Compounds and uses thereof

Abstract

The present disclosure features compounds and methods useful for treating BAF complex-associated disorders.

Description

[0001] background

[0002] This invention relates to compounds that can be used to modulate the BRG1- or BRM-related factor (BAF) complex. Specifically, this invention relates to compounds that can be used to treat disorders associated with BAF complex function.

[0003] Chromatin regulation is crucial for gene expression, and ATP-dependent chromatin remodeling is the mechanism by which such gene expression occurs. The human switch / sucrose non-fermentable (SWI / SNF) chromatin remodeling complex (also known as the BAF complex) contains two SWI2-like ATPases called BRG1 (Brahma-associated gene-1) and BRM (Brahma). The transcriptional activator BRG1 (also known as the ATP-dependent chromatin remodeling factor SMARCA4) is encoded by the SMARCA4 gene on chromosome 19. BRG1 is overexpressed in some cancer tumors and is required for cancer cell proliferation. BRM (also known as the potential global transcriptional activator SNF2L2 and / or the ATP-dependent chromatin remodeling factor SMARCA2) is encoded by the SMARCA2 gene on chromosome 9 and has been shown to be essential for tumor cell growth in cells characterized by loss-of-function mutations in BRG1. Inactivation of BRG and / or BRM leads to downstream effects in cells, including cell cycle arrest and tumor suppression. Summary of the Invention

[0004] This invention characterizes compounds that can be used to modulate the BAF complex. In some embodiments, said compounds can be used to treat disorders associated with alterations to the BAF complex, such as disorders associated with alterations to one or both of the BRG1 and BRM proteins. The compounds of this invention, alone or in combination with other pharmaceutically active agents, can be used to treat such disorders.

[0005] In one aspect, the present invention provides compounds having the structure of Formula I or pharmaceutically acceptable salts thereof:

[0006]

[0007] Formula I,

[0008] in

[0009] X is a halogen (e.g., F, Cl, Br, or I);

[0010] X 1 It does not exist or is O or NR. 1 ;

[0011] k is 0, 1, 2, or 3;

[0012] n is 0, 1, or 2;

[0013] R 1 It is H or an optional substituted C1-C6 alkyl group;

[0014] L 1 It is a C1-C6 alkylene group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group that is optionally substituted;

[0015] L 2 The absence or optional substitution of C1-C6 alkylene groups, and the optional substitution of C1-C6 alkylene groups. 20 Heteroalkyl or optionally substituted C2-C9 heterocyclic groups;

[0016] Each L 3 C1-C is independently and optionally replaced 20 Heteroalkyl groups, optionally substituted C3-C 10 Carbocyclic group, optionally substituted C3-C 10 Carbocyclic-C1-C6 alkylene, optionally substituted C2-C9 heterocyclic, optionally substituted C2-C9 heterocyclic-C1-C6 alkylene, optionally substituted C6-C 10 Aromatic, optionally substituted C6-C 10 arylene-C1-C6 alkylene, optionally substituted C2-C6 ynylene, O or NR 1 ;and

[0017] D represents the degradation portion.

[0018] In some implementations, k is 0. In some implementations, k is 1.

[0019] In some implementations, X 1 It does not exist. In some implementations, X 1 Is it O or NR? 1 .

[0020] In some implementations, R 1 It is an optionally substituted C1-C6 alkyl group. In some embodiments, R 1 It is methyl. In some embodiments, R 1 It is H.

[0021] In some implementations, L 1 It is a C1-C6 alkylene group that is optionally substituted.

[0022] In some implementations, L 1 yes

[0023]

[0024] In some implementations, L 2 It does not exist.

[0025] In some implementations, L 2 Exists. In some implementations, L 2 It is a C1-C6 alkylene group that is optionally substituted.

[0026] In some implementations, L 2 yes

[0027]

[0028] In some implementations, L 2 C1-C is arbitrarily replaced 20 Heteroalkyl groups.

[0029] In some implementations, L 2 yes

[0030]

[0031] In some implementations, n is 1. In some implementations, n is 2.

[0032] In some implementations, at least one L 3 C3-C is arbitrarily replaced. 10 Carbocyclic-C1-C6 alkylene, optionally substituted C2-C9 heterocyclic, optionally substituted C2-C9 heterocyclic-C1-C6 alkylene, optionally substituted C1-C 20 Heteroalkyl, O or NR 1 In some implementations, at least one L 3 C1-C is arbitrarily replaced 20 Heteroalkyl groups.

[0033] In some implementations, at least one L 3 yes

[0034] Phenylidene, O or NR 1 .

[0035] In some implementations, n is 0.

[0036] In some embodiments, the degradation moiety is a ubiquitin ligase-binding moiety. In some embodiments, the ubiquitin ligase-binding moiety comprises a Cereblon ligand, an IAP (inhibitor of apoptosis) ligand, a mouse dual microsome 2 homologue (MDM2), or a von Hippel-Lindau ligand.

[0037] In some embodiments, the degradation portion has the structure of formula A:

[0038]

[0039] in

[0040] Y 1 yes

[0041] R A5 It is H, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl;

[0042] R A6 It is H or optionally substituted C1-C6 alkyl; and R A7 It is H or optionally substituted C1-C6 alkyl; or R A6 and R A7 Combined with their respective bonded carbon atoms to form optionally substituted C3-C6 carbocyclic groups or optionally substituted C2-C5 heterocyclic groups; or R A6 and R A7 They combine with their respective carbon atoms to form optionally substituted C3-C6 carbocyclic groups or optionally substituted C2-C5 heterocyclic groups;

[0043] R A8 It is H, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl;

[0044] R A1 R A2 R A3 and R A4 Each of them is independently H, A 2 Halogens, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 heteroalkyl groups, optionally substituted C3-C6 alkyl groups 10 Carbocyclic group, optionally substituted C2-C9 heterocyclic group, optionally substituted C6-C 10 aryl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted -O-C3-C6 carbocyclic, hydroxyl, mercapto, or optionally substituted amino; or R A1 and RA2 R A2 and R A3 , and / or R A3 and R A4 They combine together with the carbon atoms they are attached to to form and C6-C is arbitrarily replaced. 10 aryl, optionally substituted C3-C 10 A carbocyclic group, optionally substituted C2-C9 heteroaryl or C2-C9 heterocyclic group, any one of which is optionally A 2 replace,

[0045] Where R A1 R A2 R A3 and R A4 One of them is A 2 ,or A 2 Replace; and

[0046] A 2 It is the bond between the degradation portion and the linker.

[0047] In some implementations, R A5 It is H or methyl. In some embodiments, R A5 It is H.

[0048] In some implementations, R A1 R A2 R A3 and R A4 Each of them is independently H or A 2 In some implementations, R A1 It is A 2 And R A2 R A3 and R A4 Each of them is H. In some implementations, R A2 It is A 2 And R A1 R A3 and R A4 Each of them is H. In some implementations, R A3 It is A 2 And R A1 R A2 and R A4 Each of them is H. In some implementations, R A4 It is A 2 And R A1 R A2 and R A3 Each of them is H.

[0049] In some implementations, Y 1 yes

[0050]

[0051] In some implementations, R A6 It is H.

[0052] In some implementations, R A7 It is H.

[0053] In some implementations, Y 1 yes

[0054]

[0055] In some implementations, R A8 It is H or an optionally substituted C1-C6 alkyl group. In some embodiments, R A8 It is H or methyl. In some embodiments, R A8 It is a methyl group.

[0056] In some embodiments, the degradation portion has the structure of formula A2:

[0057]

[0058] In some embodiments, the degradation portion has the structure of formula A4:

[0059]

[0060] In some embodiments, the degradation portion has the structure of formula A5:

[0061]

[0062] In some embodiments, the degradation portion has the structure of formula A6:

[0063]

[0064] In some embodiments, the degradation portion has the structure of formula A8:

[0065]

[0066] In some embodiments, the degradation portion has a structure of formula A10:

[0067]

[0068] In some embodiments, the degradation portion has the following structure:

[0069]

[0070] In some embodiments, the degradation portion has the following structure:

[0071]

[0072] In some implementations, a portion has the structure of formula C:

[0073]

[0074] in

[0075] L 4 It is -N(R) B1 (R) B2 ),

[0076] R B1 It is H, A 2 Optionally substituted C1-C6 alkyl or optionally substituted C1-C6 heteroalkyl;

[0077] R B2 It is H, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl;

[0078] R B3 It is A 2 Optionally substituted C1-C6 alkyl groups, Optionally substituted C1-C6 heteroalkyl groups, Optionally substituted C3-C 10 Carbocyclic group, optionally substituted C6-C 10 aryl, optionally substituted C1-C6 alkyl, C3-C 10 Carbocyclic group or optionally substituted C1-C6 alkyl group C6-C 10 Aryl;

[0079] R B4 H, optionally substituted C1-C6 alkyl, optionally substituted C3-C 10 Carbocyclic group, optionally substituted C6-C 10 aryl, optionally substituted C1-C6 alkyl, C3-C 10 Carbocyclic group or optionally substituted C1-C6 alkyl group C6-C 10 Aryl;

[0080] R B5 It is H, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 heteroalkyl;

[0081] v2 is 0, 1, 2, 3, or 4;

[0082] Each RB6 A is independent 2 Halogens, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 heteroalkyl groups, optionally substituted C3-C6 alkyl groups 10 Carbocyclic group, optionally substituted C2-C9 heterocyclic group, optionally substituted C6-C 10 aryl, optionally substituted C2-C9 heteroaryl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, hydroxyl, mercapto or optionally substituted amino;

[0083] R B7 and R B8 Each of these components is independently H, a halogen, an optionally substituted C1-C6 alkyl group, or an optionally substituted C6-C6 alkyl group. 10 Aryl;

[0084] R B9 It is H or optionally substituted C1-C6 alkyl; and

[0085] A 2 It is the bond between the degradation portion and the linker;

[0086] Where R B1 R B3 and R B6 One and only one of them is A 2 .

[0087] In some embodiments, the degradation portion has the structure of formula C1:

[0088]

[0089] In some embodiments, the degradation portion has the structure of formula C2:

[0090]

[0091] In some implementations, R B9 It is an optionally substituted C1-C6 alkyl group. In some embodiments, R B9 It is methyl. In some embodiments, R B9 Bonded to (S)-stereocenter. In some implementations, R B9 It is hydrogen.

[0092] In some embodiments, the degradation portion has the following structure:

[0093]

[0094] In some embodiments, the degradation portion has the following structure:

[0095]

[0096] In some embodiments, the degradation portion has the following structure:

[0097]

[0098] In some embodiments, the degradation portion has the following structure:

[0099]

[0100] In some embodiments, the degradation portion is

[0101]

[0102] In some embodiments, the degradation portion comprises the following structure

[0103] Where A 2 It is the bond between the degradation portion and the linker.

[0104] In some implementations, k is 0.

[0105] In some embodiments, the compound is selected from compounds 1 to 105 in Table 1 and their pharmaceutically acceptable salts.

[0106] Table 1. Compounds of the present invention

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] In some embodiments, the compound has at least 5 BRG1 ICs. 50 BRM IC 50 The ratio. In some embodiments, the compound has at least 7 BRG1 ICs. 50 BRM IC 50 The ratio. In some embodiments, the compound has at least 10 BRG1 IC. 50 BRM IC 50 The ratio. In some embodiments, the compound has a BRG1 IC ratio of at least 15. 50 BRM IC 50 The ratio. In some embodiments, the compound has at least 20 BRG1 IC. 50 BRM IC 50 The ratio. In some embodiments, the compound has a BRG1 IC ratio of at least 25. 50 BRM IC 50 The ratio. In some embodiments, the compound has a BRG1 IC ratio of at least 30. 50 BRM IC 50 The ratio.

[0119] In one aspect, the present invention characterizes a pharmaceutical composition comprising any of the aforementioned compounds and a pharmaceutically acceptable excipient.

[0120] In another aspect, the present invention characterizes a method for reducing the activity of the BAF complex in cells, the method comprising contacting the cells with an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0121] In some implementations, the cells are cancer cells.

[0122] In another aspect, the present invention characterizes a method for treating BAF complex-related disorders in subjects with such need, the method comprising administering to the subject an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutical composition thereof.

[0123] In some implementations, the BAF complex-related barrier is cancer.

[0124] In another aspect, the present invention characterizes a method for inhibiting BRM, the method comprising contacting cells with an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutical composition thereof.

[0125] In some implementations, the cells are cancer cells.

[0126] In another aspect, the present invention characterizes a method for inhibiting BRG1, the method comprising contacting the cells with an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0127] In some implementations, the cells are cancer cells.

[0128] In another aspect, the present invention characterizes a method for inhibiting BRM and BRG1, the method comprising contacting the cells with an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0129] In some implementations, the cells are cancer cells.

[0130] In another aspect, the present invention characterizes a method for treating impairments associated with BRG1 loss-of-function mutations in subjects in need, the method comprising administering to the subject an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutical composition thereof.

[0131] In some embodiments, the barrier associated with the BRG1 loss-of-function mutation is cancer. In other embodiments, the object is identified as having a BRG1 loss-of-function barrier, for example, being identified as having BRG1 loss-of-function cancer (e.g., the cancer has been identified as including cancer cells with BRG1 loss-of-function).

[0132] In another aspect, the present invention characterizes a method for inducing apoptosis in cells, the method comprising contacting the cells with an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutical composition thereof.

[0133] In some implementations, the cells are cancer cells.

[0134] In another aspect, the present invention characterizes a method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutical composition thereof.

[0135] In some embodiments of any of the foregoing methods, the cancer is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary origin, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, esophageal-gastric cancer, pancreatic cancer, hepatobiliary duct cancer, soft tissue sarcoma, ovarian cancer, head and neck cancer, renal cell carcinoma, bone cancer, non-Hodgkin lymphoma, small cell lung cancer, prostate cancer, embryonal tumor, germ cell tumor, cervical cancer, thyroid cancer, salivary gland cancer, gastrointestinal neuroendocrine tumor, uterine sarcoma, gastrointestinal stromal tumor, central nervous system cancer, thymic tumor, adrenocortical carcinoma, appendix cancer, small bowel cancer, or penile cancer.

[0136] In some embodiments of any of the foregoing methods, the cancer is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary origin, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, or penile cancer.

[0137] In some embodiments of any of the foregoing methods, the cancer is drug-resistant or has been resistant to prior therapies (e.g., vemurafenib, dacarbazine, CTLA4 inhibitors, PD1 inhibitors, interferon therapy, BRAF inhibitors, MEK inhibitors, radiotherapy, temozolomide, irinotecan, CAR-T therapy, etc.). Tamoxifen, Docetaxel, platinum-based drugs such as carboplatin, taxanes such as paclitaxel and docetaxel, ALK inhibitors, MET inhibitors, Giscitabine, Neratinib, PARP inhibitors, ARN810, mTOR inhibitors, topotecan, (VEGFR2 inhibitors, folate receptor antagonists, dencizumab, fortabulin, or PDL1 inhibitors) did not respond.

[0138] In some embodiments of any of the foregoing methods, the cancer has or has been identified as having a BRG1 mutation. In some embodiments of any of the foregoing methods, the BRG1 mutation is homozygous. In some embodiments of any of the foregoing methods, the cancer does not have or has been identified as not having an epidermal growth factor receptor (EGFR) mutation. In some embodiments of any of the foregoing methods, the cancer does not have or has been identified as not having an anaplastic lymphoma kinase (ALK) driver mutation. In some embodiments of any of the foregoing methods, the cancer has or has been identified as having a KRAS mutation. In some embodiments of any of the foregoing methods, the BRG1 mutation is in the ATPase catalytic domain of the protein. In some embodiments of any of the foregoing methods, the BRG1 mutation is a deletion at the C-terminus of BRG1.

[0139] In another aspect, this disclosure provides a method for treating BAF-related disorders (e.g., cancer or viral infection) in subjects with this need. The method comprises contacting cells with an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutically acceptable salt thereof, or any of the aforementioned pharmaceutical compositions. In some embodiments, the barrier is a viral infection, specifically a retroviral family such as lentiviruses (e.g., human immunodeficiency virus (HIV) and delta retroviruses (e.g., human T-cell leukemia virus I (HTLV-I), human T-cell leukemia virus II (HTLV-II)), hepatoviridae (e.g., hepatitis B virus (HBV)), flaviviridae (e.g., hepatitis C virus (HCV)), adenoviridae (e.g., human adenovirus), herpesviruses (e.g., human cytomegalovirus (HCMV), Epstein-Barr virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), herpesvirus K*, CMV, varicella-zoster virus), and papillomaviruses (e.g., human papillomavirus (HPV, HPV...). Infection with viruses belonging to the family Parvoviridae (e.g., parvovirus B19), Polyomaviridae (e.g., JC virus and BK virus), Paramyxoviridae (e.g., measles virus), and Clonorchiidae (e.g., rubella virus). In some embodiments, the barrier is CoffinSiris, neurofibromatosis (e.g., NF-1, NF-2, or schwannomatosis), or multiple meningiomas.

[0140] In another aspect, this disclosure provides a method for treating a viral infection in a subject in need. The method comprises administering to the subject an effective amount of any of the aforementioned compounds (e.g., a BRM / BRG1 dual inhibitor compound or a BRM-selective compound) or a pharmaceutically acceptable salt thereof, or any of the aforementioned pharmaceutical compositions. In some embodiments, the viral infection is a retroviral virus such as lentiviruses (e.g., human immunodeficiency virus (HIV) and delta retroviruses (e.g., human T-cell leukemia virus I (HTLV-I), human T-cell leukemia virus II (HTLV-II)), hepatoviridae (e.g., hepatitis B virus (HBV)), flaviviridae (e.g., hepatitis C virus (HCV)), adenoviridae (e.g., human adenovirus), herpesviruses (e.g., human cytomegalovirus (HCMV), Epstein-Barr virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpesvirus 6 (HHV-6), herpesvirus K*, CMV, varicella-zoster virus), or papillomaviruses (e.g., human papillomavirus (HPV, HPV...). Infection with viruses belonging to the family E1), parvoviridae (e.g., parvovirus B19), polyomaviridae (e.g., JC virus and BK virus), paramyxoviridae (e.g., measles virus), or cloacalviridae (e.g., rubella virus).

[0141] In some embodiments of any of the foregoing aspects, the compound is a BRM-selective compound. In some embodiments, the BRM-selective compound inhibits the level and / or activity of BRM by at least 10 times more than it inhibits the level and / or activity of BRG1, and / or the binding of the compound to BRM is at least 10 times more than its binding to BRG1. For example, in some embodiments, the IC50 of the BRM-selective compound is... 50 or IP 50 IC for BRG1 50 or IP s0 At most 1 / 10. In some embodiments of any of the foregoing aspects, the compound is a BRM / BRG1 dual inhibitor compound. In some embodiments, the BRM / BRG1 dual inhibitor compound has similar activity against both BRM and BRG1 (e.g., the compound's activity against BRM and BRG1 is within 10-fold (e.g., less than 5-fold, less than 2-fold). In some embodiments, the BRM / BRG1 dual inhibitor compound has greater activity against BRM. In some embodiments, the BRM / BRG1 dual inhibitor compound has greater activity against BRG1. For example, in some embodiments, the BRM / BRG1 dual inhibitor compound has an IC50 value of 1 / 10 for BRM. 50 or IP50 It is for BRG1 IC 50 or IP 50 Within 10 times.

[0142] In another aspect, the present invention characterizes a method for treating melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cancer in a subject with such need, the method comprising administering to the subject an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0143] In another aspect, the present invention characterizes a method for reducing tumor growth in subjects with a need for melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cancer, the method comprising administering to the subject an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0144] In another aspect, the present invention characterizes a method for inhibiting metastatic progression of melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cancer in subjects, the method comprising administering an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0145] In another aspect, the present invention characterizes a method for inhibiting metastatic colonization of melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cancer in a subject, the method comprising administering an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0146] In another aspect, the present invention characterizes a method for reducing the level and / or activity of BRG1 and / or BRM in melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cancer cells, the method comprising contacting the cells with an effective amount of any of the aforementioned compounds or a pharmaceutical composition thereof.

[0147] In some embodiments of any of the above aspects, the melanoma, prostate cancer, breast cancer, bone cancer, renal cell carcinoma, or hematologic cell is in the object.

[0148] In some embodiments of any of the foregoing aspects, an effective amount of the compound reduces the level and / or activity of BRG1 by at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference. In some embodiments, an effective amount of the compound reduces the level and / or activity of BRG1 by at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference. In some embodiments, an effective amount of the compound reduces the level and / or activity of BRG1 by at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0149] In some embodiments, an effective amount of the compound reduces the level and / or activity of BRG1 by at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference for at least 12 hours (e.g., 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 72 hours, or longer). In some embodiments, an effective amount of the compound reduces the level and / or activity of BRG1 by at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference for at least 4 days (e.g., 5 days, 6 days, 7 days, 14 days, 28 days, or longer).

[0150] In some embodiments of any of the foregoing aspects, the effective amount of the compound reduces the level and / or activity of BRM by at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference. In some embodiments, the effective amount of the compound reduces the level and / or activity of BRM by at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference. In some embodiments, the effective amount of the compound reduces the level and / or activity of BRM by at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0151] In some embodiments, an effective amount of the compound reduces the level and / or activity of BRM by at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference for at least 12 hours (e.g., 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 72 hours, or longer). In some embodiments, an effective amount of the compound reduces the level and / or activity of BRM by at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) compared to a reference for at least 4 days (e.g., 5 days, 6 days, 7 days, 14 days, 28 days, or longer).

[0152] In some embodiments, the object has cancer. In some embodiments, the cancer expresses BRG1 and / or BRM proteins, and / or the cells or object have been identified as expressing BRG1 and / or BRM. In some embodiments, the cancer expresses BRG1 protein, and / or the cells or object have been identified as expressing BRG1. In some embodiments, the cancer expresses BRM protein, and / or the cells or object have been identified as expressing BRM. In some embodiments, the cancer is melanoma (e.g., uveal melanoma, mucosal melanoma, or cutaneous melanoma). In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is a hematologic cancer, such as multiple myeloma, large cell lymphoma, acute T-cell leukemia, acute myeloid leukemia, myelodysplastic syndrome, immunoglobulin Aλ myeloma, diffuse mixed histiocytic and lymphocytic lymphoma, B-cell lymphoma, acute lymphoblastic leukemia (e.g., T-cell acute lymphoblastic leukemia or B-cell acute lymphoblastic leukemia), diffuse large cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the cancer is breast cancer (e.g., ER-positive breast cancer, ER-negative breast cancer, triple-positive breast cancer, or triple-negative breast cancer). In some embodiments, the cancer is bone cancer (e.g., Ewing sarcoma). In some embodiments, the cancer is renal cell carcinoma (e.g., microphthalmia transcription factor (MITF) family translocation renal cell carcinoma (tRCC)). In some embodiments, the cancer is metastatic (e.g., the cancer has spread to the liver). The metastatic cancer may include cells exhibiting migration and / or invasion of migratory cells, and / or cells exhibiting endothelial recruitment and / or angiogenesis. In other embodiments, the metastatic cancer is a cell-migrating cancer. In still other embodiments, the cell-migrating cancer is a non-metastatic cell-migrating cancer. The metastatic cancer may be cancer that has spread by seeding on the surface of the peritoneum, pleura, pericardium, or subarachnoid space. Alternatively, the metastatic cancer may be cancer that has spread via the lymphatic system or cancer that has spread via the bloodstream. In some embodiments, the effective amount of an agent that reduces the level and / or activity of BRG1 and / or BRM is an amount that effectively inhibits the metastatic colonization of cancer to the liver.

[0153] In some embodiments, the cancer contains a mutation in GNAQ. In some embodiments, the cancer contains a mutation in GNA11. In some embodiments, the cancer contains a mutation in PLCB4. In some embodiments, the cancer contains a mutation in CYSLTR2. In some embodiments, the cancer contains a mutation in BAP1. In some embodiments, the cancer contains a mutation in SF3B1. In some embodiments, the cancer contains a mutation in EIF1AX. In some embodiments, the cancer contains a TFE3 translocation. In some embodiments, the cancer contains a TFEB translocation. In some embodiments, the cancer contains a MITF translocation. In some embodiments, the cancer contains an EZH2 mutation. In some embodiments, the cancer contains a SUZ12 mutation. In some embodiments, the cancer contains an EED mutation.

[0154] In some embodiments, the method further includes administering an anticancer therapy to the subject or exposing the cells to an anticancer therapy, such as a chemotherapeutic agent or cytotoxic agent, immunotherapy, surgery, radiotherapy, thermotherapy, or photocoagulation. In some embodiments, the anticancer therapy is a chemotherapeutic agent or cytotoxic agent, such as antimetabolites, antimitotic agents, antitumor antibiotics, asparagine-specific enzymes, bisphosphonates, antitumor agents, alkylating agents, DNA repair enzyme inhibitors, histone deacetylase inhibitors, corticosteroids, demethylating agents, immunomodulators, Janus-associated kinase inhibitors, phosphatidylinositol 3-kinase inhibitors, proteasome inhibitors, or tyrosine kinase inhibitors.

[0155] In some embodiments, the compounds of the present invention are used in combination with another anticancer therapy for treating uveal melanoma (such as surgery, MEK inhibitors, and / or PKC inhibitors). For example, in some embodiments, the method further includes performing surgery before, after, or simultaneously with the administration of the compounds of the present invention. In some embodiments, the method further includes administering MEK inhibitors and / or PKC inhibitors before, after, or simultaneously with the administration of the compounds of the present invention.

[0156] In some embodiments, the anticancer therapy and the compound of the present invention are administered to each other within 28 days, and each is administered in an amount that is effective in treating the target together.

[0157] In some implementations, the object or cancer has and / or has been identified as having a BRG1 loss-of-function mutation.

[0158] In some embodiments, the cancer is resistant to one or more chemotherapeutic agents or cytotoxic agents (e.g., the cancer has been identified as resistant to chemotherapeutic agents or cytotoxic agents, such as by genetic markers, or may be resistant to chemotherapeutic agents or cytotoxic agents, such as cancers that have already become unresponsive to chemotherapeutic agents or cytotoxic agents). In some embodiments, the cancer has already become unresponsive to one or more chemotherapeutic agents or cytotoxic agents. In some embodiments, the cancer is resistant to or no longer responds to the following agents: dacarbazine, temozolomide, cisplatin, treoxazine, formustin, IMCgp100, CTLA-4 inhibitors (e.g., ipilimumab), PD-1 inhibitors (e.g., nivolumab or pembrolizumab), PD-L1 inhibitors (e.g., atezolizumab, avelumab or duvalumab), mitogen-activated protein kinase (MEK) inhibitors (e.g., selmetinib, bimetinib or trametinib), and / or protein kinase C (PKC) inhibitors (e.g., sotrastuxin or IDE196).

[0159] In some embodiments, the cancer is resistant to or unresponsive to previously administered therapeutic agents (such as MEK inhibitors or PKC inhibitors) used to treat uveal melanoma. For example, in some embodiments, the cancer is resistant to or unresponsive to mitogen-activated protein kinase (MEK) inhibitors (e.g., selmetinib, bimetinib, or trametinib) and / or protein kinase C (PKC) inhibitors (e.g., sotrastuxin or IDE196).

[0160] Chemical terminology

[0161] The terminology used in this document is for the purpose of describing a particular implementation and is not intended to be limiting.

[0162] For any of the following chemical definitions, the number following the atomic symbol indicates the total number of atoms of that element present in a particular chemical part. As will be understood, other atoms (such as H atoms) or substituent groups as described herein may be present as needed to satisfy the valence of the atoms. For example, an unsubstituted C2 alkyl group has the formula -CH2CH3. When used with groups as defined herein, references to the number of carbon atoms include divalent carbons in acetals and ketals, but not carbonyl carbons in acyl, ester, carbonate, or carbamate groups. References to the number of oxygen, nitrogen, or sulfur atoms in heteroaryl groups include only those atoms that form part of a heterocycle.

[0163] As used herein, the term "acyl" refers to an H or alkyl group attached to a parent molecule group via a carbonyl group as defined herein, and exemplifies formyl (i.e., formaldehyde), acetyl, trifluoroacetyl, propionyl, and butyryl. Exemplary unsubstituted acyl groups comprise 1 to 6, 1 to 11, or 1 to 21 carbons.

[0164] As used herein, the term "alkyl" refers to a branched or straight monovalent saturated aliphatic hydrocarbon group having 1 to 20 carbon atoms (e.g., 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms).

[0165] Alkylene is a divalent alkyl group. The term “alkenyl” as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon group having a carbon-carbon double bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2 carbon atoms).

[0166] The term "alkynyl" as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon group having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2 carbon atoms).

[0167] The term "amino" as used in this article represents -N(R) N1 )2, where each R N1 Independently, they are H, OH, NO2, and N(R) N2 2. SO2OR N2 SO2R N2 SOR N2 N-protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl or other acyl groups described herein), wherein these listed R N1 Each of the groups can be optionally substituted; or both R groups can be substituted. N1 Combining to form alkylene or heteroalkylene, wherein each R N2 Independently, it is H, alkyl, or aryl. The amino group of the present invention can be an unsubstituted amino group (i.e., -NH2) or a substituted amino group (i.e., -N(R)). N1 )2).

[0168] As used herein, the term "aryl" refers to an aromatic monocyclic or polycyclic aromatic group having 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, 1,2-dihydronaphthyl, indenyl, and 1H-indenyl.

[0169] As used herein, the term "arylalkyl" refers to an alkyl group substituted with an aryl group. Exemplary unsubstituted arylalkyl groups have 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, such as C1-C6 alkyl C6-C6). 10 Aryl, C1-C 10 Alkyl C6-C 10 Aryl or C1-C 20 Alkyl C6-C 10 Aryl groups, such as benzyl and phenethyl. In some embodiments, the alkyl and aryl groups may each be further substituted with one, two, three, or four substituent groups as defined herein with respect to the respective groups.

[0170] The term "azido" as used in this article refers to the -N3 group.

[0171] As used in this article, the term "bridged polycyclic alkyl" refers to a bridged polycyclic group with 5 to 20 carbons containing 1 to 3 bridges.

[0172] The term "cyano" used in this article refers to the -CN group.

[0173] The term "carbocyclic group" as used in this article refers to a non-aromatic C3-C group in which the ring is formed by carbon atoms. 12 Monocyclic, bicyclic, or tricyclic structures. Carbocyclic structures include cycloalkyl groups and unsaturated carbocyclic groups.

[0174] As used herein, the term "cycloalkyl" refers to a saturated, non-aromatic, and monovalent monocyclic or polycyclic group having 3 to 10, preferably 3 to 6, carbon atoms. The term is further illustrated by groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl.

[0175] As used in this article, the term "halogen" refers to a fluorine (fluorine), chlorine (chlorine), bromine (bromine), or iodine (iodine) group.

[0176] As used herein, the term "heteroalkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms of the component have been substituted with nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group may be further substituted with one, two, three, or four substituent groups as described herein with respect to alkyl groups. An example of a heteroalkyl group is "alkoxy," which, as used herein, refers to alkyl-O- (e.g., methoxy and ethoxy). Heteroalkylene is a divalent heteroalkyl group. As used herein, the term "heteroalkenyl" refers to an alkenyl group as defined herein, wherein one or more carbon atoms of the component have been substituted with nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkenyl group may be further substituted with one, two, three, or four substituent groups as described herein with respect to alkenyl groups. An example of a heteroalkenyl group is "alkenoxy," which, as used herein, refers to alkenyl-O-. Heteroalkenyl is a divalent heteroalkenyl group. As used herein, the term "heteroynyl" refers to an alkyne group as defined herein, wherein one or more carbon atoms of the component have been substituted with nitrogen, oxygen, or sulfur. In some embodiments, the heteroynyl group may be further substituted with one, two, three, or four substituent groups as described herein with respect to the ynyl group. An example of a heteroynyl group is "alkynoxy group," which, as used herein, refers to ynyl-O-. A heteroynyl group is a divalent heteroynyl group.

[0177] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or polycyclic group of 5 to 12 atoms, having at least one aromatic ring containing one, two, or three ring atoms selected from nitrogen, oxygen, and sulfur, with the remaining ring atoms being carbon. One or two ring carbon atoms of a heteroaryl group may be replaced by a carbonyl group. Examples of heteroaryl groups are pyridinyl, pyrazoyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, imidazolyl, oxaxolyl, and thiazolyl.

[0178] As used herein, the term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group. Exemplary unsubstituted heteroarylalkyl groups have 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, such as C1-C6 alkyl, C2-C9 heteroaryl, C1-C...). 10 Alkyl C2-C9 heteroaryl or C1-C 20 Alkyl C2-C9 heteroaryl). In some embodiments, the alkyl group and the heteroaryl group may each be further substituted with 1, 2, 3 or 4 substituent groups as defined herein with respect to the respective groups.

[0179] As used herein, the term "heterocyclic group" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) group having 3 to 12 atoms, having at least one non-aromatic ring containing 1, 2, 3, or 4 ring atoms selected from N, O, or S, and having no aromatic ring containing any N, O, or S atoms. Examples of heterocyclic groups include, but are not limited to, morpholino, thiomorpholino, furanyl, piperazine, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1,3-dioxanehexyl.

[0180] As used herein, the term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group. Exemplary unsubstituted heterocyclic alkyl groups have 7 to 30 carbons (e.g., 7 to 16 or 7 to 20 carbons, such as C1-C6 alkyl C2-C9 heterocyclic groups, C1-C...). 10 Alkyl C2-C9 heterocyclic or C1-C 20 Alkyl C2-C9 heterocyclic group). In some embodiments, the alkyl group and the heterocyclic group may each be further substituted with 1, 2, 3 or 4 substituent groups as defined herein with respect to the respective groups.

[0181] As used in this article, the term "hydroxyalkyl" refers to an alkyl group substituted with a -OH group.

[0182] The term "hydroxyl" used in this article refers to the -OH group.

[0183] As used herein, the term “N-protecting group” refers to those groups intended to protect amino groups from unwanted reactions during the synthetic process. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3rd edition (John Wiley & Sons, New York, 1999). N-protecting groups include, but are not limited to, acyl, aromatic, or carbamoyl groups such as formyl, acetyl, propionyl, neopentyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl; and chiral auxiliaries such as protected or unprotected D, L, or D, L-amino acids such as alanine, leucine, and phenylalanine; groups containing sulfonyl groups such as benzylsulfonyl and p-toluenesulfonyl; groups forming carbamates such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5- Dimethoxybenzyloxycarbonyl, 2,4-20-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, diphenylmethyloxycarbonyl, tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropoxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2'-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentoxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl; arylalkyl groups such as benzyl, triphenylmethyl, and benzyloxymethyl; and silyl groups such as trimethylsilyl. Preferred N-protecting groups are alloc, formyl, acetyl, benzoyl, neopentyl, tert-butylacetyl, alanyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0184] The term "nitro" as used in this article refers to the -NO2 group.

[0185] The term "thiol" used in this article refers to the -SH group.

[0186] The alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclic groups may be substituted or unsubstituted. When substituted, there will typically be 1 to 4 substituents present, unless otherwise specified. Substituents include, for example: alkyl (e.g., unsubstituted and substituted, wherein said substituents include any group described herein, e.g., aryl, halogen, hydroxyl), aryl (e.g., substituted and unsubstituted phenyl), carbocyclic (e.g., substituted and unsubstituted cycloalkyl), halogen (e.g., fluorine), hydroxyl, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, or thioalkoxy), heteroaryl, heterocyclic, amino (e.g., NH2 or mono- or di-alkylamino), azide, cyano, nitro, or mercapto. Another exemplary substituent is oxo. For example, the carbonyl group is an oxo-substituted carbon (e.g., alkyl carbon, alkenyl carbon, ynyl carbon, heteroalkyl carbon, heteroalkenyl carbon, heteroynyl carbon, carbocyclic carbon, etc.). Alternatively, sulfur can be substituted by one or two oxo groups (e.g., -SO- or -SO2- within the substituted heteroalkyl, heteroalkenyl, heteroynyl, or heterocyclic group). Aryl, carbocyclic (e.g., cycloalkyl), heteroaryl, and heterocyclic groups can also be substituted by alkyl groups (both unsubstituted and substituted, such as arylalkyl (e.g., substituted and unsubstituted benzyl)). In some embodiments, the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl groups are optionally substituted with 1, 2, 3, 4, or 5 substituents, said substituents being independently selected from aryl (e.g., substituted and unsubstituted phenyl), carbocyclic (e.g., substituted and unsubstituted cycloalkyl), halogen (e.g., fluorine), hydroxyl, heteroaryl, heterocyclic, amino (e.g., NH2 or mono- or di-alkylamino), azide, cyano, nitro, mercapto, and oxo. In some embodiments, said substituents themselves are unsubstituted.

[0187] The compounds of the present invention may have one or more asymmetric carbon atoms and may exist in the following forms: optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates, or mixtures of diastereomeric racemates. Optically active forms can be obtained, for example, by resolving racemates, by asymmetric synthesis, or by asymmetric chromatography (chromatography using chiral adsorbents or eluents). That is, some of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are paired stereoisomers whose mirror images are not superimposed, most commonly because they contain an asymmetrically substituted carbon atom as a chiral center. "Enantiomer" means one of a pair of mirror images of each other that are not superimposed. Diastereomers are stereoisomers independent of mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of the substituents surrounding one or more chiral carbon atoms. Enantiomers of compounds can be prepared, for example, by separating an enantiomer from a racemic mixture using one or more well-known techniques and methods, such as, for example, chiral chromatography and separation methods based thereon. Those skilled in the art can readily determine suitable techniques and / or methods for separating enantiomers of the compounds described herein from racemic mixtures. "Racemic mixture" or "racemic mixture" means a compound containing two enantiomers, wherein such a mixture does not exhibit optical activity; that is, they do not rotate the plane of polarized light. "Geometric isomer" means an isomer that differs in the orientation of the substituent atoms in relation to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. Atoms on each side of the carbon-carbon double bond (except H) can be in an E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented to the same side) configuration. "R", "S", "S*", "R*", "E", "Z", "cis", and "trans" indicate the configuration relative to the core molecule. Some disclosed compounds may exist in a trans-restricted isomer form. Rotation-restricted isomers are stereoisomers resulting from restricted rotation around a single bond, wherein the steric strain barrier to rotation is high enough to allow for the separation of conformational isomers. The compounds of the present invention can be prepared as single isomers through isomer-specific synthesis or resolution from mixtures of isomers. Conventional resolution techniques include using an optically active acid to form a salt of the free base of each isomer of the isomer pair (followed by stepwise crystallization and regeneration of the free base), using an optically active amine to form a salt of the acid form of each isomer of the isomer pair (followed by stepwise crystallization and regeneration of the free acid), using an optically pure acid, amine, or alcohol to form an ester or amide of each isomer of the isomer pair (followed by chromatographic separation and removal of chiral auxiliaries), or using various well-known chromatographic methods to resolve mixtures of isomers of the starting material or end product.When the stereochemistry of a disclosed compound is named or described by structure, the named or described stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight relative to other stereoisomers. When a single enantiomer is named or described by structure, the described or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% optically pure by weight. When a single diastereomer is named or described by structure, the described or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. The percentage of optical purity is the ratio of the weight of the enantiomer to the weight of the enantiomer plus the weight of its optical isomer. The diastereomeric purity by weight is the ratio of the weight of one diastereomer to the weight of all diastereomers. When the stereochemistry of the disclosed compound is named or described by structure, the named or described stereoisomer has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by molar fraction relative to other stereoisomers. When a single enantiomer is named or described by structure, the described or named enantiomer has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by molar fraction. When a single diastereomer is named or described by structure, the described or named diastereomer has a purity of at least 60%, 70%, 80%, 90%, 99%, or 99.9% by molar fraction. The purity percentage by molar fraction is the ratio of the number of moles of the enantiomer to the sum of the number of moles of its optical isomer. Similarly, the purity percentage in molar fractions is the ratio of the number of moles of diastereomers to the sum of the number of moles of diastereomers and their isomers. When the disclosed compound is named or described without indicating stereochemistry and has at least one chiral center, it should be understood that the name or structure covers the enantiomers of the compound that do not contain the corresponding optical isomer, racemic mixtures of the compound, or mixtures rich in one enantiomer relative to its corresponding optical isomer. When the disclosed compound is named or described without indicating stereochemistry and has two or more chiral centers, it should be understood that the name or structure covers the diastereomers that do not contain other diastereomers, a number of diastereomers that do not contain other diastereomer pairs, mixtures of diastereomers, mixtures of diastereomer pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomers, or mixtures of diastereomers in which one or more diastereomers are enriched relative to the other diastereomers. This invention includes all of these forms.

[0188] The compounds disclosed herein also include all isotopes of the atoms appearing in intermediate or final compounds. An "isotope" is an atom that has the same number of atoms but different mass numbers due to different numbers of neutrons in its nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0189] Unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 32 P, 33 P, 35 S, 18 F, 36 Cl、 123 I and 125 I. Isotope-labeled compounds (e.g., those labeled with...) 3 H and 14 Those labeled with C can be used in the determination of compound or substrate tissue distribution. Because of their ease of preparation and detectability, tritium (i.e.,...) 3 H) and carbon-14 (i.e., ... 14 C) Isotopes can be useful. Furthermore, heavier isotopes such as deuterium (i.e.,...) can be used. 2 H) substitution can provide certain therapeutic advantages stemming from greater metabolic stability (e.g., increased in vivo half-life or reduced dose requirement). In some embodiments, one or more hydrogen atoms are... 2 H or 3 H substitution, or one or more carbon atoms being replaced 13 C- or 14 C-enriched carbon substitution. Positron-emitting isotopes such as... 15 O、 13 N、 11 C and 18F can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. The preparation of isotopically labeled compounds is known to those skilled in the art. For example, isotopically labeled compounds can typically be prepared by replacing non-isotopically labeled reagents with isotopically labeled reagents, following procedures similar to those disclosed for the compounds of the invention described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials used in this disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples described are merely exemplary and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0190] definition

[0191] In this application, unless otherwise understood from the context, (i) the term “a” (“a”) may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; and (iii) the terms “comprising” and “including” may be understood to cover the listed components or steps, whether presented alone or together with one or more other components or steps.

[0192] As used herein, the terms “about” and “approximately” refer to values ​​within 10% above or below the described value. For example, the term “about 5 nM” indicates a range of 4.5 to 5.5 nM.

[0193] As used herein, the term "application" means administering a composition (e.g., a compound or an article comprising a compound described herein) to a subject or system. Application to an animal subject (e.g., to a human) can be by any suitable route. For example, in some embodiments, application can be bronchial (including bronchial infusion), sublingual, enteric, interdermal, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intravenous, intracardiac, mucosal, nasal, oral, rectal, subcutaneous, sublingual, local, tracheal (including intratracheal infusion), transdermal, vaginal, and vitreous.

[0194] As used in this article, the term "BAF complex" refers to the BRG1- or HRBM-related factor complex in human cells.

[0195] The term “BAF complex-related barrier” as used in this article refers to a barrier caused by or affected by the activity level of the BAF complex.

[0196] As used herein, the term "BRG1 loss-of-function mutation" refers to a mutation in BRG1 that results in reduced activity of the protein (e.g., a reduction of at least 1% in BRG1 activity, such as 2%, 5%, 10%, 25%, 50%, or 100% reduction). Exemplary BRG1 loss-of-function mutations include, but are not limited to, homozygous BRG1 mutations and deletions at the C-terminus of BRG1.

[0197] As used in this article, the term "BRG1 loss-of-function disorder" refers to a disorder (e.g., cancer) that exhibits a reduction in BRG1 activity (e.g., a reduction of at least 1%, such as a reduction of 2%, 5%, 10%, 25%, 50%, or 100%).

[0198] The term "cancer" refers to a condition caused by the proliferation of malignant neoplasms, such as tumors, growths, carcinomas, sarcomas, leukemia, and lymphomas.

[0199] As used herein, "combination therapy" or "combined administration" means administering two (or more) different agents or treatments to a subject as part of a defined treatment regimen for a specific disease or condition. The treatment regimen defines the dosage and frequency of administration of each agent, such that the effects of the various agents overlap on the subject. In some embodiments, the delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, two or more agents are not co-formulated and are administered sequentially as part of a prescribed regimen. In some embodiments, the combined administration of two or more agents or treatments results in a reduction of symptoms or other parameters related to the disorder greater than that observed with the delivery of a single agent or treatment or in the absence of other agents or treatments. The effects of the two treatments may be partially additive, completely additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent can be achieved via any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered via the same or different routes. For example, the first therapeutic agent of the combination can be administered by intravenous injection, while the second therapeutic agent of the combination can be administered orally.

[0200] "Determining the level of a protein or RNA" means detecting a protein or RNA directly or indirectly using methods known in the art. "Direct determination" means performing a process (e.g., measuring or testing a sample, or "analyzing a sample," as defined herein) to obtain a physical entity or value. "Indirect determination" means receiving a physical entity or value from another party or source (e.g., a third-party laboratory that directly obtains the physical entity or value). Methods for measuring protein levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometrics, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry, as well as determinations based on protein properties (including, but not limited to, enzyme activity or interactions with other protein couplers). Methods for measuring RNA levels are known in the art and include, but are not limited to, quantitative polymerase chain reaction (qPCR) and RNA blot analysis.

[0201] "Decrease the activity of the BAF complex" means a reduction in the activity level associated with the BAF complex or related downstream effects. A non-limiting example of decreased BAF complex activity is Sox2 activation. The activity level of the BAF complex can be measured using any method known in the art, such as that described in Kadoch et al., Cell, 2013, 153, 71-85, which is incorporated herein by reference.

[0202] As used herein, the term "degrading agent" refers to a small molecule compound that includes a degradation component, wherein the compound interacts with a protein (e.g., BRG1 and / or BRM) in a manner that leads to the degradation of the protein, for example, the binding of the compound results in a reduction of at least 5% in the protein level, for example, in a cell or object.

[0203] As used herein, the term "degradation moiety" refers to a portion of a protein that binds to and causes degradation of the protein (e.g., BRG1 and / or BRM). In one embodiment, the moiety binds to a protease or ubiquitin ligase of the metabolic protein (e.g., BRG1 and / or BRM).

[0204] "Modulating the activity of a BAF complex" means altering the activity level or associated downstream effects of a BAF complex (e.g., GBAF). The activity level of a BAF complex can be measured using any method known in the art, such as that described in Kadoch et al., Cell, 2013, 153, 71-85, which is incorporated herein by reference.

[0205] "Reducing the activity of BRG1 and / or BRM" means reducing the activity level associated with BRG1 and / or BRM or the associated downstream effects. A non-limiting example of inhibiting the activity of BRG1 and / or BRM is reducing the level of the BAF complex in cells. The activity level of BRG1 and / or BRM can be measured using any method known in the art. In some embodiments, the agent reducing the activity of BRG1 and / or BRM is a small molecule BRG1 and / or BRM degrader.

[0206] "Reducing the levels of BRG1 and / or BRM" means reducing the levels of BRG1 and / or BRM in cells or objects. The levels of BRG1 and / or BRM can be measured using any method known in the art.

[0207] “Level” refers to the level of a protein or the mRNA encoding the protein compared to a reference. This reference can be any useful reference as defined herein. A “reduced level” or “increased level” of a protein means a decrease or increase in the protein level compared to a reference (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more; a decrease or increase of more than about 10% compared to a reference). A decrease or increase of 15%, approximately 20%, approximately 50%, approximately 75%, approximately 100%, or approximately 200%; a decrease or increase of less than approximately 0.01, approximately 0.02, approximately 0.1, approximately 0.3, approximately 0.5, approximately 0.8, or less; or an increase of more than approximately 1.2, approximately 1.4, approximately 1.5, approximately 1.8, approximately 2.0, approximately 3.0, approximately 3.5, approximately 4.5, approximately 5.0, approximately 10, approximately 15, approximately 20, approximately 30, approximately 40, approximately 50, approximately 100, approximately 1000, or more. Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or as a percentage of total protein or mRNA in the sample.

[0208] As used herein, the term "BRM inhibition" refers to blocking or reducing the level or activity of the ATPase-binding domain or bromine domain of a protein. BRM inhibition can be determined using methods known in the art, such as BRM ATPase assays, Nano DSF assays, or BRM luciferase cellular assays.

[0209] As used herein, the term "pharmaceutical composition" refers to a composition containing the compounds described herein, formulated together with pharmaceutically acceptable excipients, and suitable for administration to mammals, such as humans. Typically, pharmaceutical compositions are manufactured or marketed with the approval of a government regulatory authority as part of a treatment regimen for a disease in mammals. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage forms (e.g., tablets, capsules, sacs, gel caps, or syrups); for topical administration (e.g., as creams, gels, lotions, or ointments); for intravenous administration (e.g., as a sterile solution without microparticle plugs and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation.

[0210] As used herein, “pharmaceuticalally acceptable excipient” means any component other than the compounds described herein (e.g., a medium capable of suspending or dissolving the active compound) and having substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example: anti-adhesives, antioxidants, adhesives, coatings, compression aids, disintegrants, dyes (pigments), softeners, emulsifiers, fillers (diluents), film-forming agents or coatings, flavoring agents, fragrances, flow aids (flow promoters), lubricants, preservatives, printing inks, absorbents, suspending or dispersing agents, sweeteners, and hydration water.

[0211] As used herein, the term "pharmaceuticalally acceptable salt" means any pharmaceutically acceptable salt of a compound (e.g., any compound of Formula I). ​​A pharmaceutically acceptable salt of any compound described herein may include a salt that, within reasonable medical judgment, is suitable for contact with tissues in humans and animals without undue toxicity, irritation, or allergic response, and is proportionate to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (edited by P.H. Stahl and C. G. Germuth), Wiley-VCH, 2008. Salts may be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base group with a suitable organic acid.

[0212] The compounds of the present invention may have ionizable groups to enable their preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids, or, in the case of the acidic form of the compounds of the present invention, the salts may be prepared from inorganic or organic bases. Often, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, and methods for preparing suitable salts, are well known in the art. Salts may be prepared from pharmaceutically acceptable, non-toxic acids and bases, including inorganic and organic acids and bases.

[0213] “Reference” means any useful reference used to compare protein or RNA levels. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A “reference sample” can be, for example, a control, such as a predetermined negative control value, like a “normal control” or a previous sample taken from the same subject; a sample from a normal healthy subject, such as normal cells or normal tissue; a sample from a subject without disease (e.g., cells or tissue); a sample from a subject diagnosed with a disease but not yet treated with the compounds of the present invention; a sample from a subject already treated with the compounds of the present invention; or a sample of purified protein or RNA (e.g., any of those described herein) at a known normal concentration. A “reference standard or level” means a value or number derived from a reference sample. A “normal control value” is a predetermined value indicating a non-disease state, for example, a value expected in a healthy control subject. Typically, normal control values ​​are expressed as a range (“between X and Y”), a high threshold (“not higher than X”), or a low threshold (“not lower than X”). A subject having a measurement within the normal control range for a specific biomarker is typically referred to as being "within the normal limits" for that biomarker. The normal reference standard or level can be a value or number derived from: a normal subject without disease or impairment (e.g., cancer); or a subject already treated with the compounds of the present invention. In a preferred embodiment, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of the levels of purified protein or RNA (e.g., any of those described herein) within the normal reference range can also be used as a reference.

[0214] As used herein, the term "object" refers to any organism to which the compositions according to the invention may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical objects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Objects may be seeking or needing treatment, requesting treatment, receiving treatment, or in the future receiving treatment, or may be persons or animals under the care of trained professionals for a specific disease or condition.

[0215] As used herein, the term "treat" ("treated" or "treating") means a therapeutic treatment or any measure aimed at slowing (alleviating) an undesirable physical condition, disorder, or disease, or at achieving a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to: relief of symptoms; reduction of the severity of the condition, disorder, or disease; stabilization of the condition, disorder, or disease (i.e., no worsening); delay or slowing of the onset of the progression of the condition, disorder, or disease; improvement or relief of the condition, disorder, or disease state (whether partial or complete); improvement of at least one measurable physical parameter, which may not be perceptible to the patient; or improvement or enhancement of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive side effects. Treatment also includes prolonging survival compared to the expected survival without treatment. The compounds of this invention can also be used for "preventative treatment" or "prevention" of disorders, for example, in subjects at increased risk of developing a disorder.

[0216] Details of one or more embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will become apparent from the description and from the claims.

[0217] Detailed description

[0218] This disclosure characterizes compounds that can be used to inhibit BRG1 and optionally BRM. These compounds can be used to modulate the activity of the BAF complex, for example, to treat BAF-related disorders such as cancer (e.g., BRG1-loss-of-function disorders). Exemplary compounds described herein include compounds having a structure according to Formula I or a pharmaceutically acceptable salt thereof.

[0219] Formula I:

[0220]

[0221] in

[0222] X is a halogen;

[0223] X 1 It does not exist or is O or NR. 1 ;

[0224] k is 0, 1, 2, or 3;

[0225] n is 0, 1, or 2;

[0226] R 1 It is H or an optional substituted C1-C6 alkyl group;

[0227] L 1It is a C1-C6 alkylene group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group that is optionally substituted;

[0228] L 2 The absence or optional substitution of C1-C6 alkylene groups, and the optional substitution of C1-C6 alkylene groups. 20 Heteroalkyl or optionally substituted C2-C9 heterocyclic groups;

[0229] Each L 3 C1-C is independently and optionally replaced 20 Heteroalkyl groups, optionally substituted C3-C 10 Carbocyclic group, optionally substituted C3-C 10 Carbocyclic-C1-C6 alkylene, optionally substituted C2-C9 heterocyclic, optionally substituted C2-C9 heterocyclic-C1-C6 alkylene, optionally substituted C6-C 10 Aromatic, optionally substituted C6-C 10 arylene-C1-C6 alkylene, optionally substituted C2-C6 ynylene, O or NR 1 ;and

[0230] D represents the degradation portion.

[0231] In some embodiments, the compound has the structure of any one of compounds 1 to 105 in Table 1 or a pharmaceutically acceptable salt thereof.

[0232] This document describes other implementation schemes, as well as exemplary methods for synthesizing or producing these compounds.

[0233] Drug Use

[0234] The compounds described herein can be used in the methods of the present invention, and although not theoretically limited, they are believed to exert the ability to regulate the level, state, and / or activity of the BAF complex, i.e., by inhibiting the activity of BRG1 and / or BRM proteins within the BAF complex in active mammals. Barriers associated with the BAF complex include, but are not limited to, barriers associated with loss-of-function mutations in BRG1.

[0235] One aspect of the invention relates to a method of treating a disorder associated with a BRG1 loss-of-function mutation in a subject in need, the disorder being such as cancer (e.g., non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary origin, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, or penile cancer). In some embodiments, the compound is administered in an amount and at a time that effectively results in one or more of the following (e.g., two or more, three or more, four or more): (a) reduced tumor size, (b) reduced tumor growth rate, (c) increased tumor cell death, (d) reduced tumor progression, (e) reduced number of metastases, (f) reduced metastasis rate, (g) reduced tumor recurrence, (h) increased subject survival, and (i) increased subject progression-free survival.

[0236] Cancer treatment can result in a reduction in the size or volume of a tumor. For example, after treatment, the tumor size may decrease by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) relative to its pre-treatment size. The size of a tumor can be measured using any reproducible method. For example, the size of a tumor can be measured as its diameter.

[0237] Cancer treatment can further reduce the number of tumors. For example, after treatment, the number of tumors may be reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) relative to the number before treatment. The number of tumors can be measured by any reproducible method. For example, the number of tumors can be measured by counting tumors that are visible to the naked eye or by counting them at a specific magnification (e.g., 2x, 3x, 4x, 5x, 10x, or 50x).

[0238] Cancer treatment can lead to a reduction in the number of metastatic nodules in other tissues or organs far from the primary tumor site. For example, after treatment, the number of metastatic nodules may be reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) relative to the number before treatment. The number of metastatic nodules can be measured using any reproducible measurement method. For example, the number of metastatic nodules can be measured by counting the nodules visible to the naked eye or by counting them at a specific magnification (e.g., 2x, 10x, or 50x).

[0239] Cancer treatment can lead to an increase in the mean survival time of the treated population compared to an untreated population. For example, the mean survival time may increase by more than 30 days (more than 60, 90, or 120 days). This increase in mean survival time can be measured in any reproducible manner. For example, the increase can be measured by calculating the mean survival time of the population after the onset of treatment with the compounds of the present invention. Alternatively, the increase can be measured by calculating the mean survival time of the population after completing the first round of treatment with pharmaceutically acceptable salts of the present invention.

[0240] Cancer treatment can also lead to a reduction in mortality in the treated population compared to the untreated population. For example, the mortality rate reduction may exceed 2% (e.g., exceed 5%, 10%, or 25%). This reduction in mortality can be measured in any reproducible manner, for example, by calculating the average number of disease-related deaths per unit time for the population after initiation of treatment with the pharmaceutically acceptable salt of the present invention. Alternatively, the reduction in population mortality can also be measured by calculating the average number of disease-related deaths per unit time for the population after completion of the first round of treatment with the pharmaceutically acceptable salt of the present invention.

[0241] Exemplary cancers that can be treated by this invention include, but are not limited to, non-small cell lung cancer, small cell lung cancer, colorectal cancer, bladder cancer, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, esophageal-gastric cancer, pancreatic cancer, hepatobiliary cancer, soft tissue sarcoma, ovarian cancer, head and neck cancer, renal cell carcinoma, bone cancer, non-Hodgkin lymphoma, prostate cancer, embryonal tumor, germ cell tumor, cervical cancer, thyroid cancer, salivary gland cancer, gastrointestinal neuroendocrine tumors, uterine sarcoma, gastrointestinal stromal tumors, central nervous system cancers, thymic tumors, adrenocortical carcinoma, appendix cancer, small bowel cancer, and penile cancer.

[0242] Combination formulations and their uses

[0243] The compounds of the present invention can be combined with one or more therapeutic agents. Specifically, the therapeutic agents can be those for treating or preventing any of the cancers described herein.

[0244] Combination therapy

[0245] The compounds of this invention can be used alone or in combination with other therapeutic agents (e.g., other agents for treating cancer or related symptoms) or with other types of treatments for cancer. In combination therapy, the dose of one or more therapeutic compounds can be reduced from the standard dose when administered alone. For example, the dose can be determined empirically from the combination and arrangement of drugs, or the dose can be inferred through isobolographic analysis (e.g., Black et al., Neurology 65: S3-S6, 2005). In this case, the dose of the compounds when combined should provide a therapeutic effect.

[0246] In some implementations, the second therapeutic agent is a chemotherapeutic agent (e.g., a cytotoxic agent or other chemical compound that can be used to treat cancer). These include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxin, antibiotics, L-asparaginase, topoisomerase inhibitors, interferon, platinum coordination complexes, anthrone-substituted ureas, methylhydrazine derivatives, adrenocortical inhibitors, adrenocortical steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Also included are 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. Non-limiting examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, indomethacin, and piperazine; aziridines such as benzoxotepa, carboquinone, metotipa, and urotepa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylamine, triethylphosphamide, triethylthiophosphamide, and tris(hydroxymethyl)melamine; acetogenins (especially blatazine and blatazineone); camptothecin (including its synthetic analogue topotecan); lichenin; callystatin; CC-1065 (including its synthetic analogues adoretin, calcetin, and pyrazine); cyclopeptides of Nostoc (especially Nostoc cyclopeptide 1 and Nostoc cyclopeptide 8); dorlastatin; Docalimicin (including synthetic analogs, KW-2189 and CB1-TM1); soft coral alcohol; silicoside; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, naphthylambucil, chlorphosphamide, estradiol, ifosfamide, dichloromethyldiethylamine, dichloromethyldiethylamine oxide hydrochloride, melphalan, neonitrogen mustard, benzyl mustard cholesterol, prednimustine, trazophosphatide, uracil mustard; nitrosurea such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ramustine; antibiotics such as enediyne antibiotics (e.g., calichimycin, especially calichimycin γll and calichimycin ωll (see, e.g., Agnew, Chem. Intl. Ed.) Engl. 33: 183-186 (1994)); danendomycin, including danendomycin A; bisphosphonates, such as clophosphonate; esporamycin; and new carcinogen chromophores and related chromogens of ethynylene antibiotics), aclarubicin, actinomycin, amiodarone, azoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinogen, chromomycin, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, (Doxorubicin, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, metharubicin, mitomycin such as mitomycin C, mycophenolate mofetil, nopramine, olivomycin, pepromycin, pofibromycin, puromycin, triamcinolone acetonide, rodorubicin, streptomycin, streptozotocin, tuberculin Ubenimex, fenestrated statin, zolarubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as folate, methotrexate, pteroxate, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, deoxyfluoride Uric acid, enoxabin, fluorouridine; androgens such as capprost, drotalbuterone propionate, cyclothiosterol, meandrolone, testosterone; antiadrenergics such as aminoglutethimide, mitotane, trelostan; folic acid supplements such as folinic acid; acetoglucuronolactone; aldehydephosphoramide glycoside; aminolevulinic acid; enuracil; acridine; bestrabucil; bisacodyl; edaraxazole; desphosphonamide; colchicine; diazoxide Quinones; ornithine; erythritol; epothilone; etoglucopyranoside; gallium nitrate; hydroxyurea; lentinan; chlordamine; metansine derivatives such as metansine and anthraquinone; mitoxantrone; mitoxantrone; mopidanmol; nitraerine; pentostatin; methamidophos; pirarubicin; loxoantrone; podophylloic acid; 2-ethylhydrazine; procarbazine; Polysaccharide complexes (JHS Natural Products, Eugene, Oreg.); Razosen; Rhizomycin; Sizofuran; Germonispiramine; Tenuzonic acid; Triaminoquinone; 2,2′,2″-Trichlorotriethylamine; Trichothecene derivatives (especially T-2 toxin, Verrucin A, Bacitracin A, and Serpentin); Urethan; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeusine; Piperobromide; Gacytosine; Cytarabine (“Ara-C”); Cyclophosphamide; Thiotepa; Taxanes, for example, Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) A levofloxacin-free, albumin-engineered paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Ill.), and Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorobutyrate; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; Noscholine; Teniposide; Edatraxa; Donomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoic acid such as retinoic acid; Capecitabine; and pharmaceutically acceptable salts or acids of any of the above. Two or more chemotherapeutic agents may be used in a mixture to be administered in combination with the first chemotherapeutic agent described herein. Suitable dosing regimens for combination chemotherapy are known in the art and described, for example, Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al. (2000) Lancet 355:1041-7.

[0247] In some embodiments, the second therapeutic agent is a biological agent, such as a cytokine (e.g., interferon or interleukin (e.g., IL-2)) used in cancer treatment. In some embodiments, the biological agent is an anti-angiogenic agent, such as an anti-VEGF agent, for example, bevacizumab. In some embodiments, the biological agent is an immunoglobulin-based biological agent, such as a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein, or a functional fragment thereof) that activates a target to stimulate an anticancer response or antagonizes an antigen important to cancer. Such drugs include Rituxan (rituximab); Xenipe (dacrolimus); Sulepi (baliximab); Synagis (palizumab); Remicade (infliximab); Herceptin (trastuzumab); Merotal (gem-tuzumab / ozomicin); Camppath (alenzumab); Zevalin (teimomab); Humira (adalimumab); Xolair (omaliximab); Bexxar (tosimozumab-I-131); Raptiva (efazolinumab); Erbitux (cetuximab); Avastin (bevacizumab); Tysabri (natezilumab); Actemra (tocilizumab); Vectibix (palizumab / paliximab). Monoclonal antibodies (MAbs); Lucentis (ranibizumab); Soliris (eculizumab); Cimzia (pecizumab); Simponi (golimumab); Ilaris (cananumab); Stelara (ustekinumab); Arzerra (ofumab); Prolia (deshumab); Numax (muvizumab); ABThrax (ranibizumab); Benlysta (belimumab); Yervoy (ipilimumab); Adcetris (bentoximab); Perjeta (petoximab); Kadcyla (trastuzumab-metanexine conjugate); and Gazyva (atoruzumab). Antibody-drug conjugates are also included.

[0248] The second agent can be a non-pharmacological therapeutic agent. For example, the second therapeutic agent is radiation therapy, cryotherapy, hyperthermia, and / or surgical removal of tumor tissue.

[0249] The second agent may be a checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an inhibitory antibody (e.g., a monospecific antibody such as a monoclonal antibody). The antibody may be, for example, humanized or fully human. In some embodiments, the checkpoint inhibitor is a fusion protein, such as an Fc-receptor fusion protein. In some embodiments, the checkpoint inhibitor is an agent that interacts with a checkpoint protein, such as an antibody. In some embodiments, the checkpoint inhibitor is an agent that interacts with a ligand of a checkpoint protein, such as an antibody. In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor (e.g., an inhibitory antibody or a small molecule inhibitor) (e.g., an anti-CTLA4 antibody such as ipilimumab / Yervoy or trimemumab). In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor (e.g., an inhibitory antibody or a small molecule inhibitor) (e.g., nivolumab / Pembrolizumab / Pidilizumab / CT-011). In some embodiments, the checkpoint inhibitor is a PDL1 inhibitor (e.g., an inhibitory antibody or a small molecule inhibitor) (e.g., MPDL3280A / RG7446; MEDI4736; MSB0010718C; BMS 936559). In some embodiments, the checkpoint inhibitor is a PDL2 inhibitor (e.g., an inhibitory antibody or Fc fusion protein or a small molecule inhibitor) (e.g., a PDL2 / Ig fusion protein such as AMP 224). In some embodiments, the checkpoint inhibitor is an inhibitor of B7-H3 (e.g., MGA271), B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof (e.g., an inhibitory antibody or a small molecule inhibitor).

[0250] In any of the combined implementations described herein, the first and second therapeutic agents are administered simultaneously or sequentially in any order. The first therapeutic agent may be administered immediately before or after the second therapeutic agent, at most 1 hour, at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, at most 24 hours, or at most 1-7, 1-14, 1-21, or 1-30 days later.

[0251] Pharmaceutical Composition

[0252] The compounds of the present invention are preferably formulated into pharmaceutical compositions for administration to mammals, preferably humans, in a biocompatible form suitable for in vivo administration. Therefore, in one aspect, the present invention provides a pharmaceutical composition comprising the compounds of the present invention mixed with a suitable diluent, carrier, or excipient.

[0253] The compounds of the present invention can be used in the form of free bases, salts, solvates, and prodrugs. All forms are within the scope of the invention. As will be understood by those skilled in the art, according to the method of the present invention, the described compounds, or their salts, solvates, or prodrugs, can be administered to the patient in a variety of forms, depending on the chosen route of administration. The compounds of the present invention can be administered, for example, orally, parenterally, sublingually, sublingually, nasally, rectally, via patch, pump, or transdermally, and pharmaceutical compositions are formulated accordingly. Parenterial administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and local administration modes. Parenterial administration can be performed by continuous infusion over a selected time period.

[0254] The compounds of the present invention can be administered orally, for example, with an inert diluent or with an assimilated edible carrier, or they can be encapsulated in hard or soft-shell gelatin capsules, or they can be compressed into tablets, or they can be directly mixed with food in the diet. For oral therapeutic administration, the compounds of the present invention can be mixed with excipients and used in the form of ingestible tablets, sublingual tablets, lozenges, capsules, elixirs, suspensions, syrups, and rice paper capsules. The compounds of the present invention can also be administered parenterally. Solutions of the compounds of the present invention can be prepared in water appropriately mixed with a surfactant (such as hydroxypropyl cellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, as well as in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth. The routine procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20th edition) and The United States Pharmacopeia: The National Formulary (USP 24 NF 19), published in 1999. Suitable drug forms for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be readily administered via a syringe. Compositions for nasal administration can be readily formulated as aerosols, drops, gels, and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are typically present in a sterile form in a single- or multiple-dose amount in a closed container, which may be in the form of a tube or refilled for use with a nebulizer. Alternatively, the sealed container may be a unit dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser equipped with a metering valve intended to be discarded after use. In the case of a dosage form that includes an aerosol dispenser, it will contain a propellant, which may be a compressed gas, such as compressed air, or an organic propellant, such as chlorofluorocarbons. Aerosol dosage forms may also be in the form of pump nebulizers. Compositions suitable for sublingual or oral administration include tablets, lozenges, and soft lozenges, in which the active ingredient is formulated with a carrier (such as sugar, gum arabic, tragacanth, gelatin, and glycerin). Compositions for rectal administration are conveniently in suppository form, containing a conventional suppository base, such as cocoa butter. The compounds described herein may be administered intratumorally, for example, as intratumoral injection. Intratumoral injection is direct injection into the tumor's vascular system and is particularly contemplated for discrete, solid, accessible tumors. Local, regional, or systemic administration may also be appropriate.The compounds described herein can be advantageously accessed by administering a single injection or multiple injections to the tumor, for example, at intervals of approximately 1 cm. In cases of surgical intervention, the invention can be used prior to surgery, such as to make inoperable tumors resectable. Continuous administration can also be applied where appropriate, for example, by inserting a catheter into the tumor or the tumor's vascular system.

[0255] As noted herein, the compounds of the present invention may be administered to animals, such as humans, alone or in combination with pharmaceutically acceptable carriers, in proportions determined by the solubility and chemical properties of the compounds, the chosen route of administration, and standard pharmaceutical practice.

[0256] dose

[0257] The dosage of the compounds of the present invention and / or compositions comprising the compounds of the present invention can vary according to many factors, such as the pharmacokinetic properties of the compounds; the administration mode; the recipient's age, health, and weight; the nature and severity of symptoms; the frequency of treatment and the type of concurrent treatment (if any); and the clearance of the compounds in the animals to be treated. Those skilled in the art can determine an appropriate dosage based on the above factors. The compounds of the present invention can be administered initially at a suitable dose, which can be adjusted as needed based on clinical response. Generally, satisfactory results can be obtained when the compounds of the present invention are administered to humans at daily doses, for example, from 0.05 mg to 3000 mg (measured in solid form). Dosage ranges include, for example, 10-1000 mg (e.g., 50-800 mg). In some implementations, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg of the compound is administered.

[0258] Alternatively, the patient's weight can be used to calculate the dosage. For example, the dosage of the compound or pharmaceutical composition thereof administered to the patient may be in the range of 0.1-100 mg / kg (e.g., 0.25-25 mg / kg). In exemplary, non-limiting embodiments, the dosage may be in the range of 0.5-5.0 mg / kg (e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / kg) or 5.0-20 mg / kg (e.g., 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg). Example

[0259] The following abbreviations are used in the following embodiments.

[0260] Acetyl group

[0261] ACN or MeCN acetonitrile

[0262] AcOH (acetic acid)

[0263] Ac2O Acetic anhydride

[0264] aq. water-based

[0265] Boc tert-butoxycarbonyl

[0266] Bu or n-Bu butyl

[0267] CDI 1,1′-carbonyldiimidazole

[0268] DCE or 1,2-DCE 1,2-dichloroethane

[0269] DCM dichloromethane

[0270] DIAD (Diisopropyl Azodicarbonate)

[0271] DIPEA or DIEA NN-diisopropylethylamine

[0272] DMAP 4-(dimethylamino)pyridine

[0273] DME 1,2-dimethoxyethane

[0274] DMF NN-dimethylformamide

[0275] DMSO (dimethyl sulfoxide)

[0276] EA or EtOAc (ethyl acetate)

[0277] EDCI N-(3-Dimethylaminopropyl)-N′-Ethylcarbodiimide Hydrochloride

[0278] equivalent

[0279] Et3N or TEA triethylamine

[0280] EtOH (ethanol)

[0281] FA Formic acid

[0282] h or hr hours

[0283] HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0284] HOAt 1-hydroxy-7-azabenzotriazole

[0285] HOBt or HOBT 1-hydroxybenzotriazole hydrate

[0286] iPr isopropyl

[0287] MeOH (methanol)

[0288] Me4t-BuXphos di-tert-butyl-[2,3,4,5-tetramethyl-6-(2,4,6-triisopropylphenyl)phenyl]phosphine

[0289] min minutes

[0290] MTBE tert-butyl methyl ether

[0291] n-BuLi n-Butyllithium

[0292] NMP 1-Methyl-2-pyrrolidone

[0293] OAc acetate

[0294] Pd / C carbon-supported palladium

[0295] PDC pyridinium dichromate

[0296] PdCl2(dtbpf) or dichloro[1,1′-bis(di-tert-butylphosphino)ferrocene]palladium(II)

[0297] Pd(dtbpf)Cl2

[0298] PdCl2 (dPPf) or [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride

[0299] Pd(dppf)Cl2

[0300] Pd2(dba)3 tris(dibenzylacetone)dipalladium(0)

[0301] Pd(PPh3)4 tetrakis(triphenylphosphine)palladium(0)

[0302] Pd(PPh3)2Cl2 dichlorobis(triphenylphosphine)palladium(II)

[0303] PE petroleum ether

[0304] PPh3 triphenylphosphine

[0305] Pr Propylene

[0306] Pyridine

[0307] Rac racemic

[0308] Rf retention factor

[0309] rt or rt room temperature

[0310] sat. saturated

[0311] SFC Supercritical Fluid Chromatography

[0312] t-Bu tert-butyl

[0313] tBuXphos-Pd-G3 or [2-(2-aminophenyl)phenyl]-methylsulfonyloxypalladium; di-tert-butyl

[0314] tBuXphos Pd G3 or -[2-(2,4,6-triisopropylphenyl)phenyl]phosphine

[0315] t-BuXphos-Pd(gen 3)

[0316] TFA (trifluoroacetic acid)

[0317] Tf2O trifluoromethanesulfonic anhydride

[0318] THF Tetrahydrofuran

[0319] TLC (Thin Layer Chromatography)

[0320] Xantphos-Pd-G3 [2-(2-aminophenyl)phenyl]-methylsulfonyloxy-palladium; (5-diphenylphosphine-9,9-dimethyl-xanthon-4-yl)-diphenylphosphine

[0321] XPhos Pd G3 (2-Dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)methanesulfonate

[0322] Example 1.2 Preparation of 6-amino-5-[bicyclo[1.1.1]pentan-1-yl(methyl)amino]pyridazin-3-yl)phenol

[0323]

[0324] Step 1: Preparation of N4-[bicyclo[1.1.1]pentan-1-yl]-6-chloro-N4-methylpyridazine-3,4-diamine

[0325]

[0326] DIEA (18.6 mg, 0.144 mmol) was added to a stirred mixture of 4-bromo-6-chloropyridazine-3-amine (200 mg, 0.960 mmol) and N-methylbicyclo[1.1.1]pentane-1-amine (112 mg, 1.15 mmol) in DMSO (1.00 mL) at room temperature. The resulting mixture was stirred overnight at 130 °C. The reaction mixture was purified by reversed-phase C18 rapid chromatography under the following conditions to provide N4-[bicyclo[1.1.1]pentane-1-yl]-6-chloro-N4-methylpyridazine-3,4-diamine (71 mg, 32.3%) as a white solid. LCMS (ESI) m / z: [M+H] + =225.

[0327] Step 2: Preparation of 2-(6-amino-5-[bicyclo[1.1.1]pentan-1-yl(methyl)amino]pyridazin-3-yl)phenol

[0328]

[0329] XPhos Pd G3 (11.3 mg, 0.013 mmol) and Cs2CO3 (131 mg, 0.401 mmol) were added to a stirred mixture of N4-[bicyclo[1.1.1]pentan-1-yl]-6-chloro-N4-methylpyridazine-3,4-diamine (30.0 mg, 0.134 mmol) and 2-hydroxyphenylboronic acid (36.8 mg, 0.267 mmol) in 1,4-dioxane (4.00 mL) and H2O (1.00 mL) at room temperature. The resulting mixture was stirred overnight at 100 °C. The reaction mixture was filtered through a short diatomaceous earth mat and concentrated under vacuum. The residue was purified by preparative HPLC under the following conditions (water:ACN:NH4HCO3) to provide the title compound (8 mg, 21.1%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ14.04 (s, 1H), 7.87 (dd, J=8.3, 1.6Hz, 1H), 7.67 (s, 1H), 7.25 (td, J= 7.6, 7.0, 1.6Hz, 1H), 6.94-6.86 (m, 2H), 6.38 (s, 2H), 2.76 (s, 3H), 2.45 (s, 1H), 1.97 (s, 6H). LCMS(ESI)m / z:[M+H] + =283.25.

[0330] Example 2. Preparation of (3-amino-N-methylbicyclo[1.1.1]pentane-1-carboxamide)

[0331]

[0332] Step 1: Preparation of N-[3-(methylcarbamoyl)bicyclo[1.1.1]pentan-1-yl]tert-butyl carbamate

[0333]

[0334] DIEA (3.98 g, 30.8 mmol) was added to a stirred mixture of 3-[(tert-butoxycarbonyl)amino]bicyclo[1.1.1]pentane-1-carboxylic acid (1.40 g, 6.16 mmol), HOBT (1.00 g, 7.39 mmol), and EDC·HCl (1.42 g, 7.39 mmol) in DCM (60.0 mL) at room temperature under a nitrogen atmosphere. After stirring at room temperature for 0.5 h, methylamine (2 M in THF, 15.4 mL, 30.8 mmol) was added to the reaction mixture. After stirring at room temperature for 16 h, the resulting mixture was diluted with EtOAc and quenched with ice water at 0 °C. The resulting mixture was extracted three times with EtOAc. The combined organic layers were washed twice with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography to provide N-[3-(methylcarbamoyl)bicyclo[1.1.1]pentan-1-yl]tert-butyl carbamate (800 mg, 54.0%) as a white solid. LCMS (ESI) m / z [M+H] + =241.

[0335] Step 2: Preparation of (3-amino-N-methylbicyclo[1.1.1]pentane-1-carboxamide hydrochloride)

[0336]

[0337] The solution of N-[3-(methylcarbamoyl)bicyclo[1.1.1]pentan-1-yl]carbamate tert-butyl ester (900 mg, 3.75 mmol) in HCl in 1,4-dioxane (20.00 mL) and DCM (20.00 mL) was stirred at room temperature for 3 h. The resulting mixture was filtered, and the filter cake was washed twice with DCM. The filter cake was dried under reduced pressure. (3-amino-N-methylbicyclo[1.1.1]pentan-1-carboxamide hydrochloride (750 mg, crude product) was used directly for the next step without further purification. LCMS (ESI) m / z [M+H] + =141.

[0338] Example 3. Preparation of intermediates.

[0339] The following compounds in Table A1 were prepared using common starting materials, 4-bromo-6-chloropyridazine-3-amine, and appropriate amines, according to the procedure in Example 1.

[0340] Table A1. Intermediates

[0341]

[0342] Example 4. Preparation of 2-(6-amino-5-((3-aminobicyclo[1.1.1]pentan-1-yl)amino)pyridazin-3-yl)phenol

[0343]

[0344] In an 8-mL sealed test tube, intermediate C (40.0 mg, 0.104 mmol), DCM (2.00 mL), and TFA (1.00 mL) were added. The resulting solution was stirred at room temperature for 1 h. The solid was removed by filtration and the filtrate was concentrated. The crude product was purified by preparative HPLC (water, ACN, NH4HCO3) to provide the title compound (9.3 mg, 31%) as a grayish-white solid. 1 HNMR (400MHz, DMSO-d6) 14.86 (s, 1H), δ7.69 (dd, J=8.0, 1.6Hz, 1H), 7.22 (ddd, J=8.4, 7.2, 1.6Hz, 1H), 7.00 (s, 1H), 6.95-6.81 (m, 3H), 6.30 (s, 2H), 2.15 (s, 6H). LCMS (ESI) m / z: [M+H] + =284.15.

[0345] Example 5. Preparation of 2-(6-amino-5-[[3-(aminomethyl)bicyclo[1.1.1]pentan-1-yl]amino]pyridazin-3-yl)phenol

[0346]

[0347] The title compound was prepared using tert-butyl carbamate ((3-(((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)amino)methyl)bicyclo[1.1.1]pentan-1-yl)methyl)carbamate according to the procedure in Example 4. 1 H NMR (400MHz, DMSO-d6) δ8.19 (s, 1H), 7.69 (d, J=7.2Hz, 1H), 7.24 (td, J=7.7, 1.5Hz, 1H ), 7.02 (d, J=9.3Hz, 2H), 6.89 (t, J=7.7Hz, 2H), 6.32 (s, 2H), 3.10 (s, 2H), 2.21 (s, 6H). LCMS(ESI)m / z:[M+H] + =298.1.

[0348] Example 6. Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]oxy]methyl)bicyclo[1.1.1]pentane-1-carboxylon

[0349]

[0350] Step 1: Preparation of 3-[[(3-amino-6-chloropyridazin-4-yl)oxy]methyl]bicyclo[1.1.1]pentane-1-carboxylon

[0351]

[0352] NaH (1.17 g, 0.049 mmol) was added to a stirred mixture of 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylonitrile (2.00 g, 16.2 mmol) and 4-bromo-6-chloropyridazin-3-amine (6.77 g, 0.032 mmol) in DMF (20.0 mL) at room temperature. The resulting mixture was stirred overnight at 60 °C. The residue was purified by C18 reversed-phase rapid chromatography to provide 3-[[(3-amino-6-chloropyridazin-4-yl)oxy]methyl]bicyclo[1.1.1]pentane-1-carboxylonitrile (2.3 g, 53.7%) as a brownish-yellow solid. LCMS (ESI) m / z: [M+H] + =251.1

[0353] Step 2: Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazine-4-yl]oxy]methyl)bicyclo[1.1.1]pentane-1-carboxylon

[0354]

[0355] Xphos Pd G3 (10.1 mg, 0.012 mmol) and Cs2CO3 (117 mg, 0.359 mmol) were added to a stirred mixture of 3-[[(3-amino-6-chloropyridazin-4-yl)oxy]methyl]bicyclo[1.1.1]pentane-1-carboxynitrile (30.0 mg, 0.120 mmol) and 2-hydroxyphenylboronic acid (33.0 mg, 0.239 mmol) in dioxane (3.00 mL) and H2O (0.50 mL) at room temperature. The resulting mixture was stirred overnight at 100 °C. The crude product was purified by preparative HPLC (water:ACN:FA) to provide the title compound (14 mg, 37.1%) as a white solid. 1H NMR (300MHz, DMSO-d6) δ14.34 (s, 1H), 7.93 (dd, J=8.4, 1.6Hz, 1H), 7.56 (s, 1H), 7.2 5(td, J=7.6, 1.5Hz, 1H), 6.95-6.83(m, 2H), 6.62(s, 2H), 4.29(s, 2H), 2.39(s, 6H). LCMS(ESI)m / z:[M+H] + =309.15

[0356] Example 7: Preparation of N-[3-(2-hydroxyethyl)bicyclo[1.1.1]pentan-1-yl]acetamide

[0357]

[0358] Step 1: Preparation of methyl 2-[3-acetamidobicyclo[1.1.1]pentan-1-yl]acetate

[0359]

[0360] Ac₂O (63.9 mg, 0.626 mmol) and TEA (158 mg, 1.57 mmol) were added to a stirred solution of 2-[3-aminobicyclo[1.1.1]pentan-1-yl]acetate hydrochloride (100 mg, 0.522 mmol) in DCM (10.0 mL) at room temperature. The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then quenched with water at room temperature. The resulting mixture was extracted three times with CH₂Cl₂. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to provide 2-[3-acetamidobicyclo[1.1.1]pentan-1-yl]acetate (130 mg, crude) as a grayish-white solid. LCMS (ESI) m / z: [M+H] + =198.

[0361] Step 2: Preparation of N-[3-(2-hydroxyethyl)bicyclo[1.1.1]pentan-1-yl]acetamide

[0362]

[0363] DIBAL-H (1 M, 2.8 mL, 2.8 mmol) was added to a stirred solution of 2-[3-acetamidobicyclo[1.1.1]pentan-1-yl]acetate (110 mg, 0.558 mmol) in DCM (10.0 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 h and then quenched with water. The resulting mixture was extracted three times with EtOAc. The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to provide N-[3-(2-hydroxyethyl)bicyclo[1.1.1]pentan-1-yl]acetamide (88 mg, crude) as a colorless oil. LCMS (ESI) m / z: [M+H] + =170.

[0364] Example 8. Preparation of intermediates

[0365] The following compounds in Table A2 were prepared using common starting materials, 4-bromo-6-chloropyridazine-3-amine, and appropriate alcohols, according to the procedure in Example 6.

[0366] Table A2. Intermediates

[0367]

[0368] Example 9. Preparation of 2-(6-amino-5-[[3-(aminomethyl)bicyclo[1.1.1]pentan-1-yl]methoxy]pyridazin-3-yl)phenol

[0369]

[0370] In a pressure vessel, Pd / C (10%, 100 mg) was added to a solution of 2-(6-amino-5-[[3-(cyano)bicyclo[1.1.1]pentan-1-yl]methoxy]pyridazin-3-yl)phenol (120 mg, 0.389 mmol) in MeOH (10 mL). The mixture was hydrogenated overnight at room temperature under 30 psi hydrogen pressure. The reaction mixture was filtered through a short diatomaceous earth pad and concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography under the following conditions (MeOH:water) to provide 2-(6-amino-5-[[3-(aminomethyl)bicyclo[1.1.1]pentan-1-yl]methoxy]pyridazin-3-yl)phenol (29 mg, 23.9%) as an off-white solid. LCMS (ESI) m / z [M+H] + =313.

[0371] Example 10. Preparation of N-(3-((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)amino)bicyclo[1.1.1]pentan-1-yl)acetamide

[0372]

[0373] In a 25 mL round-bottom flask, 2-(6-amino-5-[[3-(aminomethyl)bicyclo[1.1.1]pentan-1-yl]methoxy]pyridazin-3-yl)phenol (40.0 mg, 0.141 mmol), DCM (2.00 mL), DIEA (36.5 mg, 0.282 mmol), and Ac2O (14.4 mg, 0.141 mmol) were added. The resulting solution was stirred at 0 °C for 2 h in a water / ice bath. The resulting mixture was concentrated under vacuum. The residue was dissolved in 2 mL of DMF. The solid was filtered off. The crude product was purified by preparative HPLC (water:ACN:NH4HCO3) to provide the title compound (5.2 mg, 11.3%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ14.77 (s, 1H), 8.57 (s, 1H), 7.72 (dd, J=8.1, 1.6Hz, 1H), 7.22 (ddd, J=8.5, 7.2, 1.6Hz, 1H), 6.99 (d, J=13.9Hz, 2H), 6.92-6.80 (m, 2H), 6.31 (s, 2H), 2.42 (s, 6H), 1.81 (s, 3H). LCMS(ESI)m / z:[M+H] + =325.9.

[0374] Example 11. Preparation of intermediates.

[0375] Using appropriate amines, synthesize the following compounds in Table A3 according to the synthesis procedure described in Example 10.

[0376] Table A3. Intermediates

[0377]

[0378] Example 12. Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino]methyl)bicyclo[1.1.1]pentane-1-carboxylic acid

[0379]

[0380] KOH (110 mg, 1.95 mmol) was added to a solution of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino]methyl)bicyclo[1.1.1]pentane-1-carboxynitrile (10.0 mg, 0.033 mmol) in H₂O (0.20 mL) and MeOH (1.80 mL). The resulting solution was stirred at 80 °C for 8 h. The crude reaction solution was purified directly by preparative HPLC (water:MeCN:FA) to yield the title compound (10.3 mg, 31.7%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ7.87 (dd, J=8.0, 1.6Hz, 1H), 7.37-7.12 (m, 1H), 7.02 (s, 1H) ), 6.95-6.71 (m, 2H), 6.33 (s, 2H), 6.16 (s, 1H), 3.45 (d, J=5.5Hz, 2H), 1.96 (s, 6H). LCMS(ESI)m / z:[M+H] + =327.00.

[0381] Example 13. Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]oxy]methyl)bicyclo[1.1.1]pentane-1-carboxylic acid

[0382]

[0383] FHT-0015071-001 was prepared using 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]oxy]methyl)bicyclo[1.1.1]pentane-1-carboxynitrile and sodium hydroxide, according to the procedure in Example 12. 1 H NMR (300MHz, DMSO-d6) δ7.95 (dd, J=8.3, 1.6Hz, 1H), 7.58 (s, 1H), 7.25 (td, J=7 .6, 7.0, 1.5Hz, 1H), 6.95-6.84(m, 2H), 6.58(s, 2H), 4.31(s, 2H), 2.08(s, 6H). LCMS(ESI)m / z:[M+H] + =328.15.

[0384] Example 14. Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino]methyl)bicyclo[1.1.1]pentane-1-carboxamide

[0385]

[0386] H2O2 (30% in H2O, 44.3 mg, 1.30 mmol) was added to a solution of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino]methyl)bicyclo[1.1.1]pentane-1-carboxynitrile (40.0 mg, 0.130 mmol) and K2CO3 (36.0 mg, 0.260 mmol) in DMSO (2.00 mL). The resulting solution was stirred at 25 °C for 15 h. The crude reaction mixture was purified directly by preparative HPLC (water:ACN:NH4HCO3) to yield the title compound (11.2 mg, 26.3%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ15.12 (s, 1H), 7.87 (dd, J=8.0, 1.6Hz, 1H), 7.37-7.12 (m, 2H), 7.02 (s, 1H), 6.92-6.58 (m, 3H), 6.32 (s, 2H), 6.22 (t, J=5.6Hz, 1H), 3.46 (d, J=5.6Hz, 2H), 1.88 (s, 6H). LCMS(ESI)m / z:[M+H] + =326.05.

[0387] Example 15. Preparation of 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylic acid

[0388]

[0389] Step 1: Preparation of methyl 3-[2-(3-amino-6-chloropyridazin-4-yl)ethynyl]bicyclo[1.1.1]pentane-1-carboxylate

[0390]

[0391] Pd(PPh3)4 (693 mg, 0.599 mmol) and Et3N (5.00 mL) were added dropwise / partially to a stirred mixture of methyl 3-ethynylbicyclo[1.1.1]pentane-1-carboxylate (450 mg, 3.00 mmol) and 4-bromo-6-chloropyridazin-3-amine (899 mg, 4.32 mmol) in THF at 80 °C, and the mixture was stirred overnight at 80 °C. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (water:ACN:FA) to provide methyl 3-[2-(3-amino-6-chloropyridazin-4-yl)ethynyl]bicyclo[1.1.1]pentane-1-carboxylate (120 mg, 14.4%) as a white solid. LCMS (ESI) m / z: [M+H] + =278.

[0392] Step 2: Preparation of methyl 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylate

[0393]

[0394] Under a nitrogen atmosphere, Cs₂CO₃ (422 mg, 1.30 mmol) and XPhos PdG₃ (73.2 mg, 0.086 mmol) were added fractionally to a stirred mixture of methyl 3-[2-(3-amino-6-chloropyridazin-4-yl)ethynyl]bicyclo[1.1.1]pentane-1-carboxylate (120 mg, 0.432 mmol) and 2-hydroxyphenylboronic acid (179 mg, 1.30 mmol) in dioxane (4.00 mL) and H₂O (1.00 mL) at room temperature. The resulting mixture was stirred at 80 °C for 2 h under a nitrogen atmosphere. The mixture was then concentrated under reduced pressure. The crude product (mg) was purified by preparative HPLC (water:ACN:FA) to provide methyl 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylate (50 mg, 34.5%) as a white solid. LCMS (ESI) m / z: [M+H] + =336.

[0395] Step 3: Preparation of 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylic acid

[0396]

[0397] LiOH (1.07 mg, 0.045 mmol) and H₂O (0.20 mg) were added fractionally to a stirred solution of methyl 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylate (5.00 mg, 0.015 mmol) in THF under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (water:ACN:FA) to provide the title compound (8.5 mg, 29.6%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ13.20 (s, 1H), 12.40 (s, 1H), 8.22 (s, 1H), 7.88 (dd, J=8. 0, 1.6Hz, 1H), 7.24 (ddd, J=8.4, 7.2, 1.6Hz, 1H), 6.95-6.84 (m, 4H), 2.38 (s, 6H). LCMS(ESI)m / z:[M+H] + =322.20.

[0398] Example 16. Preparation of 3-(2-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethyl)bicyclo[1.1.1]pentane-1-carboxylic acid

[0399]

[0400] Step 1: Preparation of methyl 3-(2-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethyl)bicyclo[1.1.1]pentane-1-carboxylate

[0401]

[0402] Pd / C (10%, 200 mg) was added to a solution of methyl 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylate (200 mg, 0.596 mmol) in 10 MeOH (10 mL) under a nitrogen atmosphere. The mixture was hydrogenated at room temperature for 2 h under a hydrogen balloon. The mixture was filtered through a diatomaceous earth mat and concentrated under reduced pressure to provide methyl 3-(2-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethyl)bicyclo[1.1.1]pentane-1-carboxylate (150 mg, 74.1%) as a pale yellow solid. LCMS (ESI) m / z: [M+H] + =340.

[0403] Step 2: Preparation of 3-(2-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethyl)bicyclo[1.1.1]pentane-1-carboxylic acid

[0404]

[0405] Lithium hydroxide monohydrate (247 mg, 5.89 mmol) in H₂O (2.00 mL) was added to a stirred solution of methyl 3-(2-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethyl)bicyclo[1.1.1]pentane-1-carboxylate (200 mg, 0.589 mmol) in THF (10.0 mL). The resulting mixture was stirred at room temperature for 2 h. The crude product was purified by preparative HPLC under the following conditions (water:ACN:FA) to provide the title compound (4.8 mg, 2.50%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ7.94 (s, 1H), 7.89 (dd, J=8.3, 1.7Hz, 1H), 7.30-7.21 (m, 1H), 6.96-6.88 (m, 2H), 2.57-2.46 (m, 2H), 1.95-1.77 (m, 8H). LCMS(ESI)m / z:[M+H] + =326.15.

[0406] Example 17. Preparation of 3-((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)amino)bicyclo[1.1.1]pentane-1-carboxylic acid

[0407]

[0408] Add 3-([3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino)-N-methylbicyclo[1.1.1]pentane-1-carboxamide (195 mg, 0.599 mmol) to a stirred solution of HCl aqueous solution (12 N, 10.0 mL). Stir the resulting mixture at 90 °C for 2 h. Concentrate the resulting mixture under reduced pressure. Purify the residue by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide the title compound (90 mg, 48.1%) as an off-white solid. 1 H NMR (300MHz, DMSO-d6) δ13.87 (s, 1H), 7.82 (d, J=7.8Hz, 1H), 7.23-7.20 (m, 1H), 7.07(s, 1H), 7.01(s, 1H), 6.95-6.82(m, 2H), 6.32(s, 2H), 2.44(s, 6H). LCMS(ESI)m / z:[M+H] + =313.10.

[0409] Example 18. Preparation of 3-((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethynyl)bicyclo[1.1.1]pentane-1-carboxamide

[0410]

[0411] HATU (66.3 mg, 0.174 mmol) and DIEA (56.3 mg, 0.436 mmol) were added to a stirred solution of 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylic acid (28.0 mg, 0.087 mmol) and NH4Cl (14.0 mg, 0.261 mmol) in DMF (2.00 mL). The resulting mixture was stirred at room temperature for 2 h. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide the title compound (3.5 mg, 12.5%) as an off-white solid. 1 H NMR (300MHz, DMSO-d6) δ13.24 (s, 1H), 8.23 ​​(s, 1H), 7.89 (dd, J=8.0, 1.6Hz, 1H) , 7.38 (s, 1H), 7.31-7.19 (m, 1H), 7.04 (s, 1H), 6.96-6.82 (m, 4H), 2.32 (s, 6H). LCMS(ESI)m / z:[M+H] + =321.10.

[0412] Example 19. Preparation of intermediates.

[0413] Using a suitable carboxylic acid, synthesize the following compounds in Table A4 according to the synthesis procedure described in Example 18.

[0414] Table A4. Intermediates

[0415]

[0416] Example 20. Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]oxy]methyl)-N-methylbicyclo[1.1.1]pentane-1-carboxamide

[0417]

[0418] EDCI (35.1 mg, 0.184 mmol), HOBT (24.8 mg, 0.184 mmol), and DIEA (35.5 mg, 0.276 mmol) were added to a stirred mixture of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]oxy]methyl)bicyclo[1.1.1]pentane-1-carboxylic acid (30.0 mg, 0.092 mmol) and methylamine (5.69 mg, 0.184 mmol) in DMF (1.50 mL) at room temperature. The resulting mixture was stirred at room temperature for 4 h. The crude product was purified by preparative HPLC (water:ACN:FA) to provide the title compound (21 mg, 66.7%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ14.38 (s, 1H), 7.95 (dd, J=8.4, 1.6Hz, 1H), 7.70 (d, J=4.8Hz, 1H), 7.59 (s, 1H), 7.25 ( td, J=7.6, 7.0, 1.5Hz, 1H), 6.97-6.82 (m, 2H), 6.56 (s, 2H), 4.33 (s, 2H), 2.56 (d, J=4.6Hz, 3H), 2.00 (s, 6H). LCMS(ESI)m / z:[M+H] + =341.2

[0419] Example 21. Preparation of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino]methyl)-N-methylbicyclo[1.1.1]pentane-1-carboxamide

[0420]

[0421] To a stirred solution of 3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]amino]methyl)bicyclo[1.1.1]pentane-1-carboxylic acid (44.0 mg, 0.135 mmol), HOBt (36.5 mg, 0.270 mmol), and EDCI (51.7 mg, 0.270 mmol) in DMF (1 mL), methylamine hydrochloride (18.3 mg, 0.270 mmol) and DIEA (87.2 mg, 0.674 mmol) were added. The resulting mixture was stirred at 25 °C for 5 h. The crude reaction mixture was purified directly by preparative HPLC (water:ACN:NH4HCO3) to provide the title compound (7.5 mg, 15.4%) as a white solid. 1¹H NMR (300 MHz, methanol-d⁴) δ 7.74 (dd, J = 8.3, 1.6 Hz, 1H), 7.27–7.18 (m, 1H), 7.04 (s, 1H), 6.94–6.83 (m, 2H), 3.52 (s, 2H), 2.68 (s, 3H), 2.01 (s, 6H). LCMS (ESI) m / z: [M+H] + =340.05.

[0422] Example 22. Preparation of 3-((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)ethynyl)-N-methylbicyclo[1.1.1]pentane-1-carboxamide

[0423]

[0424] HATU (66.3 mg, 0.174 mmol) and DIEA (56.3 mg, 0.436 mmol) were added to a stirred solution of 3-[2-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]ethynyl]bicyclo[1.1.1]pentane-1-carboxylic acid (28.0 mg, 0.087 mmol) and methylamine hydrochloride (11.8 mg, 0.174 mmol) in DMF (2.0 mL). The resulting mixture was stirred at room temperature for 2 h. The crude product was purified by preparative HPLC (water:ACN:FA) to provide the title compound (7.6 mg, 26.1%) as a grayish-white solid. 1 H NMR (400MHz, DMSO-d6) δ13.11 (s, 1H), 8.23 ​​(s, 1H), 7.90-7.80 (m, 2H), 7.29-7. 21 (m, 1H), 7.01 (s, 2H), 6.95-6.84 (m, 2H), 2.57 (d, J=4.6Hz, 3H), 2.32 (s, 6H). LCMS(ESI)m / z:[M+H] + =335.25.

[0425] Example 23. Preparation of intermediates

[0426] The following compounds in Table A5 were synthesized using appropriate carboxylic acids and amines, following the synthesis procedure described in Example 22.

[0427] Table A5. Intermediates

[0428]

[0429]

[0430] Example 24. Preparation of (2S,4R)-1-[(2S)-2-(10-aminodecanoylamino)-3,3-dimethylbutyryl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (I-1)

[0431]

[0432] Step 1: Preparation of N-(9-[[(2S)-1-[(2S,4R)-4-hydroxy-2-([[4-(4-methyl-1,3-thiazo-5-yl)phenyl]methyl]carbamoyl)pyrrolidine-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl]nonyl)tert-butyl carbamate

[0433]

[0434] DIEA (0.90 g, 0.007 mmol) was added to a stirred mixture of (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (1.00 g, 0.002 mmol) and 10-[(tert-butoxycarbonyl)amino]decanoic acid (0.73 g, 0.003 mmol) in DCM (20.0 mL). The mixture was stirred at room temperature for 5 min, and then HATU (1.32 g, 0.003 mmol) was added. After stirring at room temperature for 2 h, water was added to the mixture, and the mixture was extracted four times with DCM. The organic fractions were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to produce a crude product, which was then purified by silica gel rapid chromatography (DCM / MeOH) to yield N-(9-[[(2S)-1-[(2S,4R)-4-hydroxy-2-([[4-(4-methyl-1,3-thiazo-5-yl)phenyl]methyl]carbamoyl)pyrrolidone-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl]nonyl)tert-butyl carbamate (1.67 g, 92.5%). LCMS (ESI) m / z [M+H] + =700.

[0435] Step 2: Preparation of (2S,4R)-1-[(2S)-2-(10-aminodecanoylamino)-3,3-dimethylbutyryl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (I-1)

[0436]

[0437] TFA (2 mL, 26.9 mmol) was added to a stirred solution of N-(9-[[(2S)-1-[(2S,4R)-4-hydroxy-2-([[4-(4-methyl-1,3-thiazo-5-yl)phenyl]methyl]carbamoyl)pyrrolidone-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl]nonyl)tert-butyl carbamate (1.67 g, 2.39 mmol) in DCM (10 mL), and the mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under vacuum. A 5% K₂CO₃ solution (MeOH / water = 5 / 2) was added to the reaction mixture, and the pH was adjusted to approximately 8-9. The mixture was stirred at room temperature for 2 h. The final mixture was concentrated under vacuum, and the crude material was purified by reversed-phase C18 rapid chromatography (water:ACN:NH4HCO3) to provide (2S,4R)-1-[(2S)-2-(10-aminodecanoylamino)-3,3-dimethylbutyryl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (I-1, 1.11 g, 73.68%). LCMS (ESI) m / z: [M+H] + =600.40.

[0438] Example 25. Preparation of intermediates

[0439] The intermediates listed in Table B1 were prepared by a similar method to that used to prepare intermediate I-1 in Example 24.

[0440] Table B1. Intermediates

[0441]

[0442]

[0443] Example 26. Preparation of 5-(2-(4-aminopiperidin-1-yl)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione TFA (I-6)

[0444]

[0445] Step 1: Preparation of 5-(2-bromoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0446]

[0447] To a solution of 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione (1.37 g, 5.00 mmol) in THF (35 mL), 2-bromoethanol (0.94 g, 7.49 mmol), PPh3 (1.97 g, 7.49 mmol), and DIAD (1.52 g, 7.49 mmol) were added at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The residue was purified by reversed-phase 18-phase rapid chromatography (water:ACN) to provide 5-(2-bromoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1.52 g, 79.82%) as a black solid. LCMS (ESI) m / z: [M+H] + =381.38.

[0448] Step 2: Preparation of tert-butyl carbamate (1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethyl)piperidin-4-yl)carbamate

[0449]

[0450] To a solution of 5-(2-bromoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1.52 g, 3.99 mmol) in ACN (35.0 mL), N-(piperidin-4-yl)carbamate tert-butyl ester (0.80 g, 3.99 mmol), KI (0.66 g, 3.99 mmol), and K₂CO₃ (1.65 g, 12.0 mmol) were added. The resulting solution was stirred at 70 °C for 2 h. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide (1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethyl)piperidin-4-yl)carbamate tert-butyl ester (1.402 g, 70.24%) as a colorless solid. LCMS(ESI)m / z:[M+H] + =501.

[0451] Step 3: Preparation of 5-(2-(4-aminopiperidin-1-yl)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione TFA (I-6)

[0452]

[0453] TFA (10.0 mL, 135 mmol) was added to a solution of tert-butyl (1.66 g, 3.32 mmol) of (1-(2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethyl)piperidin-4-yl)carbamate (10.0 mL) in DCM. The resulting solution was stirred at room temperature for 3 h and then concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to yield 5-(2-(4-aminopiperidin-1-yl)ethoxy)-2-(2,6-dioxopiridin-3-yl)isoindoline-1,3-dione carbamate (I-6, 840 mg, 62.52%) as a white solid. LCMS (ESI) m / z: [M+H] + =401.17.

[0454] Example 27. Preparation of intermediates

[0455] The intermediates listed in Table B2 were prepared using a suitable substituted bromool and N-Boc-diamine via a route similar to that described in Example 26.

[0456] Table B2. Intermediates

[0457]

[0458]

[0459] Example 28. Preparation of 4-(azacyclobutane-3-ylmethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione TFA (I-7)

[0460]

[0461] Step 1: Preparation of tert-butyl 3-((toluenesulfonyloxy)methyl)azacyclobutane-1-carboxylate

[0462]

[0463] To a stirred solution of tert-butyl 3-(hydroxymethyl)azacyclobutane-1-carboxylate (1.87 g, 9.99 mmol) in DCM (50.0 mL), DMAP (0.18 g, 1.50 mmol), TEA (2.53 g, 25.0 mmol), and p-toluenesulfonyl chloride (2.86 g, 15.0 mmol) were added at 0 °C. The resulting mixture was stirred at 0 °C for 2 h, then allowed to warm to room temperature and stirred for another 5 h. The residue was purified by silica gel column chromatography (petroleum ether / THF) to provide tert-butyl 3-((toluenesulfonyloxy)methyl)azacyclobutane-1-carboxylate (2.65 g, 77.7%) as a colorless oil. LCMS (ESI) m / z: [M+H] + =342.

[0464] Step 2: Preparation of tert-butyl 3-([[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]methyl)azacyclobutane-1-carboxylate

[0465]

[0466] To a solution of tert-butyl 3-((toluenesulfonyloxy)methyl)azacyclobutane-1-carboxylate (2.30 g, 8.39 mmol) in DMF (15.0 mL), 2-(2,6-dioxadiazin-3-yl)-4-hydroxyisoindoline-1,3-dione (2.86 g, 8.39 mmol) and Na₂CO₃ (1.33 g, 12.6 mmol) were added. The resulting mixture was stirred at 80 °C for 5 h under a dry nitrogen atmosphere. The reaction mixture was quenched with water at room temperature. The resulting mixture was extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to provide tert-butyl 3-([[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]methyl)azacyclobutane-1-carboxylate (3.37 g, 90.6%) as a pale yellow solid. LCMS (ESI) m / z: [M+H] + =444.

[0467] Step 3: Preparation of 4-(azacyclobutane-3-ylmethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione TFA (I-7)

[0468]

[0469] TFA (10.0 mL, 135 mmol) was added to a solution of tert-butyl 3-([[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-4-yl]oxy]methyl)azacyclobutane-1-carboxylate (2.39 g, 5.39 mmol) in DCM (10.0 mL). The resulting solution was stirred at room temperature for 3 h. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide 4-(azacyclobutane-3-ylmethoxy)-2-(2,6-dioxopiridin-3-yl)isoindoline-1,3-dione TFA (I-7, 1.71 g, 87.7%) as a pale yellow solid. LCMS (ESI) m / z: [M+H] + =344.12.

[0470] Example 29. Preparation of intermediates

[0471] The intermediates listed in Table B3 were prepared using appropriately substituted phenols and N-Boc-amino alcohols via a route similar to that described in Example 28.

[0472] Table B3. Intermediates

[0473]

[0474] Example 30. Preparation of 3-[5-([2-[2-(2-(2-aminoethoxy)ethoxy]ethyl]amino)-2-methyl-4-oxoquinazolin-3-yl]piperidine-2,6-dione TFA (I-11)

[0475]

[0476] Step 1: Preparation of tert-butyl carbamate (2-(2-(2-((3-(2,6-dioxopiridine-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-5-yl)amino)ethoxy)ethoxy)ethyl)carbamate)

[0477]

[0478] NaBH3CN (0.63 g, 9.99 mmol) was added to a stirred solution of 4-hydroxy-2,2-dimethyl-3,8,11-trioxa-5-azatridecane-13-aldehyde (1.25 g, 5.00 mmol) and 3-(5-amino-2-methyl-4-oxoquinazoline-3-yl)piperidine-2,6-dione (1.43 g, 5.00 mmol) in MeOH (30.0 mL), and the resulting mixture was stirred for 1 h. The reactants were quenched with a saturated aqueous NH4Cl solution at 0°C, the solvent was evaporated, and the resulting residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide tert-butyl (2-(2-(2-(2-((3-(2,6-dioxopiridin-3-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-5-yl)amino)ethoxy)ethoxy)ethyl)carbamate (1.29 g, 49.7%) as a yellow solid. LCMS (ESI) m / z [M+H] + =518.

[0479] Step 2: Preparation of 3-[5-([2-[2-(2-(2-aminoethoxy)ethoxy]ethyl]amino)-2-methyl-4-oxoquinazoline-3-yl]piperidine-2,6-dione TFA (I-11)

[0480]

[0481] A solution of tert-butyl carbamate (1.29 g, 2.48 mmol) and TFA (8.49 g, 74.5 mmol) in DCM (6.00 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated, and the resulting residue was purified by reversed-phase C18 rapid chromatography under the following conditions (water: ACN) to provide 3-[5-([2-[2-(2-aminoethoxy)ethoxy]ethyl]amino)-2-methyl-4-oxoquinazoline-3-yl]piperidin-2,6-dione TFA (I-11, 1.56 g, 95.1%) as a light brown solid. LCMS (ESI) m / z: [M+H] + =418.20.

[0482] Example 31. Preparation of intermediates

[0483] The intermediates listed in Table B4 were prepared using appropriately substituted anilines and aldehydes via a route similar to that described in Example 30.

[0484] Table B4. Intermediates

[0485]

[0486] Example 32. Preparation of 5-[(8-aminooctyl)amino]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione TFA (I-22)

[0487]

[0488] Step 1: Preparation of N-(8-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]amino]octyl)tert-butyl carbamate

[0489]

[0490] DIEA (7.02 g, 54.3 mmol) was added to a stirred mixture of 2-(2,6-dioxadipin-3-yl)-5-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (5.00 g, 18.1 mmol) and N-(8-aminooctyl)carbamate tert-butyl ester (6.64 g, 27.2 mmol) in NMP (50.0 mL) at 90 °C. After 3 h, water was added to the mixture, followed by extraction three times with EtOAc. The residue was purified by reversed-phase C18 rapid chromatography (water; ACN) to provide N-(8-[[2-(2,6-dioxadipin-3-yl)-1,3-dioxadipin-2,3-dihydro-1H-isoindole-5-yl]amino]octyl)carbamate tert-butyl ester (3.50 g, 36.69%) as a yellow solid. LCMS(ESI)m / z:[M+H] + =501.

[0491] Step 3: Preparation of 5-[(8-aminooctyl)amino]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione trifluoroacetate (I-22)

[0492]

[0493] TFA (3.00 mL) was added to a stirred mixture of N-(8-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]amino]octyl)carbamate (3.85 g, 7.69 mmol) in DCM (9.00 mL) at room temperature. The resulting mixture was stirred at room temperature for 1 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by rapid chromatography to provide I-22 (2.22 g, 56.2%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ11.08 (s, 1H), 7.80-7.72 (m, 1H), 7.72-7.67 (m, 2H), 7.55 (dd, 1H), 7.14 (s, 1H), 6.95 (d, 1H), 6.88-6.77 (m, 1H) , 5.08-4.98(m, 1H), 3.21-3.05(m, 1H), 2.95-2.75(m, 2H), 2.66-2.51(m, 2H), 2.12-1.94(m, 1H), 1.63-1.49(m, 3H), 1.45-1.31(m, 4H). LCMS(ESI)m / z:[M+H] + =401.21.

[0494] Example 33. Preparation of tert-butyl (4-((3-aminopropyl)sulfonyl)butyl)carbamate

[0495]

[0496] Step 1: Preparation of 4-methylbenzenesulfonic acid 4-((tert-butoxycarbonyl)amino)butyl ester

[0497]

[0498] DMAP (2.96 g, 24.2 mmol), TEA (40.9 g, 404 mmol), and p-toluenesulfonyl chloride (46.2 g, 242 mmol) were added to a stirred solution of N-(4-hydroxybutyl)carbamate tert-butyl ester (30.6 g, 162 mmol) in DCM (400 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h, and then at room temperature for 5 h. The solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / THF) to provide 4-methylbenzenesulfonic acid 4-((tert-butoxycarbonyl)amino)butyl ester (45.6 g, 82.2%) as a pale yellow oil. LCMS (ESI) m / z: [M+H] + =344.

[0499] Step 2: Preparation of S-(4-((tert-butoxycarbonyl)amino)butyl)thioacetic acid ester

[0500]

[0501] Thioacetic acid (15.2 g, 199 mmol) and K₂CO₃ (55.1 g, 398 mmol) were added to a stirred solution of 4-methylbenzenesulfonic acid 4-((tert-butoxycarbonyl)amino)butyl ester (45.6 g, 133 mmol) in ACN (300 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / THF) to provide thioacetic acid S-(4-((tert-butoxycarbonyl)amino)butyl) ester (28.7 g, 87.4%) as a pale yellow oil. LCMS (ESI) m / z: [M+H] + =248.

[0502] Step 3: Preparation of N-[3-([4-[(tert-butoxycarbonyl)amino]butyl]thioalkyl)propyl]carbamate benzyl ester

[0503]

[0504] To a solution of S-(4-((tert-butoxycarbonyl)amino)butyl)thioacetic acid (3.60 g, 14.6 mmol) in MeOH (90.0 mmol), benzyl (3-bromopropyl)carbamate (4.36 g, 16.0 mmol) and NaOMe (3.15 g, 58.2 mmol) were added. The resulting solution was stirred at room temperature for 3 h. The reaction mixture was quenched with water at room temperature. The resulting mixture was extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:NH4HCO3) to provide N-[3-([4-[(tert-butoxycarbonyl)amino]butyl]thioalkyl)propyl]carbamate (4.122 g, 71.42%) as a pale yellow oil. LCMS(ESI)m / z:[M+H] + =397.

[0505] Step 4: Preparation of N-(3-[4-[(tert-butoxycarbonyl)amino]butanesulfonyl]propyl)carbamate benzyl ester

[0506]

[0507] Add to a solution of N-[3-([4-[(tert-butoxycarbonyl)amino]butyl]thioalkyl)propyl]carbamate (4.13 g, 10.4 mmol) in MeOH (60.0 mL) (3.50 g, 20.8 mmol). The resulting solution was stirred at room temperature for 12 h and then concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:NH4HCO3) to provide N-(3-[4-[(tert-butoxycarbonyl)amino]butanesulfonyl]propyl)carbamate (2 g, 44.8%) as a white solid. LCMS (ESI) m / z: [M+H] + =429.

[0508] Step 5: Preparation of tert-butyl (4-((3-aminopropyl)sulfonyl)butyl)carbamate

[0509]

[0510] Ammonium formate (574 mg, 9.10 mmol) and 5% Pd(OH)₂ / C (978 mg, 6.96 mmol) were added to a solution of N-(3-[4-[(tert-butoxycarbonyl)amino]butanesulfonyl]propyl)carbamate (1.95 g, 4.55 mmol) in EtOH (30.0 mL). The resulting solution was stirred at 60 °C for 12 h under hydrogen atmosphere at 1 atm. The resulting mixture was filtered, the filter cake was washed three times with MeOH, and the filtrate was concentrated under reduced pressure to provide tert-butyl (4-((3-aminopropyl)sulfonyl)butyl)carbamate (1.12 g, crude product) as a black solid. LCMS (ESI) m / z: [M+H] + =295.

[0511] Example 34. Preparation of N-[2-(2-aminoethanesulfonyl)ethyl]carbamate tert-butyl ester

[0512]

[0513] Step 1: Preparation of N-(2-[[2-(1,3-dioxoisoindol-2-yl)ethyl]thioalkyl]ethyl)tert-butyl carbamate

[0514]

[0515] K₂CO₃ (11.7 g, 0.085 mol) was added to a stirred mixture of N-(2-thioalkylethyl)carbamate (5.00 g, 28.2 mmol) and N-(2-bromoethyl)phthalimide (7.17 g, 0.028 mol) in ACN (10.0 mL) at 70 °C under a dry nitrogen atmosphere. After 5 h, the resulting mixture was extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide N-(2-[[2-(1,3-dioxoisoindol-2-yl)ethyl]thioalkyl]ethyl)carbamate (8.20 g, 82.96%) as a white solid. LCMS (ESI) m / z: [M+H] + =351.

[0516] Step 2: Preparation of N-[2-[2-(1,3-dioxoisoindol-2-yl)ethanesulfonyl]ethyl]tert-butyl carbamate

[0517]

[0518] m-CPBA (12.1 g, 70.2 mmol) was added to a stirred mixture of N-(2-[[2-(1,3-dioxoisoindol-2-yl)ethyl]thioalkyl]ethyl]carbamate (8.20 g, 23.4 mmol) in DCM (100 mL) at room temperature under a dry nitrogen atmosphere. The reaction mixture was then quenched with a saturated aqueous solution of Na₂S₂O₃ at room temperature. A saturated aqueous solution of NaHCO₃ was added to the resulting mixture, and the mixture was extracted three times with EtOAc. The organic phase was separated and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide N-[2-[2-(1,3-dioxoisoindol-2-yl)ethanesulfonyl]ethyl]carbamate (8.40 g, 87.3%) as a white solid. LCMS(ESI)m / z:[M+H] + =383.

[0519] Step 3: Preparation of N-[2-(2-aminoethanesulfonyl)ethyl]carbamate tert-butyl ester

[0520]

[0521] Hydrazine hydrate (0.89 g, 17.8 mmol) was added to a stirred mixture of N-[2-[2-(1,3-dioxoisoindol-2-yl)ethanesulfonyl]ethyl]carbamate (3.40 g, 8.89 mmol) in EtOH (100 mL) at 80 °C under a dry nitrogen atmosphere. The resulting mixture was stirred at 80 °C for 1 h under a dry nitrogen atmosphere. The resulting mixture was filtered, the filter cake was washed with EtOH, and the filtrate was concentrated under reduced pressure to provide N-[2-(2-aminoethanesulfonyl)ethyl]carbamate (1.88 g, 77.9%) as a white solid. LCMS (ESI) m / z: [M+H] + =253.

[0522] Example 35. Preparation of tert-butyl 4-(azacyclobutane-3-ylmethyl)piperazine-1-carboxylate

[0523]

[0524] Step 1: Preparation of tert-butyl 4-([1-[)benzyloxy)carbonyl]azacyclobutane-3-yl]methyl)piperazine-1-carboxylate

[0525]

[0526] To a stirred solution of 3-formylazetane-1-carboxylate (2.0 g, 9.1 mmol) and piperazine-1-carboxylate tert-butyl ester (1.9 g, 10.2 mmol) in DMF (20 mL), NaBH(OAc)3 (7.6 g, 35.8 mmol) was added fractionally. The mixture was stirred overnight at 50 °C. An aqueous solution of NH4Cl was added to the mixture, and the mixture was extracted three times with EtOAc. The combined organic phases were concentrated to dryness under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide 4-([1-[(benzyloxy)carbonyl]azetane-3-yl]methyl)piperazine-1-carboxylate tert-butyl ester (2.8 g, 79.1%) as a colorless oil. LCMS (ESI) m / z [M+H] + =390.

[0527] Step 2: Preparation of tert-butyl 4-(azacyclobutane-3-ylmethyl)piperazine-1-carboxylate

[0528]

[0529] A solution of tert-butyl piperazine-1-carboxylate (2.8 g, 7.2 mmol) and Pd / C (0.28 g) in methanol (30 mL) was stirred at room temperature for 2 h under H2 atmosphere. The resulting mixture was filtered and the filter cake was washed with MeOH. The filtrate was concentrated under reduced pressure to provide the LCMS (ESI) m / z [M+H] of tert-butyl piperazine-1-carboxylate (1.8 g, quantitative). + =256.

[0530] Example 36. Preparation of tert-butyl 7-(3-aminopropyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate

[0531]

[0532] 7-(3-aminopropyl)-2,7-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester was prepared starting from N-(3-oxopropyl)carbamate and tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylic acid, following the synthesis protocol described in Example 35. LCMS(ESI) m / z: [M+H] + =284.

[0533] Example 37. Preparation of N-[2-[3-(2-aminoethoxy)phenoxy]ethyl]carbamate benzyl ester

[0534]

[0535] Step 1: Preparation of N-[2-(3-hydroxyphenoxy)ethyl]carbamate tert-butyl ester

[0536]

[0537] Cs₂CO₃ (88.8 g, 272 mmol) was added fractionally to a stirred solution of resorcinol (10.0 g, 90.8 mmol) and N-(2-bromoethyl)carbamate (20.4 g, 90.8 mmol) in DMF (250 mL). The resulting mixture was washed three times with EtOAc. The mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:MeOH) to provide N-[2-(3-hydroxyphenoxy)ethyl]carbamate (4.45 g, 19.3%) as a yellow oil. LCMS (ESI) m / z: [M+H] + =254.

[0538] Step 2: Preparation of N-[2-(3-[2-[(tert-butoxycarbonyl)amino]ethoxy]phenoxy)ethyl]carbamate benzyl ester

[0539]

[0540] Under a nitrogen atmosphere at room temperature, PPh3 (6.68 g, 25.5 mmol) and DIAD (5.15 g, 25.5 mmol) were added fractionally to a stirred solution of N-[2-(3-hydroxyphenoxy)ethyl]carbamate (4.30 g, 17.0 mmol) and N-(2-hydroxyethyl)carbamate (3.31 g, 17.0 mmol) in THF (25.0 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:MeOH) to provide N-[2-(3-[2-[(tert-butoxycarbonyl)amino]ethoxy]phenoxy)ethyl]carbamate (5 g, 68.4%) as a yellow oil. LCMS (ESI) m / z: [M+H] + =431.

[0541] Step 3: Preparation of N-[2-[3-(2-aminoethoxy)phenoxy]ethyl]carbamate benzyl ester

[0542]

[0543] A solution of N-[2-(3-[2-[(tert-butoxycarbonyl)amino]ethoxy]phenoxy)ethyl]carbamate (2.85 g, 6.62 mmol) and Pd / C (1.43 g, 13.4 mmol) in MeOH (15.0 mL) was stirred overnight at 50 °C under a hydrogen atmosphere. The resulting mixture was filtered and the filter cake was washed three times with MeOH. The filtrate was concentrated under reduced pressure to provide N-[2-[3-(2-aminoethoxy)phenoxy]ethyl]carbamate (2.07 g, 86.9%) as an off-white oil. LCMS (ESI) m / z: [M+H] + =297.

[0544] Example 38. Preparation of intermediates

[0545] The intermediates listed in Table B5 were prepared using appropriate amines in a manner similar to that described in the preparation of I-22 (Example 32).

[0546] Table B5. Intermediates

[0547]

[0548]

[0549]

[0550] Example 39. Preparation of 4-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy]butyric acid (I-29)

[0551]

[0552] Step 1: Preparation of tert-butyl 4-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]butyrate

[0553]

[0554] To a solution of 2-(2,6-dioxadipin-3-yl)-4-hydroxyisoindole-1,3-dione (2.00 g, 7.29 mmol) and tert-butyl 4-bromobutyrate (1.95 g, 8.752 mmol) in DMF (10.0 mL), KI (0.12 g, 0.729 mmol) and KHCO3 (1.10 g, 10.9 mmol) were added. The resulting solution was stirred at 60 °C for 5 h. The mixture was diluted with EtOAc and washed three times with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to produce a crude product. The crude product was purified by reversed-phase C18 rapid chromatography (water: ACN) to produce tert-butyl 4-[[2-(2,6-dioxadipin-3-yl)-1,3-dioxadipin-4-yl]oxy]butyrate (1.5 g, 49.4%) as a grayish-white solid. LCMS(ESI)m / z[M+H] + =417.

[0555] Step 2: Preparation of 4-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy]butyric acid (I-29)

[0556]

[0557] TFA (1 mL) was added to a stirred solution of tert-butyl 4-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]butyrate (450 mg, 1.08 mmol) in DCM (5 mL). The resulting solution was stirred at 25 °C for 2 h. The resulting mixture was concentrated. This yielded I-29 (360 mg, 92.5%) as a white solid. 1¹H NMR (400MHz, methanol-d⁴) δ 7.79 (t, J = 8.4, 7.4Hz, 1H), 7.47 (d, J = 7.8Hz, 2H), 5.12 (dd, J = 12.6, 5.5Hz, 1H), 4.30 (t, J = 6.2Hz, 2H), 2.95–2.66 (m, 3H), 2.60 (t, J = 7.3Hz, 2H), 2.25–2.18 (m, 3H). LCMS (ESI) m / z: [M+H] + =361.10.

[0558] Example 40. Preparation of intermediates

[0559] The following intermediates in Table B6 were prepared using appropriate alkyl bromides in a manner similar to that described in the preparation of intermediate I-29 (Example 39).

[0560] Table B6. Intermediates

[0561]

[0562] Example 41. Preparation of 3-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)propionic acid (I-34)

[0563]

[0564] Step 1: Preparation of tert-butyl 3-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)propionate

[0565]

[0566] To a solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoro-2,3-dihydro-1H-isoindole-1,3-dione (1.00 g, 3.62 mmol) in NMP (10.0 mL), tert-butyl 3-[2-(2-aminoethoxy)ethoxy]propionate (929 mg, 3.98 mmol) was added. The resulting mixture was stirred overnight at 90 °C. The mixture was allowed to cool to room temperature. The resulting mixture was diluted with EtOAc. The organic layer was washed five times with water and then with brine. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide tert-butyl 3-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)amino)ethoxy)ethoxy)propionate (1.14 g, 64.3%) as a yellow solid. LCMS(ESI)m / z:[M+H]+ =490.

[0567] Step 2: Preparation of 3-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)propionic acid (I-34)

[0568]

[0569] TFA (0.52 mL, 4.55 mmol) was added dropwise to a stirred solution of tert-butyl propionate (1.14 g, 2.33 mmol) in DCM (10.0 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under vacuum, and the residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide I-34 (896 mg, 70.3%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ12.15 (s, 1H), 11.09 (s, 1H), 7.63-7.55 (m, 1H), 7.15 (d, 1H), 7.05 (d, 1H), 6.61 (t, 1H ), 5.06 (dd, 1H), 3.65-3.44 (m, 8H), 2.87 (d, 1H), 2.59 (d, 2H), 2.43 (t, 2H), 2.04 (m, 1H); LCMS (ESI) m / z: [M+H] + =434.15.

[0570] Example 42. Preparation of intermediates

[0571] The intermediates listed in Table B7 are prepared from suitable aryl fluorines and amines in a manner similar to that described in the preparation of intermediate I-34 (Example 41).

[0572] Table B7. Intermediates

[0573]

[0574]

[0575] Example 43. Preparation of 2-[(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylic acid (I-42)

[0576]

[0577] Step 1: Preparation of tert-butyl 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetate

[0578]

[0579] K₂CO₃ (8.32 g, 60.2 mmol) was added to a stirred solution of 2-(2,6-dioxadipin-3-yl)-5-hydroxyisoindole-1,3-dione (5.50 g, 20.1 mmol) and 2-bromoacetic acid tert-butyl ester (3.91 g, 20.1 mmol) in DMF (15.0 mL). The resulting mixture was stirred overnight at room temperature, then taken up in water, extracted three times with EtOAc, and concentrated. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide 2-[[2-(2,6-dioxadipin-3-yl)-1,3-dioxadipindole-5-yl]oxy]acetic acid tert-butyl ester (3.2 g, 45.2%) as an off-white solid. LCMS (ESI) m / z: [M+H] + =389.

[0580] Step 2: Preparation of [[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetic acid

[0581]

[0582] A solution of tert-butyl 2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-5-yl]oxy]acetate (3.20 g, 8.24 mmol) and dry HCl in dioxane (15.0 mL, 494 mmol) was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. This yielded 1.12 g of [[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-5-yl]oxy]acetic acid as a yellow solid. LCMS (ESI) m / z: [M+H] + =289.

[0583] Step 3: Preparation of methyl 2-[(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylate]

[0584]

[0585] HATU (1.15 g, 3.03 mmol) and DIEA (782 mg, 6.05 mmol) were added dropwise over 2 h to a stirred solution of [[2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylate (670 mg, 2.02 mmol) in DMF (15.0 mL). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide methyl 2-[(2-[[2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylate (779 mg, 78.4%) as a yellow oil. LCMS(ESI)m / z:[M+H] + =444.

[0586] Step 4: Preparation of 2-[(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylic acid (I-42)

[0587]

[0588] A mixture of methyl 2-[(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylate (764 mg) and HCl / -dioxane (4 N, 5.00 mL, 20 mmol) was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography under the following conditions (water:ACN) to provide I-42 (338 mg) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ12.17 (s, 1H), 11.12 (s, 1H), 8.33 (t, 1H), 7.88 (d, 1H), 7.46 (d, 1H), 7.39 (dd, 1H), 5.13 (dd, 1H), 4.74 (s, 2H), 2.96-2.83 (m, 1H), 2.65-2.52 (m, 1H), 2.08 (s, 4H), 1.66 (td, 1H), 1.48 (h, 1H), 1.02 (td, 1H), 0.85 (dt, 1H). LCMS(ESI)m / z:[M+H] + =430.05.

[0589] Example 44. Preparation of methyl 2-[(2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetamido)methyl]cyclopropane-1-carboxylic acid (I-43)

[0590]

[0591] I-43 (423 mg, 35.74%) was prepared as a white solid from a suitable phenol in a manner similar to that described in the preparation of I-42 (Example 43). 1 H NMR (300MHz, DMSO-d6) δ12.16 (s, 1H), 11.12 (s, 1H), 8.10 (s, 1H), 7.82 (t, 1H), 7.51 (d, 1H), 7.41 (d, 1H), 5.17- 5.07 (m, 1H), 4.80 (s, 2H), 2.60 (d, 2H), 2.08 (s, 3H), 1.70-1.60 (m, 1H), 1.47 (d, 1H), 1.03 (d, 1H), 0.84 (d, 1H). LCMS(ESI)m / z:[M+H] + =430.12.

[0592] Example 45. Preparation of 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazo-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-6-oxohexanoic acid

[0593]

[0594] Step 1: Preparation of methyl 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-6-oxohexanoate

[0595]

[0596] To a solution of (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[[4-(4-methylthiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (300 mg, 0.697 mmol) and 6-methoxy-6-oxohexanoic acid (93.0 μL, 0.627 mmol) in DCM (3 mL), HATU (265 mg, 0.697 mmol) and DIEA (485 μL, 2.79 mmol) were added. The mixture was stirred at 30 °C for 2 h. The reaction mixture was diluted with water and extracted twice with DCM. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to produce a residue. The reactants were purified by reverse-phase extraction (0.1% FA conditions), and the eluent was concentrated to remove MeCN. The residue was extracted twice with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated to provide methyl 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-6-oxohexanoate (340 mg, 85.2%) as a colorless oil. LCMS (ESI) m / z = [M+H + =573.6.

[0597] Step 2: Preparation of 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-6-oxohexanoic acid

[0598]

[0599] LiOH·H2O (42.7 mg, 1.78 mmol) was added to a solution of methyl 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-6-oxohexanoate (340 mg, 0.594 mmol) in THF / MeOH / H2O (2 / 1 / 1, 4 mL). The mixture was stirred at 30 °C for 4 h. The reaction mixture was diluted with water and washed twice with EtOAc. The aqueous phase was adjusted to pH 5 with 1 N HCl. The mixture was then extracted twice with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was lyophilized under low pressure to provide 6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-6-oxohexanoic acid (185 mg, 55.9%) as a white solid. 1 H NMR (400MHz, MeOD-d4) δ8.87 (s, 1H), 7.47-7.40 (m, 4H), 4.63-4.49 (m, 5H), 4.37 (d, J=15.6Hz, 1H), 3.89-3.82 (m, 1H), 3.81-3.79 (m, 1H), 2.48 (s, 3H), 2.31-2.27 (m, 5H), 2.10-2.08 (m, 1H), 1.66-1.60 (m, 4H), 1.03 (s, 9H). LCMS(ESI)m / z=[M+H] + =559.2.

[0600] Example 46. Preparation of intermediates

[0601] The intermediates listed in Table B8 were prepared using (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[[4-(4-methylthiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide and a suitable carboxylic acid in a manner similar to that described in Example 45.

[0602] Table B8. Intermediates

[0603]

[0604]

[0605] Example 47. Preparation of (S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecane-1-acid

[0606]

[0607] Step 1: Preparation of 3-oxo-1-phenyl-2,5,8,11,14-pentahexadecane-16-acid

[0608]

[0609] Benzyl bromide (0.468 mL, 3.94 mmol) was added dropwise to a mixture of 2-[2-[2-[2-(carboxymethoxy)ethoxy]ethoxy]ethoxy]acetic acid (1 g, 3.76 mmol) and TEA (1.34 mL, 9.66 mmol) in acetone (5 mL) at 0 °C. The mixture was stirred at 20 °C for 16 h. The mixture was filtered and the filter cake was washed with acetone. The filtrate was concentrated and the residue was dissolved in water. The mixture was extracted three times with EtOAc and then treated with HCl (2 M) to adjust the pH to 3–5. The mixture was extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to provide 3-oxo-1-phenyl-2,5,8,11,14-pentahexadecane-16-acid (690 mg, 51.6% yield) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.32-7.25 (m, 5H), 5.12 (s, 2H), 4.13 (s, 2H), 4.08 (s, 2H), 3.69-3.65 (m, 4H), 3.64-3.59 (m, 8H).

[0610] Step 2: Preparation of (S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecane-1-acid benzyl ester

[0611]

[0612] HATU (795 mg, 2.09 mmol) and DIEA (728 μL, 4.18 mmol) were added to a solution of 3-oxo-1-phenyl-2,5,8,11,14-pentahexadecane-16-acid (572 mg, 1.61 mmol) in DCM (6 mL). (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[[4-(4-methylthiazolyl-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide hydrochloride (600 mg, 1.28 mmol) was added to the mixture. The mixture was stirred at 30 °C for 2 h. The mixture was concentrated under vacuum to produce a yellow solid. The residue was purified by reversed-phase rapid chromatography (FA conditions) to produce (S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecane-1-acid benzyl ester (810 mg, 72.0%) as a yellow oil. 1 HNMR (400MHz, CDCl3) δ8.70 (s, 1H), 7.41-7.34 (m, 10H), 4.77-4.76 (m, 1H), 4.63-4.47 (m, 3H), 4.38-4.33 (m, 1H), 4.22 (s, 2H), 4.13 (d, J=11.2Hz, 1H), 4.08-3.96 (m, 2H), 3.75-3.59 (m, 12H), 2.64-2.55 (m, 1H), 2.54 (s, 3H), 2.19-2.09 (m, 1H), 0.97 (s, 9H). LCMS(ESI)m / z:[M+H] + =769.4.

[0613] Step 3: Preparation of (S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecane-1-acid

[0614]

[0615] Pd / C (10%, 216 mg, 0.203 mmol) was added to a mixture of (S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecane-1-acid benzyl ester (780 mg, 1.01 mmol) in MeOH (5 mL). The mixture was purified with H2, then pressurized with H2 (15 psi) and stirred at 25 °C for 12 h, followed by stirring at 40 °C for 8 h. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography (NH4OH conditions) to produce (S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecane-1-acid (300 mg, 42.9%) as a white solid. 1 ¹H NMR (400MHz, methanol-d⁴) δ 8.90 (s, 1H), 7.55–7.39 (m, 4H), 4.62–4.51 (m, 3H), 4.42–4.35 (m, 1H), 4.16–4.12 (m, 2H), 4.08 (d, J = 3.6Hz, 2H), 3.93–3.79 (m, 2H), 3.75–3.64 (m, 13H), 2.52–2.49 (m, 3H), 2.29–2.21 (m, 1H), 2.17–2.06 (m, 1H), 1.08–1.04 (m, 9H). LCMS (ESI) m / z: [M+H] + =679.2.

[0616] Example 48. Preparation of 1-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)piperidin-4-carboxylic acid (I-61)

[0617]

[0618] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(prop-2-en-1-yloxy)isoindole-1,3-dione

[0619]

[0620] To a solution of 2-(2,6-dioxadiazin-3-yl)-5-hydroxyisoindole-1,3-dione (5.48 g, 20.0 mmol) and allyl bromide (3.63 g, 30.0 mmol) in DMF (50.0 mL), KI (332 mg, 2.00 mmol) and KHCO3 (3.00 g, 30.0 mmol) were added. The resulting mixture was stirred at 65 °C for 12 h, then diluted with water and extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / EtOAc) to provide 2-(2,6-dioxadiazin-3-yl)-5-(prop-2-en-1-yloxy)isoindole-1,3-dione (6.7 g, crude product) as a yellow-green solid. LCMS(ESI)m / z:[M+H] + =315.

[0621] Step 2: Preparation of 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetaldehyde

[0622]

[0623] To a solution of 2-(2,6-dioxadiazin-3-yl)-5-(prop-2-en-1-yloxy)isoindole-1,3-dione (3.14 g, 9.99 mmol) in dioxane (30.0 mL), NaIO4 (10.7 g, 50.0 mmol), water (3.00 mL), and 2,6-dimethylpyridine (3.21 g, 30.0 mmol) were added. K2O5O4 dihydrate (0.37 g, 0.999 mmol) was added to the mixture at room temperature. The resulting mixture was stirred at room temperature for another 2 h. The reaction mixture was quenched with water at room temperature, and the resulting mixture was extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This produces 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetaldehyde (1.83 g, 57.9%) as a light brown solid. LCMS (ESI) m / z: [M+H] + =317.

[0624] Step 3: Preparation of tert-butyl piperidine-4-carboxylate (2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)piperidin-4-carboxylate)

[0625]

[0626] To a solution of 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]acetaldehyde (1.83 g, 5.79 mmol) and tert-butyl piperidine-4-carboxylate (1.07 g, 5.79 mmol) in DMF (35.0 mL), NaBH(OAc)3 (3.68 g, 17.4 mmol) was added. The resulting mixture was stirred at room temperature for 3 h. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide 1-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)tert-butyl piperidine-4-carboxylate (1.16 g, 41.3%) as an off-white solid. LCMS (ESI) m / z: [M+H] + =401.

[0627] Step 4: Preparation of 1-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)piperidin-4-carboxylic acid (I-61)

[0628]

[0629] TFA (10.0 mL, 135 mmol) was added to a solution of 1-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)piperidin-4-carboxylic acid tert-butyl ester (1.16 g, 2.39 mmol) in DCM (10.0 mL). The resulting mixture was stirred at room temperature for 5 h. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide I-61 (845 mg, 73.4%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ11.11 (s, 1H), 8.15 (d, 1H), 7.84 (d, 1H), 7.47 (d, 1H), 7.37 (dd, 1H), 5.12 (dd, 1H), 4.31 (t, 2H), 3.02-2.85 (m , 3H), 2.79(t, 2H), 2.66-2.60(m, 1H), 2.59-2.54(m, 1H), 2.29-2.12(m, 3H), 2.15-1.99(m, 1H), 1.87-1.75(m, 2H), 1.66-1.47(m, 2H). LCMS(ESI)m / z:[M+H] + =430.15.

[0630] Example 49. Preparation of N-[2-[(2-aminoethyl)(methyl)amino]ethyl]-2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy]acetamide hydrochloride (I-63)

[0631]

[0632] Step 1: Preparation of tert-butyl 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetate

[0633]

[0634] To a solution of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindole-1,3-dione (5.50 g, 20.1 mmol) in DMF (65.0 mL), tert-butyl 2-bromoacetate (3.91 g, 20.1 mmol) and K₂CO₃ (8.32 g, 60.2 mmol) were added. The resulting solution was stirred at room temperature for 12 h. The solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide tert-butyl 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl]oxy]acetate (5.2 g, 66.8%) as a white solid. LCMS (ESI) m / z: [M+H] + =389.

[0635] Step 2: Preparation of [[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetic acid

[0636]

[0637] A solution of tert-butyl 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetate (5.32 g, 13.7 mmol) in HCl / dioxane (4 N, 50.0 mL, 200 mmol) was stirred at room temperature for 12 h. The solution was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide [[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetic acid (4.66 g, 100%) as a white solid. LCMS (ESI) m / z: [M+H] + =333.

[0638] Step 3: Preparation of N-(2-[[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetamido)ethyl](methyl)amino]ethyl)tert-butyl carbamate

[0639]

[0640] To a solution of [[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetic acid (926 mg, 2.79 mmol) in DCM (35.0 mL), N-[2-[(2-aminoethyl)(methyl)amino]ethyl]carbamate tert-butyl ester (908 mg, 4.18 mmol), HATU (1.59 g, 4.18 mmol), and DIEA (1.08 g, 8.36 mmol) were added. The resulting solution was stirred at room temperature for 3 h. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide N-(2-[[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetamido)ethyl](methyl)amino]ethyl)carbamate tert-butyl ester (1.53 g, crude product) as a pale yellow solid. LCMS(ESI)m / z:[M+H] + =532.

[0641] Step 4: Preparation of N-[2-[(2-aminoethyl)(methyl)amino]ethyl]-2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy]acetamide picoside (I-63)

[0642]

[0643] A solution of N-(2-[[2-(2-aminoethyl)(methyl)amino]ethyl]-2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]acetamido)ethyl](methyl)amino]ethyl)carbamate (600 mg, 1.13 mmol) was prepared in 4 NM HCl (123 mg, 3.39 mmol) in dioxane (20.0 mL) and stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide N-[2-[(2-aminoethyl)(methyl)amino]ethyl]-2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]oxy]acetamide (I-63, 200 mg, 41.1%) as a yellow solid. LCMS(ESI)m / z:[M+H] +=432.18.

[0644] Example 50. Preparation of intermediates

[0645] The intermediates in Table B9 are prepared by starting with appropriately substituted phenols and amines in a manner similar to that described in Example 49.

[0646] Table B9. Intermediates

[0647]

[0648] Example 51. Preparation of 4-(2-[2-[(2-aminoethyl)(methyl)amino]ethoxy]ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-29)

[0649]

[0650] Step 1: Preparation of N-[2-(2-hydroxyethoxy)ethyl]-N-methylcarbamate benzyl ester

[0651]

[0652] To a solution of 2-(2-(methylamino)ethoxy)ethane-1-ol (10.0 g, 83.9 mmol) in THF (30.0 mL), benzyl chloroformate (2.29 g, 13.4 mmol), K₂CO₃ (3.83 g, 27.7 mmol), and water (30.0 mL) were added. The resulting mixture was stirred at room temperature for 12 h and then extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na₂SO₄. After filtration, the liquid was concentrated under reduced pressure and purified by reversed-phase C18 rapid chromatography (water:ACN:NH₄HCO₃) to provide N-[2-(2-hydroxyethoxy)ethyl]-N-methylcarbamate (2.14 g, 17.5%) as a colorless oil; LCMS (ESI) m / z: [M+H] + =254.

[0653] Step 2: Preparation of N-methyl-N-(2-[2-[(4-methylbenzenesulfonyl)oxy]ethoxy]ethyl)carbamate benzyl ester

[0654]

[0655] DMAP (0.15 g, 1.27 mmol), TEA (2.14 g, 21.1 mmol), and p-toluenesulfonyl chloride (2.42 g, 12.7 mmol) were added to a stirred solution of N-[2-(2-hydroxyethoxy)ethyl]-N-methylcarbamate (2.14 g, 8.45 mmol) in DCM (30.0 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h, and then stirred at room temperature for another 5 h. The residue was purified by silica gel column chromatography (petroleum ether / THF) to provide N-methyl-N-(2-[2-[(4-methylbenzenesulfonyl)oxy]ethoxy]ethyl)carbamate (3.51 g, crude product) as a colorless oil; LCMS (ESI) m / z: [M+H] + =408.

[0656] Step 3: Preparation of N-[2-(2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]ethoxy)ethyl]-N-methylcarbamate benzyl ester

[0657]

[0658] To a solution of N-methyl-N-(2-[2-[(4-methylbenzenesulfonyl)oxy]ethoxy]ethyl)carbamate (3.51 g, 8.61 mmol) in DMF (30.0 mL), 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindole-1,3-dione (2.36 g, 8.61 mmol) and Na₂CO₃ (1.37 g, 12.9 mmol) were added. The resulting mixture was stirred at 80 °C for 12 h and then concentrated. The residue was purified by reversed-phase rapid C18 chromatography under the following conditions (water:ACN) to provide N-[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]ethoxy)ethyl]-N-methylcarbamate (1.53 g, 34.8%) as a pale yellow solid; LCMS (ESI) m / z: [M+H] + =510.

[0659] Step 4: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-[2-[2-(methylamino)ethoxy]ethoxy]isoindole-1,3-dione

[0660]

[0661] Ammonium formate (378 mg, 5.99 mmol) and Pd(OH)₂ / C (5.43 mmol) were added to a solution of N-[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl]oxy]ethoxy)ethyl]-N-methylcarbamate (1.52 g, 3.00 mmol) in EtOH (30.0 mL). The resulting suspension was stirred at 60 °C for 12 h under hydrogen at 1 atm and then filtered. The filter cake was washed three times with MeOH, and the filtrate was concentrated under reduced pressure to provide 2-(2,6-dioxopiperidin-3-yl)-4-[2-[2-(methylamino)ethoxy]ethoxy]isoindole-1,3-dione (1.06 g, 94.8%) as a pale yellow solid; LCMS(ESI) m / z [M+H] + =376.

[0662] Step 5: Preparation of N-(2-[[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]ethoxy)ethyl](methyl)amino]ethyl)tert-butyl carbamate

[0663]

[0664] To a solution of 2-(2,6-dioxopiperidin-3-yl)-4-[2-[2-(methylamino)ethoxy]ethoxy]isoindole-1,3-dione (1.06 g, 2.82 mmol) in DMF (10.0 mL), N-(2-oxoethyl)carbamate tert-butyl ester (539 mg, 3.39 mmol) and NaBH(OAc)3 (1.80 g, 8.47 mmol) were added. The resulting solution was stirred at room temperature for 2 h. After post-treatment with aqueous solution, the sample was extracted with DCM and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH) to provide N-(2-[[2-(2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]ethoxy)ethyl](methyl)amino]ethyl)carbamate tert-butyl ester (386 mg, 26.4%) as a pale yellow solid; LCMS (ESI) m / z: [M+H] + =519.

[0665] Step 6: Preparation of 4-(2-[2-[(2-aminoethyl)(methyl)amino]ethoxy]ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-29)

[0666]

[0667] TFA (5.00 mL, 67.3 mmol) was added to a solution of N-(2-[[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxy]ethoxy)ethyl](methyl)amino]ethyl)carbamate (511 mg, 0.985 mmol) in DCM (5.00 mL). The resulting solution was stirred at room temperature for 3 h. The solution was concentrated and the residue was purified by reversed-phase C18 rapid chromatography under the following conditions (water:ACN) to provide I-29 (307 mg, 73.9%) as a brown oil; 1 ¹H NMR (300MHz, DMSO-d6) δ 11.70–10.30 (m, 1H), 8.18 (s, 1H, formic acid), 7.83 (dd, 1H), 7.51 (dd, 2H), 5.09 (dd, 1H), 4.41–4.32 (m, 2H), 3.84–3.75 (m, 2H), 3.63 (d, 2H), 2.99–2.80 (m, 3H), 2.65–2.58 (m, 5H), 2.24 (s, 3H), 2.08 (s, 1H), 1.98–2.06 (m, 1H); LCMS (ESI) m / z: [M+H] + =419.

[0668] Example 52. Preparation of 5-(azacyclobutane-3-yloxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (I-33)

[0669]

[0670] Step 1: Preparation of tert-butyl 3-[(4-methylbenzenesulfonyl)oxy]azacyclobutane-1-carboxylate

[0671]

[0672] Partially added DMAP (264 mg, 2.17 mmol) and TEA (4.38 g, 43.3 mmol) to a stirred solution of 3-hydroxyazacyclobutane-1-carboxylate (2.50 g, 14.4 mmol) and p-toluenesulfonyl chloride (4.13 g, 21.7 mmol) in DCM were added to the solution. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide 3-[(4-methylbenzenesulfonyl)oxy]azacyclobutane-1-carboxylate (4.4 g, 93.1%) as a brown oil. LCMS (ESI) m / z: [M+H] + =328.

[0673] Step 2: Preparation of tert-butyl 3-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]azacyclobutane-1-carboxylate]

[0674]

[0675] To a stirred solution of 3-[(4-methylbenzenesulfonyl)oxy]azacyclobutane-1-carboxylic acid tert-butyl ester (4.40 g, 13.4 mmol) and KI (0.22 g, 1.34 mmol) in DMF, KHCO3 (4.04 g, 40.3 mmol) was added fractionally. After stirring at 100 °C for 8 h, the resulting mixture was extracted three times with EtOAc, and the organic extract was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide 3-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]azacyclobutane-1-carboxylic acid tert-butyl ester (1.73 g, 30.0%) as an off-white solid. LCMS (ESI) m / z: [M+H] + =430.

[0676] Step 3: Preparation of 5-(azacyclobutane-3-yloxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-33)

[0677]

[0678] A solution of 3-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]azacyclobutane-1-carboxylic acid tert-butyl ester (1.53 g, 3.56 mmol) and TFA (5.00 mL, 67.3 mmol) in DCM was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase C18 rapid chromatography under the following conditions (water:ACN) to provide I-33 (1.08 g, 96.4%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ11.11 (s, 1H), 8.25 (s, formic acid, 1H), 7.88 (d, 1H), 7.32 (d, 2H), 5.30 (p, 1H), 5. 13 (dd, 1H), 4.31 (dd, 2H), 3.89 (dd, 2H), 2.99-2.80 (m, 1H), 2.68-2.52 (m, 2H), 2.13-1.97 (m, 1H). LCMS(ESI)m / z:[M+H] + =330.05.

[0679] Example 53. Preparation of 5-[7-azaspiro[3.5]non-2-yloxy]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-35)

[0680]

[0681] Step 1: Preparation of tert-butyl 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]-7-azaspiro[3,5]nonane-7-carboxylate

[0682]

[0683] PPh3 (1.97 g, 7.49 mmol) was added to a solution of 2-(2,6-dioxadiidine-3-yl)-5-hydroxyisoindole-1,3-dione (1.37 g, 4.99 mmol) and 2-hydroxy-7-azaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester (1.81 g, 7.49 mmol) in THF (30.0 mL). DIAD (1.52 g, 7.49 mmol) was added dropwise to the mixture over 10 min at 0 °C. The reaction mixture was stirred at room temperature for another 5 h. The resulting mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide tert-butyl 2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]-7-azaspiro[3,5]nonane-7-carboxylate (1.96 g, 79.0%) as a white solid; LCMS (ESI) m / z: [M+H] + =498.

[0684] Step 2: Preparation of 5-[7-azaspiro[3.5]non-2-yloxy]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-35)

[0685]

[0686] TFA (10.0 mL, 135 mmol) was added to a solution of 2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]-7-azaspiro[3.5]nonane-7-carboxylic acid tert-butyl ester (1.96 g, 3.94 mmol) in DCM (10.0 mL). The resulting mixture was stirred at room temperature for 5 h, then concentrated and purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide I-35 (1.63 g, 93.1%) as a light gray solid. 1¹H NMR (300MHz, DMSO-d6) δ 8.39 (s, formic acid, 1H), 7.84 (d, 1H), 7.35–7.24 (m, 2H), 5.12 (dd, 1H), 5.00 (p, 1H), 3.00–2.81 (m, 5H), 2.67–2.43 (m, 2H), 2.51–2.43 (m, 3H), 2.16–1.95 (m, 1H), 1.95–1.82 (m, 2H), 1.65–1.78 (m, 4H); LCMS (ESI) m / z: [M+H] + =398.16.

[0687] Example 54. Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-[2-(piperazin-1-yl)ethoxy]isoindole-1,3-dione carbamate (I-36)

[0688]

[0689] Step 1: Preparation of tert-butyl piperazine-1-carboxylate

[0690]

[0691] NaBH(OAc)3 (1.26 g, 5.96 mmol) was added to a solution of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)acetaldehyde (628 mg, 1.99 mmol) and piperazine-1-carboxylic acid tert-butyl ester (370 mg, 1.99 mmol) in DMF (10.0 mL) at room temperature. The resulting mixture was stirred at room temperature for 3 h, then quenched with water, extracted into DCM, and concentrated. The residue was purified by reversed-phase C18 rapid chromatography under the following conditions (water:ACN:FA) to provide tert-butyl 4-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)piperazine-1-carboxylate (812 mg, 84.05%) as a grayish-white solid; LCMS (ESI) m / z: [M+H] + =487.

[0692] Step 2: Preparation of 2-(2,6-dioxadiazin-3-yl)-5-[2-(piperazin-1-yl)ethoxy]isoindole-1,3-dione carbamate (I-36)

[0693]

[0694] TFA (10.0 mL, 135 mmol) was added to a solution of 4-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethyl)piperazine-1-carboxylic acid tert-butyl ester (2.10 g, 4.32 mmol) in DCM (10.0 mL). The resulting mixture was stirred at room temperature for 3 h and then concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide I-36 (1.43 g, 74.2%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ11.07 (s, 1H), 8.34 (s, 1H), 7.84 (d, 1H), 7.47 (d, 1H), 7.37 (dd, 1H), 5.12 (dd, 1H), 4.30 (t, 2H), 2.99-2.86 (m, 5H), 2.77 (t, 2H), 2.67-2.55 (m, 5H), 2.13-1.96 (m, 1H).; LCMS (ESI) m / z: [M+H] + =387.16.

[0695] Example 55. Preparation of 5-[4-(azacyclobutane-3-ylmethyl)piperazin-1-yl]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-40)

[0696]

[0697] Step 1: Preparation of tert-butyl 3-([4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]methyl)azacyclobutane-1-carboxylate)

[0698]

[0699] NaBH(OAc)3 (2.04 g, 9.64 mmol) was added to a solution of 2-(2,6-dioxadiazin-3-yl)-5-(piperazin-1-yl)isoindole-1,3-dione carboxylate (1.10 g, 3.21 mmol) and tert-butyl 3-formylazetane-1-carboxylate (595 mg, 3.21 mmol) in DMF (10.0 mL). The resulting mixture was stirred at room temperature for 3 h. After aqueous posttreatment, the solution was extracted and concentrated using DCM. The residue was purified by reversed-phase C18 rapid chromatography under the following conditions (water:ACN:FA) to provide tert-butyl 3-([4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]methyl)azacyclobutane-1-carboxylate (991 mg, 60.3%) as a pale yellow solid; LCMS (ESI) m / z: [M+H] + =512.

[0700] Step 2: Preparation of 5-[4-(azacyclobutane-3-ylmethyl)piperazin-1-yl]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione carbamate (I-40)

[0701]

[0702] TFA (10.0 mL, 135 mmol) was added to a solution of tert-butyl 3-(4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]methyl)azacyclobutane-1-carboxylate (991 mg, 1.94 mmol) in DCM (10.0 mL). The resulting mixture was stirred at room temperature for 3 h and then concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:FA) to provide I-40 (702 mg, 85.3%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ11.07 (s, 1H), 8.25 (s, formic acid, 1H), 7.69 (d, 1H), 7.34 (d, 1H), 7.26 (dd, 1H), 5.08 (dd, 1H), 4.00 (t, 2H), 3.66 (dd, 2 H), 3.43 (t, 4H), 3.11-2.96 (m, 1H), 2.95-2.79 (m, 1H), 2.65-2.51 (m, 4H), 2.50-2.44 (m, 4H), 2.09-1.96 (m, 1H).; LCMS (ESI) m / z: [M+H] + =412.19.

[0703] Example 56. Preparation of intermediates

[0704] The intermediates in Table B10 are prepared from the common intermediate 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindole-1,3-dione carbamate and a suitable aldehyde in a manner similar to that described in Example 55.

[0705] Table B10. Intermediates

[0706]

[0707] Example 57. Preparation of 3-[4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]propionic acid (I-41)

[0708]

[0709] Step 1: Preparation of tert-butyl 3-[4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]propionate

[0710]

[0711] DIEA (2.8 g, 21.7 mmol) was added dropwise to a solution of 2-(2,6-dioxadipin-3-yl)-5-(piperazin-1-yl)isoindole-1,3-dione carboxylate (5.0 g, 14.6 mmol) and tert-butyl 3-bromopropionate (3.6 g, 17.2 mmol) in DMF (50 mL), and the mixture was stirred at 70 °C for 6 h. The reaction mixture was concentrated, and the residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide tert-butyl 3-[4-[2-(2,6-dioxadipin-3-yl)-1,3-dioxadipin-5-yl]piperazin-1-yl]propionate (2.1 g, 30.6%) as a yellow solid. LCMS (ESI) m / z [M+H] + =471.

[0712] Step 2: Preparation of 3-[4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]propionic acid (I-41)

[0713]

[0714] A solution of tert-butyl 3-[4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]propionate (2.1 g, 4.5 mmol) in DCM (20 mL) and TFA (5 mL) was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase C18 rapid chromatography (water:MeOH) to provide 3-[4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]piperazin-1-yl]propionate (I-41) (0.78 g, 42.2%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ12.03 (br, 1H), 11.10 (s, 1H), 7.68 (d, J = 8.5Hz, 1H), 7.35 (d, J = 1.7Hz, 1H), 7.27 (dd, J = 8.6, 2.0Hz, 1H), 5.08 (dd, J=12.8, 5.4Hz, 1H), 3.49-3.40 (m, 4H), 2.96-2.80 (m, 1H), 2.67-2.52 (m, 8H), 2.44 (t, J=6.9Hz, 2H), 2.07-1.97 (m, 1H). LCMS(ESI)m / z[M+H] + =415.10.

[0715] Example 58. Preparation of 5-(2-aminoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione trifluoroacetate (I-44)

[0716]

[0717] Step 1: Preparation of tert-butyl ((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethyl)carbamate

[0718]

[0719] DIEA (0.94 g, 7.3 mmol) was added to a stirred solution of 2-(2,6-dioxadiazol-3-yl)-5-hydroxyisoindoline-1,3-dione (1.0 g, 3.6 mmol) and tert-butyl 1,2,3-oxathiazolidin-3-carboxylate 2,2-dioxide (0.98 g, 4.4 mmol) in DMF (10 mL). After stirring at 80 °C for 2 h, water was added, followed by extraction three times with EtOAc. The combined organic layers were washed three times with water and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide tert-butyl ((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethyl)carbamate (1.7 g, 94.4%) as a yellow oil. LCMS (ESI) m / z: [M+H] + =418.

[0720] Step 2: Preparation of 5-(2-aminoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione; trifluoroacetate (I-44)

[0721]

[0722] TFA (5 mL) was added dropwise to a stirred solution of ((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)ethyl)carbamate (1.4 g, 3.4 mmol) in DCM (20 mL). After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel rapid column chromatography to provide I-44 (750 mg, 51.2%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ11.15 (s, 1H), 8.04 (br, 3H), 7.91 (d, J = 8.3Hz, 1H), 7.50 (d, J = 2.2Hz, 1H), 7.41 (dd, J = 8.3, 2.3Hz, 1H), 5 .14 ​​(dd, J=12.9, 5.3Hz, 1H), 4.38 (t, J=4.9Hz, 2H), 3.35-3.23 (m, 2H), 2.98-2.81 (m, 1H), 2.66-2.54 (m, 2H), 2.11-2.01 (m, 1H). LCMS(ESI)m / z:[M+H] + =418.10.

[0723] Example 59. Preparation of 4-(5-aminopent-1-yn-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione trifluoroacetate (I-81)

[0724]

[0725] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl trifluoromethanesulfonic acid ester

[0726]

[0727] TEA (2.28 mL, 16.4 mmol) and pyridine (2.28 mL, 28.3 mmol) were added to a stirred mixture of 2-(2,6-dioxadiidine-3-yl)-4-hydroxyisoindole-1,3-dione (2.00 g, 7.29 mmol) in DCM (21.0 mL). The reaction mixture was cooled to 0 °C, and then trifluoromethanesulfonic anhydride (3.09 g, 10.9 mmol) was added dropwise. The mixture was warmed to room temperature and stirred for 2 h. The resulting mixture was diluted with water and extracted three times with DCM. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was diluted with DCM (50 mL) and stirred at room temperature for 0.5 h. The resulting mixture was filtered, and the filter cake was washed with DCM to yield 2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl trifluoromethanesulfonic acid (1.92 g, 58.3%) as a grayish-white solid. LCMS(ESI) m / z [M+H] + =407.

[0728] Step 2: Preparation of N-[5-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]pent-4-yn-1-yl] tert-butyl carbamate

[0729]

[0730] CuI (75.0 mg, 0.394 mmol), DIEA (6.86 mL, 53.1 mmol), and Pd(PPh3)2Cl2 (276 mg, 0.394 mmol) were added to a stirred solution of 2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl trifluoromethanesulfonic acid (1.60 g, 3.94 mmol) and N-(pent-4-yn-1-yl)carbamate tert-butyl ester (2.02 g, 11.3 mmol) in THF (40 mL). The resulting mixture was stirred at 70 °C for 1 h. The reactants were filtered, and the filter cake was washed three times with EtOAc. The filtrate was concentrated under reduced pressure, and the residue was taken into water and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide N-[5-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]pent-4-yn-1-yl]carbamate tert-butyl ester (1.12 g, 64.7%) as a grayish-white solid. LCMS (ESI) m / z: [M+H] + =440.

[0731] Step 3: Preparation of 4-(5-aminopent-1-yn-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione trifluoroacetate (I-81)

[0732]

[0733] TFA (3.00 mL) was added to a stirred solution of N-[5-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]pent-4-yn-1-yl]carbamate (1.10 g, 2.50 mmol) in dichloromethane (6 mL). The resulting mixture was stirred at room temperature for 1 h, followed by concentration under reduced pressure. The residue was purified by silica gel rapid column chromatography to provide I-81 (890 mg, 91.2%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.15 (s, 1H), 7.97-7.73 (m, 6H), 5.14 (dd, 1H), 3.09-2.97 (m, 2H), 2.96-2.8 4(m, 1H), 2.67(t, 3H), 2.62-2.54(m, 1H), 2.12-2.02(m, 1H), 1.82-1.94(m, 2H). LCMS (ESI) m / z: [M+H] + =340.12.

[0734] Example 60. Preparation of tert-butyl 4-[3-(prop-2-yn-1-yloxy)propyl]piperazine-1-carboxylate

[0735]

[0736] NaH (4.4 g, 0.18 mol) was added fractionally to a stirred solution of 22.4 g (0.09 mol) of 4-(3-hydroxypropyl)piperazin-1-carboxylate in 500 mL THF at 0 °C for 30 min. Then, propargyl bromide (32.7 g, 0.27 mol) was added dropwise to the reactor at room temperature, and the mixture was stirred for 12 h. The reaction mixture was filtered, and the filter cake was washed with THF. The filtrate was concentrated to dryness, and the residue was purified by reversed-phase rapid C18 chromatography (water:ACN) to yield 14.9 g (57.6%) of 4-[3-(prop-2-yn-1-yloxy)propyl]piperazin-1-carboxylate as a grayish-white solid. LCMS (ESI) m / z: [M+H] + =283.

[0737] Example 61. Preparation of intermediates

[0738] The following compounds in Table B11 were synthesized using a suitable alkyne and in place of 2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione of 5-bromo-2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl trifluoromethanesulfonic acid, according to the scheme in Example 59.

[0739] Table B11. Intermediates

[0740]

[0741] Example 62. Preparation of 3-(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,6-diazaspiro[3.3]heptane-2-yl)propionic acid formic acid

[0742]

[0743] DIEA (8.8 g, 68.2 mmol) was added dropwise to a stirred solution of 5-[2,6-diazaspiro[3.3]heptane-2-yl]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (12.0 g, 33.9 mmol) and acrylic acid (4.9 g, 68.1 mmol) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated and the residue was purified by reversed-phase rapid C18 chromatography (water:ACN:FA) to provide 3-(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,6-diazaspiro[3.3]heptane-2-yl)propionic acid formic acid (1.8 g, 12.5%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.12 (br, 1H), 8.28 (s, 1H), 7.64 (d, J=8.3Hz, 1H), 6.79 (d, J=2.1Hz, 1H), 6.64 (dd, J=8.3, 2.1Hz, 1H), 5.06 (dd, J=12 .9, 5.4Hz, 1H), 4.10 (s, 4H), 4.10 (s, 5H), 2.95-2.81 (m, 1H), 2.61-2.5 9 (m, 2H), 2.57-2.54 (m, 1H), 2.19 (t, J=6.8Hz, 2H), 2.05-1.96 (m, 1H). LCMS(ESI)m / z:[M+H] + =427.15.

[0744] Example 63. Preparation of 5-[3-(2-aminoethoxy)azacyclobutane-1-yl]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione

[0745]

[0746] Step 1: Preparation of tert-butyl 3-(2-[[(benzyloxy)carbonyl]amino]ethoxy)azacyclobutane-1-carboxylate.

[0747]

[0748] To a solution of tert-butyl 3-(2-aminoethoxy)azacyclobutane-1-carboxylate (10.2 g, 47.3 mmol) in THF (50.0 mL), benzyl chloroformate (8.07 g, 47.3 mmol), H₂O (50.0 mL), and K₂CO₃ (19.61 g, 142 mmol) were added. The resulting solution was stirred at room temperature for 3 h. The resulting mixture was extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / EtOAc) to provide tert-butyl 3-(2-[[(benzyloxy)carbonyl]amino]ethoxy)azacyclobutane-1-carboxylate (4.23 g, 25.5%) as a colorless oil; LCMS (ESI) m / z: [M+H] + =351.

[0749] Step 2: Preparation of N-[2-(azacyclobutane-3-yloxy)ethyl]carbamate benzyl ester

[0750]

[0751] TFA (10.0 mL, 135 mmol) was added to a stirred solution of tert-butyl 3-(2-[[(benzyloxy)carbonyl]amino]ethoxy)azacyclobutane-1-carboxylate (4.23 g, 12.1 mmol) in DCM (10.0 mL). The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN:NH4HCO3) to provide N-[2-(azacyclobutane-3-yloxy)ethyl]carbamate (4.56 g) as a colorless oil; LCMS (ESI) m / z [M+H] + =251.

[0752] Step 3: Preparation of N-[2-([1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]azacyclobutane-3-yl]oxy)ethyl]carbamate]benzyl carbamate

[0753]

[0754] N-[2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (3.30 g, 11.9 mmol) and DIEA (4.63 g, 35.8 mmol) were added to a stirred solution of 2-(2,6-dioxadiazin-3-yloxy)ethyl]carbamate (2.99 g, 11.9 mmol) in DMF (45.0 mL). The resulting mixture was stirred at 90 °C for 2 h. The reaction mixture was quenched with water at room temperature. The resulting mixture was extracted three times with EtOAc. The combined organic layers were washed three times with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to provide N-[2-([1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]azacyclobutane-3-yl]oxy)ethyl]carbamate (3.41 g) as a pale yellow solid; LCMS (ESI) m / z: [M+H] + =507.

[0755] Step 4: Preparation of 5-[3-(2-aminoethoxy)azacyclobutane-1-yl]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione

[0756]

[0757] Pd / C (1.55 g, 14.6 mmol) was added to a stirred solution of N-[2-([1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]azacyclobutane-3-yl]oxy)ethyl]carbamate (3.10 g, 6.12 mmol) in ACN (45.0 mL). The resulting mixture was stirred at room temperature for 2 h under a hydrogen atmosphere. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase rapid C18 chromatography to provide the title compound (391 mg, 17.2%) as a pale yellow solid. 1 H NMR (300MHz, DMSO-d6) δ8.45-8.25 (d, 1H), 7.71-7.51 (m, 1H), 7.44-7.25 (m, 1H), 6.83 (d, 1H), 6.69 (dd, 1H), 5.12-4.99 (m, 1H), 4.61-4. 50 (m, 1H), 4.27 (dd, 2H), 3.90 (dd, 2H), 3.61-3.43 (m, 2H), 2.98-2.69 (m, 3H), 2.66-2.51 (m, 1H), 2.10-1.95 (m, 1H); LCMS (ESI) m / z: [M+H] + =373.14.

[0758] Example 64. Preparation of 5-[2-([3-[(2-aminoethoxy)methyl]phenyl]methoxy)ethoxy]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione]

[0759]

[0760] Step 1: Preparation of 2-[2-[(3-[[2-(oxecyclohexane-2-yloxy)ethoxy]methyl]phenyl)methoxy]ethoxy]oxecyclohexane

[0761]

[0762] To a solution of 2-((tetrahydro-2H-pyran-2-yl)oxy)ethane-1-ol (9.50 g, 65.0 mmol) in anhydrous THF (75.0 mL), NaH (2.34 g, 97.5 mmol) was added. The reaction mixture was stirred at 70 °C. The THF solvent was evaporated. The crude substance was dissolved in EtOAc, washed with water, washed with brine, and then dried over Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to yield compound 2-[2-[(3-[[2-(oxecyclohexane-2-yloxy)ethoxy]methyl]phenyl)methoxy]ethoxy]oxecyclohexane (13.9 g, 54.2%); LCMS (ESI) m / z: [M+H] + =395.

[0763] Step 2: Preparation of 2-([3-[(2-hydroxyethoxy)methyl]phenyl]methoxy)ethanol

[0764]

[0765] DCM (15.0 mL) and HCl (30.0 mL, 987 mmol) were added to a stirred solution of 2-[2-[(3-[[2-(oxecyclohexane-2-yloxy)ethoxy]methyl]phenyl)methoxy]ethoxy]oxecyclohexane (13.9 g, 35.2 mmol) in MeOH (15.0 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. The residue was purified by reversed-phase C18 rapid chromatography to provide 2-([3-[(2-hydroxyethoxy)methyl]phenyl]methoxy)ethanol (5.2 g, 65.2%) as a colorless oil; LCMS (ESI) m / z: [M+H] + =227.

[0766] Step 3: Preparation of 2-[[3-([2-[(4-methylbenzenesulfonyl)oxy]ethoxy]methyl)phenyl]methoxy]ethyl ester of 4-methylbenzenesulfonic acid

[0767]

[0768] DMAP (421 mg, 3.45 mmol), TEA (9.30 g, 91.9 mmol), and p-toluenesulfonyl chloride (13.1 g, 68.9 mmol) were added to a stirred solution of 2-([3-[(2-hydroxyethoxy)methyl]phenyl]methoxy)ethanol (5.20 g, 23.0 mmol) in DCM (80.0 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. The resulting mixture was stirred at room temperature for another 5 h. The residue was purified by silica gel column chromatography (petroleum ether / THF) to provide ethyl 4-methylbenzenesulfonic acid 2-[[3-([2-[(4-methylbenzenesulfonyl)oxy]ethoxy]methyl)phenyl]methoxy]ethyl ester (10.3 g, 83.4%) as a pale yellow oil; LCMS (ESI) m / z: [M+H] + =535.

[0769] Step 4: Preparation of N-(tert-butoxycarbonyl)-N-(2-[[3-([2-[(4-methylbenzenesulfonyl)oxy]ethoxy]methyl)phenyl]methoxy]ethyl)carbamate tert-butyl

[0770]

[0771] To a solution of 2-[[3-([2-[(4-methylbenzenesulfonyl)oxy]ethoxy]methyl)phenyl]methoxy]ethyl 4-methylbenzenesulfonic acid (10.3 g, 19.2 mmol) in DMF (80.0 mL), N-(tert-butoxycarbonyl)carbamate tert-butyl ester (4.58 g, 21.1 mmol) and K₂CO₃ (3.97 g, 28.8 mmol) were added. The reaction mixture was stirred at 60 °C for 9 h. The reaction mixture was then cooled to room temperature and poured into 50 mL of EtOAc. The EtOAc layer was then washed three times with 1 N HCl aqueous solution and once with brine. The EtOAc layer was dried over MgSO₄, filtered, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel rapid chromatography (hexane: EtOAc) to provide N-(tert-butoxycarbonyl)-N-(2-[[3-([2-[(4-methylbenzenesulfonyl)oxy]ethoxy]methyl)phenyl]methoxy]ethyl)carbamate tert-butyl (1.69 g, 15.2%) as a colorless oil; LCMS (ESI) m / z: [M+H] + =580.

[0772] Step 5: Preparation of N-(tert-butoxycarbonyl)-N-[2-([3-[(2-[[2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethoxy)methyl]phenyl]methoxy)ethyl]carbamate tert-butyl ester.

[0773]

[0774] Na₂CO₃ (439 mg, 4.14 mmol) was added to a solution of N-(tert-butoxycarbonyl)-N-(2-[[3-([2-[(4-methylbenzenesulfonyl)oxy]ethoxy]methyl)phenyl]methoxy]ethyl)carbamate (800 mg, 1.38 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindole-1,3-dione (799 mg, 2.91 mmol) in DMF (20.0 mL). The resulting mixture was stirred at 80 °C for 5 h. The residue was purified by reversed-phase C18 rapid chromatography (water:MeOH) to provide N-(tert-butoxycarbonyl)-N-[2-([3-[(2-[[2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethoxy)methyl]phenyl]methoxy)ethyl]carbamate as a colorless solid (920 mg, 97.8%); LCMS (ESI) m / z [M+H] + =682.

[0775] Step 6: Preparation of 5-[2-([3-[(2-aminoethoxy)methyl]phenyl]methoxy)ethoxy]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione]

[0776]

[0777] TFA (5.00 mL) was added to a solution of N-(tert-butoxycarbonyl)-N-[2-([3-[(2-[[2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethoxy)methyl]phenyl]methoxy)ethyl]carbamate (920 mg) in DCM. The resulting mixture was stirred at room temperature for 3 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography to provide the title compound (510 mg) as a pale yellow solid. 1H NMR (300MHz, DMSO-d6) δ8.40 (s, 1H), 7.85 (d, 1H), 7.48 (d, 1H), 7.42-7.24 (m, 5H), 5.13 (dd, 1H), 4.58 (s, 2H), 4.50 (s, 2H), 4.43-4.34(m, 2H), 3.83(t, 2H), 3.54(t, 2H), 2.96-2.82(m, 3H), 2.66-2.46(m, 2H), 2.12-1.98(m, 1H); LCMS (ESI) m / z: [M+H] + =482.18.

[0778] Example 65. Preparation of intermediates

[0779] The following compounds in Table B12 were prepared using the procedure in Example 64 with appropriately substituted phenols.

[0780] Table B12. Intermediates

[0781]

[0782] Example 66. Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(5-(piperazin-1-yl)pent-1-yn-1-yl)isoindoline-1,3-dione trifluoroacetic acid

[0783]

[0784] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(5-hydroxypent-1-yn-1-yl)isoindoline-1,3-dione

[0785]

[0786] DIEA (19.2 g, 14.8 mmol) and pentylo-4-yn-1-ol (3.7 g, 44.5 mmol) were added dropwise to a stirred solution of 5-bromo-2-(2,6-dioxadiazine-3-yl)isoindoline-1,3-dione (5.0 g, 14.8 mmol), Pd(PPh3)2Cl2 (2.1 g, 3.0 mmol), and cuprous iodide (I) (0.56 g, 3.0 mmol) in 40 mL of THF. The resulting mixture was stirred at 50 °C for 4 h. The mixture was filtered, and the filter cake was washed three times with THF. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / EtOAc) to provide 2-(2,6-dioxopiperidin-3-yl)-5-(5-hydroxypent-1-yn-1-yl)isoindoline-1,3-dione (1.9 g, 37.6%) as a yellow solid. LCMS (ESI) m / z: [M+H] + =341.

[0787] Step 2: Preparation of 5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)pent-4-ynealdehyde

[0788]

[0789] PCC (5.1 g, 23.5 mmol) was added fractionally to a stirred solution of 2-(2,6-dioxopiridin-3-yl)-5-(5-hydroxypentan-1-yn-1-yl)isoindoline-1,3-dione (4.0 g, 11.7 mmol) in DCM (40 mL) at room temperature. The resulting mixture was stirred at room temperature for 8 h. The mixture was filtered and the filter cake was washed three times with DCM. The filtrate was concentrated under reduced pressure to provide 5-(2-(2,6-dioxopiridin-3-yl)-1,3-dioxopiridindoline-5-yl)pentan-4-yn-aldehyde (3.6 g, 89.8%) as a yellow solid. LCMS (ESI) m / z: [M+H] + =339.

[0790] Step 3: Preparation of tert-butyl 4-(5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)pent-4-yn-1-yl)piperazine-1-carboxylate

[0791]

[0792] NaBH3CN (2.7 g, 42.6 mmol) was added fractionally to a stirred mixture of 5-(2-(2,6-dioxopiperidin-3-yl)v1,3-dioxoisoindoline-5-yl)pent-4-ynealdehyde (3.6 g, 10.6 mmol) and piperazine-1-carboxylic acid tert-butyl ester (1.98 g, 10.6 mmol) in MeOH (35.0 mL). The mixture was stirred at room temperature for 3 h. The resulting mixture was added to an aqueous solution of NH4Cl and extracted three times with EtOAc. The combined organic layers were washed twice with water and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH) to provide tert-butyl 4-(5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)pent-4-yn-1-yl)piperazine-1-carboxylate (2.1 g, 38.9%) as a yellow semi-solid. LCMS (ESI) m / z: [M+H] + =509.

[0793] Step 4: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(5-(piperazin-1-yl)pent-1-yn-1-yl)isoindoline-1,3-dione trifluoroacetic acid

[0794]

[0795] TFA (5 mL) was added dropwise to a stirred solution of tert-butyl piperazine-1-carboxylate (1.2 g, 2.4 mmol) in DCM (20 mL) at room temperature. The resulting mixture was stirred at room temperature for 3 h. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase C18 rapid chromatography under the following conditions: column (water:ACN:TFA) to provide the title compound (0.80 g, 66.7%) as a pale yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.16 (s, 1H), 9.01 (br, 2H), 7.96-7.85 (m, 3H), 5.17 (dd, J=12.8, 5.4Hz, 1H), 4.49 (br, 3H), 3.08 ( br, 3H), 2.90 (ddd, J=16.9, 13.9, 5.3Hz, 1H), 2.66-2.57 (m, 4H), 2.56-2.53 (m, 4H), 2.12-2.03 (m, 1H), 1.98-1.85 (m, 1H). LCMS(ESI)m / z:[M+H] + =409.40.

[0796] Example 67. Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-[[5-(piperazin-1-yl)pentyl]oxy]isoindole-1,3-dione

[0797]

[0798] Step 1: Preparation of 5-[4-(1,3-dioxacyclopentan-2-yl)butoxy]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione

[0799]

[0800] In an air atmosphere at 60 °C, KHCO3 (2.19 g, 21.9 mmol) and KI (272 mg, 1.64 mmol) were added fractionally to a stirred solution of 2-(2,6-dioxadiazin-3-yl)-5-hydroxyisoindole-1,3-dione (3.00 g, 10.9 mmol) and 2-(4-bromobutyl)-1,3-dioxadicyclopentane (2.74 g, 13.1 mmol) in DMF (25.0 mL). The residue was purified by reversed-phase C18 rapid chromatography (water: ACN) to provide 5-[4-(1,3-dioxadicyclopentan-2-yl)butoxy]-2-(2,6-dioxadiazin-3-yl)isoindole-1,3-dione (2.4 g, 54.5%) as an off-white solid. LCMS (ESI) m / z: [M+H] + =403.

[0801] Step 2: Preparation of 5-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]pentanal

[0802]

[0803] A solution of 5-[4-(1,3-dioxacyclopentan-2-yl)butoxy]-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione (2.40 g, 5.96 mmol) and HCl / 1,4-dioxacyclohexane (4 N, 12.0 mL, 48 mmol) and 1,4-dioxacyclohexane (12.0 mL) was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. The crude product was used directly in the next step without further purification to provide 5-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl]oxy]pentanal (1.92 g, 89.8%) as a grayish-white solid. LCMS (ESI) m / z: [M+H] + =359.

[0804] Step 3: Preparation of tert-butyl piperazine-1-carboxylate

[0805]

[0806] NaBH(OAc)3 (2.24 g, 10.5 mmol) was added fractionally to a stirred solution of 5-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-5-yl]oxy]pentanal (1.89 g, 5.27 mmol) and piperazine-1-carboxylic acid tert-butyl ester (0.98 g, 5.27 mmol) in DMF (20.0 mL). The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide 4-(5-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-5-yl]oxy]pentanyl)piperazine-1-carboxylic acid tert-butyl ester (1.46 g, 52.4%) as an off-white solid. LCMS (ESI) m / z: [M+H] + =529.

[0807] Step 4: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-[[5-(piperazin-1-yl)pentyl]oxy]isoindole-1,3-dione

[0808]

[0809] A solution of 4-(5-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-5-yl]oxy]pentyl)piperazine-1-carboxylic acid tert-butyl ester (1.40 g, 2.65 mmol) and TFA (5.00 mL, 67.3 mmol) in DCM was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase C18 rapid chromatography (water:ACN) to provide 2-(2,6-dioxopiperidin-3-yl)-5-[[5-(piperazine-1-yl)pentyl]oxy]isoindole-1,3-dione (724 mg, 63.8%) as a white solid. 1H NMR (300MHz, DMSO-d6) δ11.81 (s, 1H), 11.12 (s, 1H), 9.75 (s, 2H), 7.85 (d, 1H), 7.46-7.32 (m, 2H), 5.12 (dd, 1H), 4.20 (t, 2H), 3.66 (s, 1H), 3 .48(s, 2H), 3.40-3.30(m, 1H), 3.15(d, 2H), 2.99-2.81(m, 1H), 2.67- 2.52 (m, 1H), 2.13-1.98 (m, 1H), 1.88-1.71 (m, 4H), 1.56-1.40 (m, 2H). LCMS(ESI)m / z:[M+H] + =429.15.

[0810] Example 68. Preparation of N-[[3-([[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]oxy]methyl)bicyclo[1.1.1]pentan-1-yl]methyl]-3-[2-(2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethoxy)ethoxy]propionamide (compound 1)

[0811]

[0812] EDCI (12.3 mg, 0.064 mmol), HOBT (8.65 mg, 0.064 mmol), and DIEA (12.4 mg, 0.096 mmol) were added to a stirred mixture of 2-(6-amino-5-[[3-(aminomethyl)bicyclo[1.1.1]pentan-1-yl]methoxy]pyridazin-3-yl)phenol (10.00 mg, 0.032 mmol) and 3-[2-(2-[[2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindol-5-yl]oxy]ethoxy)ethoxy]propionic acid (13.9 mg, 0.032 mmol) in DMF (2.00 mL) at room temperature. The resulting mixture was stirred at room temperature for 4 h. The crude product was purified by preparative HPLC (water:ACN:FA) to provide compound 1 (7.6 mg, 32.0%) as a white solid. 1H NMR (300MHz, DMSO-d6) δ14.34 (s, 1H), 11.12 (s, 1H), 7.92 (dd, J=8.3, 1.6Hz, 1H), 7.85-7.75 (m, 2H), 7.57 (s, 1H), 7.44 (d, J=2.3Hz, 1H), 7.35 (dd, J=8.3, 2.3Hz, 1H), 7.30-7.19 (m, 1H), 6.94-6.83 (m, 2H), 6.55 (s, 2H), 5 .12 (dd, J=12.9, 5.3Hz, 1H), 4.30 (d, J=4.2Hz, 4H), 3.77 (t, J=4.4Hz, 2H), 3.63-3.54 (m, 4H), 3.54-3.47 (m, 2 H), 3.16 (d, J=5.7Hz, 2H), 2.96-2.82 (m, 1H), 2.66-2.53 (m, 2H), 2.31 (t, 2H), 2.11-1.98 (m, 1H), 1.70 (s, 6H). LCMS(ESI)m / z:[M+H] + =729.35.

[0813] Example 69. Preparation of 2-(6-amino-5-(2-(3-aminobicyclo[1.1.1]pentane-1-yl)ethoxy)pyridazin-3-yl)phenol hydrochloride

[0814]

[0815] HCl (gas) / 1,4-dioxane (3.00 mL) was added dropwise to a stirred solution of (3-(2-((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)oxy)ethyl)bicyclo[1.1.1]pentan-1-yl)carbamate (150, 0.364 mmol) in 1,4-dioxane (3.00 mL). The resulting mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. This yielded 2-(6-amino-5-(2-(3-aminobicyclo[1.1.1]pentan-1-yl)ethoxy)pyridazin-3-yl)phenol hydrochloride (100 mg, 88.0%) as a pale yellow solid. LCMS (ESI) m / z: [M+H] + =313.

[0816] Example 70. Preparation of the compound

[0817] The compounds in Table C1 were prepared using appropriate amines and carboxylic acids according to the scheme in Example 68.

[0818] Table C1. Compounds of the present invention

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833] Example 71. Preparation of N1-(3-(2-((3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)oxy)ethyl)bicyclo[1.1.1]pentan-1-yl)-N4-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)benzyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)succinamide (compound 42)

[0834]

[0835] HATU (8.60 mg, 0.023 mmol) and DIPEA (7.31 mg, 0.057 mmol) were added to a stirred mixture of 3-[[(2S)-1-[(2S,4R)-4-hydroxy-2-([[4-(4-methyl-1,3-thiazo-5-yl)phenyl]methyl]carbamoyl)pyrrolidone-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl]propionic acid (10.0 mg, 0.019 mmol) and 2-(6-amino-5-(2-(3-aminobicyclo[1.1.1]pentan-1-yl)ethoxy)pyridazin-3-yl)phenol hydrochloride (5.89 mg, 0.019 mmol) in DMF (1.00 mL) under a nitrogen atmosphere at room temperature. The mixture solution was purified by preparative HPLC to provide compound 42 (8.7 mg, 54.8%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ14.45 (s, 1H), 9.06 (s, 1H), 8.64 (t, J=6.0Hz, 1H), 8.39 (s, 1H), 8.05 (d d, J=8.4, 1.6Hz, 1H), 7.94 (d, J=9.3Hz, 1H), 7.67 (s, 1H), 7.54-7.41 (m, 4H), 7.38-7.26 (m, 1H), 7.03-6.92 (m, 2H), 6.52 (s, 2H), 5.20 (s, 1H), 4.63-4.39 (m, 4H), 4.38-4.23 (m, 3H), 3.71 (d, J= 7.3Hz, 2H), 2.52(s, 4H), 2.49-2.23(m, 3H), 2.18-2.04(m, 3H), 2.00-1.89(m, 7H), 0.99(s, 9H). LCMS(ESI)m / z[M+H] + =825.10.

[0836] Example 72. Preparation of the compound

[0837] The compounds in Table C2 were prepared using appropriate amines and carboxylic acids according to the scheme in Example 71.

[0838] Table C2. Compounds of the present invention

[0839]

[0840]

[0841]

[0842] Example 73. Preparation of the compound

[0843] The compounds listed in Table C3 were prepared using appropriate amines and carboxylic acids according to the scheme described in Example 22.

[0844] Table C3. Compounds of the present invention

[0845]

[0846]

[0847]

[0848]

[0849]

[0850]

[0851]

[0852]

[0853]

[0854]

[0855]

[0856]

[0857]

[0858]

[0859]

[0860] Example 74. Degradation of BRM and BRG1 by the compounds of the present invention

[0861] This embodiment demonstrates the ability of the compounds of this disclosure to degrade HiBit-BRM or HiBit-BRG1 fusion proteins in a cell-based degradation assay.

[0862] Procedure: A stable HeLa cell line expressing HiBiT-BRM was generated. On day 0, 5000 cells were seeded in 40 μL of medium into each well of a 384-well cell culture plate. On day 1, the cells were treated with 120 nL of DMSO or 120 nL of a compound diluted 3-fold serially in DMSO (two replicates for 10 spots, 30 μM as the final maximum dose). The plates were then incubated in a standard tissue culture incubator for 24 h and equilibrated at room temperature for 15 min. Freshly prepared Nano-Glo HiBiT Lytic Detection System (Promega N3050) reagents were added to each well at 20 μL. Upon addition of this lgBiT-containing reagent, HiBiT and lgBiT proteins bind to form luminescent NanoBiT luciferase. The plates were shaken at room temperature for 10 min, and the bioluminescence was read using an EnVision plate reader (PerkinElmer).

[0863] To measure BRG1 degradation, stable HeLa cell lines expressing HiBit-BRG1 and LgBit were generated. The same protocol as described above was then followed.

[0864] Calculate the degradation percentage using the following formula: %degradation = 100% - 100% x (Lum) 样品 -Lum LC ) / (Lum HC -Lum LC Cells treated with DMSO were used as a high control (HC), and cells treated with a standard of 2 μM of a known BRM / BRG1 degrader were used as a low control (LC). Data were fitted to a four-parameter nonlinear curve to calculate the IC50. 50 The (μM) values ​​are shown in Table 3.

[0865] Results: As shown in Table 2 below, the compounds of the present invention degrade both BRM and BRG1.

[0866] Table 2. BRM / BRG1 degradation activity

[0867]

[0868]

[0869]

[0870]

[0871] "+" indicates inhibition at ≥1000 nM; "++" indicates inhibition at ≥100 nM.

[0872] "+++" indicates inhibition of ≥10 nM; "++++" indicates inhibition of <10 nM.

[0873] “NC” indicates not calculated; “A” indicates maximum degradation ≥75%;

[0874] "B" indicates a maximum degradation of ≥50%; and "C" indicates a maximum degradation of <50%.

[0875] Other implementation plans

[0876] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were expressly and individually incorporated by reference in its entirety. Where a term in this application is found to have a different definition in a document incorporated herein by reference, the definition provided herein shall be used as the definition of that term.

[0877] Although the invention has been described in conjunction with its specific embodiments, it should be understood that the invention is capable of further modifications, and this application is intended to cover any variations, applications or improvements of the invention that generally follow the principles of the invention and include deviations from the disclosure that fall within the known or conventional practice of the field to which the invention pertains, and that can be applied to the basic features set forth above, as well as to the scope of the claims.

[0878] Other embodiments are described in the claims.

Claims

1. A compound having the structure of Formula I or a pharmaceutically acceptable salt thereof: in X is a halogen; X 1 is O or NR 1 ; k is 0, 1, 2, or 3; n is 0, 1, or 2; R 1 is H or Ci-C6alkyl; L 1 is C1-C6alkylene; L 2 It does not exist; it is a C1-C6 alkylene group; C1-C 20 Heteroalkyl or C2-C9 heterocyclic group; Each L 3 Independently, it is C1-C 20 Heteroalkyl, C3-C 10 subcarbocyclic group, C3-C 10 Carbocyclic-C1-C6 alkylene, C2-C9 heterocyclic, C2-C9 heterocyclic-C1-C6 alkylene, C6-C 10 Aspartic, C6-C 10 arylene-C1-C6 alkylene, C2-C6 ynylene, O or NR 1 ;and D is the degradation moiety, wherein the degradation moiety is a ubiquitin ligase-binding moiety selected from the following group: Where A 2 It is the bond between the degradation portion and the linker; and R A1 R A2 R A3 and R A4 It is hydrogen.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L 2 It is a C1-C6 alkylene group.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L 2 yes 4. The compound of claim 3 or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2.

5. The compound of claim 3 or a pharmaceutically acceptable salt thereof, wherein at least one L 3 yes Phenylidene, O or NR 1 .

6. Compounds selected from compounds 1 to 105 of the group consisting of their pharmaceutically acceptable salts:

7. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

8. Use of the compound of claim 1 in the preparation of a medicament for treating BAF complex-related disorders in subjects with such need.

9. The use of claim 8, wherein the BAF complex-related barrier is cancer or viral infection.

10. Use of the compound of claim 1 in the preparation of a medicament for treating disorders associated with loss-of-function mutations in BRG1 in subjects with such need.

11. The use as claimed in claim 10, wherein the barrier associated with the BRG1 loss-of-function mutation is cancer.

12. Use of the compound of claim 1 in the preparation of a medicament for treating cancer in subjects in need of it.

13. The use as claimed in claim 12, wherein the cancer is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary origin, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, esophageal-gastric cancer, pancreatic cancer, hepatobiliary cancer, soft tissue sarcoma, ovarian cancer, head and neck cancer, renal cell carcinoma, bone cancer, non-Hodgkin lymphoma, small cell lung cancer, prostate cancer, embryonal tumor, germ cell tumor, cervical cancer, thyroid cancer, salivary gland cancer, gastrointestinal neuroendocrine tumor, uterine sarcoma, gastrointestinal stromal tumor, central nervous system cancer, thymic tumor, adrenocortical carcinoma, appendix cancer, small bowel cancer, or penile cancer.

14. The use as claimed in claim 12, wherein the cancer is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary origin, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, or penile cancer.

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