about 500 Da to about 1 kDa, or from about 100 Da to 500 Da.
Examples of cleavable linkers include, for example, linkers comprising an amino acid sequence that is a cleavage recognition sequence for a protease. Many such cleavage recognition sequences are known in the art. In embodiments in which a compound-linker construct herein is used in conjugates that are not intended to be internalized by a cell, for example, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix in the vicinity of a target cell, such as a cancer cell, can be employed. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteases (MMPs), elastase and kallikrein-related peptidases.
In embodiments in which a compound-linker construct herein is used in conjugates intended to be internalized by a cell, a linker, L, can comprise an amino acid sequence that is recognized and cleaved by an endosomal or lysosomal protease. Examples of such proteases include, for example, cathepsins B, C, D, H, L and S, and legumain.
Cleavage recognition sequences can be, for example, dipeptides, tripeptides or tetrapeptides. Non-limiting examples of dipeptide recognition sequences that can be included in cleavable linkers described herein include, but are not limited to, Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Val-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys. Examples of tri- and tetrapeptide cleavage sequences include, but are not limited to, Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val-Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, Asn-Pro-Val, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly. In some embodiments, a linker (L) comprises a dipeptide, tripeptide, tetrapeptide, or a combination thereof. In some of these embodiments, the linker (L) comprises a dipeptide. In other embodiments, the linker (L) comprises a tripeptide. In yet other embodiments, the linker (L) comprises a tetrapeptide.
Additional examples of cleavable linkers include disulfide-containing linkers such as N-succinimydyl-4-(2-pyridyldithio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB). Disulfide-containing linkers can optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Other cleavable linkers include linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers comprising combinations of these functionalities can also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.
A further example of a cleavable linker is a linker comprising a β-glucuronide, which is cleavable by β-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf et al., 2002, Curr. Pharm. Des. 8:1391-1403, and International Patent Publication No. WO 2007/011968). β-glucuronide can also function to improve the hydrophilicity of linker, L.
Another example of a linker that is cleaved internally within a cell and improves hydrophilicity is a linker comprising a pyrophosphate diester moiety (see, for example, Kern et al., 2016, J Am Chem Soc., 138:2430-1445).
In certain embodiments, the linker, L, comprised by a compound-linker construct of Formula (A) is a cleavable linker. In some embodiments, such linker, L, comprises a cleavage recognition sequence. In some embodiments, such linker, L, can comprise an amino acid sequence that, e.g., one that is 2 or 3 residues in length, and is recognized and cleaved by a protease.
Cleavable linkers can optionally further comprise one or more additional functionalities such as self-immolative and self-elimination groups, stretchers, hydrophilic moieties, or a combination thereof.
Self-immolative and self-elimination groups that can find use in linkers herein include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, and methylated ethylene diamine (MED). Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PAB or PABC group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U.S. Pat. No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). Self-immolative/self-elimination groups are typically attached to an amino or hydroxyl group on the compound, C. Self-immolative/self-elimination groups, alone or in combination are often included in peptide-based linkers but can also be included in other types of linkers.
Stretchers that can find use in linkers for conjugates as further described herein include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers.
PEG and mPEG stretchers can also function as hydrophilic moieties within a linker of the present disclosure. For example, PEG or mPEG can be included in a linker either “in-line” or as pendant groups to increase the hydrophilicity of the linker (see, for example, U.S. Patent Application Publication No. US 2016/0310612). Various PEG-containing linkers are commercially available from companies such as Quanta BioDesign, Ltd (Plain City, OH). Other hydrophilic groups that can optionally be incorporated into linker, L, include, for example, β-glucuronide, sulfonate groups, carboxylate groups and pyrophosphate diesters.
In some embodiments, a compound-linker construct of this disclosure can comprise or consist of a compound of Formulae (I)-(IV) herein coupled to any of the linker moieties L1-L18 shown in TABLE 2A ( indicates the attachment to a compound of Formulae (I)-(IV)).
In certain embodiments, compound-linker constructs of Formula (A) can comprise a cleavable linker. In some embodiments, compound-linker constructs of Formula (A) can comprise a peptide-containing linker. In some embodiments, compound-linker constructs of Formula (A) can comprise a protease-cleavable linker.
In various embodiments, and with reference to Formula (I), a linker moiety (L) can be attached to a compound having a structure of Formula (I) at any suitable atom. Suitable groups on compounds of Formulae (I)-(IV), C, for attachment of linker, L, in either of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups and hydroxyl groups. In some embodiments of the present disclosure, linker, L, is attached to a compound of Formula (I), C, via a hydroxyl or amine group on the compound.
In some embodiments, the attachment site for the linker moiety is one of the sites shown below in Formula (I-L), based on Formula (I), with “L” indicating potential linker attachment sites:
“R3/4” indicates that when either R3 or R4 in Formula I is H, such hydrogen atom can be substituted with a linker moiety, e.g., as shown herein for MT-VC-PABC-Compound 105 and MT-VC-PABC-Compound 107. Furthermore, and in embodiments where either R3 and/or R4 in Formula I are substituents containing one or more additional nitrogen atoms suitable for attachment of a linker L, such nitrogen atom(s) may be the attachment site for the linker moiety L. Examples thereof include MT-VC-PABC-Compound 100, MT-VC-PABC-Compound 106 and MT-VC-PABC-Compound 111.
In some embodiments, a compound-linker construct herein comprising a purine-derived compound of any one of Formulae (I)-(IV) can be selected from TABLE 2B.
A compound-linker construct of Formula (A) herein can be prepared using any suitable synthetic technique known in the art, e.g., as further described herein in EXAMPLE 2.
Further disclosed herein are conjugates (also referred to herein as “immunostimulatory antibody-drug conjugates” or “ISACs”) comprising a targeting moiety (T) coupled to one or more compounds of the present disclosure, e.g., one or more compounds according to any one or more of Formulae (I)-(IV). In various embodiments, a conjugate herein comprises one or more compound-linker construct(s) of Formula (A), L-(C)p, as described herein.
In some embodiments, the conjugates of the present disclosure can comprise one or more compounds of any one of Formulae (I)-(IV) conjugated to the targeting moiety (T). In various embodiments, a conjugate of the present disclosure has a structure according to Formula (X):
T-[L-(C)p]r (X)
wherein:
In various embodiments of a conjugate of Formula (X) herein, L-(C)p is a compound-linker construct as described herein.
As further described herein, in certain embodiments, the compounds disclosed herein can be used for the preparation of conjugates that are immunostimulatory antibody-drug conjugates (ISACs) that comprise an antibody as the targeting moiety which recognizes and binds to a tumor-associated antigen (e.g., a protein that is expressed on the surface of a tumor cell to be targeted). At a target site (e.g., tumor), the immunostimulatory compound(s) of the present disclosure that are attached to the targeting moiety can be released inside an immune cell and/or within a tumor microenvironment (TME), e.g., by using a cleavable linker, thereby causing an anti-tumor effect via activation of the immune system (e.g., via TLR7-mediated signaling and/or cytokine production).
The targeting moiety, T, comprised by the conjugates of Formula (X) can be a molecule that binds, reactively associates, or complexes with a receptor, antigen or other receptive moiety associated with a given target cell population. Typically, the targeting moiety, T, functions to deliver a purine-derived compound, C, of the present disclosure to the particular target cell population with which the targeting moiety, T, reacts. Examples of targeting moieties include, but are not limited to, proteins (such as antibodies, antibody fragments and growth factors), glycoproteins, peptides (such as bombesin and gastrin-releasing peptide), lectins, vitamins (such as folic acid) and nutrient-transport molecules (such as transferrin).
Typically, the targeting moiety, T, will be bonded to linker, L, via a heteroatom of the targeting moiety, T, such as a sulfur (for example, from a sulfhydryl group), oxygen (for example, from a carbonyl, carboxyl, or hydroxyl group) or nitrogen (for example, from a primary or secondary amino group). These heteroatoms can be naturally present on targeting moiety, T, or can be introduced through engineering and/or expression, or can be introduced via chemical modification using techniques known in the art.
In some embodiments, targeting moiety, T, is an antibody. Accordingly, since the compounds of the present disclosure are capable of eliciting an immune response in a subject, various embodiments of the present disclosure relate to immunostimulatory antibody-drug conjugates (ISACs) having general Formula (X) in which the targeting moiety, T, is an antibody.
In embodiments in which the conjugate is an ISAC, the antibody included as the targeting moiety, T, can be a full-size polyclonal or monoclonal antibody, an antigen-binding antibody fragment (such as Fab, scFab, Fab′, F(ab′)2, Fv or scFv), a domain antibody (dAb) or an antibody mimetic (such as an affibody, a DARPin, an anticalin, a versabody, a duocalin, a lipocalin or an avimer). The antibody is typically directed to a particular antigen, for example, a tumor-associated antigen (TAA).
In certain embodiments in which the conjugate (X) is an ISAC, the targeting moiety, T, is a monoclonal antibody, an antigen-binding antibody fragment thereof (such as Fab, scFab, Fab′, F(ab′)2, Fv or scFv), or a domain antibody (dAb).
In certain embodiments, targeting moiety, T, can be a monoclonal antibody. The monoclonal antibody can be, for example, a non-human monoclonal antibody (such as a mouse or rabbit antibody), a human monoclonal antibody, a humanized monoclonal antibody, or a chimeric antibody (for example, a human-mouse antibody). In certain embodiments, the antibody included as a targeting moiety in a conjugate (X) herein can be a bispecific or multispecific antibody.
In certain embodiments, targeting moiety, T, comprised by the conjugate is an antibody or antigen-binding antibody fragment that binds to a tumor-associated antigen (TAA). In various embodiments, such TAA-binding antibody can comprise a functional Fc region capable of Fc-receptor binding.
As further described herein, e.g., in the “compound-linker construct” section, a linker can couple one or more immunomodulatory compounds of the present disclosure, e.g., those of any one of compounds (I)-(IV), to the targeting moiety (T). For a detailed description of linker moieties contemplated herein, it is referred to the “compound-linker construct” section above.
As further described herein, a linker, L, of a compound-linker construct herein can comprise a functional group capable of reacting with the target group or groups on a targeting moiety, T, and at least one functional group capable of reacting with a target group on a compound of the present disclosure (e.g., a compound according to any one of Formulae (I)-(IV)), C. Suitable functional groups are known in the art and include those described herein, for example, in Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). Functional groups on targeting moiety, T, and the purine-derived compound of the present disclosure, C, that can serve as target groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.
Non-limiting examples of functional groups capable of reacting with thiols include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters and tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this context are “self-stabilizing” maleimides as described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062.
Non-limiting examples of functional groups capable of reacting with amines include activated esters (such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut's reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU) and benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP).
Non-limiting examples of functional groups capable of reacting with an electrophilic group such as an aldehyde or ketone carbonyl group include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.
In certain embodiments in which targeting moiety, T, is an antibody, linker moiety, L, can include a functional group that allows for bridging of two interchain cysteines on the antibody, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), a dithioaryl(TCEP)pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).
In other embodiments, targeting moiety, T, can be modified to include a non-natural reactive group, such as an azide, that allows for conjugation to the linker via a complementary reactive group on the linker. For example, conjugation of the linker to the targeting moiety can make use of click chemistry reactions (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97), such as the azide-alkyne cycloaddition (AAC) reaction, which has been used successfully in the development of antibody-drug conjugates. The AAC reaction can be a copper-catalyzed AAC (CuAAC) reaction, which involves coupling of an azide with a linear alkyne, or a strain-promoted AAC (SPAAC) reaction, which involves coupling of an azide with a cyclooctyne.
In some embodiments, a (e.g., cleavable) linker is substantially stable in the extra-tumoral environment, e.g., in circulation. Such stable linker can be characterized in that at least about 90%, about 80%, about 70%, about 60%, about 50% or at least about 40% of the conjugates of Formula X is intact upon delivery to a tumor site and/or localization on a target cell surface and after a certain period of time. The intact conjugate can, for example, have an unchanged DAR (e.g., within about ±5%) compared to the conjugate at the time of administration. In other words, in some embodiments, the linker remains essentially uncleaved in the extra-tumoral environment during the time the conjugate is resident in this environment (e.g., in systemic circulation and/or a non-target tissue or organ). In some embodiments, such linker can be cleaved in the extra-tumoral environment but not to a degree that prevents a useful dosage of the intact conjugate being delivered to a target cell (e.g., a tumor cell). Whether a linker is not substantially sensitive to an extra-tumoral environment can be determined, e.g., by incubating the ISAC with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free compound (e.g., those molecules that were cleaved from a conjugate) present in the plasma.
Various non-cleavable linkers are known in the art for linking compounds of the present disclosure to targeting moieties and can be used in the conjugates of the present disclosure in certain embodiments. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the cell binding agent, as well as a maleimido- or haloacetyl-based moiety for reaction with the compound (e.g., any one of the compounds according to Formulae (I)-(IV)), or vice versa. An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via a maleimide group which reacts with sulfhydryls (thiols, —SH) on compound C, while the sulfo-NHS ester is reactive toward primary amines (as found in lysine and at the N-terminus of proteins or peptides) on targeting moiety T. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (“long chain” SMCC or LC-SMCC), maleimido-undecanoic acid N-succinimidyl ester (KMUA), maleimidobutyric acid N-succinimidyl ester (GMBS), maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB) and N-(p-maleimidophenyl)isocyanate (PMPI). Other examples include those comprising a haloacetyl-based functional group such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).
Conjugates of Formula (X) herein can be prepared by standard methods known in the art (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press)). Various linkers and linker components are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, for example, March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press)). In addition, various antibody drug conjugation services are available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramel Pharma Solutions (Grangemouth, UK).
Typically, preparation of the conjugates described herein comprises first preparing a compound-linker construct of Formula (A) as described herein, C-L, comprising one or more compounds of any one of Formulae (I)-(IV) and a linker, L, and then conjugating the compound-linker construct, (C-L)p, to an appropriate group on targeting moiety, T. Ligation of linker, L, to targeting moiety, T, and subsequent ligation of the targeting moiety-linker, T-L, to one or more compounds described herein, e.g., those of Formula (I), C, remains however an alternative approach that can be employed in some embodiments. Exemplary methods are described in EXAMPLE 4 herein.
Suitable groups on targeting moiety, T, for attachment of linker, L, in either of the above approaches include sulfhydryl groups (for example, on the side-chain of cysteine residues), amino groups (for example, on the side-chain of lysine residues), carboxylic acid groups (for example, on the side-chains of aspartate or glutamate residues), and carbohydrate groups (e.g., on an Fc glycan moiety). For example, targeting moiety T can comprise one or more naturally occurring sulfhydryl groups allowing targeting moiety, T, to bond to a linker, L, via the sulfur atom of a sulfhydryl group. In some embodiments, a targeting moiety, T, can comprise one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysine residues include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (“SPDP”) and 2-iminothiolane hydro-chloride (Traut's Reagent). In other embodiments, targeting moiety, T, can comprise one or more carbohydrate groups that can be chemically modified to include one or more sulfhydryl groups.
In some embodiments, carbohydrate groups on a targeting moiety, T, can also be oxidized to provide an aldehyde (—CHO) group (see, for example, Laguzza et al., 1989, J. Med. Chem. 32(3):548-55), which can subsequently be reacted with linker, L, for example, via a hydrazine or hydroxylamine group on linker, L.
A targeting moiety, T, can also be modified to include additional cysteine residues (see, for example, U.S. Pat. Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine (see, for example, Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. Chem., 25:351-361), to allow for site-specific conjugation. In some embodiments, targeting moiety, T, can be modified to include a non-natural reactive group, such as an azide, that allows for conjugation to the linker via a complementary reactive group on the linker, for example, by click chemistry (see, for example, Chio & Bane, 2020, Methods Mol. Biol., 2078:83-97).
Other protocols for the modification of proteins of which a targeting moiety (T) herein can comprise or consist of, to allow for the attachment or association of linker, L, are known in the art and include those described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002).
In those embodiments in which targeting moiety, T, is an antibody, several different reactive groups on the antibody can function as a conjugation site, including E-amino groups on lysine residues, pendant carbohydrate moieties, side-chain carboxylic acid groups on aspartate or glutamate residues, cysteine-cysteine disulfide groups and cysteine thiol groups. The amino acids used for conjugation can be part of the natural sequence of the antibody, or they can be introduced by site-specific engineering techniques known in the art, as noted herein.
Once conjugation is complete, the average number of compounds of Formulae (I)-(IV) conjugated to targeting moiety, T, (i.e., the “drug-to-antibody ratio” or DAR) can be determined by standard techniques such as UV/VIS spectroscopic analysis, ELISA-based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS. In addition, distribution of compound-linked forms (for example, the fraction of targeting moiety, T, containing zero, one, two, three, etc. compounds of Formula (I), C) can also optionally be analyzed. Various techniques are known in the art to measure DAR distribution, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF) (see, for example, Wakankar et al., 2011, mAbs, 3:161-172).
As described herein, the drug-to-antibody ratio (DAR) of a conjugate (or “p” in Formula X, if r=1, otherwise the product of q times r), as disclosed herein, refers to the ratio of the number of compound(s) C of Formulae (I)-(IV) conjugated to a targeting moiety, T.
As noted above and reflected by parameters p and r in Formula (X), a targeting moiety, “T,” can be conjugated to more than one compound(s) “C” of Formula (I). Those skilled in the art will appreciate that, while any particular targeting moiety T is conjugated to an integer number of compounds C, analysis of a preparation of the conjugate to determine the ratio of compound C to targeting moiety T can give a non-integer result, reflecting a statistical average. This ratio of compound C to targeting moiety T can generally be referred to as the drug-to-antibody ratio, or “DAR.” Accordingly, conjugate preparations having non-integer DARs are intended to be encompassed by Formula (X). One skilled in the art will appreciate that the term “DAR” can be employed to define conjugates comprising targeting moieties other than antibodies.
In some embodiments, the DAR of a conjugate of Formula (X) herein is obtained by any combination of linker-to-antibody ratio and/or drug-to-linker ratio (e.g., where r is the ratio of compound-linker constructs to antibody T, and p is the number of compounds, C, per linker, L). For example, where r=2 and p=2, the number of compounds C of Formulae (I)-(IV) in the respective antibody-drug conjugate is 4 (e.g., each tumor-targeting antibody T is coupled to 2 compound-linker constructs, with each linker construct comprising 2 immunostimulatory compounds of Formulae (I)-(IV)).
In some embodiments, the DAR of the conjugates of Formula (X) is from about 1 to about 32. In some embodiments, the DAR of the conjugates of Formula (X) is from about 1 to about 24, from about 1 to about 16, from about 1 to about 8, from about 3 to about 5, or from about 1 to about 4. In some embodiments, the DAR of the conjugates of Formula (X) is from about 2 to about 32, from about 2 to about 24, from about 2 to about 16, from about 2 to about 8 or from about 2 to about 4. In various embodiments, the DAR can be from about 1.5 to about 4.5.
In some embodiments, the DAR (i.e., the product of r×p in Formula (X)) of the conjugates of Formula (X) herein can have any numeric value from about 1 to about 8, and thus can have a value of about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or about 8.0. In some embodiments, the DAR of a conjugate of Formula (X) herein can have a value from about 2 to about 8, from about 2 to about 6, from about 2 to about 5, from about 3 to about 5, or from about 2 to about 4.
As further described elsewhere herein, all compounds (e.g., those of Formulae (I)-(IV)) of a conjugate of Formula (X) can be identical. In other embodiments, two or more compounds of a conjugate of Formula (X) can be different.
In some embodiments, a conjugate of Formula (X) herein can have an EC50 value for agonizing TLR7 from about 10 nM to about 50 pM, from about 5 nM to about 50 pM, from about 1 nM to about 50 pM, from about 750 pM to about 50 pM, from about 600 pM to about 50 pM, from about 500 pM to about 50 pM, from about 400 pM to about 50 pM, from about 300 pM to about 50 pM, from about 200 pM to about 50 pM, or from about 100 pM to about 50 pM.
Without being bound by any theory, it is assumed that anti-tumor activity of a conjugate of Formula (X) herein can be mediated by innate immune cells. The conjugate, particularly in embodiments in which the targeting moiety is an antibody or fragment thereof, can initially bind the tumor cell via the target TAA and the immune cell via FcγR engagement. Subsequently, the conjugate can be catabolized in the phagolysosome to release the TLR7 agonist, e.g., a compound of any one of Formulae (I)-(IV). Stimulation of TLR7 in the phagolysosome can induce cytokine expression, which can drive the anti-tumor response. As a result, TLR7 agonism mediated by a compound described herein can drive anti-tumor immunity in a subject.
In various embodiments, and as further described herein, a conjugate of Formula (X) of the present disclosure that comprises one or more immunomodulatory compounds can be capable of inducing production and/or release of one or more cytokines from immune cells. Such immune cells can be human or murine immune cells, such as PBMCs or splenocytes, respectively, and can be part of a cell population further comprising other cell types, such as other immune cell, tumor cells, etc. Such induction of, e.g., pro-inflammatory cytokines, can be result of an interaction between the immunostimulatory compound of the conjugate and TLR7 of immune cells.
In various embodiments, a conjugate of Formula (X) herein can induce production of one or more cytokines when contacted with a cell population comprising immune cells. In various embodiments, such cytokine belongs to the IL6 superfamily. In various embodiments, a conjugate of Formula (X) herein can induce production of IL6. Induction of IL6 can include human IL6, murine IL6, or both.
In various embodiments, a conjugate of Formula (X) herein can have an EC50 value for inducing production of a cytokine in a human or murine immune cell population of <1 nM, <500 pM, <250 pM, <100 pM, or <50 pM, or less. In some embodiments, a conjugate of Formula (X) herein can have an EC50 value for inducing production of a cytokine in a human or murine immune cell population of <1 nM, <500 nM, or <250 nM. In some embodiments, a conjugate of Formula (X) herein can have an EC50 value for inducing production of a cytokine in a human or murine immune cell population from about 1 nM to about 100 pM, from about 750 pM to about 50 pM, from about 500 pM to about 50 pM, from about 250 pM to about 50 pM, or from about 150 pM to about 50 pM. In various embodiments, the cytokine which production is induced by the conjugate is IL6. In some embodiments, induction of cytokine production of a conjugate can be determined via human and/or murine immune cell assays, which can comprise incubating the conjugate with a cell population comprising immune cells (e.g., ones that express TLR7) and tumor cells (e.g., those that express an antigen that the targeting moiety binds to), followed by measuring the amount of cytokine in the supernatant after a certain period of time, as further described herein in, e.g., EXAMPLE 5.
In various embodiments, a conjugate of Formula (X) that comprises a compound of any one of Formulae (I)-(IV) herein, can elicit an immune response in vivo. In various embodiments, such immune response can result in an anti-tumor activity when administered to a subject (e.g., a human, non-human primate, or rodent) suffering from a cancer. Such anti-tumor activity can be measured, e.g., by a reduction of tumor growth rate. In some embodiments, when administered to a group of tumor-bearing test subjects, the anti-tumor activity of a conjugate of Formula (X) herein is sufficient to reduce the volume of the tumor in one or more of the test subjects. In some embodiments, a conjugate of Formula (X) herein can reduce the volume of a tumor by at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90%, in at least one of the test subjects.
In certain embodiments, a conjugate of Formula (X) herein can demonstrate good tolerability in vivo. In some embodiments, a conjugate of Formula (X) herein can be well tolerated in vivo at doses of at least about 1 mg/kg, at least about 3 mg/kg, at least about 15 mg/kg, or about 45 mg/kg. A conjugate herein is generally well tolerated when the total body weight loss in a test subject at the end of a study period is no more than 20% relative to the baseline body weight at the start of the study, i.e., prior to treatment. A study period can be, e.g., between 10 and 16 days. In some embodiments, a conjugate of Formula (X) is well tolerated at a dose that is effective in reducing the volume of a tumor in the test subject.
Compounds of Formulae (I)-(IV) and conjugates comprising compounds of Formulae (I)-(IV) can be formulated for therapeutic use. In certain embodiments, the present disclosure further discloses pharmaceutical compositions comprising a compound of the present disclosure, e.g., a compound according to any one of Formulae (I)-(IV) (see, e.g., section above “Compounds”), or a pharmaceutically acceptable salt thereof. Further disclosed herein are pharmaceutical compositions comprising a conjugate of Formula (X) (see, e.g., section above “Conjugates”). Any such pharmaceutical composition can further comprise a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is a therapeutic composition for the treatment of a disease (e.g., a cancer) in a subject in need thereof. Such pharmaceutical compositions can be prepared by known procedures using well-known and readily available ingredients.
Pharmaceutical compositions described herein can be formulated for administration to a subject by, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or spray. The term parenteral as used herein includes subcutaneous injection, and intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. A pharmaceutical composition can be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. Pharmaceutical compositions can be provided as unit dosage formulations.
Pharmaceutical compositions intended for oral use can be prepared in either solid or fluid unit dosage forms. Fluid unit dosage forms can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents such as sweetening agents, flavouring agents, colouring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. An elixir can be prepared by using a hydroalcoholic (for example, ethanol) carrier with suitable sweeteners such as sugar and/or saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with an aqueous carrier and a suspending agent such as acacia, tragacanth, methylcellulose, and the like.
Solid formulations, such as tablets, contain the active ingredient (e.g., a compound and/or conjugate of the present disclosure) in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and/or lubricating agents, for example magnesium stearate, stearic acid or talc, as well as other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials. The tablets can be uncoated, or they can be coated by known techniques, for example, in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient (e.g., a compound and/or conjugate of the present disclosure) is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Soft gelatin capsules are typically prepared by machine encapsulation of a slurry of the active ingredient with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
Aqueous suspensions can contain the active ingredient (e.g., a compound and/or conjugate of the present disclosure) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents. Dispersing and wetting agents include, for example, naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., hepta-decaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). The aqueous suspensions can also contain one or more preservatives (e.g., ethyl, or n-propyl-p-hydroxybenzoate), one or more colouring agents, one or more flavouring agents and/or one or more sweetening agents (e.g., sucrose or saccharin).
Oily suspensions can be formulated by suspending the active ingredient (e.g., a compound and/or conjugate of the present disclosure) in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents can be added to provide palatable oral preparations. The suspensions can optionally be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water typically provide the active ingredient (e.g., a compound and/or conjugate of the present disclosure) in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. One or more additional excipients, for example sweetening, flavouring and/or colouring agents, can also be present.
Pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oil phase can be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin, or mixtures of such oils. Suitable emulsifying agents for inclusion in oil-in-water emulsions include, for example, naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soybean, lecithin), or esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate) or condensation products of such partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsions can also optionally contain sweetening and/or flavoring agents.
A pharmaceutical composition of this disclosure can be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions can be formulated using suitable dispersing or wetting agents and suspending agents such as those described above. The sterile injectable solution or suspension can comprise the active ingredient (e.g., a compound and/or conjugate of the present disclosure) in a non-toxic parentally acceptable carrier or diluent. Acceptable carriers and diluents that can be employed include, for example, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution. In addition, sterile, fixed oils can be employed as carriers. For this purpose, various bland fixed oils can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and/or buffering agents can also be included in the injectable solution or suspension.
Pharmaceutical compositions can also be formulated as suppositories for rectal administration. These compositions can be prepared by mixing the active ingredient (e.g., a compound and/or conjugate of the present disclosure) with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at physiological temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).
In certain embodiments, a pharmaceutical composition comprising a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) can be provided as part of a pharmaceutical kit or pack. Individual components of the kit can typically be packaged in separate containers. Suitable containers include, for example, bottles, blister packs, intravenous solution bags, vials, and the like, depending on the formulation of the pharmaceutical composition. In certain embodiments, the container can be in a form allowing for administration to a subject, for example, an inhaler, syringe, pipette, eye dropper, pre-soaked gauze or pad, or other such like apparatus, from which the contents can be administered to the subject.
The kit can further comprise a label or package insert on or associated with the container(s). The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. The label or package insert can further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. The label or package insert typically indicates that the compound or conjugate is for use to treat the condition of choice, for example, cancer.
If appropriate, one or more components of the kit can be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).
Certain embodiments of the present disclosure relate to the therapeutic use of the purine-derived compounds described herein, e.g., those having a structure according to any one of Formulae (I)-(IV) and conjugates comprising these compounds, e.g., conjugates of Formula (X). Some embodiments of this disclosure relate to the use of a compounds according to any one of Formulae (I)-(IV) and/or a conjugate of Formula (X) as therapeutic agents.
The TLR7-agonizing compounds of the present disclosure (e.g., those of according to any one of Formulae (I)-(IV)) can exhibit cytotoxic activity against cancer cells, and compounds of any one of Formulae (I)-(IV) and conjugates comprising these compounds, i.e., conjugates of Formula (X), are thus useful for inhibiting abnormal cancer cell or tumor cell growth, inhibiting cancer cell or tumor cell proliferation, and/or treating cancer in a patient. In certain embodiments, compounds of any one of Formulae (I)-(IV) and conjugates of Formula (X) can be used to treat cancer in a subject in need thereof. Some embodiments of the present disclosure thus relate to the use of compounds of any one of Formulae (I)-(IV) and conjugates of general Formula (X) as anti-cancer agents.
Certain embodiments of the present disclosure relate to methods of inhibiting the proliferation of cancer or tumor cells comprising contacting the cells with a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X). Some embodiments relate to a method of killing cancer or tumor cells comprising contacting the cells with a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X).
Various embodiments of this disclosure relate to methods of agonizing a TLR7 using a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) as described herein. In some embodiments, disclosed herein are methods of agonizing a TLR7 in vitro comprising contacting a cell that expresses TLR7 with a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X). In other embodiments, disclosed herein are methods of agonizing a TLR7 in a subject comprising administering to the subject a compound of any one of Formulae (I)-(IV), a conjugate of Formula (X), or a pharmaceutical composition comprising such compound or conjugate. Such method can further comprise contacting a cell expressing TLR7 in the subject with the compound of any one of Formulae (I)-(IV) or the conjugate of Formula (X).
Agonizing TLR7 on a cell in vitro or in vivo with a compound or conjugate of the present disclosure, e.g., a compound of any one of Formulae (I)-(IV) or the conjugate of Formula (X), can elicit an immune response by that cell or the subject. In some embodiments, disclosed herein are methods of eliciting an immune response in vitro, such method comprising contacting a cell with a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X). The cell can be a mammalian cell. The mammalian cell can be an immune cell, such as a lymphocyte (e.g., T cell) or a phagocyte (e.g., neutrophil, macrophage, dendritic cell, eosinophil, or monocyte). In yet other embodiments, disclosed herein are methods of eliciting an immune response in a subject in need thereof, comprising administering to the subject a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X). Such method can further comprise agonizing a TLR7 in the subject by the administered compound of any one of Formulae (I)-(IV) or the conjugate of Formula (X), thereby eliciting the immune response in the subject. In various embodiments, disclosed herein is a method for eliciting a local immune response in a subject, such method comprises administering a conjugate of Formula (X) herein, which comprises one or more compounds of any one of Formulae (I)-(IV), to the subject in need. Following administration (e.g., parenteral administration) of the conjugate of Formula (X) to the subject, such method can further comprise accumulation of the conjugate at a target site (e.g., a target organ or tissue) in the subject, and eliciting a local immune response at the target site. As used herein, the term “local immune response,” which stands in contrast to a systemic immune response, generally refers to an immune response that occurs in a subject at a specific location, e.g., organ, or within a certain tissue type present at one or more locations. As an example, a local anti-tumor immune response is generally understood herein to refer to an immune response that occurs at a tumor site comprising tumor tissue, or at multiple tumor sites, e.g., in the case of metastases. A local immune response can be measured or detected by a change in one or more physiological parameters, such as a local concentration of biomarkers, such as production/secretion of cytokines, small molecules, co-stimulatory molecules, and/or factors involved in inflammation cascades or regulation, and/or a change in immune cell populations.
In some embodiments, a method of agonizing TLR7 using a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) can further comprise activating a TLR7 signaling pathway. Such activation can cause a measurable change (i.e., an increase or decrease) in the expression of one or more intermediates in the TLR7 signaling pathway. Intermediates of the TLR7 signaling pathway include, e.g., MyD88, IRAK4, IRAK1, IRAK2, TRAF6, TAK1, IKK, NF-κB, FADD, Caspase 8, Caspase 3, and/or IRF7. See, e.g., Chi H et al. Front Pharmacol. 2017; 8:304. Examples of inflammatory cytokines that can be modulated in response to incubation or contact with a compound and/or a conjugate of the present disclosure include, but are not limited to, tumor necrosis factor (TNF; also known as TNFα or cachectin), interleukin (IL)-1α, IL1β, IL2; IL5, IL6, IL8, IL15, IL18, interferon γ (IFN-γ); platelet-activating factor (PAF), thromboxane; soluble adhesion molecules; vasoactive neuropeptides; phospholipase A2; plasminogen activator inhibitor (PAI-1); free radical generation; neopterin; CD14; prostacyclin; neutrophil elastase; protein kinase; monocyte chemotactic proteins 1 and 2 (MCP-1, MCP-2); macrophage migration inhibitory factor (MIF), and high mobility group box protein 1 (HMGB-1).
Methods of assessing one or more of these physiological parameters are known in the art. For example, a cytokine can be directly detected, e.g., by ELISA. Other suitable methods include liquid chromatography and tandem mass spectrometry. Quantitative changes of the biological molecules (e.g., cytokines) can be measured in a biological sample such as organ, tissue, urine, or plasma. Detection of the biological molecules can be performed directly on a sample taken from a subject, or the sample can be treated between sample collection and analysis.
Some embodiments of this disclosure relate to methods of treating a subject having a cancer by administering to the subject a compound of Formula (I) or a conjugate of Formula (X), or a pharmaceutical composition comprising such compound or conjugate. In this context, treatment with a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) can result in one or more of a reduction in the size of a tumor, the slowing or prevention of an increase in the size of a tumor, an increase in the disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of a subsequent occurrence of a tumor (for example, metastasis), an increase in the time to progression, reduction of one or more adverse symptom(s) associated with a tumor, and/or an increase in the overall survival time of a subject having the cancer.
Certain embodiments relate to the use of a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) in a method of inhibiting proliferation of and/or killing cancer cells in vitro. Some embodiments relate to the use of a compound of any one of Formulae (I)-(IV) or a conjugate of Formula (X) in a method of inhibiting proliferation of and/or killing cancer cells in vivo in a subject having a cancer.
Examples of cancers which can be treated in certain embodiments of this disclosure using a compound or conjugate disclosed herein include hematologic neoplasms, including leukemias, myelomas and lymphomas; carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as “solid tumors.” Examples of commonly occurring solid tumors that can be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, uterine cancer, non-small cell lung cancer (NSCLC) and colorectal cancer. Various forms of lymphoma also can result in the formation of a solid tumor and, therefore, can also be considered to be solid tumors in certain situations.
In other embodiments, a compound of any one of Formulae (I)-(IV), or a conjugate of Formula (X) herein, can be used as a vaccine adjuvant. In yet other embodiments, a compound of any one of Formulae (I)-(IV), or a conjugate of Formula (X) herein, can be used in a method of treating a viral infection in a subject in need thereof. Such method can comprise administering an effective amount of the compound or conjugate to the subject, thereby treating the viral infection in the subject.
In certain embodiments, the present disclosure relates to any one or more of embodiments 1-140.
Embodiment 1. A compound of Formula (I):
Embodiment 2. The compound of embodiment 1, wherein R2 is halogen, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy.
Embodiment 3. The compound of any one of embodiments 1-2, wherein R2 is halogen or optionally substituted C1-C6 alkoxy.
Embodiment 4. The compound of any one of embodiments 1-3, wherein R2 is halogen or unsubstituted C1-C6 alkoxy.
Embodiment 5. The compound of any one of embodiments 1-4, wherein R2 is fluorine or methoxy.
Embodiment 6. The compound of any one of embodiments 1-5, wherein X is O.
Embodiment 7. The compound of any one of embodiments 1-5, wherein X is NH.
Embodiment 8. The compound of any one of embodiments 1-7, wherein R1 is optionally substituted C2-C6 alkyl.
Embodiment 9. The compound of any one of embodiments 1-7, wherein R1 is branched and optionally substituted C3-C8 hydroxyalkyl.
Embodiment 10. The compound of any one of embodiments 1-9, wherein R3 and R4 are independently H, or Q-R5.
Embodiment 11. The compound of any one of embodiments 1-10, wherein R3 and R4 are both H, or R3 and R4 are both Q-R5.
Embodiment 12. The compound of any one of embodiments 1-10, wherein R3 is H and R4 is Q-R5.
Embodiment 13. The compound of embodiment 12, wherein Q is a bond and R5 is optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 14. The compound of embodiment 12, wherein Q is optionally substituted C1-C6-alkyl and R5 is H, OH, NR6R7, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 15. The compound of embodiment 12, wherein Q is optionally substituted C2-C6-alkenyl and R5 is H, OH, NR6R7, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 16. The compound of any one of embodiments 1-12, wherein Q is:
and wherein:
Embodiment 17. The compound of embodiment 16, wherein Q is selected from the group consisting of:
wherein a and b are each independently 1, 2, or 3.
Embodiment 18. The compound of any one of embodiments 1-12 or 14-17, wherein R5 is NR6R7 and R6 and R7 are both H.
Embodiment 19. The compound of any one of embodiments 1-12 or 14-17, wherein R5 is NR6R7, R6 is H and R7 is optionally substituted C1-C6 alkyl.
Embodiment 20. The compound of any one of embodiments 1-12 or 14-17, wherein R5 is NR6R7 and R6 and R7 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 21. The compound of any one of embodiments 1-9, wherein R3 and R4 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 22. The compound of any one of embodiments 1-21, wherein n is 1.
Embodiment 23. The compound of any one of embodiments 1-22, wherein m is an integer from 0 to 3 or from 1 to 3.
Embodiment 24. The compound of embodiment 1, wherein R2 is H and R1 is branched and optionally substituted C3-C8 hydroxyalkyl.
Embodiment 25. The compound of embodiment 24, wherein R1 is branched and unsubstituted C3-C8 hydroxyalkyl.
Embodiment 26. The compound of embodiment 1, wherein R2 is H, X is NH, and R1 is optionally substituted C5-C6 alkyl.
Embodiment 27. The compound of embodiment 26, wherein R1 is unsubstituted C5-C6 alkyl.
Embodiment 28. The compound of embodiment 1, wherein R2 is H, m is 0 or 1, and R3 and R4 together with the N form an unsubstituted piperazinyl ring or an optionally substituted C6-C10-heterospirocycloalkyl ring.
Embodiment 29. The compound of embodiment 28, wherein R3 and R4 together with the N form an unsubstituted piperazinyl ring.
Embodiment 30. The compound of embodiment 28, wherein R3 and R4 together with the N form an optionally substituted C6-C10-heterospirocycloalkyl ring.
Embodiment 31. The compound of any one of embodiments 28-30, wherein m is 0.
Embodiment 32. The compound of any one of embodiments 28-30, wherein m is 1.
Embodiment 33. The compound of any one of embodiments 1-32, wherein each alkyl, alkenyl, cycloalkyl, spirocycloalkyl, heterocycloalkyl, heterospirocycloalkyl, aryl and heteroaryl group is optionally substituted —NH2, —CO2H, —OH, carbonyl, halogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 carboxyalkyl, (C0-C4 alkyl)-cycloalkyl, (C0-C4 alkyl)-heterocycloalkyl, (C0-C4 alkyl)-spirocycloalkyl, (C0-C4 alkyl)-heterospirocycloalkyl, (C0-C4 alkyl)-aryl, and (C0-C4 alkyl)-heteroaryl, and wherein each of the alkyl, hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 carboxyalkyl, cycloalkyl, heterocycloalkyl, spirocycloalkyl, heterospirocycloalkyl, aryl or heteroaryl group can itself be substituted with one or more of: —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl, or unsubstituted C1-C4 carboxyalkyl.
Embodiment 34. The compound of embodiment 33, wherein each alkyl, alkenyl, cycloalkyl, spirocycloalkyl, heterocycloalkyl, heterospirocycloalkyl, aryl and heteroaryl group is optionally substituted with one or more substituents selected from the group consisting of —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl and unsubstituted C1-C4 carboxyalkyl.
Embodiment 35. The compound of embodiment 1, wherein the compound is selected from any one of compounds 100-195 listed in TABLE 1.
Embodiment 36. The compound of embodiment 1, having the structure of Formula (II):
wherein:
Embodiment 37. The compound of embodiment 36, wherein X is O and R1 is optionally substituted C2-C6 alkyl.
Embodiment 38. The compound of any one of embodiments 36-37, wherein R1 is unsubstituted C2-C6 alkyl.
Embodiment 39. The compound of any one of embodiments 36-38, wherein Y is CH.
Embodiment 40. The compound of any one of embodiments 36-39, wherein m is 0 or 1, 1 or 2, or 1 or 3.
Embodiment 41. The compound of any one of embodiments 36-40, wherein R3 and R4 are both H.
Embodiment 42. The compound of any one of embodiments 36-40, wherein R3 and R4 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 43. The compound of any one of embodiments 36-40, wherein R3 and R4 are either both H or Q-R5, or R3 is H and R4 is Q-R5.
Embodiment 44. The compound of embodiment 43, wherein Q is a bond and R5 is optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 45. The compound of embodiment 43, wherein Q is optionally substituted C1-C6-alkyl and R5 is H, OH, NR6R7, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 46. The compound of embodiment 43, wherein Q is optionally substituted C2-C6-alkenyl and R5 is H, OH, NR6R7, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 47. The compound of any one of embodiments 36-43 or 45-46, wherein R5 is NR6R7 and R6 and R7 are both H.
Embodiment 48. The compound of any one of embodiments 36-43 or 45-46, wherein R5 is NR6R7, R6 is H and R7 is optionally substituted C1-C6 alkyl.
Embodiment 49. The compound of any one of embodiments 36-43 or 45-46, wherein R6 and R7 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 50. The compound of any one of embodiments 36-49, wherein m is 1 or 2, or 1 or 3.
Embodiment 51. The compound of embodiment 36, wherein the compound is selected from SUBTABLE 1A.
Embodiment 52. The compound of any one of embodiments 1-9, 21, or 42, wherein R3 and R4 together with the N form a ring selected from the group consisting of:
wherein:
Embodiment 53. The compound of embodiment 52, wherein R3 and R4 together with the N form a ring selected from the group consisting of:
Embodiment 54. The compound of any one of embodiments 1-9 or 21, wherein the compound is selected from SUBTABLE 1B.
Embodiment 55. The compound of any one of embodiments 1-11, wherein R3 and R4 are both Q-R5.
Embodiment 56. The compound of embodiment 55, wherein the compound is:
Embodiment 57. The compound of embodiment 1-11, wherein R3 and R4 are both H.
Embodiment 58. The compound of embodiment 57, wherein the compound is:
Embodiment 59. The compound of embodiment 13, wherein the compound is selected from SUBTABLE 1C.
Embodiment 60. The compound of embodiment 15 or embodiment 46, wherein the compound is:
Embodiment 61. The compound of any one of embodiments 1-10 or 14, wherein the compound is selected from SUBTABLE 1D.
Embodiment 62. The compound of any one of embodiments 1-10 or 16-17, wherein R6 is H and R7 is H, optionally substituted C1-C6 alkyl or R6 and R7 together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 63. The compound of embodiment 62, wherein the compound is selected from SUBTABLE 1E.
Embodiment 64. The compound of any one of embodiments 1-9 or 36, wherein the compound is:
Embodiment 65. The compound of embodiments 1 or 24-25, wherein the compound is:
Embodiment 66. A compound of Formula (III):
Embodiment 67. The compound of embodiment 66, wherein R1a is optionally substituted C2-C6 alkyl.
Embodiment 68. The compound of any one of embodiments 66-67, wherein R1a is unsubstituted C2-C6 alkyl.
Embodiment 69. The compound of any one of embodiments 66-68, wherein R2a is halogen or optionally substituted C1-C6 alkoxy.
Embodiment 70. The compound of any one of embodiments 66-69, wherein R2a is halogen or unsubstituted C1-C6 alkoxy.
Embodiment 71. The compound of any one of embodiments 66-70, wherein R2a is fluorine or methoxy.
Embodiment 72. The compound of any one of embodiments 66-71, wherein R3a and R4a together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 73. The compound of any one of embodiments 66-71, wherein R3a and R4a are both H.
Embodiment 74. The compound of any one of embodiments 66-71, wherein R3a and R4a are independently H, or Q-R5.
Embodiment 75. The compound of embodiment 74, wherein R3a is H and R4a is Q-R5a.
Embodiment 76. The compound of any one of embodiments 74-75, wherein Q is a bond and R5a is optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 77. The compound of any one of embodiments 74-75, wherein Q is optionally substituted C1-C6-alkyl and R5a is H, OH, NR6aR7a, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 78. The compound of any one of embodiments 74-75, wherein Q is optionally substituted C2-C6-alkenyl and R5a is H, OH, NR6aR7a, optionally substituted C3-C6-carboxyalkyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C2-C6-heterocycloalkyl, optionally substituted C3-C7-heterobicycloalkyl, optionally substituted C6-C10-heterospirocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
Embodiment 79. The compound of any one of embodiments 66-75, wherein Q is:
and wherein:
Embodiment 80. The compound of embodiment 79, wherein Q is selected from the group consisting of:
wherein a and b are independently 1, 2, or 3.
Embodiment 81. The compound of any one of embodiments 66-75 or 77-80, wherein R6a and R7a are both H.
Embodiment 82. The compound of any one of embodiments 66-75 or 77-80, wherein R6a is H and R7a is optionally substituted C1-C6 alkyl.
Embodiment 83. The compound of any one of embodiments 66-75 or 77-80, wherein R6a and R7a together with the N form an optionally substituted C2-C6-heterocycloalkyl ring, optionally substituted C3-C7-heterobicycloalkyl ring, optionally substituted C6-C10-heterospirocycloalkyl ring or an optionally substituted heteroaryl ring.
Embodiment 84. The compound of any one of embodiments 66-83, wherein u is 1.
Embodiment 85. The compound of any one of embodiments 66-84, wherein v is an integer from 0 to 4, from 1 to 4, 1 to 3, or from 1 to 2.
Embodiment 86. The compound of any one of embodiments 66-72, wherein R3a and R4a together with the N form a ring selected from the group consisting of:
wherein:
Embodiment 87. The compound of embodiment 86, wherein R3a and R4a together with the N form a ring selected from the group consisting of:
Embodiment 88. The compound of embodiment 66, having the structure of Formula (IV):
wherein:
Embodiment 89. The compound of embodiment 66 or embodiment 88, wherein each alkyl, alkenyl, cycloalkyl, spirocycloalkyl, heterocycloalkyl, heterospirocycloalkyl, aryl and heteroaryl group is optionally substituted —NH2, —CO2H, —OH, carbonyl, halogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 carboxyalkyl, (C0-C4 alkyl)-cycloalkyl, (C0-C4 alkyl)-heterocycloalkyl, (C0-C4 alkyl)-spirocycloalkyl, (C0-C4 alkyl)-heterospirocycloalkyl, (C0-C4 alkyl)-aryl, and (C0-C4 alkyl)-heteroaryl, and wherein each of the alkyl, hydroxyalkyl, C1-C4 aminoalkyl, C1-C4 carboxyalkyl, cycloalkyl, heterocycloalkyl, spirocycloalkyl, heterospirocycloalkyl, aryl or heteroaryl group can itself be substituted with one or more of: —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl, or unsubstituted C1-C4 carboxyalkyl.
Embodiment 90. The compound of embodiment 89, wherein each alkyl, alkenyl, cycloalkyl, spirocycloalkyl, heterocycloalkyl, heterospirocycloalkyl, aryl and heteroaryl group is optionally substituted with one or more substituents selected from the group consisting of —NH2, —CO2H, —OH, carbonyl, halogen, unsubstituted C1-C4 alkyl, unsubstituted C1-C4 hydroxyalkyl, unsubstituted C1-C4 aminoalkyl and unsubstituted C1-C4 carboxyalkyl.
Embodiment 91. The compound of embodiment 66 or embodiment 88, wherein the compound is selected from SUBTABLE 1A.
Embodiment 92. The compound of embodiment 66 or embodiment 88, wherein the compound is:
Embodiment 93. The compound of any one of embodiments 1-92, wherein the compound has an EC50 value for agonizing human or murine TLR7 of <500 nM, <250 nM, or <100 nM as determined in a reporter gene assays, and/or an EC50 value for inducing IL6 production in a human or murine immune cell population of <5 μM, <1 μM, <500 nM, or <100 nM, as determined in an immune cell assay.
Embodiment 94. A pharmaceutical composition comprising a compound of any one of embodiments 1-92, and a pharmaceutically acceptable carrier or diluent.
Embodiment 95. A method of agonizing TLR7, the method comprising contacting a cell that expresses TLR7 with a compound of any one of embodiments 1-92, thereby agonizing TLR7.
Embodiment 96. A method of inducing release of a cytokine from a cell, the method comprising contacting the cell with a compound of any one of embodiments 1-92, thereby inducing release of the cytokine from the cell.
Embodiment 97. A method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of any one of embodiments 1-92.
Embodiment 98. The method of embodiment 97, wherein the compound agonizes TLR7 in the subject, thereby stimulating the immune response in the subject.
Embodiment 99. A method of inhibiting the proliferation of cancer cells, the method comprising contacting a cell population comprising the cancer cells with an effective amount of the compound of any one of embodiments 1-92.
Embodiment 100. A method of killing cancer cells, the method comprising contacting a cell population comprising the cancer cells with an effective amount of the compound of any one of embodiments 1-92.
Embodiment 101. The method of any one of embodiments 99-100, wherein the cell population comprises immune cells.
Embodiment 102. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of embodiments 1-92.
Embodiment 103. The method of embodiment 102, wherein the compound agonizes TLR7 in the subject, thereby treating the cancer in the subject.
Embodiment 104. A compound of any one of embodiments 1-92 for use in therapy.
Embodiment 105. A compound of any one of embodiments 1-92 for use in the treatment of cancer.
Embodiment 106. Use of a compound of any one of embodiments 1-92 in the manufacture of a medicament for the treatment of cancer.
Embodiment 107. A compound-linker construct having Formula (A):
L-(C)p (A)
wherein:
Embodiment 108. The compound-linker construct of embodiment 107, wherein L is a cleavable linker.
Embodiment 109. The compound-linker construct of embodiment 107, wherein L is a non-cleavable linker.
Embodiment 110. The compound-linker construct of any one of embodiments 107-108, wherein L comprises a dipeptide, tripeptide, tetrapeptide, or a combination thereof.
Embodiment 111. The compound-linker construct of any one of embodiments 107-110, wherein L comprises a polyethylene glycol (PEG) moiety.
Embodiment 112. The compound-linker construct of any one of embodiments 107-108, wherein L is a protease cleavable linker.
Embodiment 113. The compound-linker construct of any one of embodiments 107-112, wherein C is the compound of embodiment 36.
Embodiment 114. The compound-linker construct of any one of embodiments 107-112, wherein C is the compound of embodiment 66.
Embodiment 115. The compound-linker construct of embodiment 107, wherein the compound-linker is selected from TABLE 2B.
Embodiment 116. The compound-linker construct of any one of embodiments 107-115, wherein p is 1, 2, or 3.
Embodiment 117. A conjugate having Formula (X):
T-[L-(C)p]r (X)
wherein:
Embodiment 118. The conjugate of embodiment 117, wherein L-(C), is the compound-linker construct of any one of embodiments 107-116.
Embodiment 119. The conjugate of any one of embodiments 117-118, wherein r is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
Embodiment 120. The conjugate of embodiment 117, wherein C is the compound of embodiment 36.
Embodiment 121. The conjugate of embodiment 117, wherein C is the compound of embodiment 66 or embodiment 88.
Embodiment 122. The conjugate of embodiment 117, wherein C is selected from SUBTABLE 1F.
Embodiment 123. The conjugate of any one of embodiments 117-122, wherein T binds to a tumor associated antigen (TAA).
Embodiment 124. The conjugate of any one of embodiments 117-123, wherein T is an antibody or antigen-binding antibody fragment.
Embodiment 125. The conjugate of embodiment 124, wherein the antibody is a bispecific or multispecific antibody.
Embodiment 126. The conjugate of any one of embodiments 124-125, wherein the antibody or antigen-binding antibody fragment binds to a TAA.
Embodiment 127. The conjugate of any one of embodiments 117-126, wherein the conjugate has an EC50 value for inducing production of a cytokine in a human or murine immune cell population of <1 nM, <500 pM, or <100 pM, as determined in a reporter gene assay.
Embodiment 128. A pharmaceutical composition comprising the conjugate of any one of embodiments 117-126, and a pharmaceutically acceptable carrier or diluent.
Embodiment 129. A method of agonizing TLR7, the method comprising contacting a cell that expresses TLR7 with a conjugate of any one of embodiments 117-126, thereby agonizing TLR7.
Embodiment 130. A method of inducing release of a cytokine from a cell, the method comprising contacting the cell with a conjugate of any one of embodiments 117-126, thereby inducing release of the cytokine from the cell.
Embodiment 131. A method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate of any one of embodiments 117-126.
Embodiment 132. The method of embodiment 132, wherein the conjugate agonizes TLR7 in the subject, thereby stimulating the immune response in the subject.
Embodiment 133. A method of inhibiting the proliferation of cancer cells, the method comprising contacting a cell population comprising the cancer cells with an effective amount of the conjugate of any one of embodiments 117-126.
Embodiment 134. A method of killing cancer cells, the method comprising contacting a cell population comprising the cancer cells with an effective amount of the conjugate of any one of embodiments 117-126.
Embodiment 135. The method of any one of embodiments 133-134, wherein the cell population comprises immune cells.
Embodiment 136. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate of any one of embodiments 117-126.
Embodiment 137. The method of embodiment 136, wherein the conjugate agonizes TLR7 in the subject, thereby treating the cancer in the subject.
Embodiment 138. A conjugate of any one of embodiments 117-126 for use in therapy.
Embodiment 139. A conjugate of any one of embodiments 117-126 for use in the treatment of cancer.
Embodiment 140. Use of a conjugate of any one of embodiments 117-126 in the manufacture of a medicament for the treatment of cancer.
The following Examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.
The following Examples provide illustrative methods of making and using compounds of the present disclosure, e.g., a compound of any one of Formulae (I)-(IV). It is understood that one skilled in the art can be able to synthesize these compounds by similar methods or by combining other methods known in the art. Preparation of other compounds of Formulae (I)-(IV) not specifically illustrated herein could be achieved by one skilled in the art using the methods described herein or similar methods with appropriate starting components and modification of the parameters of the synthesis as needed. In general, starting components and materials can be obtained from commercial sources such as Sigma Aldrich (Merck KGaA), Alfa Aesar and Maybridge (Thermo Fisher Scientific Inc.), Matrix Scientific, Tokyo Chemical Industry Ltd. (TCI) and Fluorochem Ltd., and/or synthesized according to sources known to those skilled in the art (see, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7th edition, John Wiley & Sons, Inc., 2013) or prepared as described herein.
The following abbreviations are used throughout the Examples: DCM=dichloromethane; DIPEA=N,N-diisopropylethylamine; DMA=dimethylacetamide; DMF=dimethylformamide; DMSO=dimethylsulphoxide; IL6=interleukin 6; LC/MS=Liquid Chromatography/Mass Spectrometry; LC/MSD=Liquid Chromatography/Mass Selective Detector; SEC=Size-exclusion chromatography; HIC=hydrophobic interaction chromatography; RP-UPLC=reverse-phase ultra performance liquid chromatography; HPLC=high-performance liquid chromatography; MT=maleimidotriethylene glycolate; PABC=p-aminobenzyloxycarbonyl; PBMC=peripheral blood mononuclear cell; PNP=p-nitrophenol; rt=room temperature; TCEP=tris(2-carboxyethyl)phosphine; TFA=trifluoroacetic acid; TNF-α=tumor necrosis factor α; VC=valine-citrulline; UHPLC=ultra high-performance liquid chromatograph.
To the chloride compound in DMF (50-100 mg/mL) was added a primary or secondary amine (2-5 eq.) followed by DIPEA (3-5 eq.), and the solution was heated to 50-75° C. with stirring. Upon completion (typically 1-18 h), the reaction mixture was adjusted to pH 2 with 6 M HCl and purified by reverse-phase HPLC to provide the desired product after lyophilization.
To a stirring solution of the Boc protected amine compound in dichloromethane (0.1 M) was added TFA (10% by volume). Upon completion (typically 1 h), the reaction mixture was concentrated in vacuo and co-evaporated with DCM several times to provide a crude solid or was purified by preparative HPLC to provide the desired product after lyophilization.
The alcohol compound was dissolved 10% SOCl2/DCM (0.05-0.1 M). The solution was stirred at room temperature until completion (typically 12-24 h), then concentrated in vacuo. The residue was co-evaporated (2×10 mL dichloromethane) to give the crude chloride compound, which was typically used without further purification.
To the primary or secondary amine compound in dimethylformamide (0.05-0.1 M) was added the compound of Example 2.5 (MT-VC-PABC-PNP) (1-1.1 eq.) followed by DIPEA (3 eq.). Upon completion (typically 1-4 h), the reaction mixture was acidified with aqueous HCl (1 M) then purified by reverse-phase HPLC to provide the desired drug-linker after lyophilization.
Flash Chromatography: Crude reaction products were purified with Biotage® Snap Ultra columns (10, 25, 50, or 100 g) (Biotage, Charlotte, NC), and eluting with linear gradients of ethyl acetate/hexanes or methanol/dichloromethane on a Biotage® Isolera™ automated flash system (Biotage, Charlotte, NC). Alternatively, reverse-phase flash purification was conducted using Biotage® Snap Ultra C18 columns (12, 30, 60, or 120 g) and eluting with linear gradients of CH3CN+0.1% TFA/H2O+0.1% TFA. Purified compounds were isolated by either removal of organic solvents by rotavap or lyophilization of acetonitrile/water mixtures.
Preparative HPLC: Reverse-phase HPLC of crude compounds was performed using a Kinetex® 5-μm EVO C18 100 Å (250×21.2 mm) column (Phenomenex) on an Agilent 1260 Infinity II preparative LC-MSD system (Agilent Technologies, Inc., Santa Clara, CA), and eluting with linear gradients of CH3CN+0.1% TFA/H2O+0.1% TFA. Purified compounds were isolated by lyophilization of acetonitrile/water mixtures.
LC-MS: Reactions were monitored for completion and purified compounds were analyzed using a Kinetex® 2.6-μm C18 100 Å (30×3 mm) column (Phenomenex) on an Agilent 1290 HPLC/6120 single quad LC-MS system (Agilent Technologies, Inc., Santa Clara, CA), and eluting with a with a 10 to 100% CH3CN+0.1% TFA/H2O+0.1% TFA linear gradient.
NMR: 1H NMR spectra were collected with a Bruker AVANCE III 300 Spectrometer (300 MHz) (Bruker Corporation, Billerica, MA). Chemical shifts are in parts per million (ppm).
To the primary or secondary amine compound in dimethylformamide (0.05-0.1 M) was added the compound of Example 2.38 (MT-VK(Boc)-PABC-PNP) (1.0-1.1 eq.) followed by DIPEA (3-5 eq.). HOBt (1.0 eq.) was used for some reactions as indicated. Upon completion (typically 1-4 h), the reaction mixture was acidified with aqueous HCl (1 M) then purified by reverse-phase HPLC to provide the Boc protected drug-linker after lyophilization. The Boc intermediate was then deprotected according to General Procedure 2. Upon completion (typically 1 h), the reaction mixture was concentrated in vacuo and co-evaporated with DCM several times to provide a crude solid or was purified by preparative HPLC to provide the desired product after lyophilization.
To the chloride compound in DMF (50-100 mg/mL) was added a primary or secondary amine (2-5 eq.) and NaI (0.1-1.0 eq.) followed by DIPEA (3-5 eq.), and the solution was heated to 80° C. with stirring. Upon completion (typically 1-18 h), the reaction mixture was adjusted to pH 2 with 6 M HCl and purified by reverse-phase HPLC to provide the desired product after lyophilization.
The antibody (1-10 mg/mL in phosphate buffered saline, pH 7.4) was reduced with TCEP (1-10 mM in dH2O) (1.0-3.0 eq.) in the presence of 1 mM DTPA. The solution was mixed thoroughly and incubated at 37° C. for 120 min before cooling on ice. The reduced antibody solution was then further buffer exchanged into 10 mM sodium acetate buffer, pH 4.5 by passage over a Zeba™ Spin Desalting Columns (40 KDa MWCO; Thermo Scientific™). To the reduced protein solution stored on ice was added the maleimide functionalized compound-linker construct (10 mM in DMSO) (12-20 eq.). In some embodiments, propylene glycol (10-30 percent v/v %) was added to the reduced protein solution prior to the addition of compound-linker construct. The conjugation reaction was immediately mixed thoroughly by pipetting and conjugation was allowed to proceed at room temperature for 120 to 180 min. Once complete, reaction mixture was purified by passage over Zeba™ Spin Desalting Columns (40 KDa MWCO; Pierce) pre-equilibrated with 10 mM sodium acetate, pH 4.5. The purified conjugates were stored at 4° C. and analyzed for total protein content (bicinchoninic acid assay, Pierce micro-BCA protocol, catalogue #23225), characterized by HPLC-HIC, SEC, and/or RP-UPLC-MS. The average DAR and drug distribution was derived from interpretation of HIC and/or LC-MS data. Average DAR estimates of the synthesized conjugates were in the range of about 1.5 to 5, or more specifically from 1.9 to 4.3, as shown further herein. Endotoxin levels were assessed using Endosafe® LAL test cartridges (Charles River Catalogue #PTS20005F), with a sensitivity of 0.005 EU/mL on an Endosafe® nexgen-PTS™ testing system. Residual free compound and compound-linker construct levels were assessed by RP-UPLC-MS, with a threshold set at 1% ((free compound+compound-linker construct)/(conjugated compound-linker construct)).
To a solution of 2,6-dichloro-9H-purine (11 g, 56 mmol, 1.0 eq.) in 100 mL EtOAc was added dihydropyran (7.0 g, 83 mmol, 1.5 eq.) and p-TsOH (96 mg, 0.56 mmol, 0.010 eq.). The mixture was heated to 50° C. for 3 h then concentrated in vacuo to yield the crude titled product 1 as a yellow solid (quantitative yield). LC-MS: Calc'd m/z=272.0 for C10H10Cl2N4O. found [M+H]+=273.0.
To a solution of NH4OH in iPrOH (100 mL, 2.0 M) was added compound 1 (15 g, 55 mmol). The mixture was heated to 55° C. for 18 h then poured into H2O (30 mL) and allowed to cool to room temperature. The resulting suspension was filtered, and the filter cake was washed with iPrOH (100 mL) to give the titled product 2 as a white solid (13 g, 51 mmol, 93%). LC-MS: Calc'd m/z=253.1 for C10H10ClN5O. found [M+H]+=254.1.
To a solution of 2-chloroadenine (7.0 g, 41 mmol, 1.0 eq.) in DMSO (80 mL) was added K2CO3 (5.7 g, 41 mmol, 1.0 eq.) then benzyl bromide (7.4 g, 43 mmol, 1.1 eq.) and the resulting mixture stirred at room temperature for 18 h. The reaction was then concentrated in vacuo, the residue taken up in H2O (100 mL) and EtOAc (50 mL) and the resulting mixture stirred vigorously for 3 h, after which the EtOAc was removed in vacuo and the suspension filtered. The filter cake was then washed with H2O (2×50 mL) and air dried for 18 h to give the titled product 3 as a solid (7.2 g, 28 mmol, 67%). LC-MS: Calc'd m/z=259.1 for C12H10ClN5. found [M+H]+=260.2.
To a solution of pentane-1,2-diol (2.9 g, 28 mmol, 1.3 eq.) and imidazole (1.5 g, 21 mmol, 1.0 eq.) in DCM (10 mL) at 0° C. was added TBDPS-Cl and the mixture stirred for 1 h. The reaction was then allowed to warm to room temperature and stirred for an additional 36 h, after which it was concentrated in vacuo and the residue taken up in EtOAc (30 mL). The resulting organic solution was then washed with brine (2×10 mL), dried over MgSO4 and concentrated in vacuo to give the titled product 4 as a clear and colourless oil (7.0 g, 20.4 mmol, 95%). 1H NMR (300 MHz, CDCl3) δ 7.88-7.70 (m, 5H), 7.57-7.35 (m, 5H), 3.89-3.53 (m, 3H), 3.03 (s, 1H), 1.61-1.31 (m, 4H), 1.17 (s, 9H), 1.05-0.92 (m, 3H).
To a solution of compound 2 (20.0 g, 78.8 mmol, 1.00 eq.) in EtOH (250 mL) was added KOtBu (17.7 g, 157 mmol, 2.0 eq.). The resulting solution was stirred at 60° C. for 18 h, after which the solvent was removed in vacuo. The resulting solid was suspended in H2O (150 mL) and extracted with EtOAc (3×250 mL). The aqueous layer was further extracted with 10% iPrOH/EtOAc (100 mL). The pooled organics were washed with brine (50 mL) then dried over MgSO4 and concentrated in vacuo to yield the titled compound 5 as an orange solid (20.0 g, 76.0 mmol, 96.4%). LC-MS: Calc'd m/z=263.1 for C12H17N5O2. found [M+H]+=264.2.
To a suspension of compound 5 (20.0 g, 76.0 mmol, 1.00 eq.) in DCM (200 mL) was added NBS (20.3 g, 91.2 mmol, 1.20 eq.) in portions over the course of 3 mins. The resulting mixture was stirred at room temperature for 18 h, after which the reaction was quenched by the addition of 1 M NaHSO3 (50 mL) and rapidly stirred for 30 mins. The resulting mixture was diluted with DCM (100 mL) and the separated organics washed with 1 M NaHSO3 (2×50 mL). The pooled organics were dried over MgSO4, filtered then concentrated in vacuo to yield the titled compound 6 as an orange solid (23.5 g, 68.7 mmol, 90.4%). LC-MS: Calc'd m/z=341.1 for C12H16BrN5O2. found [M+H]+=342.2.
To a solution of compound 6 (23 g, 67 mmol, 1.0 eq.) in MeOH (150 mL) was added NaOMe (9.1 g, 170 mmol, 2.5 eq.). The resulting mixture was stirred at room temperature for 72 h. Additional NaOMe (3.6 g, 66 mmol, 1.0 eq.) was added and the mixture was heated to reflux for 72 h. The solvent was removed in vacuo and crude material redissolved in EtOAc (250 mL), followed by washing with 1 M NaH2PO4 (150 mL). The aqueous layer was extracted with EtOAc (3×150 mL), the pooled organics dried over MgSO4, and the solution concentrated in vacuo to yield title compound 7 as a red solid (17 g, 58 mmol, 86%). LC-MS: Calc'd m/z=293.2 for C13H19N5O3. found [M+H]+=294.2.
Compound 7 (13 g, 44 mmol, 1.0 eq.) was dissolved in 15% TFA/MeOH (40 mL) and the resulting solution stirred at room temperature for 72 h. The solvent was removed in vacuo to yield the titled compound 8 as a yellow solid (14 g, 40 mmol, 91%). LC-MS: Calc'd m/z=209.1 for C8H11N5O2. found [M+H]+=210.1.
To a solution of compound 8 (0.70 g, 3.4 mmol, 1.0 eq.) and methyl 4-(bromomethyl)-3-methoxybenzoate (0.87 g, 3.4 mmol, 1.0 eq.) in DMF (8 mL) was added CsCO3 (1.1 g, 3.4 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The titled compound 9 was obtained as a white solid (0.65 g, 1.7 mmol, 50%). LC-MS: Calc'd m/z=387.2 for C18H21N5O5. found [M+H]+=388.2.
To a stirring solution of compound 9 (250 mg, 0.65 mmol, 1.0 eq.) in anhydrous THF (5 mL) cooled to 0° C. was added lithium aluminum hydride (24 mg, 0.65 mmol, 1.0 eq.) in small portions over 5 mins. The resulting suspension was allowed to warm to room temperature and stirred for 15 mins, after which it was quenched with H2O (1 mL) and diluted with MeOH (100 mL) then filtered through a celite plug. The filtrate was concentrated in vacuo and redissolved in 50% CH3CN/H2O (25 mL) to yield the titled compound 10 after lyophilization (230 mg, 0.63 mmol, 97%). LC-MS: Calc'd m/z=359.2 for C17H21N5O4. found [M+H]+=360.2.
The title compound 11 was prepared according to General Procedure 3 using compound 10 (210 mg, 0.58 mmol, 1.0 eq.) and 10% SOCl2/DCM (10 mL). The titled compound was obtained as a yellow solid (assumed quantitative yield). LC-MS: Calc'd m/z=363.1 for C16H18ClN5O3. found [M+H]+=364.2.
The titled compound was prepared according to General Procedure 1 from compound 11 (0.040 g, 0.11 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (61 mg, 0.33 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 100 as a white solid (55 mg, 0.073 mmol, 66%). LC-MS: Calc'd m/z=413.2 for C20H27N7O3. found [M+H]+=414.3. 1H NMR (300 MHz, MeOD) δ 7.14 (s, 1H), 7.10 (d, J=7.7 Hz, 1H), 6.98 (d, J=7.6, 1H), 5.04 (s, 2H), 4.38 (q, J=7.0 Hz, 2H), 4.17 (s, 2H), 3.89 (s, 3H), 3.50-3.42 (m, 4H), 3.33-3.26 (m, 4H), 1.34 (t, J=7.1 Hz, 3H).
The title compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and 1-amino-2-methylpropan-2-ol (7.0 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 101 as a white solid (11.7 mg, 0.0181 mmol, 66.0%). LC-MS: Calc'd m/z=416.2 for C20H28N6O4. found [M+H]+=417.3. 1H NMR (300 MHz, CD3CN) δ 7.14 (s, 1H), 7.07 (d, J=7.8 Hz, 1H), 6.98 (d, J=7.7 1H), 4.97 (s, 2H), 4.33 (q, J=7.1 Hz, 2H), 4.18 (s, 2H), 3.86 (s, 3H), 2.89 (s, 2H), 1.29 (t, J=7.1 Hz, 3H), 1.20 (s, 6H).
The title compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and diethylamine (7.0 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 102 as a white solid (10 mg, 0.026 mmol, 95%). LC-MS: Calc'd m/z=400.2 for C20H28N6O3. found [M+H]+=401.2. 1H NMR (300 MHz, CD3CN) δ 7.13-7.05 (m, 2H), 6.97 (dd, J=7.7, 1.6 Hz, 1H), 4.97 (s, 2H), 4.31 (q, J=7.0 Hz, 2H), 4.19 (s, 2H), 3.87 (s, 3H), 3.11 (septet, J=7.2 Hz, 4H), 1.30-1.23 (m, 9H).
The title compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and 5-aminopentan-1-ol (9.0 mg, 0.03 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 103 as a white solid (10 mg, 0.015 mmol, 95%). LC-MS: Calc'd m/z=430.2 for C21H30N6O4. found [M+H]+=431.2. 1H NMR (300 MHz, CD3CN) δ 7.11-7.01 (m, 2H), 6.94 (dd, J=7.7, 1.6 Hz, 1H), 4.97 (s, 2H), 4.32 (q, J=7.0 Hz, 2H), 4.09 (s, 2H), 3.86 (s, 3H), 3.49 (t, J=6.2 Hz, 2H), 3.01-2.90 (m, 2H), 1.66 (p, J=7.7 Hz, 2H), 1.49 (p, J=6.6 Hz, 2H), 1.43-1.30 (m, 2H), 1.29 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and 1,4-piperazinedipropanamine (17 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 104 as a white solid (14.8 mg, 0.0150 mmol, 54.7%). LC-MS: Calc'd m/z=527.3 for C26H41N9O3. found [M+H]+=528.3. 1H NMR (300 MHz, CD3CN) δ 7.14-7.03 (m, 2H), 6.96 (dd, J=7.9, 1.5 Hz, 1H), 4.97 (s, 2H), 4.35 (q, J=7.1 Hz, 2H), 4.13 (s, 2H), 3.85 (s, 3H), 3.13-2.99 (m, 8H), 2.06 (qd, J=7.9, 2.8 Hz, 4H), 1.30 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl (4-(aminomethyl)benzyl)carbamate (0.020 g, 0.084 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 105 as a white solid (4.8 mg, 0.0059 mmol, 43%). LC-MS: Calc'd m/z=463.2 for C24H29N7O3. found [M+H]+=464.3. 1H NMR (300 MHz, CD3CN) δ 7.46 (s, 4H), 7.12-7.01 (m, 2H), 6.99-6.91 (m, 1H), 4.96 (s, 2H), 4.33 (q, J=7.1 Hz, 2H), 4.18 (s, 2H), 4.17 (s, 2H), 4.11 (s, 2H), 3.85 (s, 3H), 1.29 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl (4-aminobutyl)carbamate (24 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 106 as a white solid (5.6 mg, 0.0050 mmol, 36%). LC-MS: Calc'd m/z=415.2 for C20H29N7O3. found [M+H]+=416.3. 1H NMR (300 MHz, CD3CN) δ 7.13-6.99 (m, 2H), 6.95 (dd, J=7.8, 1.6 Hz, 1H), 4.96 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 4.11 (s, 2H), 3.86 (s, 3H), 3.04-2.86 (m, 4H), 1.75-1.60 (dq, J=12.5, 6.5 Hz, 4H), 1.29 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and benzylamine (9.0 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 107 as a white solid (14 mg, 0.021 mmol, 76%). LC-MS: Calc'd m/z=434.2 for C23H26N6O3. found [M+H]+=435.3. 1H NMR (300 MHz, CD3CN) δ 7.45 (s, 5H), 7.11-6.99 (m, 2H), 6.95 (dd, J=7.7, 1.6 Hz, 1H), 4.96 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 4.17 (s, 2H), 4.15 (s, 2H), 3.86 (s, 3H), 1.29 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and picolamine (15 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 193 as a white solid (15 mg, 0.020 mmol, 72%). LC-MS: Calc'd m/z=435.2 for C22H25N7O3. found [M+H]+=436.3. 1H NMR (300 MHz, CD3CN) δ 8.80 (s, 1H), 8.74-8.68 (m, 1H), 8.36 (dd, J=8.1, 2.1 Hz, 1H), 7.82 (dd, J=8.1, 5.4 Hz, 1H), 7.11 (d, J=1.6 Hz, 1H), 7.05 (d, J=7.8 Hz, 1H), 6.97 (d, J=7.8, 1H), 4.96 (s, 2H), 4.37-4.29 (m, 4H), 4.24 (s, 2H), 3.84 (s, 3H), 1.29 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and cyclobutylamine (0.010 g, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 108 as a white solid (9.0 mg, 0.014 mmol, 52%). LC-MS: Calc'd m/z=398.2 for C20H26N6O3. found [M+H]+=399.3. 1H NMR (300 MHz, CD3CN) δ 7.09-7.00 (m, 2H), 6.92 (dd, J=7.8, 1.6 Hz, 1H), 4.96 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 3.99 (s, 2H), 3.86 (s, 3H), 3.69 (p, J=8.2 Hz, 1H), 2.28-2.09 (m, 4H), 1.95-1.73 (m, 2H), 1.29 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and 3,3-difluorocyclobutylamine (15 mg, 0.12 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 109 as a white solid (12 mg, 0.018 mmol, 66%). LC-MS: Calc'd m/z=434.2 for C20H24F2N6O3. found [M+H]+=435.3. 1H NMR (300 MHz, MeOD) δ 7.14 (s, 1H), 7.08 (d, J=7.6 Hz, 1H), 7.00 (d, J=8.2 Hz, 1H), 5.05 (s, 2H), 4.31 (q, J=5.2 Hz, 2H), 4.19 (s, 2H), 3.93 (s, 3H), 3.87-3.75 (m, 1H), 3.18-2.76 (m, 4H), 1.33 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (15.0 mg, 0.0412 mmol, 1.00 eq.) and 4-Boc-aminopiperidine (25 mg, 0.12 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 110 as a white solid (8.2 mg, 0.011 mmol, 52%). LC-MS: Calc'd m/z=427.2 for C21H29N7O3. found [M+H]+=428.3. 1H NMR (300 MHz, CD3CN) δ 7.14-7.11 (m, 2H), 6.97 (dd, J=7.7, 1.6 Hz, 1H), 4.99 (s, 2H), 4.37 (q, J=7.1 Hz, 2H), 4.21 (s, 2H), 3.86 (s, 3H), 3.50-3.41 (m, 4H), 3.07-2.96 (m, 2H), 2.25-2.18 (m, 2H), 1.30 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (15 mg, 0.041 mmol, 1.0 eq.) and tert-butyl (2-aminoethyl)carbamate (20 mg, 0.12 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 111 as a white solid (11.0 mg, 0.0151 mmol, 73.1%). LC-MS: Calc'd m/z=387.2 for C18H25N7O3. found [M+H]+=388.3. 1H NMR (300 MHz, CD3CN) δ 7.11 (m, 2H), 6.97 (dd, J=7.8, 1.6 Hz, 1H), 4.98 (s, 2H), 4.39 (q, J=7.0 Hz, 2H), 4.18 (s, 2H), 3.85 (s, 3H), 3.43-3.29 (m, 4H), 1.31 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (15 mg, 0.041 mmol, 1.0 eq.) and tert-butyl (2-(piperazin-1-yl)ethyl)carbamate (28 mg, 0.12 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 112 as a white solid (17 mg, 0.019 mmol, 91%). LC-MS: Calc'd m/z=456.2 for C22H32N8O3. found [M+H]+=457.3. 1H NMR (300 MHz, CD3CN) δ 7.20-7.07 (m, 1H), 7.12 (s, 1H), 6.99 (d, J=7.5 Hz, 1H), 4.99 (s, 2H), 4.40 (q, J=7.1, 2H), 4.24 (s, 2H), 3.84 (s, 3H), 3.42-3.35 (m, 8H), 3.07 (q, J=7.0 Hz, 2H), 2.89-2.73 (m, 2H), 1.31 (t, J=7.0 Hz, 3H).
To a solution of Example 2.26 (35 mg, 0.046 mmol, 1.0 eq.) in DMF (500 μL) and CH3CN (500 μL) was added H2O (2 mL) then L-cysteine (17 mg, 0.14 mmol, 3.0 eq.) and 0.5 M Na2HPO4 (500 μL). The resulting solution was stirred at room temperature for 1 h, after which 10 M NaOH was added (600 μL) and the reaction mixture stirred at room temperature for an additional 2 h. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 12 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 113 as a white solid (35 mg, 0.031 mmol, 68%). LC-MS: Calc'd m/z=675.3 for C29H14N9O8S. found [M+H]+=676.4. 1H NMR (300 MHz, MeOD) δ 7.19-7.06 (m, 2H), 6.99 (d, J=7.7 Hz, 1H), 5.05 (s, 2H), 4.51-4.21 (m, 3H), 4.19 (d, J=4.0 Hz, 2H), 3.90 (s, 3H), 3.88-3.71 (m, 1H), 3.64-2.99 (m, 14H), 2.87 (dd, J=16.4, 7.1 Hz, 1H), 2.77-2.50 (m, 1H), 1.35 (t, J=7.1 Hz, 3H).
To a solution of Nα-Boc-L-lysine (32 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.027 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 114 as a white solid (7.9 mg, 0.010 mmol, 37%). LC-MS: Calc'd m/z=473.2 for C22H31N7O5. found [M+H]+=474.3. 1H NMR (300 MHz, MeOD) δ 7.18-7.04 (m, 2H), 7.00 (d, J=7.7 Hz, 1H), 5.05 (s, 2H), 4.34 (q, J=7.1 Hz, 2H), 4.20 (s, 2H), 3.98 (t, J=6.3 Hz, 1H), 3.93 (s, 3H), 3.13-3.01 (m, 2H), 2.05-1.48 (m, 6H), 1.34 (t, J=7.1 Hz, 4H).
To a solution of 6-aminohexanoic acid (18 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 115 as a white solid (6.7 mg, 0.013 mmol, 48%). LC-MS: Calc'd m/z=458.2 for C22H30N6O5. found [M+H]+=459.2. 1H NMR (300 MHz, MeOD) δ 7.14 (d, J=7.5 Hz, 2H), 7.01 (dd, J=7.6, 1.6 Hz, 1H), 5.06 (s, 2H), 4.46-4.33 (m, 2H), 4.19 (s, 2H), 3.93 (s, 3H), 3.11-2.99 (m, 2H), 2.34 (t, J=7.2 Hz, 2H), 1.79-1.61 (m, 4H), 1.52-1.25 (m, 5H).
To a solution of carnosine (31 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 116 as a white solid (8.5 mg, 0.0090 mmol, 35%). LC-MS: Calc'd m/z=553.2 for C25H31N9O6. found [M+H]+=554.3. 1H NMR (300 MHz, MeOD) δ 8.83 (d, J=1.4 Hz, 1H), 7.37 (s, 1H), 7.18-6.98 (m, 3H), 5.06 (s, 2H), 4.80 (dd, J=8.4, 5.3 Hz, 1H), 4.38 (q, J=7.1 Hz, 2H), 4.22 (s, 2H), 3.93 (s, 3H), 3.41-3.05 (m, 4H), 2.72 (dd, J=7.6, 5.7 Hz, 2H), 1.36 (t, J=7.1 Hz, 3H).
To a solution of L-phenylalanine (22 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 117 as a white solid (6.2 mg, 0.0086 mmol, 32%). LC-MS: Calc'd m/z=492.2 for C25H28N6O5. found [M+H]+=493.3. 1H NMR (300 MHz, MeOD) δ 7.39-7.24 (m, 5H), 7.12-7.07 (m, 2H), 6.98 (d, J=7.7 Hz, 1H), 5.06 (s, 2H), 4.37 (q, J=7.1 Hz, 2H), 4.28-4.19 (m, 3H), 3.91 (s, 3H), 3.29-3.21 (m, 2H), 1.35 (t, J=7.2 Hz, 3H).
To a solution of L-phenylalaninol (21 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 118 as a white solid (11 mg, 0.015 mmol, 55%). LC-MS: Calc'd m/z=478.2 for C25H30N6O4. found [M+H]+=479.3. 1H NMR (300 MHz, MeOD) δ 7.41-7.11 (m, 7H), 7.04 (dd, J=7.8, 1.6 Hz, 1H), 5.07 (s, 2H), 4.45-4.22 (m, 4H), 3.93 (s, 3H), 3.78 (dd, J=12.1, 3.3 Hz, 1H), 3.58 (dd, J=12.2, 4.7 Hz, 1H), 3.50-3.35 (m, 1H), 3.11 (dd, J=13.5, 4.7 Hz, 1H), 2.99 (dd, J=13.5, 10.3 Hz, 1H), 1.36 (t, J=7.1 Hz, 3H).
To a solution of L-serine (14 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 119 as a white solid (8.7 mg, 0.013 mmol, 48%). LC-MS: Calc'd m/z=432.2 for C19H24N6O6. found [M+H]+=433.3. 1H NMR (300 MHz, MeOD) δ 7.22-7.12 (m, 2H), 7.04 (dd, J=7.7, 1.6 Hz, 1H), 5.08 (s, 2H), 4.43 (q, J=7.1 Hz, 2H), 4.31 (s, 2H), 4.15-3.98 (m, 3H), 3.93 (s, 3H), 1.38 (t, J=7.1 Hz, 3H).
To a solution of iminodiacetic acid (18 mg, 0.14 mmol, 5.0 eq.) in aqueous NaHCO3 (0.275 mL, 1.14 M, 12.5 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 4 h then acidified with aqueous HCl (5 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 120 as a white solid (7.9 mg, 0.011 mmol, 42%). LC-MS: Calc'd m/z=460.2 for C20H24N6O7. found [M+H]+=461.3. 1H NMR (300 MHz, MeOD) δ 7.22-6.99 (m, 3H), 5.07 (s, 2H), 4.51 (s, 2H), 4.40-4.34 (m, 2H), 4.16-4.08 (m, 4H), 3.94 (s, 3H), 1.35 (t, J=7.1, 3H).
To a solution of (2S,3S)-2-amino-3-hydroxypentanoic acid (37 mg, 0.28 mmol, 10 eq.) in aqueous NaHCO3 (0.362 mL, 1.14 M, 15.0 eq.) was added a solution of compound 11 (0.010 g, 0.014 mmol, 1.0 eq.) in 100 μL DMA. The resulting solution was heated to 50° C. for 18 h then acidified with aqueous HCl (1 M). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 121 as a white solid (7.9 mg, 0.012 mmol, 42%). LC-MS: Calc'd m/z=458.2 for C22H30N6O5. found [M+H]+=459.2. 1H NMR (300 MHz, MeOD) δ 7.19-7.13 (m, 1H), 7.12-6.96 (m, 2H), 5.05 (s, 2H), 4.40-4.17 (m, 4H), 3.96-3.85 (m, 1H), 2.10-1.93 (m, 1H), 1.72-1.53 (m, 1H), 1.53-1.38 (m, 1H), 1.34 (t, J=7.1 Hz, 3H), 1.06-0.92 (m, 6H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.027 mmol, 1.0 eq.) and (2R,3S)-2-aminopentane-1,3-diol (16 mg, 0.14 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 122 as a white solid (9.8 mg, 0.015 mmol, 53%). LC-MS: Calc'd m/z=444.2 for C22H32N6O4. found [M+H]+=445.3. 1H NMR (300 MHz, MeOD) δ 7.18 (s, 1H), 7.12-6.98 (m, 2H), 5.04 (s, 2H), 4.41-4.22 (m, 4H), 3.94-3.81 (m, 1H), 3.75 (dd, J=12.1, 7.1 Hz, 1H), 3.19-3.08 (m, 1H), 1.94-1.80 (m, 1H), 1.54-1.18 (m, 5H), 0.98 (d, J=6.9 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.027 mmol, 1.0 eq.) and ammonia/DMF (15 μL, 2 M, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 123 as a white solid (1.7 mg, 0.0030 mmol, 11%). LC-MS: Calc'd m/z=344.2 for C6H20N6O3. found [M+H]+=345.2. 1H NMR (300 MHz, MeOD) δ 7.13-6.99 (m, 2H), 6.95 (d, J=7.5 Hz, 1H), 5.04 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 4.10 (s, 2H), 3.93 (s, 3H), 1.33 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.027 mmol, 1.0 eq.) and ethylamine (9.3 μL, 0.14 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 124 as a white solid (6.4 mg, 0.011 mmol, 39%). LC-MS: Calc'd m/z=372.2 for C6H20N6O3. found [M+H]+=373.2. 1H NMR (300 MHz, MeOD) δ 7.17-7.04 (m, 2H), 7.00 (dd, J=7.7, 1.6 Hz, 1H), 5.05 (s, 2H), 4.34 (q, J=7.1 Hz, 2H), 4.18 (s, 2H), 3.93 (s, 3H), 3.12 (q, J=7.3 Hz, 2H), 1.34 (t, J=7.2 Hz, 6H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.027 mmol, 1.0 eq.) and dipropylamine (19 μL, 0.14 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 125 as a white solid (2.5 mg, 0.0038 mmol, 14%). LC-MS: Calc'd m/z=428.3 for C22H32N6O3. found [M+H]+=429.3. 1H NMR (300 MHz, MeOD) δ 7.15 (s, 1H), 7.12-6.97 (m, 2H), 5.06 (s, 2H), 4.38-4.24 (m, 4H), 3.95 (s, 3H), 3.09 (t, J=8.5 Hz, 4H), 1.92-1.65 (m, 4H), 1.33 (t, J=7.1 Hz, 3H), 1.00 (t, J=7.3 Hz, 6H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl N-[(2E)-4-aminobut-2-en-1-yl]carbamate (15 mg, 0.082 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 126 as a white solid (3.0 mg, 0.0070 mmol, 26%). LC-MS: Calc'd m/z=413.2 for C20H27N7O3. found [M+H]+=414.3. 1H NMR (300 MHz, MeOD) δ 7.13 (d, J=1.6 Hz, 1H), 7.08-6.94 (m, 2H), 6.15-5.92 (m, 2H), 5.04 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 4.19 (s, 2H), 3.93 (s, 3H), 3.76 (d, J=5.8 Hz, 2H), 3.66 (d, J=5.4 Hz, 2H), 1.32 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl-3-aminopiperidine-1-carboxylate (30 mg, 0.15 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 127 as a white solid (3.8 mg, 0.0088 mmol, 63%). LC-MS: Calc'd m/z=427.2 for C21H29N7O3. found [M+H]+=428.2. 1H NMR (300 MHz, MeOD) δ 7.18 (s, 1H), 7.10-6.97 (m, 2H), 5.04 (s, 2H), 4.38-4.22 (m, 4H), 3.94 (s, 3H), 3.92-3.70 (m, 2H), 3.62-3.38 (m, 2H), 3.15-2.91 (m, 2H), 2.45-2.35 (m, 1H), 2.21-2.10 (m, 1H), 1.88-1.55 (m, 1H), 1.33 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl-(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (27 mg, 0.15 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 128 as a white solid (8.0 mg, 0.010 mmol, 71%). LC-MS: Calc'd m/z=425.2 for C21H27N7O3. found [M+H]+=426.3. 1H NMR (300 MHz, MeOD) δ 7.20 (s, 1H), 7.10-6.98 (m, 2H), 5.04 (s, 2H), 4.52 (s, 1H), 4.42-4.27 (m, 4H), 4.24 (d, J=13.0 Hz, 1H), 3.93 (s, 3H), 3.75 (d, J=12.8 Hz, 1H), 3.54-3.35 (m, 3H), 2.58 (d, J=12.7 Hz, 1H), 2.16 (d, J=12.6 Hz, 1H), 1.34 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl 3-aminoazetidine-1-carboxylate (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 129 as a white solid (7.0 mg, 0.0094 mmol, 67%). LC-MS: Calc'd m/z=399.2 for C19H25N7O3. found [M+H]+=400.3. 1H NMR (300 MHz, MeOD) δ 7.16 (s, 1H), 7.04 (d, J=7.7 Hz, 1H), 7.01-6.93 (m, 1H), 5.04 (s, 2H), 4.37-4.20 (m, 6H), 4.15 (s, 2H), 3.94 (s, 3H), 1.44-1.28 (m, 4H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl 6-amino-2-azaspiro[3.3]heptane-2-carboxylate (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 130 as a white solid (7.9 mg, 0.010 mmol, 72%). LC-MS: Calc'd m/z=439.2 for C22H29N7O3. found [M+H]+=440.3. 1H NMR (300 MHz, MeOD) δ 7.25-7.02 (m, 2H), 6.97 (dd, J=7.7, 1.6 Hz, 1H), 5.05 (s, 2H), 4.34 (q, J=7.1 Hz, 2H), 4.17 (s, 2H), 4.09 (d, J=3.2 Hz, 4H), 3.93 (s, 3H), 3.78-3.65 (m, 1H), 2.81-2.62 (m, 2H), 2.58-2.45 (m, 2H), 1.35 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 131 as a white solid (0.010 g, 0.011 mmol, 72%). LC-MS: Calc'd m/z=470.3 for C23H34N8O3. found [M+H]+=471.4. 1H NMR (300 MHz, MeOD) δ 7.28-6.97 (m, 3H), 5.06 (s, 2H), 4.45-4.28 (m, 2H), 4.22 (s, 2H), 3.93 (s, 3H), 3.32-3.23 (m, 4H), 3.27-3.10 (m, 2H), 2.97-2.82 (m, 4H), 2.71 (t, J=6.8 Hz, 2H), 2.08-1.89 (m, 2H), 1.36 (t, J=7.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl N-(piperidin-4-ylmethyl)carbamate (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 132 as a white solid (7.9 mg, 0.010 mmol, 72%). LC-MS: Calc'd m/z=441.3 for C22H31N7O3. found [M+H]+=442.4. 1H NMR (300 MHz, MeOD) δ 7.20-6.97 (m, 3H), 5.06 (s, 2H), 4.41-4.28 (m, 4H), 3.94 (s, 3H), 3.57 (d, J=12.4 Hz, 2H), 3.11-2.96 (m, 2H), 2.91 (d, J=6.7 Hz, 2H), 2.25-1.86 (m, 3H), 1.65-1.51 (m, 2H), 1.34 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl (3R)-3-aminopyrrolidine-1-carboxylate carbamate (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 133 as a white solid (0.010 g, 0.013 mmol, 93%). LC-MS: Calc'd m/z=413.2 for C20H27N7O3. found [M+H]+=414.3. 1H NMR (300 MHz, MeOD) δ 7.21 (s, 1H), 7.15-6.99 (m, 2H), 5.06 (s, 2H), 4.41-4.27 (m, 4H), 4.14 (p, J=7.1 Hz, 1H), 3.94 (s, 3H), 3.81-3.68 (m, 1H), 3.67-3.48 (m, 2H), 3.48-3.33 (m, 1H), 2.68-2.50 (m, 1H), 2.39-2.21 (m, 1H), 1.35 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 134 as a white solid (3.6 mg, 0.0039 mmol, 28%). LC-MS: Calc'd m/z=456.3 for C22H32N8O3. found [M+H]+=457.3. 1H NMR (300 MHz, MeOD) δ 7.18 (d, J=1.5 Hz, 1H), 7.14-6.98 (m, 2H), 5.06 (s, 2H), 4.35 (q, J=7.1 Hz, 2H), 4.25 (s, 2H), 3.94 (s, 3H), 3.32-3.14 (m, 6H), 2.79-2.66 (m, 6H), 1.35 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and N-aminoethylmorpholine (0.010 g, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 135 as a white solid (3.1 mg, 0.0039 mmol, 28%). LC-MS: Calc'd m/z=457.3 for C22H31N7O4. found [M+H]+=458.4. 1H NMR (300 MHz, MeOD) δ 7.16 (d, J=1.5 Hz, 1H), 7.09 (d, J=7.7 Hz, 1H), 7.02 (dd, J=7.7, 1.6 Hz, 1H), 5.05 (s, 2H), 4.32 (q, J=7.1 Hz, 2H), 4.26 (s, 2H), 3.94 (s, 3H), 3.85-3.76 (m, 4H), 3.04 (t, J=6.4 Hz, 2H), 2.90-2.84 (m, 4H), 1.48 (s, 2H), 1.34 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and 1-amino-3-(morpholin-4-yl)propan-2-ol (20 mg, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 136 as a white solid (2.9 mg, 0.0036 mmol, 25%). LC-MS: Calc'd m/z=487.3 for C23H33N7O5. found [M+H]+=488.4. 1H NMR (300 MHz, MeOD) δ 7.19 (d, J=1.6 Hz, 1H), 7.12-6.97 (m, 2H), 5.05 (s, 2H), 4.50-4.41 (m, 1H), 4.34 (q, J=7.1 Hz, 2H), 4.27 (s, 2H), 4.03-3.85 (m, 4H), 3.94 (s, 3H), 3.41-3.35 (m, 4H), 3.28-3.10 (m, 3H), 3.04 (dd, J=12.9, 9.1 Hz, 1H), 1.35 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (0.010 g, 0.028 mmol, 1.0 eq.) and 2-(thiomorpholin-4-yl)ethanamine (0.010 g, 0.09 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 137 as a white solid (3.5 mg, 0.0043 mmol, 31%). LC-MS: Calc'd m/z=473.2 for C22H31N7O3S. found [M+H]+=473.3. 1H NMR (300 MHz, MeOD) δ 7.19-6.97 (m, 3H), 5.05 (s, 2H), 4.39-4.22 (m, 4H), 3.94 (s, 3H), 3.36-3.30 (m, 2H), 3.15-2.98 (m, 6H), 2.83 (q, J=4.6 Hz, 4H), 1.34 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (12 mg, 0.033 mmol, 1.0 eq.) and tert-butyl [4,4′-bipiperidine]-1-carboxylate (35 mg, 0.13 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 138 as a white solid (8.0 mg, 0.0096 mmol, 58%). LC-MS: Calc'd m/z=495.3 for C26H37N7O3. found [M+H]+=496.4. 1H NMR (300 MHz, MeOD) δ 7.16 (d, J=1.5 Hz, 1H), 7.12-6.96 (m, 2H), 5.05 (s, 2H), 4.43-4.24 (m, 4H), 3.93 (s, 3H), 3.53 (d, J=12.1 Hz, 2H), 3.43 (d, J=12.6 Hz, 2H), 2.95 (q, J=10.1 Hz, 4H), 2.00 (t, J=12.4 Hz, 4H), 1.58-1.44 (m, 6H), 1.33 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (12 mg, 0.033 mmol, 1.0 eq.) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (33 mg, 0.13 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 139 as a white solid (7.7 mg, 0.0093 mmol, 56%). LC-MS: Calc'd m/z=481.3 for C25H35N7O3. found [M+H]+=482.4. 1H NMR (300 MHz, MeOD) δ 7.16 (d, J=1.5 Hz, 1H), 7.12-6.96 (m, 2H), 5.05 (s, 2H), 4.38-4.25 (m, 4H), 3.94 (s, 3H), 3.38 (d, J=12.7 Hz, 2H), 3.24-3.18 (m, 6H), 2.08-1.96 (m, 2H), 1.96-1.90 (m, 2H), 1.72-1.66 (m, 4H), 1.33 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 11 (30 mg, 0.083 mmol, 1.0 eq.) and tert-butyl N-[2-(piperidin-4-yl)ethyl]carbamate (94 mg, 0.41 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 140 as a white solid (7.7 mg, 0.0097 mmol, 23%). LC-MS: Calc'd m/z=455.3 for C23H33N7O3. found [M+H]+=456.3. 1H NMR (300 MHz, MeOD) δ 7.17 (d, J=1.5 Hz, 1H), 7.09 (d, J=7.7 Hz, 1H), 7.01 (dd, J=7.7, 1.5 Hz, 1H), 5.06 (s, 2H), 4.40-4.26 (m, 4H), 3.94 (s, 3H), 3.51 (d, J=12.6 Hz, 2H), 2.97 (d, J=7.7 Hz, 5H), 2.08-1.94 (m, 2H), 1.64 (dd, J=14.4, 6.6 Hz, 2H), 1.54-1.40 (m, 2H), 1.34 (t, J=7.1 Hz, 3H).
The title compound 12 was prepared according to General Procedure 1 from compound 11 (80 mg, 0.22 mmol, 1.0 eq.) and (4-(aminomethyl)phenyl)methanol (90 mg, 0.7 mmol, 3 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 0 to 40% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (75 mg, 0.16 mmol, 73%). LC-MS: Calc'd m/z=464.2 for C24H28N6O4. found [M+H]+=465.3.
The title compound 13 was prepared according to General Procedure 3 from compound 12 (75 mg, 0.16 mmol, 1.0 eq.) and 10% SOCl2/DCM (4 mL). The titled compound was obtained as a yellow solid (65 mg, 0.14 mmol, 83%). LC-MS: Calc'd m/z=482.2 for C24H27ClN6O3. found [M+H]+=483.3.
The titled compound was prepared according to General Procedure 1 from compound 13 (8.0 mg, 0.017 mmol, 1.0 eq.) and dipropylamine (11 μL, 0.083 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 141 as a white solid (2.7 mg, 0.0030 mmol, 18%). LC-MS: Calc'd m/z=547.3 for C30H41N7O3. found [M+H]+=548.3. 1H NMR (300 MHz, MeOD) δ 7.63 (s, 4H), 7.15 (d, J=1.3 Hz, 1H), 7.02 (q, J=7.8 Hz, 2H), 5.04 (s, 2H), 4.42 (s, 2H), 4.36-4.22 (m, 6H), 3.93 (s, 3H), 3.15-2.98 (m, 4H), 1.85-1.71 (m, 4H), 1.32 (t, J=7.1 Hz, 3H), 0.99 (t, J=7.3 Hz, 6H).
The titled compound was prepared according to General Procedure 1 from compound 13 (8.0 mg, 0.017 mmol, 1.0 eq.) and morpholine (7.2 μL, 0.083 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 142 as a white solid (4.1 mg, 0.0047 mmol, 28%). LC-MS: Calc'd m/z=533.3 for C28H35N7O4. found [M+H]+=534.3. 1H NMR (300 MHz, MeOD) δ 7.62 (s, 4H), 7.14 (d, J=1.5 Hz, 1H), 7.11-6.95 (m, 2H), 5.04 (s, 2H), 4.42 (s, 2H), 4.39-4.23 (m, 6H), 3.92 (s, 3H), 4.14-3.67 (m, 8H), 1.33 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 13 (8.0 mg, 0.017 mmol, 1.0 eq.) and tert-butyl-N-(piperidin-4-yl)carbamate (17 mg, 0.083 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 143 as a white solid (8.6 mg, 0.0083 mmol, 49%). LC-MS: Calc'd m/z=646.4 for C34H46N8O5. found [M+H]+=647.5. 1H NMR (300 MHz, MeOD) δ 7.61 (s, 4H), 7.14 (s, 1H), 7.09-6.95 (m, 2H), 5.04 (s, 2H), 4.39-4.23 (m, 8H), 3.92 (s, 3H), 3.63-3.57 (m, 1H), 3.56-3.46 (m, 2H), 3.18-3.04 (m, 2H), 2.20-2.09 (m, 2H), 1.77-1.63 (m, 2H), 1.45 (s, 9H), 1.33 (t, J=7.1 Hz, 3H).
Compound 143 (4.5 mg, 0.0045 mmol, 1.0 eq.) was deprotected according to General Procedure 2 to give the title compound 144 as a white solid (4.3 mg, 0.0043 mmol, 96%). LC-MS: Calc'd m/z=546.3 for C29H38N8O3. found [M+H]+=547.4. 1H NMR (300 MHz, MeOD) δ 7.60 (s, 4H), 7.14 (s, 1H), 7.02 (q, J=7.8 Hz, 2H), 5.04 (s, 2H), 4.41-4.20 (m, 9H), 3.92 (s, 3H), 3.59 (d, J=12.7 Hz, 2H), 3.29-3.10 (m, 2H), 2.26 (d, J=13.9 Hz, 2H), 2.08-1.93 (m, 2H), 1.32 (t, J=7.1 Hz, 3H).
The title compound 14 was prepared according to General Procedure 1 from compound 11 (8 mg, 0.017 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (15 mg, 0.083 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (9.0 mg, 0.0092 mmol, 54%). LC-MS: Calc'd m/z=632.3 for C33H44N8O5. found [M+H]+=633.4. 1H NMR (300 MHz, MeOD) δ 7.62 (s, 4H), 7.17-7.11 (m, 1H), 7.10-6.95 (m, 2H), 5.04 (s, 2H), 4.41 (s, 2H), 4.38-4.23 (m, 6H), 3.92 (s, 3H), 3.72-3.66 (m, 4H), 3.30-3.24 (m, 4H), 1.49 (s, 9H), 1.33 (t, J=7.0 Hz, 3H).
Compound 14 (5.0 mg, 0.0051 mmol, 1.0 eq.) was deprotected according to General Procedure 2 to give the title compound 145 as a white solid (4.8 mg, 0.0049 mmol, 95%). LC-MS: Calc'd m/z=532.3 for C28H36N8O3. found [M+H]+=533.4. 1H NMR (300 MHz, MeOD) δ 7.49 (s, 4H), 7.16-6.95 (m, 3H), 5.04 (s, 2H), 4.32 (q, J=7.1 Hz, 2H), 4.26-4.20 (m, 4H), 3.92 (s, 3H), 3.76 (s, 2H), 3.33-3.24 (m, 4H), 2.86-2.76 (m, 4H), 1.32 (t, J=6.7 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 13 (8.0 mg, 0.017 mmol, 1.0 eq.) and tert-butyl (4-(aminomethyl)benzyl)carbamate (20 mg, 0.083 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 146 as a white solid (5.4 mg, 0.0052 mmol, 61%). LC-MS: Calc'd m/z=582.3 for C32H38N8O3. found [M+H]+=583.4. 1H NMR (300 MHz, MeOD) δ 7.58 (d, J=4.0 Hz, 8H), 7.13 (s, 1H), 7.07-6.94 (m, 2H), 5.03 (s, 2H), 4.36-4.21 (m, 10H), 4.18 (s, 2H), 3.92 (s, 3H), 1.32 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 13 (8.0 mg, 0.017 mmol, 1.0 eq.) and benzylamine (9.1 μL, 0.083 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 147 as a white solid (2.7 mg, 0.0030 mmol, 18%). LC-MS: Calc'd m/z=553.3 for C31H35N7O3. found [M+H]+=554.4. 1H NMR (300 MHz, MeOD) δ 7.59 (s, 4H), 7.49 (s, 5H), 7.13 (d, J=1.3 Hz, 1H), 7.08-6.94 (m, 2H), 5.03 (s, 2H), 4.37-4.22 (m, 10H), 3.92 (s, 3H), 1.32 (t, J=7.1 Hz, 3H).
To a solution of iminodiacetic acid (14 mg, 0.10 mmol, 5.0 eq.) in aqueous NaHCO3 (0.272 mL, 1.14 M, 15.0 eq.) was added a solution of compound 11 (0.010 g, 0.021 mmol, 1.0 eq.) in 100 μL DMF. The resulting solution was heated to 60° C. for 18 h. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 151 as a white solid (1.5 mg, 0.0026 mmol, 26%). LC-MS: Calc'd m/z=579.2 for C28H33N7O7. found [M+H]+=580.3. 1H NMR (300 MHz, MeOD) δ 7.63-7.47 (m, 4H), 7.13 (s, 1H), 7.10-6.95 (m, 2H), 5.04 (s, 2H), 4.37-4.21 (m, 8H), 3.93 (s, 3H), 3.79 (s, 4H), 1.33 (t, J=7.1 Hz, 3H).
To a solution of compound 100 (0.020 g, 0.048 mmol, 1.0 eq.) in DMF (500 μL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (16 mg, 0.053 mmol, 1.1 eq.) then DIPEA (42 μL, 0.24 mmol, 5.0 eq.). The resulting solution was stirred at room temperature for 18 h, after which L-cysteine (12 mg, 0.096 mmol, 2.0 eq.) was added and the resulting solution was stirred at room temperature for 1 h. H2O (200 μL) was then added and the reaction mixture stirred at room temperature for an additional 2 h. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 5 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 148 as a white solid (5.1 mg, 0.0047 mmol, 9.8%). LC-MS: Calc'd m/z=745.3 for C33H47N9O9S. found [M+H]+=746.4.
To a solution of compound 100 (0.020 g, 0.048 mmol, 1.0 eq.) in DMF (500 μL) was added compound 56 (16 mg, 0.053 mmol, 1.1 eq.) then DIPEA (42 μL, 0.24 mmol, 5.0 eq.). The resulting solution was stirred at room temperature for 18 h, after which L-cysteine (12 mg, 0.096 mmol, 2.0 eq.) was added and the resulting solution was stirred at room temperature for 1 h. H2O (200 μL) was then added and the reaction mixture stirred at room temperature for an additional 2 h. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 5 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 149 as a white solid (0.5 mg, 0.0004 mmol, 1%). LC-MS: Calc'd m/z=835.4 for C36H53N9O12S. found [M+H]+=836.5.
To a solution of compound 8 (0.75 g, 1.7 mmol, 1.0 eq.) and methyl 4-(bromomethyl)benzoate (0.39 g, 1.7 mmol, 1.0 eq.) in DMF (8 mL) was added CsCO3 (0.56 g, 1.7 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 15 was obtained as a white solid (0.65 g, 1.4 mmol, 81%). LC-MS: Calc'd m/z=357.1 for C17H19N5O4. found [M+H]+=358.2.
To a stirring solution of compound 15 (0.20 g, 0.56 mmol, 1.0 eq.) in anhydrous THF (15 mL) cooled to 0° C. was added lithium aluminum hydride (64 mg, 1.7 mmol, 3.0 eq.) in small portions over 5 mins. The resulting suspension was allowed to warm to room temperature and stirred for 15 mins, after which it was quenched with H2O (10 mL) and diluted with MeOH (50 mL) then filtered through a celite plug. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 16 as a white solid (0.12 g, 0.36 mmol, 65%). LC-MS: Calc'd m/z=329.2 for C11H19N5O3. found [M+H]+=330.2.
The title compound 17 was prepared according to General Procedure 3 from compound 16 (0.10 g, 0.30 mmol, 1.0 eq.) and 10% SOCl2/DCM (5 mL) and obtained as a yellow solid (assumed quantitative yield). LC-MS: Calc'd m/z=333.1 for C15H16ClN5O2. found [M+H]+=333.2.
The titled compound was prepared according to General Procedure 1 from compound 17 (16 mg, 0.048 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (28 mg, 0.14 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 150 as a white solid (5.1 mg, 0.0070 mmol, 30%). LC-MS: Calc'd m/z=383.2 for C19H25N7O2. found [M+H]+=383.3. 1H NMR (300 MHz, MeOD) δ 7.45 (d, J=8.1 Hz, 2H), 7.37 (d, J=8.1 Hz, 2H), 5.02 (s, 2H), 4.39 (q, J=7.1 Hz, 2H), 3.80 (s, 2H), 3.31-3.25 (m, 4H), 2.91-2.82 (m, 4H), 1.38 (t, J=7.1 Hz, 3H).
To a solution of compound 8 (430 mg, 2.0 mmol, 1.0 eq.) and methyl 4-(bromomethyl)-3-fluorobenzoate (0.50 g, 2.0 mmol, 1.0 eq.) in DMF (5 mL) was added CsCO3 (660 mg, 2.0 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 18 was obtained as a white solid (0.50 g, 1.3 mmol, 66%). LC-MS: Calc'd m/z=375.1 for C17H18FN5O4. found [M+H]+=376.2.
To a stirring solution of compound 18 (0.50 g, 1.3 mmol, 1.0 eq.) in anhydrous THF (4 mL) cooled to 0° C. was added lithium aluminum hydride (51 mg, 1.3 mmol, 1.0 eq.). The resulting suspension was allowed to warm to room temperature and stirred for 15 mins, after which it was diluted with MeOH (50 mL) then filtered through a celite plug. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 40% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 19 as a white solid (0.40 g, 1.2 mmol, 86%). LC-MS: Calc'd m/z=347.1 for C16H18FN5O3. found [M+H]+=348.2.
The title compound 20 was prepared according to General Procedure 3 from compound 19 (0.20 g, 0.58 mmol, 1.0 eq.) and 10% SOCl2/DCM (5 mL) and obtained as a yellow solid (assumed quantitative yield). LC-MS: Calc'd m/z=351.1 for C15H15ClFN5O2. found [M+H]+=352.2.
The titled compound was prepared according to General Procedure 1 from compound 20 (0.020 g, 0.057 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (32 mg, 0.17 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 152 as a white solid (12 mg, 0.016 mmol, 57%). LC-MS: Calc'd m/z=401.2 for C19H24FN7O2. found [M+H]+=402.3. 1H NMR (300 MHz, MeOD) δ 7.34 (t, J=7.7 Hz, 1H), 7.28-7.14 (m, 2H), 5.10 (s, 2H), 4.41 (q, J=7.1 Hz, 2H), 3.80 (s, 2H), 3.30 (d, J=5.3 Hz, 4H), 2.88 (t, J=5.3 Hz, 4H), 1.38 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 20 (0.020 g, 0.057 mmol, 1.0 eq.) and tert-butyl N-(4-aminobutyl)carbamate (34 mg, 0.17 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 153 as a white solid (0.010 g, 0.013 mmol, 46%). LC-MS: Calc'd m/z=415.2 for C19H24FN7O2. found [M+H]+=416.3. 1H NMR (300 MHz, MeOD) δ 7.48-7.27 (m, 3H), 5.13 (s, 2H), 4.43-4.30 (m, 4H), 3.65-3.54 (m, 2H), 3.49-3.39 (m, 1H), 3.16 (t, J=12.6 Hz, 2H), 2.32-2.21 (m, 2H), 2.05-1.88 (m, 2H), 1.36 (t, J=7.1 Hz, 3H).
To a solution of compound 2 (9.8 g, 39 mmol, 1.0 eq.) in nBuOH (100 mL) was added KOtBu (8.7 g, 77 mmol, 2.0 eq.). The resulting solution was stirred at 100° C. for 18 h, after which the solvent was removed in vacuo. The resulting solid was dissolved in EtOH (100 mL) and H2O (20 mL) then extracted with EtOAc (2×50 mL). The pooled organics were washed with brine (5 mL) then dried over MgSO4 and concentrated in vacuo to yield the title compound 21 as an orange solid (assumed quantitative yield). LC-MS: Calc'd m/z=291.2 for C14H21N5O2. found [M+H]+=292.2.
To a suspension of compound 21 (11 g, 39 mmol, 1.0 eq.) in DCM (50 mL) was added NBS (6.9 g, 39 mmol, 1.0 eq.). The resulting mixture was stirred at room temperature for 18 h, after which the reaction was quenched by the addition of 1 M NaHSO3 (50 mL) and rapidly stirred for 30 mins. The resulting mixture was diluted with DCM (100 mL) and the separated organics washed with 1 M NaHSO3 (2×50 mL). The pooled organics were dried over MgSO4, filtered then concentrated in vacuo to yield the title compound 22 as an orange solid (13 g, 34 mmol, 88%). LC-MS: Calc'd m/z=369.1 for C14H20BrN5O2. found [M+H]+=370.2.
To a solution of compound 22 (13 g, 34 mmol, 1.0 eq.) in MeOH (100 mL) was added NaOMe (5.47 g, 101 mmol, 3.00 eq.). The resulting mixture was stirred at room temperature for 72 h. Additional NaOMe (3.6 g, 66 mmol, 1.0 eq.) was added and the mixture was heated to reflux for 72 h. The solvent was removed in vacuo and crude material redissolved in EtOAc (250 mL) then washed with 1 M NaH2PO4 (150 mL). The aqueous layer was then extracted with EtOAc (3×150 mL), the pooled organics dried over MgSO4, and the solution concentrated in vacuo to yield the title compound 23 as a red solid (11 g, 34 mmol, 100%). LC-MS: Calc'd m/z=321.2 for C15H23N5O3. found [M+H]+=322.3.
Compound 23 (11 g, 34 mmol, 1.0 eq.) was dissolved in 10% TFA/MeOH (50 mL) and the resulting solution stirred at room temperature for 2 weeks. The solvent was removed in vacuo and the residue co-evaporated with MeOH (2×20 mL) to yield the title compound 24 as an orange solid (9.8 g, 28 mmol, 83%). LC-MS: Calc'd m/z=237.1 for C10H15N5O2. found [M+H]+=238.2.
To a solution of compound 24 (570 mg, 2.4 mmol, 1.0 eq.) and methyl 4-(bromomethyl)-3-methoxybenzoate (620 mg, 2.4 mmol, 1.00 eq.) in DMF (5 mL) was added CsCO3 (620 mg, 2.4 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 25 was obtained as a white solid (240 mg, 0.58 mmol, 24%). LC-MS: Calc'd m/z=415.2 for C20H25N5O5. found [M+H]+=416.3.
To a stirring solution of compound 25 (240 mg, 0.58 mmol, 1.0 eq.) in anhydrous THF (5 mL) cooled to 0° C. was added lithium aluminum hydride (22 mg, 0.58 mmol, 1.0 eq.). The resulting suspension was allowed to warm to room temperature and stirred for 15 mins, after which it was diluted with MeOH (25 mL) then filtered through a celite plug. The filtrate was concentrated in vacuo to yield the title compound 26 (220 mg, 0.57 mmol, 98%). LC-MS: Calc'd m/z=387.2 for C19H25N5O4. found [M+H]+=388.3.
The title compound 27 was prepared according to General Procedure 3 from compound 26 (220 mg, 0.57 mmol, 1.0 eq.) and 15% SOCl2/DCM (5 mL). The titled compound was obtained as a yellow solid (210 mg, 0.577 mmol, 98.8%). LC-MS: Calc'd m/z=391.1 for C18H22ClN5O3. found [M+H]+=392.2.
The titled compound was prepared according to General Procedure 1 from compound 27 (28 mg, 0.071 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (53 mg, 0.28 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 154 as a white solid (6.8 mg, 0.0087 mmol, 24%). LC-MS: Calc'd m/z=441.2 for C22H31N7O3. found [M+H]+=442.3. 1H NMR (300 MHz, MeOD) δ 7.07-6.94 (m, 2H), 6.90 (d, J=7.7 Hz, 1H), 5.03 (s, 2H), 4.28 (t, J=6.6 Hz, 2H), 3.90 (s, 3H), 3.76 (s, 2H), 3.34-3.24 (m, 4H), 2.84 (s, 4H), 1.72 (p, J=6.7 Hz, 2H), 1.46 (h, J=7.4 Hz, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (25 mg, 0.064 mmol, 1.0 eq.) and tert-butyl N-(4-aminobutyl)carbamate (51 mg, 0.26 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 155 as a white solid (6.1 mg, 0.0076 mmol, 24%). LC-MS: Calc'd m/z=455.3 for C23H33N7O3. found [M+H]+=456.3. 1H NMR (300 MHz, MeOD) δ 7.15 (s, 1H), 7.03 (q, J=7.9 Hz, 2H), 5.05 (s, 2H), 4.35-4.20 (m, 4H), 3.94 (s, 3H), 3.61 (d, J=12.9 Hz, 2H), 3.45 (s, 1H), 3.14 (t, J=13.1 Hz, 2H), 2.27 (d, J=14.7 Hz, 2H), 1.93 (d, J=13.3 Hz, 2H), 1.78-1.63 (m, 2H), 1.46 (h, J=7.4 Hz, 2H), 0.98 (t, J=7.4 Hz, 2H).
The titled compound was prepared according to General Procedure 1 from compound 27 (30 mg, 0.077 mmol, 1.0 eq.) and tert-butyl N-(piperidin-4-ylmethyl)carbamate (49 mg, 0.23 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 156 as a white solid (8.0 mg, 0.0099 mmol, 13%). LC-MS: Calc'd m/z=469.3 for C24H35N7O3, found [M+H]+=470.4. 1H NMR (300 MHz, MeOD) δ 7.16 (s, 1H), 7.10-6.96 (m, 2H), 5.05 (s, 2H), 4.34-4.20 (m, 4H), 3.94 (s, 3H), 3.62-3.56 (m, 2H), 3.12-2.96 (m, 2H), 2.95-2.87 (m, 2H), 2.14-1.87 (m, 4H), 1.79-1.63 (m, 2H), 1.60-1.35 (m, 3H), 0.96 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (30 mg, 0.077 mmol, 1.0 eq.) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (58 mg, 0.23 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 157 as a white solid (0.010 g, 0.012 mmol, 15%). LC-MS: Calc'd m/z=509.3 for C27H39N7O3. found [M+H]+=510.3. 1H NMR (300 MHz, MeOD) δ 7.15 (s, 1H), 7.03 (q, J=7.7 Hz, 2H), 5.05 (s, 2H), 4.32 (s, 2H), 4.25 (t, J=6.6 Hz, 2H), 3.95 (s, 3H), 3.24-3.19 (m, 8H), 3.19-3.08 (m, 2H), 2.08-1.97 (m, 2H), 1.96-1.90 (m, 2H), 1.69 (s, 6H), 1.55-1.35 (m, 2H), 0.96 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (30 mg, 0.077 mmol, 1.0 eq.) and tert-butyl N-[2-(piperidin-4-yl)ethyl]carbamate (52 mg, 0.23 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 158 as a white solid (9.0 mg, 0.011 mmol, 14%). LC-MS: Calc'd m/z=483.3 for C25H37N7O3. found [M+H]+=484.3. 1H NMR (300 MHz, MeOD) δ 7.15 (s, 1H), 7.02 (q, J=7.9 Hz, 2H), 5.05 (s, 2H), 4.32-4.20 (m, 4H), 3.95 (s, 3H), 3.50 (s, 2H), 3.05-2.93 (m, 6H), 2.06-1.96 (m, 2H), 1.78-1.60 (m, 4H), 1.53-1.35 (m, 3H), 0.96 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (30 mg, 0.077 mmol, 1.0 eq.) and 1-amino-2-methylpropan-2-ol (0.020 g, 0.23 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 159 as a white solid (0.010 g, 0.030 mmol, 39%). LC-MS: Calc'd m/z=444.2 for C22H32N6O4. found [M+H]+=445.3. 1H NMR (300 MHz, MeOD) δ 7.18 (s, 1H), 7.08-6.96 (m, 2H), 5.04 (s, 2H), 4.30-4.19 (m, 4H), 3.94 (s, 3H), 2.93-2.83 (m, 2H), 1.78-1.63 (m, 2H), 1.55-1.33 (m, 2H), 1.29 (s, 6H), 0.96 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (30 mg, 0.077 mmol, 1.0 eq.) and tert-butyl N-(4-aminobutyl)carbamate (43 mg, 0.23 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 160 as a white solid (0.010 g, 0.013 mmol, 17%). LC-MS: Calc'd m/z=443.3 for C22H33N7O3. found [M+H]+=444.3. 1H NMR (300 MHz, MeOD) δ 7.14 (s, 1H), 7.08-6.94 (m, 2H), 5.04 (s, 2H), 4.30-4.17 (m, 4H), 3.94 (s, 3H), 3.10 (t, J=7.6 Hz, 2H), 2.98 (t, J=7.3 Hz, 2H), 1.89-1.62 (m, 8H), 1.55-1.37 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (30 mg, 0.077 mmol, 1.0 eq.) and (2R,3S)-2-aminopentane-1,3-diol (27 mg, 0.23 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 161 as a white solid (1.9 mg, 0.0027 mmol, 3.5%). LC-MS: Calc'd m/z=472.3 for C24H36N6O4. found [M+H]+=473.4. 1H NMR (300 MHz, MeOD) δ 7.19 (s, 1H), 7.04 (s, 2H), 5.05 (s, 2H), 4.42-4.19 (m, 4H), 3.94 (s, 3H), 3.88 (dd, J=12.1, 3.9 Hz, 1H), 3.76 (dd, J=12.1, 7.2 Hz, 1H), 3.15 (dt, J=7.3, 4.2 Hz, 1H), 1.88 (s, 1H), 1.78-1.63 (m, 2H), 1.56-1.38 (m, 3H), 1.38-1.25 (m, 1H), 1.04-0.87 (m, 9H).
The titled compound was prepared according to General Procedure 1 from compound 27 (13 mg, 0.033 mmol, 1.0 eq.) and ammonia/DMF (83 μL, 2 M, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 162 as a white solid (1.6 mg, 0.0027 mmol, 8.1%). LC-MS: Calc'd m/z=372.2 for C18H24N6O3. found [M+H]+=373.3. 1H NMR (300 MHz, MeOD) δ 7.10 (d, J=1.6 Hz, 1H), 7.03 (d, J=7.8 Hz, 1H), 6.95 (dd, J=7.7, 1.6 Hz, 1H), 5.04 (s, 2H), 4.25 (t, J=6.5 Hz, 2H), 4.10 (s, 2H), 3.93 (s, 3H), 1.79-1.63 (m, 2H), 1.58-1.37 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (13 mg, 0.033 mmol, 1.0 eq.) and methylamine hydrochloride (11.2 mg, 0.17 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 163 as a white solid (2.6 mg, 0.0042 mmol, 13%). LC-MS: Calc'd m/z=386.2 for C19H26N6O3. found [M+H]+=387.3. 1H NMR (300 MHz, MeOD) δ 7.11 (s, 1H), 7.08-6.93 (m, 2H), 5.04 (s, 2H), 4.24 (t, J=6.5 Hz, 2H), 4.17 (s, 2H), 3.94 (s, 3H), 2.73 (s, 3H), 1.70 (p, J=6.7 Hz, 2H), 1.46 (dq, J=14.5, 7.3 Hz, 2H), 0.96 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (13 mg, 0.033 mmol, 1.0 eq.) and cyclobutylamine (14 μL, 0.17 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 164 as a white solid (5.3 mg, 0.0081 mmol, 25%). LC-MS: Calc'd m/z=426.2 for C22H30N6O3. found [M+H]+=427.3. 1H NMR (300 MHz, MeOD) δ 7.19-6.92 (m, 3H), 5.05 (s, 2H), 4.26 (t, J=6.5 Hz, 2H), 4.07 (s, 2H), 3.94 (s, 3H), 3.80 (p, J=8.0 Hz, 1H), 2.50-2.02 (m, 4H), 2.03-1.77 (m, 2H), 1.78-1.61 (m, 2H), 1.60-1.37 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 27 (13 mg, 0.033 mmol, 1.0 eq.) and benzylamine (18 μL, 0.17 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 165 as a white solid (7.8 mg, 0.011 mmol, 34%). LC-MS: Calc'd m/z=462.2 for C25H30N6O3. found [M+H]+=463.3. 1H NMR (300 MHz, MeOD) δ 7.48 (s, 5H), 7.13 (d, J=1.5 Hz, 1H), 7.10-6.94 (m, 2H), 5.04 (s, 2H), 4.32-4.16 (m, 6H), 3.93 (s, 3H), 1.86-1.62 (m, 2H), 1.57-1.36 (m, 2H), 0.96 (t, J=7.4 Hz, 3H).
To a solution of compound 24 (0.90 g, 3.8 mmol, 1.0 eq.) and methyl 4-(bromomethyl)-3-methoxybenzoate (0.87 g, 3.8 mmol, 1.0 eq.) in DMF (8 mL) was added CsCO3 (1.3 g, 3.8 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 28 was obtained as a white solid (0.23 g, 0.60 mmol, 16%). LC-MS: Calc'd m/z=385.2 for C19H23N5O4. found [M+H]+=386.3.
To a stirring solution of compound 28 (0.23 g, 0.60 mmol, 1.0 eq.) in anhydrous THF (8 mL) cooled to 0° C. was added lithium aluminum hydride (23 mg, 0.60 mmol, 1.0 eq.). The resulting suspension was stirred for 5 mins then allowed to warm to room temperature and stirred for a further 15 mins, after which it was diluted with MeOH (50 mL) then filtered through a celite plug. The filtrate was concentrated in vacuo to yield the title compound 29 as a yellow solid (0.20 g, 0.56 mmol, 94%). LC-MS: Calc'd m/z=357.2 for C18H23N5O3. found [M+H]+=358.3.
The title compound 30 was prepared according to General Procedure 3 from compound 29 (200 mg, 0.56 mmol, 1.0 eq.) and 10% SOCl2/DCM (10 mL). The titled compound was obtained as a yellow solid (assumed quantitative yield). LC-MS: Calc'd m/z=361.1 for C7H20ClN5O2. found [M+H]+=396.2.
The titled compound was prepared according to General Procedure 1 from compound 30 (30 mg, 0.083 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (77 mg, 0.42 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 166 as a white solid (0.010 g, 0.014 mmol, 33%). LC-MS: Calc'd m/z=411.2 for C21H29N7O2. found [M+H]+=412.3. 1H NMR (300 MHz, MeOD) δ 7.07-6.94 (m, 2H), 6.90 (d, J=7.7 Hz, 1H), 5.03 (s, 2H), 4.28 (t, J=6.6 Hz, 2H), 3.90 (s, 3H), 3.76 (s, 2H), 3.34-3.24 (m, 4H), 2.84 (s, 4H), 1.72 (p, J=6.7 Hz, 2H), 1.46 (h, J=7.4 Hz, 2H), 0.97 (t, J=7.4 Hz, 3H).
To a solution of compound 24 (0.70 g, 3.0 mmol, 1.0 eq.) and 2-chloro-5-(chloromethyl)pyridine (0.48 g, 3.0 mmol, 1.0 eq.) in DMF (5 mL) was added CsCO3 (0.96 g, 3.0 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 31 was obtained as a white solid (0.65 g, 1.8 mmol, 61%). LC-MS: Calc'd m/z=362.1 for C16H19ClN6O2. found [M+H]+=363.2.
A DMF (0.40 mL) solution of compound 31 (15 mg, 0.041 mmol, 1.0 eq.), tert-butyl piperazine-1-carboxylate (9.2 mg, 0.050 mmol, 1.2 eq.), KOtBu (7.0 mg, 0.062 mmol, 1.5 eq.) and Pd-PEPPSI™-iPr in a crimp-sealable microwave vial was thoroughly degassed and sealed under N2 then heated to 85° C. for 2 h. The reaction mixture was then diluted with CH3CN/H2O (1 mL) and reverse phase flash purification was accomplished as described in General Procedure 5 using a 12 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 167 as a white solid (0.010 g, 0.025 mmol, 60%). LC-MS: Calc'd m/z=398.2 for C19H26N8O2. found [M+H]+=399.3. 1H NMR (300 MHz, MeOD) δ 8.31 (d, J=2.3 Hz, 1H), 7.79 (dd, J=8.8, 2.4 Hz, 1H), 6.95 (d, J=8.8 Hz, 1H), 4.94 (s, 2H), 4.36 (t, J=6.5 Hz, 2H), 3.86-3.76 (m, 4H), 3.33-3.27 (m, 4H), 1.85-1.70 (m, 2H), 1.52 (dq, J=14.5, 7.4 Hz, 2H), 1.01 (t, J=7.4 Hz, 3H).
To a solution of tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (280 mg, 1.1 mmol, 10 eq.) in DMF (0.50 mL) and DIPEA (50 μL) was added compound 31 (0.040 g, 0.11 mmol, 1.0 eq.) and the resulting mixture heated to 140° C. for 18 h. The reaction was then adjusted to pH 1 with 6 M HCl and preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 168 as a white solid (0.7 mg, 0.0009 mmol, 0.8%). LC-MS: Calc'd m/z=466.3 for C24H34N8O2. found [M+H]+=467.4. 1H NMR (300 MHz, MeOD) δ 8.09 (s, 1H), 8.01 (d, J=8.2 Hz, 1H), 7.25 (d, J=9.4 Hz, 1H), 4.93 (s, 2H), 4.32 (t, J=6.5 Hz, 2H), 4.12 (q, J=7.0 Hz, 4H), 3.67 (t, J=5.9 Hz, 6H), 3.23 (t, J=5.9 Hz, 6H), 1.78-1.62 (m, 2H), 1.58-1.35 (m, 2H), 1.00 (t, J=7.4 Hz, 3H).
To a solution of compound 24 (580 mg, 2.4 mmol, 1.0 eq.) and 4-bromo-1-(bromomethyl)-2-methoxybenzene (680 mg, 2.4 mmol, 1.0 eq.) in DMF (8 mL) was added CsCO3 (0.80 g, 2.4 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 32 was obtained as a white solid (0.50 g, 1.1 mmol, 47%). LC-MS: Calc'd m/z=435.1 for C18H22BrN5O3. found [M+H]+=436.2.
A DMF (0.50 mL) solution of compound 32 (0.020 g, 0.046 mmol, 1.0 eq.), tert-butyl piperazine-1-carboxylate (11 mg, 0.060 mmol, 1.3 eq.), KOtBu (18 mg, 0.16 mmol, 3.5 eq.) and Pd2dba3 (4.2 mg, 0.0046 mmol, 0.10 eq.) and RuPhos (11 mg, 0.023 mmol, 0.50 eq.) in a crimp-sealable microwave vial was thoroughly degassed and sealed under N2 then heated to 100° C. for 18 h. The reaction mixture was then diluted with EtOAc (25 mL), filtered through celite and concentrated in vacuo. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (500 μL), H2O (500 μL) and 6 M HCl (100 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 169 after lyophilization (0.010 g, 0.015 mmol, 33%). LC-MS: Calc'd m/z=427.2 for C21H29N7O3. found [M+H]+=428.3. 1H NMR (300 MHz, MeOD) δ 6.98 (d, J=8.3 Hz, 1H), 6.64 (d, J=2.1 Hz, 1H), 6.57-6.44 (m, 1H), 4.94 (s, 2H), 4.30 (t, J=6.5 Hz, 2H), 3.88 (s, 3H), 3.39 (q, J=6.1 Hz, 8H), 1.80-1.69 (m, 2H), 1.56-1.36 (m, 2H), 0.96 (t, J=7.4 Hz, 3H).
A DMF (0.50 mL) solution of compound 32 (15 mg, 0.034 mmol, 1.0 eq.), 4-Boc-aminopiperidine (10 mg, 0.052 mmol, 1.5 eq.), KOtBu (15 mg, 0.14 mmol, 4.0 eq.) and Pd2dba3 (6.3 mg, 0.0068 mmol, 0.20 eq.) and RuPhos (12 mg, 0.028 mmol, 0.80 eq.) in a crimp-sealable microwave vial was thoroughly degassed and sealed under N2 then heated to 100° C. for 18 h. The reaction mixture was then diluted with EtOAc (25 mL), filtered through celite and concentrated in vacuo. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (500 μL) and 6 M HCl (250 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 170 after lyophilization (1.8 mg, 0.0027 mmol, 7.9%). LC-MS: Calc'd m/z=441.3 for C22H31N7O3. found [M+H]+=442.4. 1H NMR (300 MHz, MeOD) δ 6.96 (d, J=8.3 Hz, 1H), 6.62 (s, 1H), 6.52 (dd, J=8.4, 2.2 Hz, 1H), 4.96 (s, 2H), 4.31 (t, J=6.5 Hz, 2H), 3.84 (s, 3H), 3.90-3.75 (m, 3H), 2.86 (t, J=12.4 Hz, 2H), 2.12-2.07 (m, 2H), 1.84-1.65 (m, 4H), 1.56-1.38 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
A DMF (0.50 mL) solution of compound 32 (13 mg, 0.030 mmol, 1.0 eq.), tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (12 mg, 0.046 mmol, 1.5 eq.), KOtBu (14 mg, 0.12 mmol, 4.0 eq.) and Pd2dba3 (5.9 mg, 0.0060 mmol, 0.20 eq.) and RuPhos (11 mg, 0.024 mmol, 0.80 eq.) in a crimp-sealable microwave vial was thoroughly degassed and sealed under N2 then heated to 100° C. for 18 h. The reaction mixture was then diluted with EtOAc (25 mL), filtered through celite and concentrated in vacuo. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (500 μL) and 6 M HCl (250 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 171 after lyophilization (6.4 mg, 0.0088 mmol, 29%). LC-MS: Calc'd m/z=495.3 for C26H37N7O3. found [M+H]+=496.4. 1H NMR (300 MHz, MeOD) δ 7.20-7.10 (m, 2H), 7.02 (dd, J=8.2, 2.2 Hz, 1H), 5.03 (s, 2H), 4.31 (t, J=6.5 Hz, 2H), 3.94 (s, 3H), 3.56 (t, J=5.7 Hz, 4H), 3.29-3.23 (m, 4H), 2.08-1.85 (m, 8H), 1.81-1.65 (m, 2H), 1.56-1.38 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
A solution of compound 32 (380 mg, 0.87 mmol, 1.0 eq.), Pd(OAc)2 (20 mg, 0.087 mmol, 0.10 eq.), triphenylphosphine (110 mg, 0.44 mmol, 0.50 eq.), DIPEA (0.91 mL, 5.2 mmol, 6.0 eq.) and CuI (33 mg, 0.17 mmol, 0.20 eq.) in anhydrous DMF (4 mL) was sealed in a round-bottomed flask with a rubber septum and thoroughly degassed with N2. Anhydrous propargyl alcohol (75 μL, 1.3 mmol, 1.5 eq.) was added via syringe and the reaction mixture heated to 75° C. for 18 h under N2, after which it was diluted with H2O (50 mL) and extracted with EtOAc (3×50 mL). The pooled organics were then washed with brine (1×50 mL), dried over Na2SO4 and concentrated in vacuo to give the crude product. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 75% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 33 as a deep red solid (130 mg, 0.32 mmol, 36%). LC-MS: Calc'd m/z=411.2 for C21H25N5O4. found [M+H]+=412.3.
A solution of compound 33 (150 mg, 0.37 mmol, 1.0 eq.) in MeOH (5 mL) was degassed with N2, after which 10% Pd/C (39 mg, 0.037 mmol, 0.10 eq.) was added and the reaction vessel sealed and purged with additional N2. The reaction vessel was then purged with H2 and the mixture stirred under H2 at room temperature for 18 h. The suspension was filtered through celite and the filtrate concentrated in vacuo to give the title product 34 as a red oily solid, which was carried forward without additional purification (130 mg, 0.30 mmol, 83%; carried). LC-MS: Calc'd m/z=415.2 for C21H29N5O4. found [M+H]+=416.3.
To a solution of compound 34 (0.050 g, 0.12 mmol, 1.0 eq.) dissolved in anhydrous DCM (5 mL) was added DIPEA (63 μL, 0.36 mmol, 3.0 eq.) then methanesulfonyl chloride (17 mg, 0.14 mmol, 1.2 eq.) and the resulting solution stirred at room temperature for 1 h. The solvent was removed in vacuo to give the titled product 35, which was carried forward without additional purification (assumed quantitative yield). LC-MS: Calc'd m/z=479.2 for C21H29N5O6S. found [M+H]+=480.3.
The titled compound was prepared according to General Procedure 8 from compound 35 (0.010 g, 0.020 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (15 mg, 0.081 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (400 μL) and 6 M HCl (200 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 173 after lyophilization (4.6 mg, 0.0057 mmol, 28%). LC-MS: Calc'd m/z=469.3 for C24H35N7O3. found [M+H]+=470.4. 1H NMR (300 MHz, MeOD) δ 7.02-6.85 (m, 2H), 6.83-6.71 (m, 1H), 5.01 (s, 2H), 4.30 (t, J=6.5 Hz, 2H), 4.16 (s, 2H), 3.86 (s, 3H), 3.56-3.43 (m, 4H), 3.06-2.93 (m, 2H), 2.71 (t, J=7.4 Hz, 2H), 2.10-1.94 (m, 2H), 1.77-1.64 (m, 2H), 1.63-1.28 (m, 4H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 8 from compound 35 (5.1 mg, 0.010 mmol, 1.0 eq.) and 4-Boc-aminopiperidine (8.3 mg, 0.042 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (400 μL) and 6 M HCl (200 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 172 after lyophilization (1.0 mg, 0.0012 mmol, 12%). LC-MS: Calc'd m/z=483.3 for C25H37N7O3. found [M+H]+=484.4. 1H NMR (300 MHz, MeOD) δ 6.96-6.85 (m, 2H), 6.76 (d, J=7.7 Hz, 1H), 5.00 (s, 2H), 4.27 (t, J=6.5 Hz, 2H), 3.88 (s, 3H), 3.75-3.65 (m, 2H), 3.47-3.41 (m, 1H), 3.19-3.01 (m, 2H), 2.77-2.66 (m, 2H), 2.33-2.22 (m, 2H), 2.11-2.05 (m, 2H), 1.98-1.88 (m, 2H), 1.79-1.63 (m, 2H), 1.48-1.34 (m, 4H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 8 from compound 35 (0.010 g, 0.020 mmol, 1.0 eq.) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (0.020 g, 0.081 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (400 μL) and 6 M HCl (200 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 174 after lyophilization (4.6 mg, 0.0052 mmol, 26%). LC-MS: Calc'd m/z=537.3 for C29H43N7O3. found [M+H]+=538.4. 1H NMR (300 MHz, MeOD) δ 6.95-6.85 (m, 2H), 6.76 (d, J=7.8 Hz, 1H), 5.00 (s, 2H), 4.27 (t, J=6.6 Hz, 2H), 3.88 (s, 3H), 3.51-3.41 (m, 2H), 3.28-3.18 (m, 4H), 3.15-3.09 (m, 2H), 2.77-2.66 (m, 2H), 2.13-1.93 (m, 6H), 1.93-1.87 (m, 4H), 1.72-1.66 (m, 4H), 1.56-1.37 (m, 2H), 0.95 (q, J=9.9 Hz, 3H).
The titled compound was prepared according to General Procedure 8 from compound 35 (0.010 g, 0.020 mmol, 1.0 eq.) and tert-butyl N-[2-(piperidin-4-yl)ethyl]carbamate (19 mg, 0.081 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (400 μL) and 6 M HCl (200 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 175 after lyophilization (1.6 mg, 0.0019 mmol, 9.4%). LC-MS: Calc'd m/z=511.3 for C27H41N7O3. found [M+H]+=512.4. 1H NMR (300 MHz, MeOD) δ 6.94-6.85 (m, 2H), 6.76 (d, J=7.7 Hz, 1H), 5.00 (s, 2H), 4.25 (t, J=6.6 Hz, 2H), 3.88 (s, 3H), 3.64-3.58 (m, 2H), 3.21-2.85 (m, 6H), 2.74-2.66 (m, 2H), 2.21-1.93 (m, 4H), 1.80-1.57 (m, 5H), 1.53-1.39 (m, 2H), 1.36-1.28 (m, 2H), 0.96 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 8 from compound 35 (0.010 g, 0.020 mmol, 1.0 eq.) and 1-Boc-aminopiperidine carbamate (19 mg, 0.081 mmol, 4.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the Boc/8-MeO intermediate. This intermediate was then dissolved in CH3CN (400 μL) and 6 M HCl (200 μL) and the mixture heated to 55° C. for 3 h. The reaction mixture was cooled to room temperature to give the title product 176 after lyophilization (1.5 mg, 0.0018 mmol, 9.1%). LC-MS: Calc'd m/z=483.3 for C25H37N7O3. found [M+H]+=484.4. 1H NMR (300 MHz, MeOD) δ 7.02-6.84 (m, 2H), 6.76 (dd, J=7.8, 1.5 Hz, 1H), 5.01 (s, 2H), 4.30 (t, J=6.5 Hz, 2H), 3.87 (s, 3H), 3.56-3.41 (m, 4H), 3.31-3.25 (m, 5H), 3.06-2.92 (m, 2H), 2.71 (t, J=7.4 Hz, 2H), 2.09-1.93 (m, 2H), 1.84-1.64 (m, 2H), 1.56-1.28 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
To a solution of (3-methoxy-4-methylphenyl)acetic acid (5.0 g, 28 mmol, 1.0 eq.) in EtOH (30 mL) was added concentrated H2SO4 (0.3 mL). The resulting mixture was heated to reflux for 1.5 h then concentrated in vacuo and redissolved in EtOAc (50 mL) The resulting organic solution was extracted with H2O (1×20 mL) then brine (1×10 mL), dried over MgSO4 and concentrated in vacuo to yield the crude title product 36 as a yellow oil, which was carried forward without additional purification (assumed quantitative yield). LC-MS: Calc'd m/z=208.1 for C12H16O3. found [M+H]+=209.2.
To a solution of compound 36 (2.7 g, 13 mmol, 1.0 eq.) in DCM (10 mL) was added NBS (2.5 g, 14 mmol, 1.0 eq.) followed by 30% H2O2 (1.5 g, 13 mmol, 1.0 eq.). The resulting solution was stirred at room temperature for 18 h, concentrated in vacuo and redissolved in CH3CN (5 mL) and H2O (1 mL). Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 20 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by normal phase flash purification as described in General Procedure 5 using a 50 g silica column, eluting with a 10 to 100% EtOAc/hexanes gradient to give the title compound 37 (350 mg, 1.2 mmol, 9.4%). LC-MS: Calc'd m/z=286.0 for C12H15BrO3, found [M+H]+=287.0.
To a solution of compound 24 (470 mg, 2.0 mmol, 1.0 eq.) and compound 37 (560 mg, 2.0 mmol, 1.0 eq.) in DMF (3 mL) was added CsCO3 (640 mg, 2.0 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 38 was obtained as a white solid (160 mg, 0.35 mmol, 18%). LC-MS: Calc'd m/z=443.2 for C22H29N5O5. found [M+H]+=444.3.
To a stirring solution of compound 38 (380 mg, 0.68 mmol, 1.0 eq.) in anhydrous THF (2 mL) cooled to 0° C. was added lithium aluminum hydride (86 mg, 1.4 mmol, 2.0 eq.). The resulting suspension was allowed to warm to room temperature and stirred for 15 mins, after which it was diluted with MeOH (5 mL) and H2O (1 mL) then filtered through a celite plug. The filtrate was then concentrated in vacuo and reverse phase flash purification was accomplished as described in General Procedure 5 using a 30 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 39 as a white solid (0.20 g, 0.39 mmol, 57%). LC-MS: Calc'd m/z=401.2 for C20H27N5O4. found [M+H]+=402.3.
The title compound 40 was prepared according to General Procedure 3 from compound 39 (0.10 g, 0.19 mmol, 1.0 eq.) and 10% SOCl2/DCM (10 mL). The titled compound was obtained as a yellow solid (72 mg, 0.16 mmol, 84%). LC-MS: Calc'd m/z=405.2 for C19H24ClN5O3. found [M+H]+=406.3.
The titled compound was prepared according to General Procedure 1 from compound 40 (9.7 mg, 0.024 mmol, 1.0 eq.), NaI (0.5 mg, 0.003 mmol, 0.1 eq.) and tert-butyl piperazine-1-carboxylate (9.0 mg, 0.048 mmol, 2.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 180 as a white solid (7.0 mg, 0.010 mmol, 43%). LC-MS: Calc'd m/z=455.3 for C23H33N7O3. found [M+H]+=456.4. 1H NMR (300 MHz, MeOD) δ 7.23 (dd, J=8.5, 2.2 Hz, 1H), 7.03-6.97 (m, 2H), 5.04 (s, 2H), 4.35 (td, J=6.5, 4.1 Hz, 2H), 3.86 (d, J=2.0 Hz, 3H), 3.58-3.43 (m, 6H), 3.32-3.19 (m, 4H), 2.96 (dd, J=10.0, 6.1 Hz, 2H), 1.74 (dq, J=8.5, 6.6 Hz, 2H), 1.56-1.38 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 40 (9.7 mg, 0.024 mmol, 1.0 eq.), NaI (0.5 mg, 0.003 mmol, 0.1 eq.) and 4-Boc-aminopiperidine (0.010 g, 0.048 mmol, 2.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 181 as a white solid (6.1 mg, 0.0087 mmol, 37%). LC-MS: Calc'd m/z=469.3 for C24H35N7O3. found [M+H]+=470.4. 1H NMR (300 MHz, MeOD) δ 7.23 (dd, J=8.3, 2.3 Hz, 1H), 7.04-6.93 (m, 2H), 5.04 (s, 2H), 4.35 (t, J=2.2 Hz, 2H), 3.85 (s, 3H), 3.79-3.39 (m, 3H), 3.33-3.05 (m, 4H), 2.98 (dd, J=10.3, 6.4 Hz, 2H), 2.38-1.89 (m, 4H), 1.82-1.58 (m, 2H), 1.56-1.38 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 40 (9.7 mg, 0.024 mmol, 1.0 eq.), NaI (4 mg, 0.02 mmol, 1 eq.) and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (18 mg, 0.071 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 177 as a white solid (11 mg, 0.015 mmol, 63%). LC-MS: Calc'd m/z=523.3 for C28H41N7O3. found [M+H]+=524.4. 1H NMR (300 MHz, MeOD) δ 7.23 (dd, J=8.4, 2.2 Hz, 1H), 7.04-6.96 (m, 2H), 5.04 (s, 2H), 4.37 (t, J=6.5 Hz, 2H), 3.86 (s, 3H), 3.55-3.45 (m, 2H), 3.34-3.05 (m, 8H), 3.03-2.91 (m, 2H), 2.08-1.86 (m, 4H), 1.83-1.64 (m, 6H), 1.60-1.29 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 40 (9.7 mg, 0.024 mmol, 1.0 eq.), NaI (4 mg, 0.02 mmol, 1 eq.) and tert-butyl N-[2-(piperidin-4-yl)ethyl]carbamate (16 mg, 0.071 mmol, 3.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 178 as a white solid (13 mg, 0.018 mmol, 76%). LC-MS: Calc'd m/z=497.3 for C26H39N7O3. found [M+H]+=498.4. 1H NMR (300 MHz, MeOD) δ 7.26-7.10 (m, 1H), 7.04-6.88 (m, 1H), 6.94 (s, 1H), 5.03 (t, J=2.4 Hz, 2H), 4.40-4.24 (m, 2H), 3.83 (s, 3H), 3.72-3.58 (m, 2H), 3.26 (dd, J=10.4, 6.3 Hz, 2H), 2.97 (dt, J=18.0, 7.2 Hz, 4H), 2.71 (t, J=7.1 Hz, 2H), 2.02 (d, J=14.1 Hz, 1H), 1.81-1.59 (m, 6H), 1.45 (p, J=7.5 Hz, 2H), 1.34-1.28 (m, 2H), 0.97 (t, J=7.4 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 40 (9.7 mg, 0.024 mmol, 1.0 eq.), NaI (4 mg, 0.02 mmol, 1 eq.) and 1-Boc-aminopiperidine (0.010 g, 0.048 mmol, 2.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Deprotection was accomplished according to General Procedure 2 to give the title compound 179 as a white solid (12 mg, 0.015 mmol, 62%). LC-MS: Calc'd m/z=469.3 for C24H35N7O3. found [M+H]+=470.4. 1H NMR (300 MHz, MeOD) δ 7.23 (dd, J=8.4, 2.2 Hz, 1H), 7.00 (dd, J=5.3, 3.1 Hz, 2H), 5.04 (s, 2H), 4.36 (t, J=6.5 Hz, 2H), 3.85 (s, 3H), 3.61-3.34 (m, 3H), 3.24 (dd, J=9.2, 6.6 Hz, 2H), 3.08 (td, J=13.2, 2.8 Hz, 2H), 2.91 (dd, J=9.2, 6.5 Hz, 2H), 2.33 (d, J=13.6 Hz, 2H), 1.99-1.67 (m, 4H), 1.56-1.38 (m, 2H), 1.33 (s, 3H).
A mixture of compound 2 (5.0 g, 19 mmol, 1.0 eq.), compound 4 (20.3 g, 59.1 mmol, 3.0 eq.) and CsCO3 (6.4 g, 19 mmol, 1.0 eq.) was heated at 170° C. for 18 h. K2CO3 (20 mg) was then added, and the reaction stirred at 170° C. for an additional 72 h. The reaction was diluted with DCM (1000 mL), filtered, and concentrated in vacuo to give the crude product as a brown solid. Normal phase flash purification was accomplished as described in General Procedure 5 using a 100 g silica column, eluting with a 0 to 100% EtOAc/hexanes gradient, followed by reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 100% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 41 as a white solid (790 mg, 1.4 mmol, 7.2%). LC-MS: Calc'd m/z=559.3 for C31H41N5O3Si, found [M+H]+=560.4.
Compound 41 (210 mg, 0.38 mmol, 1.0 eq.) was dissolved in 15% TFA/MeOH (5 mL) and the resulting solution stirred at room temperature for 18 h, after which the solvent was removed in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 42 as a solid (57 mg, 0.097 mmol, 26%). LC-MS: Calc'd m/z=475.3 for C26H33N5O2Si. found [M+H]+=476.4.
To a solution of compound 42 (41 mg, 0.14 mmol, 1.0 eq.) and tert-butyl (4-(aminomethyl)benzyl)carbamate (0.080 g, 0.14 mmol, 1.0 eq.) in DMF (2 mL) was added CsCO3 (44 mg, 0.14 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 12 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 43 was obtained as a white solid (120 mg, 0.13 mmol, 91%). LC-MS: Calc'd m/z=694.4 for C39H50N6O4Si. found [M+H]+=695.5.
A solution of compound 43 (120 mg, 0.17 mmol, 1.0 eq.) and TBAF (0.70 mL, 1 M in THF, 1.0 eq.) was stirred at 40° C. for 18 h. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 12 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 44 was obtained as a white solid (65 mg, 0.14 mmol, 84%). LC-MS: Calc'd m/z=456.3 for C23H32N6O4. found [M+H]+=457.4.
To a suspension of compound 44 (52 mg, 0.11 mmol, 1.0 eq.) and CsCO3 (37 mg, 0.11 mmol, 1.0 eq.) in DCM (3 mL) was added NBS (0.020 g, 0.11 mmol, 1.0 eq.) and the resulting mixture stirred at room temperature for 2 h. The reaction mixture was then quenched with 1 M NaHSO3 (0.50 mL), diluted with H2O (5 mL) and extracted with EtOAc (2×10 mL). The pooled organics were then dried over MgSO4 and concentrated in vacuo to yield the crude product as an orange solid. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 12 g C18 column, eluting with a 10 to 100% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 45 was obtained as a solid (17 mg, 0.032 mmol, 28%). LC-MS: Calc'd m/z=534.2 for C23H31BrN6O4. found [M+H]+=535.2.
Compound 45 (7.0 mg, 0.013 mmol, 1.0 eq.) was dissolved in MeOH (5 mL) followed by the addition of NaOMe (2.8 mg, 0.052 mmol, 4.0 eq.) and the resulting mixture heated at 80° C. for 36 h, after which the solvent was removed in vacuo to give the crude intermediate. This intermediate was then dissolved in 3 M HCl (3 mL) and heated to 60° C. for 3 h. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 182 as a white solid (4.1 mg, 0.011 mmol, 85%). LC-MS: Calc'd m/z=372.2 for C18H24N6O3. found [M+H]+=373.3. 1H NMR (300 MHz, MeOD) δ 7.54-7.38 (m, 4H), 5.03 (s, 2H), 4.14-4.07 (m, 3H), 3.70 (d, J=5.0 Hz, 2H), 1.76-1.63 (m, 2H), 1.59-1.37 (m, 2H), 0.96 (t, J=7.3 Hz, 3H).
To a solution of compound 2 (2.0 g, 7.9 mmol, 1.0 eq.) in ethylene glycol (10 mL) was added amylamine (3.6 mL, 32 mmol, 4.0 eq.). The resulting solution was stirred at 120° C. for 18 h, after which it was cooled and taken up in EtOAc (100 mL). The resulting organic solution was washed with H2O (2×50 mL), dried over MgSO4, and concentrated in vacuo to yield the title compound 46 as a viscous brown oil (2.4 g, 7.9 mmol, 100%). LC-MS: Calc'd m/z=304.2 for C15H24N6O. found [M+H]+=305.2.
To a suspension of compound 46 (2.4 g, 7.9 mmol, 1.0 eq.) in DCM (50 mL) at 0° C. was added NBS (1.5 g, 8.3 mmol, 1.1 eq.). The resulting mixture was allowed to warm to room temperature and stirred for 30 mins. The reaction mixture was then washed with H2O (2×50 mL) and the remaining organics dried over MgSO4 then concentrated in vacuo to yield the title product 47 (2.9 g, 7.6 mmol, 96%). LC-MS: Calc'd m/z=382.1 for C15H23BrN6O. found [M+H]+=383.2.
To a solution of compound 47 (2.9 g, 7.6 mmol, 1.0 eq.) in MeOH (50 mL) was added NaOMe (4.1 g, 76 mmol, 10 eq.) and the resulting mixture heated at 70° C. for 36 h, after which the solvent was removed in vacuo and the residue taken up in EtOAc (50 mL). The resulting organic solution was washed with H2O (2×50 mL) and then concentrated in vacuo to give the crude THP-protected 8-methoxy intermediate. This intermediate was then dissolved in 15% TFA/MeOH (50 mL) and the resulting solution stirred at room temperature for 72 h. The solvent was removed in vacuo to yield the title compound 48 (2.2 g, 6.0 mmol, 80%). LC-MS: Calc'd m/z=250.2 for C11H16N6O. found [M+H]+=251.3.
To a solution of compound 48 (2.2 g, 6.0 mmol, 1.0 eq.) and methyl-4-(bromomethyl)benzoate (1.4 g, 6.0 mmol, 1.0 eq.) in DMF (50 mL) was added CsCO3 (5.9 g, 18 mmol, 3.0 eq.). The resulting suspension was stirred at room temperature for 18 h then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 30 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 49 was obtained as a white solid (910 mg, 2.3 mmol, 38%). LC-MS: Calc'd m/z=398.2 for C20H26N6O3. found [M+H]+=399.3.
To a stirring solution of compound 49 (910 mg, 2.3 mmol, 1.0 eq.) in anhydrous THF (20 mL) cooled to 0° C. was added lithium aluminum hydride (4.5 mL, 1 M in THF, 2.0 eq.). The resulting suspension was allowed to warm to room temperature and stirred for 1 h, after which it was quenched with wetted Na2SO4 (1 g) and H2O (2 mL) then filtered through a celite plug. The celite plug was washed with MeOH (50 mL) and the combined filtrate concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 50 as a white solid (480 mg, 1.3 mmol, 57%). LC-MS: Calc'd m/z=370.2 for C19H26N6O2. found [M+H]+=371.3.
To a solution of compound 50 (0.10 g, 0.27 mmol, 1.0 eq.) in DCM (5 mL) was added SOCl2 (0.10 mL, 1.4 mmol, 5.0 eq.) and the resulting solution stirred at room temperature for 3 h. The solvent was then removed in vacuo and the residue co-evaporated with toluene (2×10 mL) to give the title compound 51 as a yellow solid (assumed quantitative yield). LC-MS: Calc'd m/z=374.2 for C18H23ClN6O. found [M+H]+=375.3.
To compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) was added NH4OH (1 mL, 1 M in MeOH) and the resulting solution heated to 60° C. for 18 h, after which the solvent was removed in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18, eluting with a 10 to 40% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title product 183 as a white solid (2.1 mg, 0.0036 mmol, 13%). LC-MS: Calc'd m/z=355.2 for C18H25N7O. found [M+H]+=356.3. 1H NMR (300 MHz, MeOD) δ 7.55-7.38 (m, 4H), 5.02 (s, 2H), 4.11 (s, 2H), 3.41 (t, J=7.1 Hz, 2H), 1.64 (t, J=7.1 Hz, 2H), 1.44-1.28 (m, 4H), 1.00-0.90 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and 1-amino-2-methylpropan-2-ol (20 mg, 0.23 mmol, 8.5 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 184 as a white solid (8.5 mg, 0.013 mmol, 49%). LC-MS: Calc'd m/z=427.3 for C22H33N7O2. found [M+H]+=428.4. 1H NMR (300 MHz, MeOD) δ 7.52 (d, J=2.0 Hz, 4H), 5.04 (s, 2H), 4.24 (s, 2H), 3.42 (t, J=7.1 Hz, 2H), 2.90 (s, 2H), 1.65 (t, J=7.1 Hz, 2H), 1.39 (h, J=3.3 Hz, 4H), 1.26 (s, 6H), 1.00-0.89 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and diethylamine (28 μL, 0.27 mmol, 10 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 185 as a white solid (8.3 mg, 0.013 mmol, 49%). LC-MS: Calc'd m/z=411.3 for C22H33N7O. found [M+H]+=412.4. 1H NMR (300 MHz, MeOD) δ 7.60-7.46 (m, 4H), 5.05 (s, 2H), 4.34 (s, 2H), 3.42 (t, J=7.1 Hz, 2H), 3.28-3.13 (m, 4H), 1.70-1.57 (m, 2H), 1.44-1.29 (m, 10H), 0.95 (td, J=5.7, 3.0 Hz, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and 3,3-difluorocyclobutylamine (20 mg, 0.18 mmol, 6.7 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 186 as a white solid (6.4 mg, 0.0095 mmol, 35%). LC-MS: Calc'd m/z=445.2 for C22H29F2N7O. found [M+H]+=446.3. 1H NMR (300 MHz, MeOD) δ 7.51 (q, J=8.3 Hz, 4H), 5.04 (s, 2H), 4.21 (s, 2H), 3.79 (dt, J=14.6, 7.5 Hz, 1H), 3.41 (t, J=7.1 Hz, 2H), 3.14-2.75 (m, 4H), 1.72-1.59 (m, 2H), 1.45-1.32 (m, 4H), 1.00-0.89 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (20 mg, 0.10 mmol, 3.7 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 186 as a yellow solid (3.8 mg, 0.0050 mmol, 37%). LC-MS: Calc'd m/z=424.3 for C22H32N8O. found [M+H]+=425.4. 1H NMR (300 MHz, MeOD) δ 7.48 (d, J=8.0 Hz, 2H), 7.41 (d, J=7.9 Hz, 2H), 5.01 (s, 2H), 3.91 (s, 2H), 3.44 (t, J=7.1 Hz, 2H), 3.39-3.31 (m, 4H), 3.04-2.94 (m, 4H), 1.71-1.60 (m, 2H), 1.44-1.36 (m, 4H), 1.00-0.90 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and 4-Boc-aminopiperidine (20 mg, 0.096 mmol, 3.6 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 188 as a white solid (4.1 mg, 0.0053 mmol, 39%). LC-MS: Calc'd m/z=438.3 for C23H34N8O. found [M+H]+=439.4. 1H NMR (300 MHz, MeOD) δ 7.53 (q, J=8.1 Hz, 4H), 5.05 (s, 2H), 4.34 (s, 2H), 3.59 (d, J=13.0 Hz, 2H), 3.48-3.38 (m, 3H), 3.15 (t, J=13.2 Hz, 2H), 2.30-2.20 (m, 2H), 2.04-1.90 (m, 2H), 1.71-1.60 (m, 2H), 1.44-1.28 (m, 4H), 1.00-0.89 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and 1-Boc-aminopiperidine (20 mg, 0.096 mmol, 3.6 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 194 as a white solid (6.0 mg, 0.0077 mmol, 57%). LC-MS: Calc'd m/z=438.3 for C23H34N8O. found [M+H]+=439.4. 1H NMR (300 MHz, MeOD) δ 7.52 (d, J=1.2 Hz, 4H), 5.04 (s, 2H), 4.29 (s, 2H), 3.63-3.37 (m, 5H), 3.12 (td, J=13.2, 2.9 Hz, 2H), 2.43 (d, J=13.7 Hz, 2H), 1.94 (qd, J=13.2, 4.2 Hz, 2H), 1.71-1.60 (m, 2H), 1.44-1.28 (m, 4H), 1.01-0.90 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and tert-butyl (2-aminoethyl)carbamate (20 mg, 0.12 mmol, 4.6 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 189 as a white solid (4.4 mg, 0.0059 mmol, 44%). LC-MS: Calc'd m/z=398.3 for C20H30N8O. found [M+H]+=399.4. 1H NMR (300 MHz, MeOD) δ 7.53 (d, J=2.3 Hz, 4H), 5.04 (s, 2H), 4.30 (s, 2H), 3.48-3.35 (m, 4H), 3.40-3.34 (m, 2H), 1.66 (t, J=7.1 Hz, 2H), 1.44-1.34 (m, 4H), 1.00-0.90 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and tert-butyl (4-aminobutyl)carbamate (20 mg, 0.11 mmol, 3.9 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 190 as a white solid (4.1 mg, 0.0053 mmol, 40%). LC-MS: Calc'd m/z=426.3 for C22H34N8O. found [M+H]+=427.4. 1H NMR (300 MHz, MeOD) δ 7.57-7.47 (m, 4H), 5.01 (d, J=13.8 Hz, 2H), 4.21 (s, 2H), 3.42 (t, J=7.1 Hz, 2H), 3.10 (t, J=7.5 Hz, 2H), 2.98 (t, J=7.2 Hz, 2H), 1.88-1.56 (m, 6H), 1.44-1.28 (m, 4H), 1.00-0.89 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and tert-butyl (4-(aminomethyl)benzyl)carbamate (20 mg, 0.084 mmol, 3.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 191 as a white solid (4.3 mg, 0.0053 mmol, 39%). LC-MS: Calc'd m/z=474.3 for C26H34N8O. found [M+H]+=475.4. 1H NMR (300 MHz, MeOD) δ 7.63-7.44 (m, 8H), 5.03 (s, 2H), 4.31-4.23 (m, 4H), 4.17 (s, 2H), 3.42 (t, J=7.1 Hz, 2H), 1.65 (t, J=7.1 Hz, 2H), 1.43-1.28 (m, 4H), 0.99-0.89 (m, 3H).
The titled compound was prepared according to General Procedure 1 from compound 51 (0.010 g, 0.027 mmol, 1.0 eq.) and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (20 mg, 0.087 mmol, 3.2 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the titled compound as a white solid (4.5 mg, 0.0056 mmol, 41%). LC-MS: Calc'd m/z=467.3 for C24H37N9O. found [M+H]+=468.4. 1H NMR (300 MHz, MeOD) δ 7.52 (s, 4H), 5.04 (s, 2H), 4.26 (s, 2H), 3.43 (t, J=7.1 Hz, 2H), 3.26 (dt, J=10.1, 5.1 Hz, 6H), 3.19 (t, J=5.8 Hz, 2H), 2.79-2.69 (m, 4H), 1.71-1.60 (m, 2H), 1.44-1.28 (m, 4H), 1.00-0.89 (m, 3H).
To a solution of methyl 5-methylpyridine-2-carboxylate (750 mg, 5.0 mmol, 1.0 eq.) and AIBN (81 mg, 0.50 mmol, 0.10 eq.) in CCl4 (5 mL) at 55° C. was added NBS (970 mg, 5.5 mmol, 1.1 eq.) dissolved in CCl4 (5 mL). The resulting mixture was heated to reflux for 18 h then concentrated in vacuo and redissolved in DCM (150 mL). The resulting solution was extracted with H2O (1×100 mL) and brine (1×50 mL) then dried over MgSO4 and concentrated in vacuo to give the crude product. Normal phase flash purification was accomplished as described in General Procedure 5 using a 50 g silica column, eluting with a 0 to 50% hexanes/EtOAc gradient to give the title product 52 as an off-white solid (0.35 g, 1.5 mmol, 31%). LC-MS: Calc'd m/z=229.0 for C9H14O5S. found [M+H]+=230.0. 1H NMR (300 MHz, CDCl3) δ 8.77 (dd, J=2.2, 0.8 Hz, 1H), 8.15 (dd, J=8.0, 0.9 Hz, 1H), 7.91 (dd, J=8.1, 2.3 Hz, 1H), 4.53 (s, 2H), 4.04 (s, 3H).
To a solution of compound 8 (310 mg, 1.5 mmol, 1.0 eq.) and compound 52 (340 mg, 1.5 mmol, 1.0 eq.) in DMF (8 mL) was added CsCO3 (480 mg, 1.5 mmol, 1.0 eq.). The resulting suspension was stirred at room temperature for 18 h and then concentrated in vacuo. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. The title compound 53 was obtained as a white solid (230 mg, 0.64 mmol, 43%). LC-MS: Calc'd m/z=358.1 for C18H22N6O4. found [M+H]+=359.2.
To a solution of compound 53 (230 mg, 0.64 mmol, 1.0 eq.) in anhydrous THF (8 mL) cooled to 0° C. was added lithium aluminum hydride (24 mg, 0.64 mmol, 1.0 eq.) over 5 mins. The resulting suspension was allowed to warm to room temperature and stirred for 15 mins, after which it was diluted with MeOH (50 mL) then filtered through a celite plug. The filtrate was concentrated in vacuo to yield the title compound 54, which was carried forward without additional purification (0.20 g, 0.61 mmol, 94%). LC-MS: Calc'd m/z=330.1 for C15H18N6O3. found [M+H]+=331.2.
The title compound 55 was prepared according to General Procedure 3 from compound 54 (0.20 g, 0.61 mmol, 1.0 eq.) and 15% SOCl2/DCM (5 mL). The titled compound was obtained as a yellow solid (assumed quantitative yield). LC-MS: Calc'd m/z=334.1 for C14H15ClN6O2. found [M+H]+=335.2.
The titled compound was prepared according to General Procedure 1 from compound 55 (70 mg, 0.21 mmol, 1.0 eq.) and tert-butyl piperazine-1-carboxylate (0.20 g, 1.0 mmol, 5.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2 to give the title compound 192 as a yellow solid (3.1 mg, 0.0065 mmol, 6.2%). LC-MS: Calc'd m/z=484.2 for C18H24N8O2. found [M+H]+=385.3. 1H NMR (300 MHz, MeOD) δ 8.73 (s, 1H), 8.10 (dd, J=8.1, 2.3 Hz, 1H), 7.67 (d, J=8.1 Hz, 1H), 5.11 (s, 2H), 4.38 (q, J=7.1 Hz, 2H), 3.88 (s, 2H), 3.33-3.23 (m, 4H), 2.82 (t, J=5.2 Hz, 4H), 1.38 (t, J=7.1 Hz, 3H).
Title compound 56 was prepared according to the procedure described in International Patent Publication No. WO2017/054080. LC-MS: Calc'd m/z=449.4 for C19H19F4O7. found [M+H]+=450.4. 1H NMR (300 MHz, MeOD) δ 7.42 (tt, J=10.5, 7.2 Hz, 1H), 6.81 (s, 2H), 3.87 (t, J=6.0 Hz, 2H), 3.73-3.56 (m, 12H), 2.98 (t, J=6.0 Hz, 2H).
Title compound 57 was prepared according to the procedure described in International Patent Publication No. WO 2005/112919. LC-MS: Calc'd m/z=379.2 for C18H29N5O4. found [M+H]+=380.2.
To a solution of compound 57 (2.37 g, 6.26 mmol, 1.00 eq.) in DMF (15 mL) was added compound 56 (2.81 g, 6.26 mmol, 1.00 eq.) followed by DIPEA (2.73 mL, 15.6 mmol, 2.50 eq.). The resulting mixture stirred at room temperature for 18 h, after which the reaction was adjusted to pH 1 with 1 M HCl. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 120 g C18 column, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 58 as a white solid (4.00 g, 6.04 mmol, 96.5%). LC-MS: Calc'd m/z=662.3 for C31H46N6O10. found [M+H]+=663.2.
To a solution of compound 58 (2.50 g, 3.77 mmol, 1.00 eq.) in DMF (5 mL) was added bis(4-nitrophenyl) carbonate (1.26 g, 4.15 mmol, 1.50 eq.) followed by DIPEA (1.98 mL, 11.3 mmol, 3.00 eq.). The resulting mixture stirred at room temperature for 18 h, after which the reaction was adjusted to pH 1 with 1 M HCl. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 120 g C18 column, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 59 as a white solid (2.50 g, 3.02 mmol, 80.1%). LC-MS: Calc'd m/z=827.3 for C38H49N7O14. found [M+H]+=828.4. 1H NMR (300 MHz, MeOD) δ 8.39-8.29 (m, 2H), 7.68 (d, J=8.1 Hz, 2H), 7.54-7.39 (m, 4H), 6.83 (s, 2H), 5.28 (s, 2H), 4.54 (dd, J=9.0, 4.7 Hz, 1H), 4.22 (d, J=6.9 Hz, 1H), 3.82-3.51 (m, 12H), 3.28-3.08 (m, 5H), 2.57 (t, J=6.0 Hz, 2H), 2.21-1.49 (m, 4H), 1.01 (dd, J=6.8, 4.9 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 100 (26.5 mg, 0.0640 mmol, 1.00 eq.) and compound 59 (59 mg. 0.071 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (49.0 mg, 0.0443 mmol, 69.2%). LC-MS: Calc'd m/z=1101.5 for C52H71N13O14. found [M+H]+=1102.8. 1H NMR (300 MHz, MeOD) δ 7.69-7.55 (m, 2H), 7.39-7.30 (m, 2H), 7.19-7.10 (m, 2H), 7.02 (dd, J=7.7, 1.5 Hz, 1H), 6.82 (s, 2H), 5.12 (s, 2H), 5.06 (s, 2H), 4.54-4.48 (m J=9.2, 4.9 Hz, 1H), 4.38 (q, J=7.1 Hz, 2H), 4.34 (s, 2H), 4.22 (d, J=6.9 Hz, 1H), 3.92 (s, 3H), 3.82-3.67 (m, 2H), 3.72-3.59 (m, 4H), 3.57 (d, J=2.4 Hz, 8H), 3.29-3.08 (m, 1H), 2.57 (t, J=6.0 Hz, 2H), 2.13 (h, J=6.7 Hz, 1H), 1.94 (dd, J=12.9, 5.6 Hz, 1H), 1.84-1.69 (m, 1H), 1.60 (h, J=7.2 Hz, 2H), 1.35 (t, J=7.1 Hz, 3H), 1.00 (dd, J=6.8, 4.0 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 107 (8.8 mg, 0.013 mmol, 1.0 eq.) and compound 59 (12 mg. 0.015 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 40 to 52% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (2.7 mg, 0.0022 mmol, 17%). LC-MS: Calc'd m/z=1122.5 for C55H70N12O14. found [M+H]+=1123.7. 1H NMR (300 MHz, MeOD) δ 7.71-6.95 (m, 12H), 6.81 (s, 2H), 5.26-5.06 (m, 2H), 5.04-4.98 (m, 2H), 4.62-4.30 (m, 7H), 4.22 (d, J=6.8 Hz, 1H), 3.85-3.49 (m, 17H), 3.29-2.89 (m, 2H), 2.59 (t, 2H), 2.31-1.50 (m, 5H), 1.46-1.25 (m, 3H), 1.12-0.89 (m, 6H).
The titled compound was prepared according to General Procedure 4 from the Boc intermediate of compound 105 (17 mg, 0.021 mmol, 1.0 eq.) and compound 59 (19 mg. 0.023 mmol, 1.1 eq.) followed by deprotection of the crude intermediate according to General Procedure 3. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (8.7 mg, 0.0063 mmol, 30%). LC-MS: Calc'd m/z=1151.5 for C56H73N13O14. found [M+H]+=1152.7. 1H NMR (300 MHz, MeOD) δ 7.58-7.52 (m, 2H), 7.41-7.04 (m, 9H), 6.81 (s, 2H), 5.16 (s, 2H), 4.98 (s, 2H), 4.55-4.40 (m, 3H), 4.36-4.17 (m, 3H), 4.10 (s, 2H), 3.86-3.50 (m, 19H), 3.26-3.09 (m, 2H), 2.59 (t, J=6.0 Hz, 2H), 2.21-2.09 (m, 1H), 1.97-1.91 (m, 1H), 1.83-1.74 (m, 1H), 1.66-1.51 (m, 2H), 1.31 (t, J=7.1 Hz, 3H), 1.07-0.97 (m, 6H).
The titled compound was prepared according to General Procedure 4 from compound 108 (15 mg, 0.024 mmol, 1.0 eq.) and compound 59 (0.020 g. 0.024 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 45% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (14 mg, 0.012 mmol, 51%). LC-MS: Calc'd m/z=1086.5 for C52H70N12O14. found [M+H]+=1087.7. 1H NMR (300 MHz, MeOD) δ 7.49-7.43 (m, 4H), 7.05 (d, J=7.6 Hz, 1H), 6.81 (s, 2H), 6.73-6.64 (m, 2H), 5.02 (s, 4H), 4.54 (s, 3H), 4.36 (q, J=7.1 Hz, 2H), 4.22 (d, J=6.9 Hz, 1H), 3.88-3.47 (m, 19H), 3.35-3.07 (m, 2H), 2.59 (t, J=6.0 Hz, 2H), 2.11 (s, 4H), 2.01-1.71 (m, 2H), 1.60 (dt, J=15.2, 7.7 Hz, 4H), 1.32 (t, J=7.0 Hz, 3H), 1.02 (dd, J=6.8, 4.3 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 141 (15 mg, 0.017 mmol, 1.0 eq.) and compound 59 (15 mg. 0.018 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 30 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (5.0 mg, 0.0032 mmol, 19%). LC-MS: Calc'd m/z=1235.6 for C62H85N13O14. found [M+H]+=1236.8. 1H NMR (300 MHz, MeOD) δ 7.66-7.11 (m, 9H), 6.94-6.88 (m, 1H), 6.81 (s, 2H), 6.68-6.62 (m, 1H), 5.16 (s, 2H), 4.99 (s, 2H), 4.54 (d, J=20.3 Hz, 5H), 4.34 (s, 4H), 4.21 (d, J=6.8 Hz, 1H), 3.90-3.49 (m, 19H), 3.29-2.98 (m, 6H), 2.59 (t, J=6.0 Hz, 2H), 2.14 (p, J=6.8 Hz, 1H), 2.04-1.49 (m, 4H), 1.32 (t, 3H), 1.07-0.94 (m, 14H).
The titled compound was prepared according to General Procedure 4 from compound 142 (15 mg, 0.017 mmol, 1.0 eq.) and compound 59 (16 mg. 0.019 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 30 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (5.0 mg, 0.0032 mmol, 19%). LC-MS: Calc'd m/z=1221.6 for C60H79N13O15. found [M+H]+=1222.8. 1H NMR (300 MHz, MeOD) δ 7.59-7.53 (m, 2H), 7.48-7.04 (m, 8H), 6.95-6.89 (m, 1H), 6.81 (s, 2H), 5.16 (s, 2H), 4.98 (s, 2H), 4.50 (s, 4H), 4.32 (q, J=6.9 Hz, 3H), 4.21 (d, J=6.7 Hz, 1H), 4.07 (s, 2H), 3.89-3.45 (m, 17H), 3.29-3.10 (m, 2H), 3.04-2.98 (m, 4H), 2.91-2.85 (m, 4H), 2.59 (t, J=6.0 Hz, 2H), 2.22-2.09 (m, 1H), 2.01-1.70 (m, 2H), 1.66-1.55 (m, 2H), 1.31 (t, J=7.1 Hz, 3H), 1.03 (dd, J=6.8, 4.1 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 143 (15 mg, 0.015 mmol, 1.0 eq.) and compound 59 (14 mg. 0.017 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 30 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (7.6 mg, 0.0045 mmol, 30%). LC-MS: Calc'd m/z=1334.7 for C66H90N14O16. found [M+H]+=1335.8. 1H NMR (300 MHz, MeOD) δ 7.59-7.53 (m, 2H), 7.45-7.04 (m, 8H), 6.99-6.91 (m, 1H), 6.81 (s, 2H), 5.16 (s, 2H), 4.99 (s, 2H), 4.53-4.47 (m, 4H), 4.42-4.15 (m, 5H), 3.87-3.42 (m, 20H), 3.29-2.98 (m, 7H), 2.59 (t, J=6.0 Hz, 2H), 2.25-1.53 (m, 7H), 1.45 (s, 9H), 1.32 (t, J=7.1 Hz, 3H), 1.03 (dd, J=6.8, 4.1 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 112 (15 mg, 0.033 mmol, 1.0 eq.) and compound 59 (0.030 g. 0.036 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (2.0 mg, 0.0013 mmol, 4.1%). LC-MS: Calc'd m/z=1144.6 for C54H76N14O14. found [M+H]+=1147.8. 1H NMR (300 MHz, MeOD) δ 7.63 (d, J=8.1 Hz, 2H), 7.34 (d, J=8.1 Hz, 2H), 7.11-6.99 (m, 2H), 6.95 (d, J=5.3 Hz, 1H), 6.83 (s, 2H), 5.09-5.01 (m, 4H), 4.50 (s, 1H), 4.32 (q, J=7.1 Hz, 2H), 4.19 (s, 1H), 3.99 (s, 2H), 3.92 (s, 3H), 3.85-3.50 (m, 18H), 3.37 (s, 2H), 3.23-2.80 (m, 8H), 2.57 (t, J=6.0 Hz, 2H), 2.17-1.47 (m, 5H), 1.33 (t, J=7.0 Hz, 3H), 0.99 (dd, J=6.9, 2.8 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 150 (15 mg, 0.039 mmol, 1.0 eq.) and compound 59 (36 mg. 0.043 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (8.7 mg, 0.0080 mmol, 20%). LC-MS: Calc'd m/z=1071.5 for C51H69N13O13. found [M+H]+=1072.7. 1H NMR (300 MHz, MeOD) δ 7.65-7.57 (m, 2H), 7.53 (d, J=8.2 Hz, 2H), 7.46 (d, J=8.2 Hz, 2H), 7.32 (d, J=8.5 Hz, 2H), 6.80 (s, 2H), 5.10 (s, 2H), 5.04 (s, 2H), 4.51 (dd, J=9.1, 4.8 Hz, 1H), 4.36 (dd, J=13.9, 6.8 Hz, 4H), 4.27-4.15 (m, 1H), 3.80-3.50 (m, 18H), 3.27-3.04 (m, 6H), 2.55 (t, J=6.0 Hz, 2H), 2.20-2.00 (m, 1H), 1.96-1.83 (m, 1H), 1.82-1.68 (m, 1H), 1.67-1.52 (m, 2H), 1.36 (t, J=7.1 Hz, 3H), 0.98 (dd, J=6.8, 4.0 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 106 (15 mg, 0.036 mmol, 1.0 eq.) and compound 59 (33 mg. 0.040 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (3.2 mg, 0.0024 mmol, 6.7%). LC-MS: Calc'd m/z=1103.5 for C52H73N13O14. found [M+H]+=1104.7. 1H NMR (300 MHz, MeOD) δ 7.59 (d, J=8.2 Hz, 2H), 7.31 (d, J=8.3 Hz, 2H), 7.14-7.02 (m, 2H), 6.83 (s, 2H), 5.08-5.01 (m, 4H), 4.54-4.48 (m, 1H), 4.31 (q, J=7.1 Hz, 2H), 4.21 (d, J=6.9 Hz, 1H), 4.14 (s, 2H), 3.94 (s, 3H), 3.80-3.49 (m, 14H), 3.24-3.11 (m, 3H), 3.08-3.02 (m, 3H), 2.57 (t, J=6.0 Hz, 2H), 2.19-2.07 (m, 1H), 1.95-1.89 (m, 1H), 1.74-1.68 (m, 3H), 1.61-1.55 (m, 5H), 1.33 (t, J=7.1 Hz, 3H), 1.00 (dd, J=6.8, 3.6 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 111 (15 mg, 0.039 mmol, 1.0 eq.) and compound 59 (35 mg. 0.043 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (4.8 mg, 0.0037 mmol, 9.4%). LC-MS: Calc'd m/z=1075.5 for C50H69N13O14. found [M+H]+=1076.7. 1H NMR (300 MHz, MeOD) δ 7.60 (d, J=8.1 Hz, 2H), 7.32 (d, J=8.2 Hz, 2H), 7.17-7.04 (m, 2H), 6.98 (d, J=7.8 Hz, 1H), 6.82 (s, 2H), 5.10-5.03 (m, 4H), 4.56-4.48 (m, 1H), 4.33 (q, J=7.1 Hz, 2H), 4.25-4.19 (m, 3H), 3.93 (s, 3H), 3.85-3.41 (m, 16H), 3.27-3.09 (m, 4H), 2.57 (t, J=6.0 Hz, 2H), 2.14 (dt, J=13.6, 6.8 Hz, 1H), 1.96-1.90 (m, 1H), 1.82-1.69 (m, 1H), 1.67-1.54 (m, 2H), 1.34 (t, J=7.1 Hz, 3H), 1.01 (dd, J=6.8, 3.8 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 144 (0.010 g, 0.011 mmol, 1.0 eq.) and compound 59 (8.3 mg. 0.010 mmol, 0.90 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (4.9 mg, 0.0031 mmol, 31%). LC-MS: Calc'd m/z=1234.6 for C61H82N14O14. found [M+H]+=1235.7. 1H NMR (300 MHz, MeOD) δ 7.64-7.58 (m, 6H), 7.32 (d, J=8.2 Hz, 2H), 7.14 (s, 1H), 7.11-6.96 (m, 2H), 6.83 (s, 2H), 5.04 (s, 4H), 4.51 (s, 1H), 4.42-4.12 (m, 9H), 3.92 (s, 3H), 3.79-3.45 (m, 18H), 3.16 (ddt, J=20.8, 13.8, 7.4 Hz, 4H), 2.57 (t, J=6.0 Hz, 2H), 2.22-2.02 (m, 3H), 1.94 (d, J=7.5 Hz, 1H), 1.83-1.56 (m, 4H), 1.33 (t, J=7.1 Hz, 3H), 1.00 (dd, J=6.8, 3.8 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 145 (0.010 g, 0.011 mmol, 1.0 eq.) and compound 59 (8.5 mg. 0.010 mmol, 0.90 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 40 to 52% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (4.7 mg, 0.0030 mmol, 29%). LC-MS: Calc'd m/z=1220.6 for C60H80N14O14. found [M+H]+=1221.7. 1H NMR (300 MHz, MeOD) δ 7.72-7.52 (m, 6H), 7.35 (d, J=8.5 Hz, 2H), 7.12 (s, 1H), 7.09-6.95 (m, 2H), 6.83 (s, 2H), 5.13 (s, 2H), 5.04 (s, 2H), 4.57-4.47 (m, 1H), 4.40 (s, 2H), 4.38-4.17 (m, 7H), 3.92 (s, 3H), 3.82-3.52 (m, 14H), 3.29-3.04 (m, 10H), 2.57 (t, J=6.0 Hz, 2H), 2.11 (td, J=13.5, 6.7 Hz, 1H), 1.93 (s, 1H), 1.83-1.68 (m, 1H), 1.67-1.54 (m, 2H), 1.33 (t, J=7.1 Hz, 3H), 1.01 (dd, J=6.8, 3.7 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 152 (8.4 mg, 0.013 mmol, 1.0 eq.) and compound 59 (11 mg. 0.013 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (0.010 g, 0.00076 mmol, 57%). LC-MS: Calc'd m/z=1089.5 for C51H68FN13O13. found [M+H]+=1090.7. 1H NMR (300 MHz, MeOD) δ 7.69-7.57 (m, 2H), 7.51-7.32 (m, 1H), 7.38-7.25 (m, 4H), 6.82 (s, 2H), 5.17-5.09 (m, 4H), 4.59-4.31 (m, 5H), 4.22 (d, J=6.9 Hz, 1H), 3.86-3.48 (m, 14H), 3.31-3.02 (m, 10H), 2.57 (t, J=6.1 Hz, 2H), 2.12 (hept, J=6.6 Hz, 1H), 1.94 (td, J=13.0, 7.8 Hz, 1H), 1.77 (dtd, J=14.0, 9.3, 5.1 Hz, 1H), 1.59 (qd, J=13.9, 6.9 Hz, 2H), 1.37 (t, J=7.1 Hz, 3H), 1.00 (dd, J=6.8, 4.0 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 154 (2.2 mg, 0.0057 mmol, 1.0 eq.) and compound 59 (4.7 mg. 0.0057 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (2.3 mg, 0.0020 mmol, 36%). LC-MS: Calc'd m/z=1129.6 for C54H75N13O14. found [M+H]+=1130.7. 1H NMR (300 MHz, MeOD) δ 7.67-7.58 (m, 2H), 7.35 (d, J=8.5 Hz, 2H), 7.18-6.96 (m, 3H), 6.82 (s, 2H), 5.13 (s, 2H), 5.05 (s, 2H), 4.52 (dt, J=9.9, 5.2 Hz, 1H), 4.37-4.17 (m, 5H), 3.94 (s, 3H), 3.85-3.47 (m, 14H), 3.27-3.08 (m, 10H), 2.57 (t, J=6.1 Hz, 2H), 2.13 (h, J=6.7 Hz, 1H), 1.92 (dd, J=13.7, 8.4 Hz, 1H), 1.86-1.37 (m, 7H), 1.08-0.91 (m, 9H).
The titled compound was prepared according to General Procedure 4 from compound 166 (2.2 mg, 0.0034 mmol, 1.0 eq.) and compound 59 (2.8 mg. 0.0034 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (2.3 mg, 0.00017 mmol, 50%). LC-MS: Calc'd m/z=1099.6 for C53H73N13O13. found [M+H]+=1100.7. 1H NMR (300 MHz, MeOD) δ 8.24 (d, J=7.6 Hz, OH), 7.67-7.44 (m, 7H), 7.34 (d, J=8.5 Hz, 2H), 6.82 (s, 2H), 5.12 (s, 2H), 5.06 (s, 2H), 4.56-4.46 (m, 1H), 4.38-4.27 (m, 4H), 4.21 (d, J=6.9 Hz, 1H), 3.87-3.49 (m, 14H), 3.29-3.08 (m, 10H), 2.57 (t, J=6.0 Hz, 2H), 2.22-2.05 (m, 1H), 1.98-1.85 (m, 1H), 1.83-1.67 (m, 3H), 1.67-1.57 (m, 1H), 1.57-1.40 (m, 2H), 1.05-0.94 (m, 9H).
The titled compound was prepared according to General Procedure 4 from compound 155 (3.4 mg, 0.0050 mmol, 1.0 eq.) and compound 59 (4.1 mg. 0.0050 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (2.6 mg, 0.0026 mmol, 38%). LC-MS: Calc'd m/z=1143.6 for C55H77N13O14. found [M+H]+=1144.7. 1H NMR (300 MHz, MeOD) δ 7.61 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.5 Hz, 2H), 7.17-6.96 (m, 3H), 6.83 (s, 2H), 5.06 (s, 4H), 4.56-4.45 (m, 1H), 4.38-4.16 (m, 5H), 3.95 (s, 3H), 3.81-3.44 (m, 18H), 3.27-3.03 (m, 7H), 2.57 (t, J=6.1 Hz, 2H), 2.30-1.36 (m, 9H), 1.05-0.91 (m, 9H).
The titled compound was prepared according to General Procedure 4 from compound 132 (4.4 mg, 0.0066 mmol, 1.0 eq.) and compound 59 (5.5 mg. 0.0066 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (5.0 mg, 0.0040 mmol, 61%). LC-MS: Calc'd m/z=1129.6 for C54H75N13O14. found [M+H]+=1130.7. 1H NMR (300 MHz, MeOD) δ 7.68-7.56 (m, 2H), 7.31 (d, J=8.4 Hz, 2H), 7.17-7.06 (m, 2H), 7.05-6.96 (m, 1H), 6.83 (s, 2H), 5.09-5.00 (m, 4H), 4.52 (dd, J=9.1, 5.0 Hz, 1H), 4.42-4.17 (m, 5H), 3.94 (s, 3H), 3.81-3.41 (m, 18H), 3.29-2.86 (m, 7H), 2.57 (t, J=6.0 Hz, 2H), 2.23-1.26 (m, 10H), 1.00 (dd, J=6.8, 3.7 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 140 (4.5 mg, 0.0066 mmol, 1.0 eq.) and compound 59 (5.5 mg. 0.0066 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (5.3 mg, 0.0039 mmol, 59%). LC-MS: Calc'd m/z=1143.6 for C55H77N13O14. found [M+H]+=1144.7. 1H NMR (300 MHz, MeOD) δ 7.62 (d, J=8.2 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H), 7.17-7.06 (m, 2H), 7.05-6.97 (m, 1H), 6.83 (s, 2H), 5.09-5.00 (m, 4H), 4.50 (dd, J=9.1, 4.9 Hz, 1H), 4.40-4.14 (m, 5H), 3.94 (s, 3H), 3.85-3.39 (m, 18H), 3.15 (dt, J=16.9, 6.8 Hz, 8H), 2.88 (t, J=12.6 Hz, 1H), 2.57 (t, J=6.0 Hz, 2H), 2.20-1.39 (m, 7H), 1.34 (t, J=7.1 Hz, 3H), 0.99 (dd, J=6.9, 2.7 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 139 (6.2 mg, 0.0087 mmol, 1.0 eq.) and compound 59 (7.2 mg. 0.0087 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (6.1 mg, 0.0044 mmol, 50%). LC-MS: Calc'd m/z=1169.6 for C57H79N13O14. found [M+H]+=1170.7. 1H NMR (300 MHz, MeOD) δ 7.66-7.57 (m, 2H), 7.33 (d, J=8.5 Hz, 2H), 7.18-7.07 (m, 2H), 7.01 (dd, J=7.7, 1.5 Hz, 1H), 6.83 (s, 2H), 5.11-5.03 (m, 4H), 4.51 (dd, J=9.1, 5.0 Hz, 1H), 4.41-4.27 (m, 4H), 4.21 (d, J=6.9 Hz, 1H), 3.94 (s, 3H), 3.83-3.42 (m, 18H), 3.27-3.08 (m, 8H), 2.57 (t, J=6.0 Hz, 2H), 2.22-2.04 (m, 1H), 1.97 (d, J=14.7 Hz, 4H), 1.89-1.47 (m, 4H), 1.45-1.29 (m, 5H), 1.00 (dd, J=6.8, 4.1 Hz, 6H).
The titled compound was prepared according to General Procedure 4 from compound 110 (18 mg, 0.027 mmol, 1.0 eq.) and compound 59 (23 mg. 0.027 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (13 mg, 0.010 mmol, 35%). LC-MS: Calc'd m/z=1115.5 for C53H73N13O14. found [M+H]+=1116.7. 1H NMR (300 MHz, MeOD) δ 7.60 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.2 Hz, 2H), 7.18-7.07 (m, 2H), 7.01 (dd, J=7.7, 1.5 Hz, 1H), 6.82 (s, 2H), 5.09-5.01 (m, 4H), 4.58-4.43 (m, 1H), 4.44-4.18 (m, 5H), 3.93 (s, 3H), 3.81-3.45 (m, 18H), 3.29-3.01 (m, 7H), 2.57 (t, J=6.0 Hz, 2H), 2.24-1.50 (m, 5H), 1.35 (t, J=7.1 Hz, 3H), 1.00 (dd, J=6.8, 4.0 Hz, 6H).
To a suspension of 1-(2-hydroxyethyl)pyrrole-2,5-dione (0.10 g, 0.71 mmol, 15 eq.) in DCM (10 mL) was added Dess-Martin periodinane (0.30 g, 0.71 mmol, 15 eq.). The resulting mixture was stirred at room temperature for 18 h then filtered through a celite plug. The filtrate was concentrated in vacuo to give the crude intermediate aldehyde which was carried forward without additional purification. The aldehyde was then dissolved in 3:1 EtOAc/AcOH (10 mL) along with Compound 100 (35 mg, 0.046 mmol, 1.0 eq.) followed by the addition of Na(OAc)3BH (98 mg, 0.46 mmol, 10 eq.). The resulting mixture was stirred at room temperature for 3 h then concentrated in vacuo to give the crude product. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (16 mg, 0.021 mmol, 46%). LC-MS: Calc'd m/z=536.3 for C26H32N8O5. found [M+H]+=537.4. 1H NMR (300 MHz, MeOD) δ 7.16 (d, J=8.6 Hz, 2H), 7.01 (d, J=7.7 Hz, 1H), 6.85 (s, 2H), 5.07 (s, 2H), 4.42 (q, J=7.1 Hz, 2H), 4.23 (s, 2H), 3.92 (s, 3H), 3.79-3.69 (m, 2H), 3.26-3.20 (m, 4H), 3.05-2.99 (m, 4H), 2.90-2.84 (m, 2H), 1.37 (t, J=7.1 Hz, 3H).
To a solution of compound 100 (7.0 mg, 0.0093 mmol, 1.0 eq.) in DMF (300 μL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (3.5 mg, 0.011 mmol, 1.2 eq.) then DIPEA (4.7 μL, 0.027 mmol, 3.0 eq.) and the resulting solution stirred at room temperature for 1 h. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (4.6 mg, 0.0055 mmol, 59%). LC-MS: Calc'd m/z=606.3 for C30H38N8O6. found [M+H]+=607.4. 1H NMR (300 MHz, MeOD) δ 7.17 (d, J=1.5 Hz, 1H), 7.11 (d, J=7.7 Hz, 1H), 7.03 (dd, J=7.7, 1.5 Hz, 1H), 6.82 (s, 2H), 5.06 (s, 2H), 4.33 (dd, J=14.3, 7.2 Hz, 4H), 3.95 (s, 3H), 3.52 (t, J=6.9 Hz, 2H), 3.47-2.97 (m, 8H), 2.44 (t, J=7.4 Hz, 3H), 1.73-1.54 (m, 6H), 1.34 (t, J=7.1 Hz, 2H).
To a solution of compound 100 (0.010 g, 0.012 mmol, 1.0 eq.) and compound 56 (5.4 mg, 0.012 mmol, 1.0 eq.) in DMF (400 μL) was added DIPEA (6.1 μL, 0.035 mmol, 3.0 eq.) and the resulting solution stirred at room temperature for 1 h. Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (6.3 mg, 0.0068 mmol, 57%). LC-MS: Calc'd m/z=696.3 for C33H44N8O9. found [M+H]+=697.4. 1H NMR (300 MHz, MeOD) δ 7.21-7.08 (m, 2H), 7.04 (dd, J=7.7, 1.5 Hz, 1H), 6.80 (s, 2H), 5.06 (s, 2H), 4.42-4.29 (m, 4H), 3.94 (s, 3H), 3.81-3.54 (m, 18H), 3.49-3.03 (m, 4H), 2.71 (s, 2H), 1.35 (t, J=7.1 Hz, 3H).
The titled compound was prepared according to General Procedure 4 from compound 178 (3.4 mg, 0.0050 mmol, 1.0 eq.) and compound 59 (4.2 mg. 0.0050 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 20 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (2.8 mg, 0.0020 mmol, 41%). LC-MS: Calc'd m/z=1143.6 for C55H77N13O14. found [M+H]+=1144.8.
The titled compound was prepared according to General Procedure 4 from compound 172 (5.0 mg, 0.0070 mmol, 1.0 eq.) and compound 59 (5.8 mg. 0.0050 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 20 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (1.2 mg, 0.00086 mmol, 12%). LC-MS: Calc'd m/z=1171.6 for C57H81N13O14. found [M+H]+=1172.8.
The titled compound was prepared according to General Procedure 4 from compound 174 (5.0 mg, 0.0072 mmol, 1.0 eq.) and compound 59 (5.9 mg. 0.0072 mmol, 1.0 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 20 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (3.3 mg, 0.0024 mmol, 33%). LC-MS: Calc'd m/z=1157.6 for C56H79N13O14. found [M+H]+=1158.8.
The titled compound was prepared according to General Procedure 4 from compound 187 (20 mg, 0.048 mmol, 1.0 eq.) and compound 59 (44 mg. 0.053 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (24.4 mg, 0.018 mmol, 38%). LC-MS: Calc'd m/z=1112.6 for C54H76N14O12. found [M+H]+=1113.8. 1H NMR (300 MHz, MeOD) δ 7.67-7.46 (m, 6H), 7.34 (d, J=8.5 Hz, 2H), 6.82 (s, 2H), 5.12 (s, 2H), 5.05 (s, 2H), 4.58-4.47 (m, 1H), 4.36 (s, 2H), 4.26-4.16 (m, 1H), 3.85-3.52 (m, 18H), 3.42 (t, J=7.1 Hz, 2H), 3.18 (dtd, J=20.4, 13.5, 6.7 Hz, 6H), 2.57 (t, J=6.0 Hz, 2H), 2.20-2.02 (m, 1H), 1.98-1.88 (m, 1H), 1.83-1.73 (m, 1H), 1.70-1.56 (m, 4H), 1.43-1.32 (m, 4H), 1.05-0.89 (m, 9H).
The titled compound was prepared according to General Procedure 4 from compound 188 (25 mg, 0.057 mmol, 1.0 eq.) and compound 59 (52 mg. 0.063 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled compound as a white solid (21 mg, 0.015 mmol, 27%). LC-MS: Calc'd m/z=1126.6 for C55H78N14O12. found [M+H]+=1127.8. 1H NMR (300 MHz, MeOD) δ 7.64-7.45 (m, 6H), 7.31 (d, J=8.2 Hz, 2H), 6.82 (s, 2H), 5.04 (s, 4H), 4.54-4.48 (m, 1H), 4.30 (s, 2H), 4.21 (dd, J=7.0, 2.9 Hz, 1H), 3.84-3.38 (m, 21H), 3.29-3.01 (m, 6H), 2.57 (t, J=6.0 Hz, 2H), 2.26-1.86 (m, 3H), 1.84-1.52 (m, 4H), 1.43-1.32 (m, 4H), 1.08-0.89 (m, 9H).
The titled compound was prepared according to General Procedure 4 from the Boc intermediate of compound 191 (37 mg, 0.046 mmol, 1.0 eq.) and compound 59 (42 mg. 0.051 mmol, 1.1 eq.). Preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 30 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. Half of the resulting Boc intermediate was reserved and deprotection of the remaining material was accomplished according to General Procedure 2. Final preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient. to give the titled compound as a white solid (10 mg, 0.0072 mmol, 31%). LC-MS: Calc'd m/z=1162.6 for C58H78N14O12. found [M+H]+=1163.8. 1H NMR (300 MHz, MeOD) δ 7.60-7.07 (m, 12H), 6.81 (s, 2H), 5.14 (s, 2H), 4.99 (s, 2H), 4.62-4.39 (m, 4H), 4.19 (d, J=6.8 Hz, 1H), 4.11 (s, 2H), 3.77 (q, J=5.5 Hz, 1H), 3.70-3.49 (m, 14H), 3.44-3.34 (m, 2H), 3.27-3.09 (m, 4H), 2.60 (t, J=6.1 Hz, 2H), 2.22-2.09 (m, 1H), 1.99-1.93 (m, 1H), 1.85-1.75 (m, 1H), 1.66-1.58 (m, 4H), 1.41-1.28 (m, 4H), 1.03 (dd, J=6.8, 3.3 Hz, 4H), 0.95-0.89 (m, 3H).
To a solution of 2,5-dioxopyrrolidin-1-yl-(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (14 g, 33 mmol, 1.0 eq.) and N6-(tert-butoxycarbonyl)-L-lysine (8.9 g, 36 mmol, 1.1 eq.) in THF (100 mL) was added saturated NaHCO3 solution (43 mL, 49 mmol, 1.5 eq.) and H2O (50 mL) and the resulting mixture stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo, redissolved in 5:1 toluene/iPrOH (300 mL) and heated to reflux for 30 mins then cooled to room temperature. The reaction mixture was then dried over MgSO4, filtered, and concentrated in vacuo to give the crude product as an oily foam solid, which was then suspended in 3:1 EtOAc/hexanes (100 mL). The resulting suspension was sonicated for 20 mins then stirred at room temperature for 18 h, after which the title product 60 was isolated as a solid cake after filtration (12 g, 21 mmol, 63%). LC-MS: Calc'd m/z=567.3 for C31H41N3O7. found [M+H]+=568.4.
A mixture of compound 60 (1.1 g, 2.0 mmol, 1.0 eq.), EEDQ (0.98 g, 3.9 mmol, 2.0 eq.) and P-aminobenzylalcohol (0.34 g, 2.8 mmol, 1.4 eq.) in THF (25 mL) was stirred at room temperature for 18 h in a tinfoil wrapped flask. The reaction mixture was then concentrated in vacuo and normal phase flash purification was accomplished as described in General Procedure 5 using a 50 g silica column, eluting with a 0 to 10% DCM/MeOH gradient to give the title compound 61 as a yellow solid (1.2 g, 1.8 mmol, 90%). LC-MS: Calc'd m/z=672.4 for C38H48N4O7. found [M+H]+=673.5.
Compound 61 (0.080 g, 1.8 mmol, 1.0 eq.) was dissolved in 20% piperidine/DMF (2 mL) and the resulting solution stirred for 30 mins, after which the solvent was removed in vacuo and the residue co-evaporated with DMF (1×3 mL) to give the crude intermediate. A mixture of the crude intermediate and compound 56 (0.080 g, 0.18 mmol, 1.0 eq.) was then dissolved in DMF (1 mL), followed by the addition of DIPEA (93 mL, 0.54 mmol, 3.0 eq.). The resulting solution was stirred at room temperature for 1 h, after which bis(4-nitrophenyl) carbonate (0.11 g, 0.36 mmol, 2.0 eq.) was added. The resulting mixture was stirred at room temperature for an additional 30 mins, after which the reaction was adjusted to pH 1 with 1 M HCl. Reverse phase flash purification was accomplished as described in General Procedure 5 using a 60 g C18 column, eluting with a 10 to 90% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the title compound 62 as a white solid (140 mg, 0.16 mmol, 87%). LC-MS: Calc'd m/z=898.4 for C43H56N6O15. found [M+H-Boc]+=799.5.
The titled compound was prepared according to General Procedure 7 from compound 100 (0.010 g, 0.024 mmol, 1.0 eq.) and compound 62 (24 mg. 0.027 mmol, 1.1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2. Final preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled product as a white solid (11 mg, 0.0075 mmol, 31%). LC-MS: Calc'd m/z=1072.5 for C52H72N12O3. found [M+H]+=1073.7. 1H NMR (300 MHz, MeOD) δ 7.64-7.54 (m, 2H), 7.38-7.29 (m, 2H), 7.17-7.03 (m, 2H), 6.99 (dd, J=7.7, 1.5 Hz, 1H), 6.82 (s, 2H), 5.11 (s, 2H), 5.03 (s, 2H), 4.53 (td, J=8.9, 4.6 Hz, 1H), 4.32 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 4.14-4.03 (m, 1H), 3.92 (s, 3H), 3.85-3.50 (m, 18H), 3.26 (s, 4H), 2.95 (t, J=7.3 Hz, 2H), 2.55 (t, J=6.0 Hz, 2H), 2.16-1.98 (m, 1H), 2.02-1.87 (m, 1H), 1.90-1.76 (m, 1H), 1.73-1.67 (m, 2H), 1.62-1.50 (m, 2H), 1.31 (t, J=7.1 Hz, 3H), 1.00 (dd, J=6.8, 3.6 Hz, 6H).
The titled compound was prepared according to General Procedure 7 from compound 107 (7.0 mg, 0.018 mmol, 1.0 eq.) and compound 62 (17 mg. 0.019 mmol, 1.1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2. Final preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled product as a white solid (3.1 mg, 0.0023 mmol, 13%). LC-MS: Calc'd m/z=1093.5 for C55H71N11O13. found [M+H]+=1094.7. 1H NMR (300 MHz, MeOD) δ 7.54 (s, 2H), 7.27 (s, 7H), 7.17 (s, 2H), 6.98 (s, 1H), 6.81 (s, 2H), 5.15 (s, 2H), 5.01 (s, 2H), 4.43 (t, J=23.4 Hz, 7H), 4.22 (d, J=6.8 Hz, 1H), 3.81-3.52 (m, 17H), 3.05 (t, J=7.3 Hz, 2H), 2.58 (t, J=6.0 Hz, 2H), 2.21-2.08 (m, 1H), 1.98-1.89 (m, 1H), 1.79-1.73 (m, 1H), 1.55-1.49 (m, 2H), 1.45-1.40 (m, 2H), 1.40-1.28 (m, 3H), 1.02 (dd, J=6.7, 4.7 Hz, 6H).
The titled compound was prepared according to General Procedure 7 from compound 108 (7.0 mg, 0.018 mmol, 1.0 eq.) and compound 62 (17 mg. 0.019 mmol, 1.1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2. Final preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 50% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled product as a white solid (3.1 mg, 0.0024 mmol, 13%). LC-MS: Calc'd m/z=1057.5 for C52H71N11O13. found [M+H]+=1058.7. 1H NMR (300 MHz, MeOD) δ 7.62-6.97 (m, 6H), 6.89 (s, 1H), 6.83 (s, 1H), 5.07 (s, 2H), 5.00 (s, 2H), 4.61-4.52 (m, 3H), 4.30 (q, J=7.1 Hz, 2H), 4.18-4.07 (m, 1H), 3.90-3.46 (m, 18H), 2.98 (t, J=7.3 Hz, 2H), 2.58 (t, J=5.9 Hz, 2H), 2.21-1.47 (m, 14H), 1.30 (t, J=7.0 Hz, 3H), 1.03 (dd, J=6.7, 2.6 Hz, 6H).
The titled compound was prepared according to General Procedure 7 from compound 101 (20 mg, 0.049 mmol, 1.0 eq.), compound 62 (48 mg. 0.054 mmol, 1.1 eq.) and HOBt (7 mg, 0.05 mmol, 1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 20 to 65% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2. Final preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled product as a white solid (19.2 mg, 0.015 mmol, 30%). LC-MS: Calc'd m/z=1075.5 for C52H73N11O14. found [M+H]+=1076.7. 1H NMR (300 MHz, MeOD) δ 7.60 (d, J=8.3 Hz, 1H), 7.48 (d, J=8.2 Hz, 2H), 7.37 (d, J=8.2 Hz, 1H), 7.10 (d, J=8.2 Hz, 2H), 6.96 (d, J=8.0 Hz, 1H), 6.83 (s, 2H), 5.09-4.99 (m, 4H), 4.67 (s, 2H), 4.63-4.51 (m, 1H), 4.41-4.28 (m, 3H), 4.18-4.08 (m, 2H), 3.86-3.50 (m, 17H), 2.97 (t, J=7.3 Hz, 2H), 2.58 (t, J=5.9 Hz, 2H), 2.19-2.04 (m, 1H), 2.04-1.90 (m, 1H), 1.93-1.78 (m, 1H), 1.78-1.65 (m, 2H), 1.61-1.55 (m, 2H), 1.31 (t, J=6.9 Hz, 3H), 1.18 (s, 6H), 1.02 (dd, J=6.7, 1.8 Hz, 6H).
The titled compound was prepared according to General Procedure 7 from compound 159 (5.0 mg, 0.011 mmol, 1.0 eq.), compound 62 (9.0 mg. 0.011 mmol, 1.0 eq.) and HOBt (2 mg, 0.01 mmol, 1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2 to give the titled product as a white solid (1.0 mg, 0.00075 mmol, 6.7%). LC-MS: Calc'd m/z=1103.6 for C54H77N11O14. found [M+H]+=1104.8. 1H NMR (300 MHz, MeOD) δ 7.63 (dd, J=20.2, 8.5 Hz, 2H), 7.49-7.31 (m, 2H), 7.10 (d, J=8.1 Hz, 1H), 6.97-6.89 (m, 2H), 6.84 (s, 2H), 5.08 (s, 2H), 5.01 (s, 2H), 4.61-4.55 (m, 2H), 4.24 (d, J=8.8 Hz, 1H), 4.10 (t, J=7.2 Hz, 3H), 3.90-3.49 (m, 17H), 3.02-2.89 (m, 4H), 2.57 (t, J=5.9 Hz, 2H), 1.94-1.50 (m, 7H), 1.46-1.25 (m, 4H), 1.25-1.12 (m, 3H), 1.08-0.84 (m, 12H).
The titled compound was prepared according to General Procedure 7 from compound 161 (7.5 mg, 0.016 mmol, 1.0 eq.), compound 62 (14 mg. 0.016 mmol, 1.0 eq.) and HOBt (2 mg, 0.02 mmol, 1 eq.). Preparative HPLC purification of the Roe intermediate was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O++0.1% TFA gradient, followed by deprotection as described in General Procedure 2 to give the titled product as a white solid (1.2 mg, 0.00082 mmol, 5.6%). LC-MS: Calc'd m/z=1131.6 for C56H81N11O14. found [M+H]+=1132.8.
The titled compound was prepared according to General Procedure 7 from compound 167 (2.8 mg, 0.0070 mmol, 1.0 eq.), compound 62 (6.3 mg. 0.0070 mmol, 1.0 eq.) and HOBt (1 mg, 0.007 mmol, 1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2 to give the titled product as a white solid (3.4 mg, 0.0026 mmol, 38%). LC-MS: Calc'd m/z=1057.5 for C51H71N13O12. found [M+H]+=1058.7. 1H NMR (300 MHz, MeOD) δ 8.19-8.05 (m, 2H), 7.71-7.57 (m, 2H), 7.47-7.28 (m, 3H), 6.84 (s, 2H), 5.14 (s, 2H), 4.98 (s, 2H), 4.55 (dd, J=9.1, 4.2 Hz, 1H), 4.36 (t, J=6.5 Hz, 2H), 4.15-4.06 (m, 1H), 3.88-3.51 (m, 22H), 2.97 (t, J=7.3 Hz, 2H), 2.57 (t, J=5.8 Hz, 2H), 2.26-1.42 (m, 11H), 1.06-0.95 (m, 9H).
The titled compound was prepared according to General Procedure 8 from compound 182 (1.5 mg, 0.0040 mmol, 1.0 eq.) and compound 62 (3.6 mg. 0.0040 mmol, 1.0 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2 to give the titled product as a white solid (2.5 mg, 0.0020 mmol, 50%). LC-MS: Calc'd m/z=1031.5 for C50H69N11O13. found [M+H]+=1032.7. 1H NMR (300 MHz, MeOD) δ 7.57 (d, J=8.2 Hz, 2H), 7.41-7.20 (m, 6H), 6.83 (s, 2H), 5.22-5.12 (m, 1H), 5.05 (s, 2H), 4.99 (s, 2H), 4.57 (s, 1H), 4.27 (s, 2H), 4.11 (t, J=7.2 Hz, 1H), 3.87-3.46 (m, 16H), 2.97 (t, J=7.3 Hz, 2H), 2.57 (t, J=5.9 Hz, 2H), 2.20-1.25 (m, 11H), 1.06-0.88 (m, 9H).
The titled compound was prepared according to General Procedure 8 from compound 169 (7.0 mg, 0.016 mmol, 1.0 eq.) and compound 62 (16 mg. 0.018 mmol, 1.1 eq.). Preparative HPLC purification of the Boc intermediate was accomplished as described in General Procedure 5, eluting with a 10 to 60% CH3CN+0.1% TFA/H2O+0.1% TFA gradient, followed by deprotection as described in General Procedure 2. Final preparative HPLC purification was accomplished as described in General Procedure 5, eluting with a 10 to 55% CH3CN+0.1% TFA/H2O+0.1% TFA gradient to give the titled product as a white solid (2.3 mg, 0.0017 mmol, 11%). LC-MS: Calc'd m/z=1086.55 for C53H74N12O13. found [M+H]+=1087.7. 1H NMR (300 MHz, MeOD) δ 7.61 (d, J=8.3 Hz, 2H), 7.37 (d, J=8.5 Hz, 2H), 6.98 (d, J=8.4 Hz, 1H), 6.83 (s, 2H), 6.61 (d, J=2.1 Hz, 1H), 6.50 (dd, J=8.4, 2.2 Hz, 1H), 5.13 (s, 2H), 4.96 (s, 2H), 4.63-4.51 (m, 1H), 4.32 (t, J=6.5 Hz, 2H), 4.16-4.05 (m, 1H), 3.91-3.51 (m, 21H), 3.20-3.14 (m, 4H), 2.97 (t, J=7.3 Hz, 2H), 2.57 (t, J=5.9 Hz, 2H), 2.19-1.38 (m, 11H), 1.06-0.91 (m, 9H).
The ability of immunostimulatory compounds of Formulae (I)-(IV) herein to agonize human and mouse TLR7 and initiate downstream immunomodulatory activity, e.g., cytokine production, was assessed by reporter gene assays (RGAs) and cytokine release assays from primary immune cells, respectively.
1.5×104 HEK-Blue™ hTLR7 (Invivogen, San Diego, CA) or HEK-Blue™ mTLR7 (Invivogen, San Diego, CA) reporter cells were treated with a titration series of test compound (4-fold titration from 15 pM, 11 points). Compounds were initially diluted to 1 mM in DMSO and subsequently serial-diluted in assay media (HEK-Blue™ Detection; Invivogen, San Diego, CA). Cells with test compound were incubated overnight at 37° C.+5% CO2, and the amount of secreted embryonic alkaline phosphatase (SEAP) was determined by measuring absorbance (620 nm) using a Synergy™ H1 microplate reader. Dose response curves were plotted on PRISM (GraphPad Software, San Diego, CA) and EC50 values were calculated as the concentration of the compound required to produce 50% maximal effect.
Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh Leukopak using magnetic separation. Briefly, erythrocytes and granulocytes were magnetically labeled with Erythrocyte Depletion MicroBeads (Miltenyi Biotec) and Granulocyte Depletion MicroBeads (Miltenyi Biotec), respectively. The cell suspension was loaded onto a Multi-24 Column Block (Miltenyi Biotec) and placed in the magnetic field of a MultiMACS Cell24 Separator Plus (Miltenyi Biotec). The unlabelled PBMCs were collected in the run through. PBMCs were resuspended in CryoStor® CS10 (STEMCELL) and frozen. On the day before the assay, PBMCs were thawed in warm assay buffer (RPMI+10% FBS) and resuspended in assay buffer+10 ng/ml hIL3 (R&D Systems). Cells were rested at 37° C.+5% CO2 overnight. The following day, cells were spun down and resuspended in fresh assay buffer. 300,000 cells were added to each well of plates containing test compound. For test compound preparation, each compound was initially diluted to 1 mM in DMSO and further diluted in assay buffer. A 4-fold 11-point titration starting from 15 μM was performed. Cells were incubated with test compound at 37° C.+5% CO2 for 24 hours before supernatant was harvested. Human interleukin 6 (hIL6), human tumor necrosis factor alpha (hTNFα), and human interferon alpha (hIFNα) were quantified in the supernatant using homogeneous time resolved fluorescence (Cisbio). Dose response curves were plotted on PRISM (GraphPad Software, San Diego, CA) and EC50 values were calculated as the concentration of the compound required to produce 50% maximal effect.
Mouse splenocytes were isolated from fresh mouse spleens. Briefly, spleens were harvested from female balb/c nude mice and transferred to a 70 μm cell strainer. The cell strainer was subsequently placed in a 6-well suspension culture plate containing 5 mL media (RPMI+10% FBS). The spleens were crushed against the cell strainer using the flat end of a 10 mL syringe plunger and the cell strainer was washed with 5 mL media. The cell strainer was discarded, and the cell suspension was washed in PBS. Red blood cells were removed using PharmaLyse (BD). The cell suspension was washed in media, resuspended in CryoStor® CS10 (STEMCELL) and frozen. On assay day, mouse splenocytes were thawed in warm assay buffer (RPMI+10% FBS). Cells were spun down and resuspended in fresh assay buffer. 100,000 cells were added to each well of plates containing test compound. For test compound preparation, each compound was initially diluted to 1 mM in DMSO and further diluted in assay buffer. A 4-fold 11-point titration starting from 15 μM was performed. Cells were incubated with test compound at 37° C.+5% CO2 for 24 hours before supernatant was harvested. Murine IL6 (mIL6) and murine TNFα (mTNFα) were quantified in the supernatant using homogeneous time resolved fluorescence (Cisbio). Dose response curves were plotted on PRISM (GraphPad Software, San Diego, CA) and EC50 values were calculated as the concentration of the compound required to produce 50% maximal effect.
Each assay included a positive control compound (referred to herein as “benchmark,” see, e.g., TABLE 3 below, and taken from International Patent Publication No. WO2017/072662, structure shown below) and a negative control (growth medium). Dose response curves were plotted on PRISM (GraphPad Software, San Diego, CA) and EC50 values were calculated as the concentration of the compound required to produce 50% maximal effect (IL6 induction or NF-κB activation measured by SEAP for PBMC and HEK Blue hTLR7 assays, respectively). The structure of the benchmark compound from International Patent Publication No. WO 2017/072662 is as follows:
The results of the Reporter Gene Assay (RGA, for human (h) and mouse (in) TLR7) experiments as well as the compounds' ability to induce production of cytokines in PBMCs and mouse splenocytes are shown and summarized below in TABLE 3.
1For PBMC IFNα, EC50 values were approximated and divided into the following groups: (i) “−” = >10 nM, (ii) 10 − 1 nM, (iii) 1 − 0.1 nM, or (iv) <0.1 nM.
The results demonstrate that the majority of the tested immunostimulatory compounds have TLR7 agonist activity and/or are capable of inducing cytokine release in these assays. Notably, many of the compounds comprising a substituted (e.g., with methoxy, fluorine, etc.) aryl moiety attached to the N9 atom of the purine moiety, showed double or single digit nanomolar EC50 values for targeting human and/or mouse TLR7, and for inducing cytokine production in human PBMCs and/or mouse splenocytes.
This example describes the preparation of immunostimulatory antibody conjugates comprising an immunostimulatory compound of the present disclosure.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed in the presence of 30 percent v/v propylene glycol with 17.4 equivalents of compound-linker construct (MT-VC-PABC-Compound 105) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (ME2-Compound 100) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of LC-MS data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed in the presence of 30 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 107) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 108) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 101) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 112) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 106) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 111) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 144) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 187) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 188) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 150) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 145) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 152) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 154) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 166) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 155) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 140) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 139) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 110) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 172) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 161) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 182) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 1.1 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 159) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 1.1 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 167) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 1.1 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 180) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 1.1 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v propylene glycol with 20 equivalents of compound-linker construct (MT-VK-PABC-Compound 169) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 141) with an incubation time of 120 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 142) with an incubation time of 120 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 143) with an incubation time of 120 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 15 equivalents of compound-linker construct (MT-VC-PABC-Compound 191) with an incubation time of 120 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 132) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 1.1 equivalents of TCEP. The conjugation was performed in the presence of 10 percent v/v % propylene glycol with 20 equivalents of compound-linker construct (MT-VC-PABC-Compound 173) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 15 equivalents of compound-linker construct (MT-VC-PABC-Compound 100) with an incubation time of 130 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.15 equivalents of TCEP. The conjugation was performed with 15 equivalents of compound-linker construct (MT-VK-PABC-Compound 100) with an incubation time of 150 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 19.4 equivalents of compound-linker construct (MC-Compound 100) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
This conjugate was obtained following the General Procedure 9, using Trastuzumab as the antibody reduced with 2.2 equivalents of TCEP. The conjugation was performed with 19.4 equivalents of compound-linker construct (MT-Compound 100) with an incubation time of 180 min. The average DAR and drug distribution was derived from interpretation of HPLC-HIC data and is shown in TABLE 4 herein.
10 μL of conjugated ISAC samples at 1 mg/ml were deglycosylated using EndoS (1 μg) after a 1 h incubation at room temperature. 2 μL of 500 mM TCEP (in water) was added to 11 μl sample and incubated at 70° C. for 30 min for reduction and denaturation. Samples were then run on an LC-MS-QTOF using a 1 μl injection each, following the analytical method described below:
MS data analysis was performed as described below. For cysteine conjugates, integrated TIC at two regions (Light Chain (LC) region between 4 to 8 mins, Heavy Chain (HC) region between 8 to 13 mins). Deconvolution Parameters:
The average degree of conjugation of compound-linker construct to antibody (DAR) was assessed by hydrophobic interaction chromatography (HIC). These techniques are described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, vol. 1045, pp. 275-284. L. Ducry, Ed. Typically, the measured DAR values ranged from 3.6 to 4.2. Briefly, ISACs were subjected to HIC on a TSKgel® Butyl-NPR column (Tosoh Bioscience; 4.6 mm×3 mm i.d.; 2.5-micron particle size) connected to an Agilent 1100 series HPLC. Samples were injected (5 μL) at or above 2 mg/mL. A linear gradient elution was employed starting at 95% mobile phase A/5% mobile phase B, transitioning to 5% mobile phase A/95% mobile phase B over a period of 12 min (mobile phase A: 1.5 M ammonium sulfate+25 mM sodium phosphate, pH 6.95; mobile phase B: 25% isopropanol, 75% 25 mM sodium phosphate, pH 6.95). Injection of unmodified antibody provided a means of identifying the peak with DAR=0. Antibodies were detected on the basis of absorbance at 280 nm.
Samples were run on an Agilent 1290 UHPLC system equipped with a quaternary pump and a DAD detector, using a 5 μl injection volume for each sample following the analytical method described below:
This example demonstrates the ability of the tested ISACs produced as described in EXAMPLE 4 to induce the production of human IL6 (hIL6) from PBMCs and/or murine IL6 (mIL6) from mouse splenocytes, and/or to agonize TLR7 as determined by secreted embryonic alkaline phosphatase (SEAP) from RAW-Blue™ cells as part of a reporter gene assay.
Human PBMCs and mouse splenocytes were isolated as described above in EXAMPLE 3. On assay day, human PBMCs or mouse splenocytes were thawed in warm assay media (RPMI+10% FBS). Cells were spun down and resuspended in fresh assay buffer. 100,000 cells were added to each well of plates containing an ISAC. For ISAC preparation, a 5-fold 7-point titration from 100 nM in assay media was performed. For tumor cell preparation, NCI_N87 tumor cells were propagated in tissue culture plates until they reach 90% confluency and detached from tissue culture plates with TrypLE™ Express Enzyme (ThermoFisher). 10,000 tumor cells were added to wells containing antibody drug conjugate with human PBMCs or with mouse splenocytes. The co-culture was incubated with ISAC at 37° C.+5% CO2 for 24 hours before supernatant was harvested. hIL6 and mIL6 were quantified from the human PBMC+NCI_N87 co-culture supernatant and mouse splenocyte+NCI_N87 co-culture supernatant, respectively, using homogeneous time resolved fluorescence (Cisbio). Dose response curves were plotted on PRISM (GraphPad Software, San Diego, CA) and EC50 values were calculated as the concentration of the compound required to produce 50% maximal effect.
The in vitro activity of ISACs was also assessed in a reporter gene assay using mouse RAW-Blue™ cells (Invivogen, San Diego, CA). Briefly, RAW-Blue™ cells were maintained in growth media recommended by the vendor until assay day. On assay day, RAW-Blue™ cells were scraped from tissue culture flasks and resuspended in assay media (DMEM+10% FBS). 100,000 RAW-Blue™ cells were added to each well of plates containing an ISAC. For ISAC preparation, a 5-fold 7-point titration from 100 nM in assay media was performed. For tumor cell preparation, NCI_N87 tumor cells were propagated in tissue culture plates until they reach 90% confluency and detached from tissue culture plates with TrypLE™ Express Enzyme (ThermoFisher). 100,000 tumor cells were added to wells containing ISAC with RAW-Blue™ cells. The co-culture was incubated with ISAC at 37° C.+5% CO2 for 48 hours before 10 μl supernatant was harvested. To the supernatant, 90 ul QUANTI-Blue™ Solution (Invivogen, San Diego, CA) was added and the amount of secreted embryonic alkaline phosphatase (SEAP) was determined by measuring absorbance (620 nm) using a Synergy™ H1 microplate reader. Dose response curves were plotted on PRISM (GraphPad Software, San Diego, CA) and EC50 values were calculated as the concentration of the compound required to produce 50% maximal effect.
Trastuzumab coupled to the benchmark compound (e.g., without the cysteine-capped hemisuccinimide moiety, which can be a result of in vivo metabolism and constitute an active metabolite upon cleavage from the targeting moiety) was used as a positive control in this study. The following “activated” version of the benchmark was used for coupling to Trastuzumab:
The EC50 values that were determined for tested ISACs to induce the production of (i) hIL6 from PBMCs, (ii) mIL6 from mouse splenocytes, and (iii) secreted embryonic alkaline phosphatase (SEAP) from RAW-Blue™ cells is shown in TABLE 5 (Trastuzumab is abbreviated with “Tras”).
These data show that certain compounds that showed strong immunomodulatory activity as a free molecule (i.e., not in form of a conjugate), such as compounds 100, 101, 105, 106, 132, 139, 140, 154, 155 and 182, also demonstrated strong immunomodulatory activity when tested in an ISAC format in these assays. Some of the tested conjugates, e.g., Tras-ME2-Compound 100, Tras-MT-VK-PABC-Compound 101, Tras-MT-VC-PABC-Compound 106, outperformed the benchmark conjugate.
This example demonstrates the ability selected ISACs produced as described in EXAMPLE 4 to provide anti-tumor activity in an NCI-N87 HER2high xenograft model of gastric cancer. Based on the results obtained and described in EXAMPLE 5, 12 different ISACs were selected and further tested in vivo.
Tumor cell suspensions (107 cells in a 1:1 mix of PBS and matrigel) were implanted subcutaneously into balb/c nude mice. When mean tumor volume reached ˜160 mm3 the animals were randomly assigned to groups (n=6 per group) and treated as shown in TABLE 6 (Trastuzumab is abbreviated with “Tras”). Briefly, ISACs and antibody control were intravenously (iv) administered once on the study start date (day 0) using a dose of 2.5 mg/kg. Tumor volume and body weight were measured twice weekly with a study duration of 30 days.
The results for in vivo anti-tumor activity and weight loss are shown in
Notably, the conjugates Tras-ME2-Compound 100 and Tras-MT-VK-PABC-Compound 101 each resulted in at least 50% (i.e., 3/6) complete responses, which significantly outperformed the benchmark ISAC (i.e., 0/6) complete responses, and lead to a significant reduction in tumor size of at least about 50%.
In addition, the benchmark control ISAC, Trastuzumab-benchmark compound, resulted in transient mean body weight loss of between 5-10% at 3 days post dose, which fully recovered by 7 days post dose. All other tested ISACs resulted in minor or no body weight loss indicating that they were well tolerated at the tested dose.
This example shows results of a tolerability study performed in vivo using selected ISACs described herein to more specifically determine the in vivo applicability of the compounds and conjugates disclosed herein, as well as to determine the in vivo tolerability of ISACs described herein relative to previously disclosed benchmark compounds.
Hence, tolerability of ISACs was assessed following a single iv injection of a solution containing the respective ISAC to balb/c mice (n=5) at the beginning of the study (study day 0) at doses outlined in TABLE 7. Body weights were recorded daily, where possible, until study termination at day 16 post dose for doses 3 mg/kg and 15 mg/kg, and day 11 post dose for doses of 45 mg/kg. Animals were sacrificed if body weight loss exceeded 20% from baseline. Vehicle and Trastuzumab at 45 mg/kg were administered as controls and were both well tolerated.
The changes in body weight (shown as %-change relative to baseline) over the course of this study and following administration of all tested ISACs at various dose levels are shown in
The data demonstrate that while the benchmark conjugate was well tolerated at 3 mg/kg, it was not tolerated at 15 mg/kg with all animals being terminated at day 7.
In contrast, Tras-MT-VK-PABC-Compound 101 was well tolerated at all dose levels tested, with only minor transient body weight loss (BWL) observed and survival of all mice at doses as high as 45 mg/kg, which was a significant improvement when compared to the benchmark conjugate.
The conjugate Tras-ME2-Compound 100, which comprised a non-cleavable linker, was not tolerated at any dose tested. At 3 mg/kg, 2/5 mice were terminated by Day 11. Remaining mice showed minor to strong BWL that recovered by 2 weeks post dose. At 15 mg/kg, animals showed 10-14% BWL at 2 days post dose that partially recovered before further BWL, but no survival was observed beyond study day 7. At 45 mg/kg, rapid BWL resulted in lethality, with 4/5 animals not surviving past day 3 of the study. It should be noted, however, that this conjugate was well tolerated at 2.5 mg/kg, which was a dose level that was effective in reducing tumor volume (see Example 6).
Doses of the ISAC Tras-MT-VC-PABC-Compound 154 of up to 45 mg/kg did not result in any loss of animals from the study. At doses of 3 mg/kg, minor BWL was observed at 3 days post dose that fully recovered. At doses of 15 mg/kg, rapid BWL loss of 5-10% was observed in all animals at day 2 that rapidly recovered by day 3. A secondary moderate reduction in BW was observed in most animals at day 6 that rapidly recovered. At 45 mg/kg, rapid BWL was observed at 1 day post dose of between 12-14% that partially recovered by˜day 5, before a secondary minor BWL at day 6-7 was observed, followed by recovery and survival of all animals on study.
The ISAC Tras-MT-VC-PABC-Compound 155 resulted in only minor BWL in most animals at doses of 3 mg/kg between day 1-3 post dose, followed by recovery. At doses of 15 mg/kg, rapid BWL of between 3-7% was observed at 1 day post dose, followed by rapid recovery and a secondary BWL at day 7 of between 14-17% with rapid recovery of 4/5 animals and loss of 1 animal.
Finally, the ISAC Tras-MT-VK-PABC-Compound 106 resulted in moderate BWL of between 5-8% at doses of 3 mg/kg at day 1 post dose, followed by recovery. At doses of 15 mg/kg, rapid BWL was observed at 1 day post dose of between 11-23%, resulting in loss of 1/5 animals from study, while 4/5 animals recovered. At 45 mg/kg, rapid BWL of between 14-20% resulted in 2/5 animals not surviving past day 2, with recovery of the remaining animals.
This is a Continuation of PCT/CA2023/051006, filed Jul. 26, 2023, which claims priority to U.S. Provisional Patent Application No. 63/392,329, filed Jul. 26, 2022, both of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63392329 | Jul 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CA2023/051006 | Jul 2023 | WO |
| Child | 19033344 | US |
