Low molecular weight protein degrading agents and their use

Abstract

The present invention relates to compounds of formula (Ia), (Ib), (Ic) and (II) and their use in methods of treating cancer. These compounds can be used in combination with existing anti-cancer therapies to improve their efficacy.

Description

Technical Field

[0001] This invention relates to compounds that regulate the cellular concentration of various disease-related proteins, such as transcription factor SALL4 and translation termination factor GSPT1, and their applications. Background Technology

[0002] The ubiquitin-proteasome system (UPS) is responsible for maintaining a healthy and well-balanced proteome. During ubiquitination, ubiquitin units are covalently linked to proteins, forming polyubiquitin chains, which mark the protein's degradation by the proteasome. Ubiquitination is crucial for the regulation of almost all cellular processes and is itself tightly regulated. Ubiquitin ligases such as cereblon (CRBN) promote the ubiquitination of various proteins in the body and contribute to the precise regulation of the system. Upon recognition, ubiquitin ligases mediate the binding of the ubiquitin moiety to the target protein, thus tagging the target protein for degradation by the proteasome.

[0003] The idea of ​​selective target protein degradation (TPD) by modulating UPS was first described in 1999 (US2002173049 A1 (PROTEINIX INC) November 21, 2002). The implementation of this concept has been demonstrated in clinically approved thalidomide analogs, as thalidomide analogs are compatible with CRL4. CRBN The binding of E3 ligases induces the recruitment of the selected target protein, leading to its ubiquitination and subsequent proteasome degradation. Recently, Faust TB et al. Annu. Rev. Cancer Biol. 2021. 5:181–201 reviewed the latest scientific and clinical advances in TPD.

[0004] Cereblon modulators in cancer treatment

[0005] Cereblon (CRBN) is a protein associated with DDB1 (DNA damage-binding protein 1), CUL4 (Cullin-4), and RBX1 (RING-Box protein 1). These proteins together form a ubiquitin ligase complex, which belongs to the CullinRING ligase (CRL) protein family and is referred to as CRL4. CRBN Thalidomide, a drug approved in the late 1990s for the treatment of multiple myeloma, binds to cereblon and modulates CRL4. CRBN Substrate specificity of ubiquitin ligase complex. This mechanism is the basis for the pleiotropic effects of thalidomide on immune cells and cancer cells (Lu G et al. Science. 2014 Jan 17; 343(6168): 305-9).

[0006] Cereblon modulators have been clinically proven effective in many hematologic malignancies, such as multiple myeloma, myelodysplastic syndromes, lymphoma, and leukemia (Le Roy A et al. Front Immunol. 2018; 9:977). The antitumor activity of CMA is mediated by the following:

[0007] ● It inhibits cancer cell proliferation and induces apoptosis.

[0008] ●Disruption of nutritional support for the tumor stroma,

[0009] ● Stimulation of immune cells leads to the proliferation of T cells, the production of cytokines, and the activation of NK (natural killer) cells.

[0010] The success of thalidomide in cancer treatment spurred efforts to develop analogues with higher potency and fewer harmful side effects. This resulted in a number of drug candidates, including lenalidomide, pomalidomide, CC-220, CC-122, CC-885, and CC-90009. These compounds are collectively known as Cereblon modulators (CMAs). For a discussion of these compounds, see, for example, US 5635517 (B2), WO2008039489 (A2), WO2017197055 (A1), WO2018237026 (A1), WO2017197051 (A1), US 8518972 (B2), EP2057143 (B1), WO2019014100 (A1), WO2004103274 (A2) and Surka Ch et al. Blood. 2021 Feb 4;137(5):661-677.

[0011] Novel substrate degradation patterns of cereblon modulators mediate phenotypes and clinical outcomes in an environment-specific manner. For example, downregulation of lymphokine transcription factors IKZF1 (KAROS family zinc finger protein 1) and IKZF3 (KAROS family zinc finger protein 3) mediates the clinical efficacy of lenalidomide and pomalidomide in multiple myeloma. Meanwhile, downregulation of IKZF1 and IKZ3 has been shown to contribute to the occurrence of side effects, reducing the dosage that can be administered to patients with myelodysplastic syndromes. Side effects occurring during lenalidomide treatment include neutropenia, leukopenia, thrombocytopenia, anemia, and bleeding disorders (Stahl M et al. Cancer. 2017 May 15;123(10):1703-1713). Therefore, it is hoped that the development of cereblon modulators can be advanced to achieve the desired CRL4. CRBN The substrate specificity of ubiquitin ligase complexes allows for the achievement of desired therapeutic and safety profiles based on clinical context (Sievers QL et al. Science. 2018Nov 2; 362(6414).

[0012] SALL4-targeted strategy to eliminate tumor cells

[0013] The expression of the Sal-like protein 4 (SALL4) transcription factor is primarily detected in embryonic stem cells (ESCs), adult germ cells, and blood progenitor cell populations, where it acts as a core controller regulating cellular "stem cell nature" during developmental events. However, SALL4 is reactivated and misregulated in a variety of cancers, including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), germ cell tumors, breast cancer, hepatocellular carcinoma (HCC), lung cancer, glioma, and gastric cancer. Aberrant expression of SALL4 has also been detected in patients with myelodysplastic syndromes (MDS), and its expression level is associated with disease progression. Furthermore, SALL4 expression is associated with poorer survival and prognosis in hepatocellular carcinoma and is also associated with metastasis in, for example, endometrial cancer, colorectal cancer, and esophageal squamous cell carcinoma (Yong KJ et al. The New England Journal of Medicine, 2013, Forghanifard MM et al. Journal of Biomedical Science, 2013).

[0014] Downregulation of SALL4 leads to increased apoptosis and cell cycle arrest (Gao C et al. Transfusion, 2013; Ma Y et al. Blood, 2006; Cao D et al. The American Journal of Surgical Pathology, 2009; Kobayashi D et al. International Journal of Oncology, 2011; Oikawa T et al. Hepatology, 2013; Morita S et al. The American Journal of Surgical Pathology, 2013; Zhang L et al.: Journal of Neuro-Oncology, 2015; Wang F et al. Journal of Hematology Oncology, 2013; Zhang L et al.: Oncogene, 2013). SALL4-derived peptides block protein-protein interactions with nucleosome remodeling and histone deacetylation (NuRD) complexes, leading to significant leukemia cell death, but have no cytotoxic effect on normal CD34+ HSCs / HPCs (GaoC et al. Blood, 2013).

[0015] Studies have shown that upregulating SALL4 in cancer cells can promote their proliferation and invasion, as well as tumor drug resistance, while downregulating SALL4 can inhibit cancer cell growth. Another approach to finding more effective and efficient methods for cancer cell elimination is to combine existing practices. Here, downregulating SALL4 is one way to sensitize tumor cells to standard care cancer therapies such as surgery, chemotherapy, hormone therapy, radiation therapy and / or biotherapy, and immunotherapy.

[0016] GSPT1-targeted strategy to eliminate tumor cells

[0017] GSPT1 is a translation termination factor, and its downregulation may activate the integrated stress response leading to cancer cell death. GSPT1 deficiency has been shown to play a crucial functional role in the anti-AML activity of CC-90009, which is currently in clinical development. GSPT1 degradation activates the GCN1 / GCN2 / eIF2α / ATF4 axis of the integrated stress response and subsequently induces acute apoptosis in AML (Surka Ch et al. Blood. 2021 Feb 4;137(5):661-677). Summary of the Invention

[0018] This invention provides compounds that can regulate the levels of target disease-related proteins (e.g., SALL4 and GSPT1) both in vitro and in vivo. The compounds of this invention exhibit preferential degradation of the target proteins, thereby producing unique phenotypic characteristics.

[0019] The present invention also provides a method for treating cancer, the method comprising administering to a patient a pharmaceutical composition comprising a compound of the present invention.

[0020] This invention relates to the development of drug candidates for inhibiting cancer development and / or increasing the effectiveness of currently available therapies. The efficacy of small molecule drugs depends on the induced degradation of preferentially targeted proteins. An example of a protein preferentially targeted by the compounds of this invention is SALL4, which plays an important role in the carcinogenesis process and its progression. Another protein preferentially targeted by the compounds of this invention is GSPT1.

[0021] This invention provides a method for regulating the expression levels of therapeutic proteins (e.g., to increase efficacy and / or reduce side effects). This invention provides compounds that induce preferential degradation of specific targets (e.g., SALL4), thereby providing a novel mechanism for the therapeutic activity of proteins that are not normally susceptible to the effects of small molecule compounds.

[0022] The compounds of this invention effectively inhibit the growth of several cancer types: hepatocellular carcinoma (HEP3B, SNU-398), neuroblastoma (Kelly), leukemia (KG-1, KG-1a, UOC-M1, MOLT-3, MOLT-4, MOLM-13, MOLM-1, MOLM-6), prostate cancer (22Rv1), and multiple myeloma (MOLP-2). Furthermore, the compounds of this invention did not exhibit activity against H929 and several other cell lines (Tables 10 and 12), making them unique compared to known compounds such as CC-90009, lenalidomide, pomalidomide, CC-122, and CC-220. This surprising effect makes the compounds clinically attractive because their enhanced selectivity may correspond to a therapeutic window for specific cancer types such as HCC.

[0023] The developed SALL4 degradation candidate drug could be used to treat new cancer types for which IMiD is known to be inapplicable.

[0024] The compounds of this invention can eliminate the side effects that occur in patients taking lenalidomide. Since these effects of lenalidomide are caused by the degradation of IKZF1 / IKZF3, they can be eliminated by using the compounds of this invention.

[0025] To minimize the potential adverse side effects caused by IKZF1 or IKZF3 degradation, candidate drugs that preferentially target SALL4 are provided, which have no or low activity against the target protein compared to current IMiD drugs.

[0026] The compounds of this invention exhibit high priority for the degradation of SALL4 protein and can effectively and rapidly induce protein degradation, which will significantly improve the prognosis of cancer patients.

[0027] In a first aspect, the present invention provides compounds of formula (Ia), (Ib) or (Ic):

[0028]

[0029] Or its pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug.

[0030] in

[0031] L is selected from hydrogen, alkyl, alkenyl, benzyl, aryl, heteroaryl, haloalkyl, haloalkenyl, and -CH2OC(O). t Bu, -CH2C(O)OR'', -C(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)NR''2, -OR'', -NR''2, -S(O)2R'' or P(O)(OR'')(OR'');

[0032] Each R'' is independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl;

[0033] Each R 14 Independently selected from deuterium and hydrogen;

[0034] R 15 Selected from hydrogen, deuterium, and C1-C4 alkyl groups;

[0035] R g It is CR a R b R c ,

[0036] R h Selected from H and C1-C4 alkyl groups;

[0037] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0038] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0039] R c Selected from NR1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0040] Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0041] n is 0, 1, or 2;

[0042] X is selected from F, Cl, Br, and I;

[0043] R 1 Selected from H and C1-C3 alkyl groups,

[0044] R 2 Selected from H, C1-C3 alkyl, -COR 3 and -COOR 3 ,

[0045] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0046] R 3 Selected from:

[0047] Unsubstituted C1-C4 alkyl groups;

[0048] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 -OH, OR 5 OCOR 5 Unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole group, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, -OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); and wherein R 4 Not X;

[0049] C2-C substituted with halophenyl groups10 alkyl;

[0050] A 6-membered aryl group substituted by one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl);

[0051] Unsubstituted 5-membered or 6-membered heterocyclic groups;

[0052] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted with one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered aryl, 6-membered aryl, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0053] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2;

[0054] In equation (Ia):

[0055] When R a R b R 1 and R 2 When each is H, then n is 0 or 1;

[0056] When R a R b R d R e R f R h R 1 R 2 R 14 And L are each H, and R 15 If it is H or C1-C4 alkyl, then n is 0;

[0057] When R a R b and R 1 Each is H and R 2 It is -COR 3 When n is 0 or 1, then n is either 0 or 1; and

[0058] When R a R b R d R e R f and R 1 Each is H and R 3 When the alkyl group is unsubstituted (C1-C4), then n is 0.

[0059] In some implementation schemes, R 3 Selected from:

[0060] Unsubstituted C1-C4 alkyl groups;

[0061] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 -OH, OR 5 OCOR 5 Unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole group, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); and wherein R 4 Not X;

[0062] A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and

[0063] Unsubstituted 5-membered or 6-membered heterocyclic groups.

[0064] In some implementation schemes, R 3 Selected from:

[0065] Unsubstituted C1-C4 alkyl groups;

[0066] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 -OH, OR 5 OCOR 5 Unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole group, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, -OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); wherein R 4 It is not X, and where C1-C 10 When alkyl groups are substituted with indole, C1-C 10 The alkyl group is also affected by at least one additional R 4 replace;

[0067] A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and

[0068] Unsubstituted 5-membered or 6-membered heterocyclic groups.

[0069] In a second aspect, the present invention provides compounds of formula (II):

[0070]

[0071] Or its pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug.

[0072] in:

[0073] Each R 1 Independently selected from H and C1-C4 alkyl groups;

[0074] R 11 Is it OH or OR? 5a ;as well as

[0075] R 5a It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0076] In some implementations of any of the above aspects, R 11 It is OH.

[0077] In some implementations of any of the above aspects, NR 1 R 1 It is NH2. In some embodiments of any of the above aspects, R h It is H. In other implementations, R h It is a methyl group.

[0078] In some implementations of any of the above aspects, R a and R b Each is H. In other implementations, R a and R b Each is a deuterium. In other implementations, R a It is H and R b It is a methyl group.

[0079] In some implementations of any of the above aspects, R c Selected from NHR 2 and OH.

[0080] In some embodiments of any of the above aspects, the compound is selected from:

[0081]

[0082] And its pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs.

[0083] In some implementations of any of the above aspects, R c It is NHR 2 .

[0084] In some implementations of any of the above aspects, R 2 Selected from H, -COR 3 and -COOR 3

[0085] In some implementations of any of the above aspects, R 3 It is by one or more R 4 Replacement C1-C 10 Alkyl group. In some embodiments, each R 4 Independently selected from NH2, OCOR 5 substituted or unsubstituted meta-dioxanepentenyl, indole, and 6-membered aryl groups substituted with one or more -OCO (C1-C4 alkyl); wherein R 4 It's not X.

[0086] In some embodiments of any of the above aspects, the compound is selected from compounds 51, 2, 22, 3, 24, 6, 23, 52, and 37:

[0087]

[0088] And its pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs.

[0089] In a third aspect, the present invention provides a pharmaceutical composition comprising a compound of any of the embodiments described above.

[0090] In a fourth aspect, the present invention provides a compound for use in a method of treating cancer, the method comprising administering the compound to a subject in need of it, wherein the compound is:

[0091] (i) Compounds of formula (Ia), (Ib), or (Ic):

[0092]

[0093]

[0094] Or its pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug.

[0095] in

[0096] L is selected from hydrogen, alkyl, alkenyl, benzyl, aryl, heteroaryl, haloalkyl, haloalkenyl, and -CH2OC(O). t Bu, -CH2C(O)OR'', -C(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)NR''2, -OR'', -NR''2, -S(O)2R'' or P(O)(OR'')(OR'');

[0097] Each R'' is independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl;

[0098] Each R 14 Independently selected from deuterium and hydrogen;

[0099] R 15 Selected from hydrogen, deuterium, and C1-C4 alkyl groups;

[0100] R g Selected from -COOH and CR a R b R c ,

[0101] R h Selected from H and C1-C4 alkyl groups;

[0102] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0103] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0104] R c Selected from NR 1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0105] Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0106] n is 0, 1, or 2;

[0107] X is selected from F, Cl, Br, and I;

[0108] R 1 Selected from H and C1-C4 alkyl groups,

[0109] R 2 Selected from H, C1-C4 alkyl, -COR 3 and -COOR 3 ,

[0110] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0111] R 3 Selected from:

[0112] Unsubstituted C1-C4 alkyl groups;

[0113] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 -OH, OR 5 OCOR 5 Unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole group, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); and wherein R 4 Not X;

[0114] C2-C substituted with halophenyl groups 10 alkyl;

[0115] A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and

[0116] Unsubstituted 5-membered or 6-membered heterocyclic groups

[0117] R5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted with one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered aryl, 6-membered aryl, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0118] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2;

[0119] Where R a R b and R 1 Each is H and R 2 Is it H or -COR? 3 When n is 0 or 1, then n is either 0 or 1.

[0120] or

[0121] (ii) Compounds of formula (II):

[0122]

[0123] Or its pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug.

[0124] in:

[0125] Each R 1 Independently selected from H and C1-C4 alkyl groups;

[0126] R 11 Is it OH or OR? 5a ;as well as

[0127] R 5a It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0128] In a fifth aspect, the present invention provides a pharmaceutical composition for use in a method of treating cancer, the method comprising administering the pharmaceutical composition to a subject in need of it, wherein the pharmaceutical composition comprises:

[0129] (i) Compounds of formula (Ia), (Ib), or (Ic):

[0130]

[0131]

[0132] Or its pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug.

[0133] in

[0134] L is selected from hydrogen, alkyl, alkenyl, benzyl, aryl, heteroaryl, haloalkyl, haloalkenyl, and -CH2OC(O). t Bu, -CH2C(O)OR'', -C(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)NR''2, -OR'', -NR''2, -S(O)2R'' or P(O)(OR'')(OR'');

[0135] Each R'' is independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, or benzyl;

[0136] Each R 14 Independently selected from deuterium and hydrogen;

[0137] R 15 Selected from hydrogen, deuterium, and C1-C4 alkyl groups;

[0138] R g Selected from -COOH and CR a R b R c ,

[0139] R h Selected from H and C1-C4 alkyl groups;

[0140] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0141] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0142] R c Selected from NR 1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0143] Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0144] n is 0, 1, or 2;

[0145] X is selected from F, Cl, Br, and I;

[0146] R 1 Selected from H and C1-C4 alkyl groups,

[0147] R 2Selected from H, C1-C4 alkyl, -COR 3 and -COOR 3 ,

[0148] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0149] R 3 Selected from:

[0150] Unsubstituted C1-C4 alkyl groups;

[0151] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 OH, OR 5 OCOR, unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, -OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); and wherein R 4 Not X;

[0152] C2-C substituted with halophenyl groups 10 alkyl;

[0153] A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and

[0154] Unsubstituted 5-membered or 6-membered heterocyclic groups;

[0155] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted with one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered aryl, 6-membered aryl, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0156] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2;

[0157] Where R a R b and R 1 Each is H and R 2 Is it H or -COR? 3 When n is 0 or 1, then n is either 0 or 1.

[0158] or

[0159] (ii) Compounds of formula (II):

[0160]

[0161] Or its pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug.

[0162] in:

[0163] Each R 1 Independently selected from H and C1-C4 alkyl groups;

[0164] R 11 Is it OH or OR? 5a ;as well as

[0165] R 5a It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0166] In a sixth aspect, the present invention provides a method for treating cancer, the method comprising administering to a subject in need of the compound or pharmaceutical composition described in either the fourth or fifth aspect.

[0167] In some implementation schemes of any of aspects four through six, R 3 Selected from:

[0168] Unsubstituted C1-C4 alkyl groups;

[0169] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 -OH, OR 5 OCOR 5Unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole group, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); and wherein R 4 Not X;

[0170] A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and

[0171] Unsubstituted 5-membered or 6-membered heterocyclic groups.

[0172] In some implementation schemes of any of aspects four through six, R 3 Selected from:

[0173] Unsubstituted C1-C4 alkyl groups;

[0174] by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOR 5 NHCOOR 5 -OH, OR 5 OCOR 5 Unsubstituted 5-membered heterocyclic group, substituted or unsubstituted meta-dioxolaneyl group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group, 6-membered heteroaryl group, indole group, and 6-membered aryl group substituted by one or more substituents independently selected from C1-C4 alkyl, -OH, -CH2-OH, -OCO (C1-C4 alkyl), and CH2OCO (C1-C4 alkyl); wherein R 4 It is not X, and where C1-C 10 When alkyl groups are substituted with indole, C1-C 10 The alkyl group is also affected by at least one additional R 4 replace;

[0175] A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and

[0176] Unsubstituted 5-membered or 6-membered heterocyclic groups.

[0177] In some implementation schemes of any of aspects four through six, R 11 It is OH.

[0178] In some implementation schemes of any of aspects four through six, NR1 R 1 It is NH2.

[0179] In some implementation schemes of any of aspects four through six, R h It is H. In other implementations, R h It is a methyl group.

[0180] In some implementation schemes of any of aspects four through six, R a and R b Each is H. In other implementations, R a and R b Each is a deuterium. In other implementations, R a It is H and R b It is a methyl group.

[0181] In some implementation schemes of any of aspects four through six, R c Selected from NHR 2 and OH.

[0182] In some embodiments of any of aspects four through six, the compound is selected from the compounds in Table 1:

[0183] Table 1.

[0184]

[0185] And its pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs.

[0186] In some implementation schemes of any of aspects four through six, R c It is NHR 2 .

[0187] In some implementation schemes of any of aspects four through six, R 2 Selected from H, -COR 3 and -COOR 3 .

[0188] In some implementation schemes of any of aspects four through six, R 3 It is by one or more R 4 Replacement C1-C 10 alkyl.

[0189] In some implementation schemes of any of aspects four through six, each R 4 Independently selected from NH2, OCOR 5 Indole and 6-membered aryl groups substituted with one or more -OCO (C1-C4 alkyl); wherein R 4 It's not X.

[0190] In some embodiments of any of aspects four through six, the compound is selected from compounds 51, 2, 22, 3, 24, 6, 23, 52, 37, and 1:

[0191] .

[0192] In some embodiments of any of aspects four through six, the cancer is hepatocellular carcinoma, neuroblastoma, leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), multiple myeloma, breast cancer, prostate cancer, bladder cancer, kidney cancer, muscle cancer, ovarian cancer, skin cancer, pancreatic cancer, breast cancer, colon cancer, blood cancer, connective tissue cancer, placental cancer, bone cancer, uterine cancer, cervical cancer, choriocarcinoma, endometrial cancer, gastric cancer, or lung cancer. In some embodiments, the cancer is hepatocellular carcinoma, neuroblastoma, leukemia, prostate cancer, or multiple myeloma.

[0193] In some embodiments of any of aspects four through six, the cancer is hepatocellular carcinoma. In some such embodiments, the compound:

[0194] (a) Selected from compounds 6, 3, 36, 42, 26, 23, 24, 1, 52, 28, 27, 37, 39, 38 and 5;

[0195] (b) Selected from compounds 6, 3, 36, 42, 26, 23, 24, 1 and 52.

[0196] In some embodiments of any of aspects four through six, the cancer is neuroblastoma. In some such embodiments, the compound is selected from compounds 3, 36, 42, 37, 28, 27, and 1.

[0197] In some embodiments of any of aspects four through six, the cancer is leukemia. In some such embodiments, the compound is selected from compounds 3, 36, 42, 37, 28, 27, 24, and 1.

[0198] In some embodiments of any of aspects four through six, the method of treating cancer further includes administering a second cancer therapy to the subject. In some embodiments, the second cancer therapy is chemotherapy, radiotherapy, or immunotherapy. In some embodiments, the second cancer therapy includes administering an agent selected from therapeutic antibodies that specifically bind to cancer antigens, hematopoietic growth factors, cytokines, anticancer agents, antibiotics, Cox-2 inhibitors, immunomodulators, immunosuppressants, corticosteroids, or pharmacologically active mutants or derivatives thereof.

[0199] In some embodiments of any of the fourth to sixth aspects, the method includes orally administering the compound or the pharmaceutical composition to the subject.

[0200] In some embodiments of any of the fourth to sixth aspects, the cancer is associated with one or more proteins selected from the group consisting of SALL4 or GSPT1.

[0201] In some embodiments of any of the first to sixth aspects, the compound has formula (Ia) or formula (II).

[0202] In some embodiments of any of the first to sixth aspects, the compound has formula (Ib).

[0203] In some embodiments of any of the first to sixth aspects, the compound has formula (Ic).

[0204] In some embodiments of any of the first to sixth aspects, the compound has formula (Ia) or formula (Ic).

[0205] In some embodiments of any of the first to sixth aspects, the compound has formula (II).

[0206] In some embodiments of any of the first to sixth aspects, L is selected from hydrogen, alkyl, alkenyl, benzyl, aryl, heteroaryl, haloalkyl, haloalkenyl, -CH2OC(O). t Bu, -CH2C(O)OR'', -C(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)NR''2, -OR'', -NR''2, -S(O)2R''. In some embodiments, L is alkyl, benzyl, -CH2OC(O)Me, or -CH2OC(O). t Bu. In other embodiments, L is hydrogen.

[0207] In some embodiments of any of the first to sixth aspects, n is 1. In other embodiments, n is 0.

[0208] In some implementation schemes of any of the first to sixth aspects, each R 14 It is deuterium. In other implementations, each R 14 It is hydrogen.

[0209] In some implementation schemes of any of the first to sixth aspects, R 15 It is deuterium. In other implementations, R 15 It is hydrogen.

[0210] In some implementation schemes of any of the first to sixth aspects, R e It is X.

[0211] In some implementation schemes of any of the first to sixth aspects, R 1 Selected from H and methyl. In some embodiments, R 1 It is H.

[0212] In some implementation schemes of any of the first to sixth aspects, R 2 Selected from H, methyl, -COR 3 and -COOR 3 In some implementations, R 2 It is H or methyl. In some embodiments, R 2 It is -COR 3 or -COOR 3 .

[0213] In some implementation schemes of any of the first to sixth aspects, R 1 It is H, R 2 It is H. In other implementations, R 1 It is methyl, R 2 It is methyl. In other embodiments, R 1 It is H, R 2 It is -COR 3 or -COOR 3 .

[0214] In some embodiments of any of the first to sixth aspects, administration of the compound or the pharmaceutical composition to a subject reduces the level of the target protein in the subject.

[0215] In some implementations, the target protein is selected from SALL-4 and GSPT1.

[0216] In some embodiments of any of the first to sixth aspects, administration of the compound or the pharmaceutical composition to the subject induced a minimal or substantially no reduction in the level of IKZF1 or IKZF3 protein. Attached Figure Description

[0217] Figure 1 Representative results of the SALL4 degradation assay in the Kelly cell line are shown. Cells were treated for 24 hours with compounds 1 and 44 of the present invention at concentrations ranging from 0.01 μM to 1 μM, as well as the reference compounds thalidomide and lenalidomide.

[0218] Figure 2 shows the SALL4 degradation-time process in the Kelly cell line. Cells were treated with 0.1 μM lenalidomide, compound 1, and compound 44 for 3, 6, 12, 24, 48, and 72 hours. A) WB membrane, B) Percentage of optical density of DMSO control, normalized to loading control.

[0219] Figure 3 Representative results from the GSPT1 degradation assay in the Hep3B cell line are shown. Cells were treated with compounds 52, 5, 7, and 54 of the present invention at a concentration of 10 μM for 24 hours.

[0220] Figure 4 shows representative results of the Ikaros (IKZF1) degradation assay in the H929 cell line. A) Cells were treated for 24 hours with compounds 1, 44, 28, and 27 of the present invention at concentrations of 1 μM and 10 μM, as well as the reference compound lenalidomide. B) Cells were treated for 24 hours with compounds 4, 52, 5, 7, and 54 at concentrations of 10 μM, as well as the reference compounds 100, CC-90009, and pomalidomide.

[0221] Figure 5 Representative results from the Aiolos (IKZF3) degradation assay in the H929 cell line are shown. Cells were treated for 24 hours with compounds 1, 44, 28, and 27 of the present invention at concentrations of 1 μM and 10 μM, as well as the reference compound lenalidomide.

[0222] Figure 6 shows the effects of various compounds on cell viability. Luminescence (RLU) values ​​were normalized to DMSO control. A) Hep3B cells were treated for 72 h with compounds 1, 2, 3, 6, 23, 37, and 52 of the present invention at concentrations ranging from 0.001 μM to 50 μM. B) H929 cells were treated for 72 h with compounds 1, 3, 37, and 52 of the present invention at concentrations ranging from 0.001 μM to 50 μM, and with reference compound CC-90009 and pomalidomide. C) SNU-398 cells were treated for 72 h with compound 3 of the present invention at concentrations ranging from 0.001 μM to 50 μM, and with reference compound CC-90009.

[0223] Figure 7 shows the effects of various compounds on cell survival. A) Kelly and B) Hep3B cells were treated with compound 1 or lenalidomide at concentrations ranging from 0.1 μM to 10 μM. Crystal violet staining was performed after 9 to 10 days of culture. Detailed Implementation

[0224] The present invention provides compounds of formulas (Ia), (Ib), (Ic) and (II) as defined above, and pharmaceutical compositions comprising these compounds.

[0225] The present invention also provides a method for treating cancer, the method comprising administering the compound or pharmaceutical composition of the present invention to a subject in need of such treatment.

[0226] The compounds of this invention induce efficient degradation of SALL4 protein in Kelly (neuroblastoma) cell lines over a wide concentration range (see [link]). Figure 1 Therefore, the compounds of the present invention can be used as anticancer drug candidates. The compounds of the present invention possess unique degradation properties because they induce efficient degradation of selected oncogenic proteins such as SALL4 and GSPT1 proteins, but are inactive or have low potency against Ikaros (IKZF1) and Aiolos (IKZF3). Considering the properties of existing degrading agents such as thalidomide and lenalidomide, the unique degradation properties of these compounds are surprising (see [link to relevant documentation]). Figures 1-5 Furthermore, the kinetics of SALL4 degradation were evaluated (see Figure 2). The compounds of the present invention degrade SALL4 more rapidly and efficiently than lenalidomide, indicating that the compounds of the present invention can be administered at lower doses than the reference compounds.

[0227] The compounds of this invention effectively inhibit the growth of several cancer types: hepatocellular carcinoma (HEP3B, SNU-398), neuroblastoma (Kelly), leukemia (KG-1, KG-1a, UOC-M1, MOLT-3, MOLT-4, MOLM-13, MOLM-1, MOLM-6), prostate cancer (22Rv1), and multiple myeloma (MOLP-2). Furthermore, the compounds of this invention did not exhibit activity against H929 and several other cell lines (Tables 10 and 12), making them unique compared to known compounds such as CC-90009, lenalidomide, pomalidomide, CC-122, and CC-220. This surprising effect makes the compounds clinically attractive because their enhanced selectivity may correspond to a therapeutic window for specific cancer types such as HCC.

[0228] Furthermore, the ability of the compounds of this invention to affect the survival of cancer cell lines was evaluated (see [link]). Figure 7A and Figure 7B In cell lines expressing SALL4 (Kelly, Hep3B), the compounds of this invention impaired cell survival, while lenalidomide was inactive against them.

[0229] The compounds of the present invention also exhibit particularly favorable pharmacokinetic properties.

[0230] The compounds of the present invention (or compounds for use according to the present invention) include:

[0231]

[0232] *Available for commercial purchase

[0233] The compound may be in the form of a pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate (e.g., hydrate), amino acid conjugate, or prodrug.

[0234] As used herein, and unless otherwise stated, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid (including inorganic and organic acids). Suitable non-toxic acids include inorganic and organic acids, such as, but not limited to, acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinylsulfonic acid, formic acid, fumaric acid, furoic acid, gluconic acid, glutamic acid, glucuronic acid, galacturonic acid, glycidic acid, hydrobromic acid, hydrochloric acid, hydroxyethylsulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucilage, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, propionic acid, phosphoric acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc. Suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, and sulfuric acid. As used herein, unless otherwise stated, the term "solvent" refers to a compound or salt thereof of the present invention that also includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0235] As used herein, unless otherwise stated, the term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide said compound. Examples of prodrugs include, but are not limited to, compounds comprising a biohydrolyzable moiety, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acylureas, and biohydrolyzable phosphate ester analogs. Other examples of prodrugs include compounds comprising a -NO, -NO2, -ONO, or -ONO2 moiety. Prodrugs can typically be prepared using methods well-known to the public, such as those described in Burger's Medicinal Chemistry and Drug Discovery, 172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995) and Design of Prodrugs (H. Bundgaard ed., Elselvier, New York 1985).

[0236] As used herein, unless otherwise stated, the term "amino acid conjugate" refers to a conjugate of a compound (e.g., compounds of formula (I), (II), or (III) disclosed herein) with any suitable amino acid. Suitable amino acids may include, for example, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, arginine, histidine, lysine, aspartic acid, and glutamic acid. Particularly suitable amino acids include, but are not limited to, valine, threonine, tyrosine, tryptophan, and arginine.

[0237] As used herein, unless otherwise stated, the terms "biohydrolyzable carbamate," "biohydrolyzable carbonate," "biohydrolyzable acylurea," and "biohydrolyzable phosphate" refer to the carbamate, carbonate, acylurea, and phosphate ester of a compound that meets any of the following criteria: 1) does not interfere with the biological activity of the compound, but may impart beneficial properties to the compound in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive, but is converted into a biologically active compound in vivo. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic amines and heteroaromatic amines, and polyetheramines.

[0238] As used herein, the term "optical isomer" refers to a selected isomer of an optically active compound that exists as at least two pairs of isomers (defined by the chiral center) that rotate plane-polarized light in opposite directions.

[0239] As used herein, unless otherwise stated, the term "stereoisomer" includes all enantiomerically pure and enantiomerically enriched compounds of the present invention.

[0240] As used herein, unless otherwise stated, the term "steroisomerally pure" refers to a composition containing one stereoisomer of a compound and substantially free of other stereoisomers of the compound. For example, a stereoisomerally pure composition of a compound having one chiral center will substantially free of the opposite enantiomer of the compound. A stereoisomerally pure composition of a compound having two chiral centers will substantially free of other diastereomers of the compound. A typical stereoisomerally pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, even more preferably more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, and most preferably more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound.

[0241] As used herein, unless otherwise stated, the term "stereoisomer-enriched" refers to a composition comprising more than about 55% by weight of one stereoisomer of the compound, more than about 60% by weight of one stereoisomer of the compound, preferably more than about 70% by weight of one stereoisomer of the compound, and more preferably more than about 80% by weight of one stereoisomer of the compound. As used herein, unless otherwise stated, the term "enantiomer-pure" refers to a composition comprising pure stereoisomers of a compound having a chiral center. Similarly, the term "enantiomer-enriched" refers to a composition comprising stereoisomers enriched in a compound having a chiral center.

[0242] As used herein, a compound that induces a “minimum reduction” in specific protein levels refers to a reduction of less than 25% in protein levels after incubating test cells with a 10 μM compound for 24 hours. A compound that induces a “substantially no reduction” in specific protein levels refers to a reduction of less than 25% in protein levels after incubating test cells with a 20 μM compound for 24 hours.

[0243] Example

[0244] General Program

[0245] The compounds of the present invention are advantageous in terms of their synthetic feasibility. The synthesis of these compounds can be summarized by the general steps listed below:

[0246] Example Method 1: Coupling of Amine and Acid

[0247]

[0248] Reaction scheme 1: Coupling of amine and acid

[0249] Under an inert atmosphere, towards amine (R) 2 NH2, hydrochloride, 1 eq), and a suitable acid (R in the above reaction scheme) 1 COOH (1.2 eq) and DMAP (0–0.1 eq) were added to a solution of anhydrous DMF, along with DIPEA (2.2–5 eq) and HATU (1.2–2.5 eq) in anhydrous DMF. The reaction mixture was stirred overnight at room temperature. The crude product was purified by preparative HPLC and / or preparative TLC.

[0250] Example Method 2: Hydrolysis of lactone

[0251]

[0252] Reaction scheme 2. Hydrolysis of lactone

[0253] To a solution of substituted phthalide in a mixture of MeOH, THF (or 2-MeTHF), and H₂O (1:1:1), NaOH (4 eq) was added, and the reaction mixture was stirred at room temperature for 1 to 18 hours. After completion, the mixture was diluted with water, acidified with 10% KHSO₄, and the product was extracted with 2-MeTHF. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give the product as a free acid.

[0254] Example Method 3: Oxidation of Hydroxy Acids

[0255]

[0256] Reaction scheme 3. Oxidation of hydroxy acids

[0257] Pyridine Chlorochromate (1.5 eq) A solution of hydroxy acid (1 eq) in DCM was added to the suspension in anhydrous DCM. The reaction mixture was stirred at room temperature for 1 to 3 hours, diluted with diethyl ether, filtered through diatomaceous earth, and concentrated under reduced pressure. The product was purified by rapid column chromatography.

[0258] Example Method 4: Reductive Amination with Amines

[0259]

[0260] Reaction scheme 4. Reductive amination

[0261] To hydroxyfuranone (1 eq) and amine (R 2 NH2 (hydrochloride, 1.5 eq) was added to a solution of NaBH(OAc)3 (2.5–5 eq) in DMF, followed by the addition of TFA or AcOH (0–50 eq). The reaction mixture was stirred at room temperature for 18 hours, concentrated under reduced pressure, and the crude product was purified by rapid column chromatography and / or preparative HPLC and / or preparative TLC.

[0262] Example Method 5: Reaction of o-(bromomethyl)aryl esters with amines

[0263]

[0264] Reaction scheme 5. Reaction of o-(bromomethyl)aryl esters with amines

[0265] To 2-(bromomethyl)aryl esters (1 eq) and amines (R 2 Add DIPEA (2-5 eq) to a mixture of NH2 (hydrochloride, 1.0-1.2 eq) in DMF or ACN, and stir the mixture at 90 °C for 6 to 18 hours. Concentrate the reaction mixture under reduced pressure, and purify the crude product by preparative HPLC and / or preparative TLC.

[0266] Example Method 6: Deprotection of tert-butyl carbamate

[0267]

[0268] Reaction scheme 6. Deprotection of tert-butyl carbamate

[0269] Program A

[0270] The Boc-protected amine (pure amine or solution in DCM) was treated with TFA at room temperature. The reaction mixture was stirred at room temperature for 1 to 24 hours and concentrated under reduced pressure to give the product. 0.01 M HCl was added to convert it to an HCl salt.

[0271] Program B

[0272] Amines protected by Boc at room temperature in 1,4-di Concentrated HCl was added to the mixture of alkane / H2O. The reaction mixture was stirred at room temperature for 1 to 24 hours and then concentrated to obtain the product.

[0273] Example 1: Synthesis of 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione (1)

[0274]

[0275] Step 1. Dissolve 181.2 g (1 mol) of 2-methyl-4-nitrobenzoic acid in 500 mL of methanol, add SOCl2 (119 g, 73 mL, 1 mmol), and reflux the mixture for 6 hours. Evaporate the solvent under reduced pressure. Add NaHCO3 to the residue. 3（aq） The product was extracted into CHCl3 and concentrated under reduced pressure to give 181.5 g of methyl 2-methyl-4-nitrobenzoate (93% yield).

[0276] Step 2. Methyl 2-methyl-4-nitrobenzene (195.2 g, 1 mol) was dissolved in CCl4 (600 mL), and N-bromosuccinimide (178.0 g, 1 mol) was added. The mixture was stirred for 30 minutes. A catalytic amount of benzoyl peroxide was added, and the mixture was refluxed for 3 hours. The mixture was cooled to room temperature and filtered. The mother liquor was evaporated to obtain a mixture of methyl 2-(bromomethyl)-4-nitrobenzene and methyl 2-(dibromomethyl)-4-nitrobenzene, which was used in the next step without purification.

[0277] Step 3. A mixture of methyl 2-(bromomethyl)-4-nitrobenzene and methyl 2-(dibromomethyl)-4-nitrobenzene was dissolved in THF (400 mL), and diethyl phosphite (1 eq) and DIPEA (1 eq) were added. The reaction mixture was stirred at room temperature for 12 hours. The solvent was removed, and the residue was dissolved in EtOAc (300 mL), filtered, and the filtrate was washed with water (3 × 150 mL). The organic layer was separated, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give 184 g of 2-(bromomethyl)-4-nitrobenzene (67% yield, two steps).

[0278] Step 4. DIPEA (259 g, 350 mL, 2 mol) was added to a mixture of 2-(bromomethyl)-4-nitrobenzene ester (274 g, 1 mol) and 3-aminoglutamylimide hydrochloride (198 g, 1.200 mol) in DMF (150 mL), and the mixture was stirred at 90 °C for 6 hours, cooled, and diluted with water (300 mL). The precipitate was filtered and washed with water to give 209 g of 3-(5-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione (72% yield).

[0279] Step 5. 145 g (500 mmol) of 3-(5-nitro-1-oxoisoindoline-2-yl)piperidine-2,6-dione was dissolved in acetic acid (150 mL), 5% Pd / C (10 mmol) was added, and the reaction mixture was stirred at 50 °C for 12 h under a hydrogen atmosphere (30 bar). The mixture was filtered, washed with EtOAc (2 × 100 mL), and the combined filtrates were evaporated under reduced pressure. The residue was purified by column chromatography to give 108 g of 3-(5-amino-1-oxoisoindoline-2-yl)piperidine-2,6-dione (83% yield).

[0280] Step 6. A suspension of 3-(5-amino-1-oxoisoindoline-2-yl)piperidine-2,6-dione (25.92 g, 100 mmol) in acetic acid (100 mL) was cooled to +15 °C and treated dropwise with a solution of NaNO2 (8.3 g, 120 mmol) in 50 mL of water. The suspension was stirred at room temperature for 2 hours, and then a solution of CuCN (134.5 g, 1.5 mol) and NaCN (49 g, 1 mol) in water (75 mL) was added dropwise over 30 minutes. The mixture was stirred at room temperature for 3 hours, and then heated at 60 °C for 2 hours. The precipitate was filtered, washed with water, and crystallized from DMF / i-PrOH (1:1) to give 14 g of 2-(2,6-dioxopiperidine-3-yl)-1-oxoisoindoline-5-nitriles (52% yield).

[0281] Step 7. Add 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-nitriles (2.69 g, 10 mmol) to a 1,4-dioxopiperidin-3-yl)-1-oxoisoindoline-5-nitriles. Acetic acid (10 mL) was added to the solution in alkane (150 mL), and the mixture was stirred at 50 °C for 10 hours under a hydrogen atmosphere (60 bar). After completion, the mixture was filtered, washed with EtOAc (2 × 100 mL), and the combined filtrates were evaporated under reduced pressure. The residue was purified by column chromatography to give 2.08 g of 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione hydrochloride (67% yield).

[0282] .

[0283] Example 2: 3-(5-(aminomethyl- d 2 Synthesis of 1-oxoisoindoline-2-yl)piperidine-2,6-dione (2)

[0284]

[0285] 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-nitriles (0.1 g, 0.37 mmol) and PtO2 (0.05 g) were prepared in... i The mixture of -PrOD (3 mL), DMF (1 mL), and 4 M DCl (0.3 mL) was degassed, and the reaction mixture was stirred at room temperature for 24 hours under D2 gas (1 bar). The reaction mixture was filtered and concentrated. The crude product was purified by preparative HPLC to give 2 mg of 3-(5-(aminomethyl-) d 2 1-O-isoindoline-2-yl)piperidine-2,6-dione acetate (20% yield).

[0286] .

[0287] Example 3: 3-[5-(aminomethyl)-6-fluoro-1-oxo-2,3-dihydro-1- H [-isoindol-2-yl]piperidine-2,6-di Synthesis of ketones (3)

[0288]

[0289] Step 1. At room temperature, NaBrO3 (4.0 g, 25.86 mmol) and NaHSO3 (2.7 g, 25.86 mmol) were added to a solution of 4-bromo-5-fluoro-2-methylbenzoic acid (2.0 g, 8.62 mmol) in an EtOAc / H2O (25 / 20 mL) mixture, and the reaction mixture was stirred for 48 hours. The mixture was washed with water, dried over anhydrous Na2SO4, concentrated, and purified by rapid column chromatography to give 0.8 g of 5-bromo-6-fluoroisobenzofuran-1 ( 3H )-ketone (40% yield).

[0290] Step 2. To 5-bromo-6-fluoroisobenzofuran-1 ( 3H Zn(CN)₂ (384 mg, 3.26 mmol) was added to a solution of 6-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (47.6%) in DMF (7 mL), and the mixture was purged with argon for 10 min. Then, Pd(PPh₃)₄ (227 mg, 0.2 mmol) was added to the reaction mixture, and the mixture was purged with argon for 10 min. The reaction mixture was stirred in a sealed tube at 90 °C for 16 h. After the reaction was complete, the mixture was filtered through a diatomaceous earth bed and washed with EtOAc. The filtrate was diluted with EtOAc and washed with water. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography to give 110 mg of 6-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (47.6% yield) as a pale yellow solid.

[0291] Step 3. To a solution of 6-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (630 mg, 3.56 mmol) in ethanol (10 mL), Raney nickel and Boc-anhydride (3.3 mL, 14.26 mmol) were added, and the reaction mixture was stirred at room temperature under H2 atmosphere for 16 hours. After the reaction was complete, the mixture was filtered through a diatomaceous earth bed and washed with ethanol. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography to give 600 mg of ((6-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate tert-butyl ester (59.9% yield) as a grayish-white solid.

[0292] Step 4. At 0 °C, NaOH (227 mg, 5.65 mmol) was added to a solution of tert-butyl (6-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (500 mg, 1.88 mmol) in a mixture of THF (10 mL) and water (8.0 mL). The reaction mixture was then stirred at room temperature for 16 hours. The evaporation was evaporated under reduced pressure, and the residue was dissolved in water. Extraction was performed with EtOAc (20 mL), followed by acidification of the aqueous phase with 1 (N) HCl while cooling. The combined organic layers were washed with water and then with a brine solution after extraction with EtOAc. The organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give 450 mg of 4-(((tert-butoxycarbonyl)amino)methyl)-5-fluoro-2-(hydroxymethyl)benzoic acid (87% yield).

[0293] Step 5. At -10 °C, TMS-diazomethane (11 mL, 20.06 mmol) (in 2 M ether) was added dropwise to a solution of 4-(((tert-butoxycarbonyl)amino)methyl)-5-fluoro-2-(hydroxymethyl)benzoic acid (600 mg, 2.0 mmol) in methanol (8 mL) and EtOAc (8 mL). The reaction mixture was then stirred at room temperature for 3 hours. The reaction mixture was then quenched by adding water and extracted with EtOAc. The combined organic layers were washed with water and then with a brine solution. The organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give crude 600 mg of methyl 4-(((tert-butoxycarbonyl)amino)methyl)-5-fluoro-2-(hydroxymethyl)benzoate, which could be used for the next step of the synthesis without further purification.

[0294] Step 6. At 0 °C, PPh3 (1.51 g, 5.75 mmol) and CBr4 (1.91 g, 5.75 mmol) were added to a solution of methyl 4-(((tert-butoxycarbonyl)amino)methyl)-5-fluoro-2-(hydroxymethyl)benzoate (900 mg, 2.87 mmol) in THF (20 mL). The reaction mixture was then stirred at room temperature for 16 hours under a nitrogen atmosphere. The reaction mixture was quenched by adding water and extracted with EtOAc. The combined organic layers were washed with water and then with a saline solution. The organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a crude product. The crude product was purified by rapid column chromatography to give 360 ​​mg of methyl 2-(bromomethyl)-4-(((tert-butoxycarbonyl)amino)methyl)-5-fluorobenzoate (31% yield) as a white solid.

[0295] Step 7. Using the general procedure shown in reaction scheme 5 and method 5 of Example 5 above, and starting with methyl 2-(bromomethyl)-4-({[(tert-butoxy)carbonyl]amino}methyl)-5-fluorobenzoate (50.0 mg, 0.133 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (1.200 eq), synthesize N -{[2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl} tert-butyl carbamate (yield 73.8%).

[0296] .

[0297] Step 8. Using the general procedure shown in Procedure B of Reaction Scheme 6 and Method 6 of Example 6, and with N -{[2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1-yl] H tert-butyl 3-[5-(aminomethyl)-6-fluoro-1-oxo-2,3-dihydro-1-carbamate (7.2 mg, 0.018 mmol) was used as a starting material to synthesize 3-[5-(aminomethyl)-6-fluoro-1-oxo-2,3-dihydro-1-carbamate]. H -isoindol-2-yl]piperidine-2,6-dione (yield 97.7%).

[0298] .

[0299] Example 4: 3-[5-(aminomethyl)-6-methyl-1-oxo-2,3-dihydro-1-yl] H -isoindol-2-yl]piperidine-2,6- Synthesis of diketone (4)

[0300]

[0301] Step 1. K₂HPO₄ (6.07 g, 34.88 mmol) and Pd(OAc)₂ (261 mg, 1.163 mmol) were added to a solution of 4-bromo-3-methylbenzoic acid (2.5 g, 11.628 mmol) in CH₂Br₂ (25 mL). The reaction mixture was stirred at 140 °C for 48 hours in a sealed tube under an inert atmosphere. The mixture was filtered, concentrated, and purified by rapid column chromatography to give 750 mg of 5-bromo-6-methylisobenzofuran-1 (3... H )-ketone (28% yield).

[0302] Step 2. To 5-bromo-6-methylisobenzofuran-1(3 H Zn(CN)₂ (1.933 g, 16.52 mmol) was added to a solution of 6-methyl-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (1.5 g, 6.60 mmol) in DMF (15 mL), followed by Pd(PPh₃)₄ (0.763 g, 0.661 mmol). The reaction mixture was heated at 100 °C for 16 hours under an inert atmosphere. The reaction was quenched with ice water, and the product was extracted into EtOAc. The organic layer was dried over Na₂SO₄, concentrated, and purified by rapid column chromatography to give 900 mg of 6-methyl-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (78% yield).

[0303] Step 3. To a solution of 6-methyl-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (400 mg, 2.30 mmol) in ethanol (5 mL), Boc2O (1.056 mL, 4.598 mmol) was added, followed by Raney nickel (80 mg), and the reaction mixture was stirred under a hydrogen atmosphere (1 bar) for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by rapid column chromatography to give 360 ​​mg of tert-butyl ((6-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (56% yield).

[0304] Step 4. Using the general procedure shown in reaction scheme 2 and method 2 of example above, and with N 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-(hydroxymethyl)-5-methylbenzoic acid (82% yield) was synthesized from tert-butyl carbamate (30.0 mg, 0.108 mmol) as the starting material.

[0305] Step 5. Using the general procedure shown in reaction scheme 3 and method 3 of Example 3 above, and starting with 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-(hydroxymethyl)-5-methylbenzoic acid (26.4 mg, 0.089 mmol), synthesize N -[(3-hydroxy-6-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)methyl] tert-butyl carbamate (87% yield).

[0306] Step 6. Using the general procedure shown in reaction scheme 4 and method 4 of example above, and with N Synthesis using tert-butyl carbamate (22.9 mg, 0.070 mmol) as the starting material N -{[2-(2,6-dioxopiperidin-3-yl)-6-methyl-1-oxo-2,3-dihydro-1H-isoindol-5-yl]methyl} tert-butyl carbamate (21% yield).

[0307] Step 7. Use the general procedure shown in Procedure A of Reaction Scheme 6 and Method 6 of Example 6, and with N -{[2-(2,6-dioxopiridine-3-yl)-6-methyl-1-oxo-2,3-dihydro-1-yl] H 3-[5-(aminomethyl)-6-methyl-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione (100% yield) was synthesized from tert-butyl carbamate (5.1 mg, 0.013 mmol) as the starting material.

[0308] .

[0309] Example 5: 3-[5-(aminomethyl)-4-fluoro-1-oxo-2,3-dihydro-1- H [-isoindol-2-yl]piperidine-2,6-di Synthesis of ketones (5)

[0310]

[0311] Step 1. To 5-bromo-4-fluoroisobenzofuran-1(3 HZn(CN)₂ (1.24 g, 10.87 mmol) was added to a solution of 4-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (1.00 g, 4.35 mmol) in DMF (10 mL), followed by Pd(PPh₃)₄ (0.753 g, 0.652 mmol). The reaction mixture was heated at 90 °C for 16 hours under an inert atmosphere. The reaction was quenched with ice water, and the product was extracted into EtOAc. The organic layer was dried over Na₂SO₄, concentrated, and purified by rapid column chromatography to give 500 mg of 4-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (65% yield).

[0312] Step 2. To a solution of 4-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (500 mg, 2.80 mmol) in ethanol (10 mL), Boc2O (1.29 mL, 5.61 mmol) was added, followed by Raney nickel (100 mg), and the reaction mixture was stirred under a hydrogen atmosphere (1 bar) for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by rapid column chromatography to give 395 mg of tert-butyl ((6-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (50% yield).

[0313] Step 3. Using the general procedure shown in reaction scheme 2 and method 2 of example above, and with N 4-({[(tert-butoxy)carbonyl]amino}methyl)-3-fluoro-2-(hydroxymethyl)benzoic acid (98.4% yield) was synthesized from tert-butyl carbamate (30.0 mg, 0.107 mmol) as the starting material.

[0314] Step 4. Using the general procedure shown in reaction scheme 3 and method 3 of Example 3 above, and starting with 4-({[(tert-butoxy)carbonyl]amino}methyl)-3-fluoro-2-(hydroxymethyl)benzoic acid (31.4 mg, 0.105 mmol), synthesize N -[(4-fluoro-3-hydroxy-1-oxo-1,3-dihydro-2-benzofuran-5-yl)methyl] tert-butyl carbamate (58% yield).

[0315] Step 5. Using the general procedure shown in reaction scheme 4 and method 4 of example above, and with N Synthesis using tert-butyl carbamate (20.2 mg, 0.061 mmol) as the starting material N-{[2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl}tert-butyl carbamate (32% yield).

[0316] Step 6. Using the general steps shown in procedure A of reaction scheme 6 and method 6 of example above, and with N -{[2-(2,6-dioxopiperidin-3-yl)-4-fluoro-1-oxo-2,3-dihydro-1-yl] H 3-[5-(aminomethyl)-4-fluoro-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione (100% yield) was synthesized from tert-butyl carbamate (3.8 mg, 0.010 mmol) as the starting material.

[0317] .

[0318] Example 6: 3-[5-(aminomethyl)-4-methyl-1-oxo-2,3-dihydro-1-methyl] H -isoindol-2-yl]piperidine-2,6- Synthesis of diketone (6)

[0319]

[0320] Step 1. At 0 °C, thallium(III) trifluoroacetate (8.081 mg, 14.871 mmol) was added to a solution of (3-bromo-2-methylphenyl)methanol (2.3 g, 11.439 mmol) in TFA (10 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, azeotropically reacted with DCE (twice), and dissolved in degassed methanol (12 mL). MgO (968 mg, 24.023 mmol), LiCl (970 mg, 22.879 mmol), and PdCl2 (203 mg, 1.144 mmol) were added, and the reaction mixture was stirred under a CO atmosphere (1 bar) for 4 hours. The mixture was filtered, concentrated, and purified by rapid column chromatography to give 1.55 g of 5-bromo-4-methylisobenzofuran-1(3-) H )-ketone (60% yield).

[0321] Step 2. To 5-bromo-4-methylisobenzofuran-1(3 HZn(CN)₂ (1.933 g, 16.52 mmol) was added to a solution of 4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (1.5 g, 6.60 mmol) in DMF (15 mL), followed by Pd(PPh₃)₄ (0.763 g, 6.61 mmol). The reaction mixture was heated at 100 °C for 16 hours under an inert atmosphere. The reaction was quenched with ice water, and the product was extracted into EtOAc. The organic layer was dried over Na₂SO₄, concentrated, and purified by rapid column chromatography to give 700 mg of 4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-nitrile (61% yield).

[0322] Step 3. To a solution of 4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (1.5 g, 8.67 mmol) in ethanol (15 mL), Boc2O (3.98 mL, 17.34 mmol) was added, followed by Raney nickel (250 mg), and the reaction mixture was stirred under a hydrogen atmosphere (1 bar) for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by rapid column chromatography to give 360 ​​mg of tert-butyl ((4-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (45% yield).

[0323] Step 4. Using the general procedure shown in reaction scheme 2 and method 2 of example above, and with N 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-(hydroxymethyl)-3-methylbenzoic acid (100.0% yield) was synthesized from tert-butyl carbamate (30.0 mg, 0.108 mmol) as the starting material.

[0324] Step 5. Using the general procedure shown in reaction scheme 3 and method 3 of Example 3 above, and starting with 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-(hydroxymethyl)-3-methylbenzoic acid (36.1 mg, 0.116 mmol), synthesize N -[(3-hydroxy-4-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)methyl] tert-butyl carbamate (52% yield).

[0325] Step 6. Using the general procedure shown in reaction scheme 4 and method 4 of example above, and with N Synthesis using tert-butyl carbamate (36.1 mg, 0.064 mmol) as the starting material N-{[2-(2,6-dioxopiridine-3-yl)-4-methyl-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl}tert-butyl carbamate (15% yield).

[0326] Step 7. Use the general procedure shown in Procedure A of Reaction Scheme 6 and Method 6 of Example 6, and with N -{[2-(2,6-dioxopiridine-3-yl)-4-methyl-1-oxo-2,3-dihydro-1-yl] H 3-[5-(aminomethyl)-4-methyl-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione (22.6% yield) was synthesized from tert-butyl carbamate (11.8 mg, 0.031 mmol) as the starting material.

[0327] .

[0328] Example 7: 3-[5-(aminomethyl)-7-fluoro-1-oxo-2,3-dihydro-1- H [-isoindol-2-yl]piperidine-2,6-di Synthesis of ketones (7)

[0329]

[0330] Step 1. To 5-bromo-7-fluoroisobenzofuran-1(3 H )-keto (250 mg, 1.082 mmol) in di Zn(CN)₂ (254 mg, 2.165 mmol) was added to a solution of alkane (7 mL), followed by Pd₂dba₃ (99 mg g, 0.11 mmol) and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (Xantphos) (94 mg, 0.162 mmol). The reaction mixture was heated at 100 °C for 16 hours under an inert atmosphere. The reaction mixture was filtered, concentrated, and purified by rapid column chromatography to give 100 mg of 7-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (52% yield).

[0331] Step 2. To a solution of 7-fluoro-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (500 mg, 2.825 mmol) in ethanol (20 mL), Boc2O (739 mg, 3.39 mmol) was added, followed by Raney nickel (500 mg), and the reaction mixture was stirred under a hydrogen atmosphere (1 bar) for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by rapid column chromatography to give 300 mg of tert-butyl ((7-fluoro-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (37% yield).

[0332] Step 3. Using the general procedure shown in reaction scheme 2 and method 2 of example above, and with N 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-fluoro-6-(hydroxymethyl)benzoic acid (92.4% yield) was synthesized from tert-butyl carbamate (30.0 mg, 0.107 mmol) as the starting material.

[0333] Step 4. Using the general procedure shown in reaction scheme 3 and method 3 of Example 3 above, and starting with 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-fluoro-6-(hydroxymethyl)benzoic acid (29.5 mg, 0.089 mmol), synthesize N -[(7-fluoro-3-hydroxy-1-oxo-1,3-dihydro-2-benzofuran-5-yl)methyl] tert-butyl carbamate (77% yield).

[0334] Step 5. Using the general procedure shown in reaction scheme 4 and method 4 of example above, and with N Synthesis using tert-butyl carbamate (20.3 mg, 0.061 mmol) as the starting material N -{[2-(2,6-dioxopiperidin-3-yl)-7-fluoro-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl} tert-butyl carbamate (17.9% yield).

[0335] Step 6. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Method 6 of Example 6, and with N The compound was synthesized from tert-butyl carbamate (4.3 mg, 0.011 mmol) as the starting material (100.0% yield).

[0336] .

[0337] Example 8: N -[(1 S )-1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H-isoindole- 5-yl]ethyl]carbamate tert-butyl ester (9) and 3-{5-[(1 S )-1-aminoethyl]-1-oxo-2,3-dihydro-1 H -Isoin Synthesis of dolo-2-ylpiperidine-2,6-dione (8)

[0338]

[0339] Step 1. At 0°C, to 5-acetylisobenzofuran-1(3 H )-ketone (3.0 g, 17.04 mmol) and (S Ti(OEt)4 (7.14 mL, 34.08 mmol) was added to a solution of 2-methylpropane-2-sulfinamide (2.27 g, 18.74 mmol) in THF (50 mL), and the reaction mixture was stirred at 70 °C for 20 hours. Then, the reaction mixture was added dropwise to a suspension of NaBH4 (2.57 g, 68.16 mmol) in THF at -60 °C, and the mixture was slowly heated to room temperature. The reaction mixture was quenched with MeOH (10 mL), poured into a saline solution, filtered, and diluted with water. The product was extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by rapid column chromatography to give 2.8 g of ( S )-2-methyl- N -(( S )-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)propane-2-sulfinamide (58% yield) is a white solid.

[0340] Step 2. At 10℃, to ( S )-2-methyl- N -(( S )-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)propane-2-sulfinamide (510 mg, 1.815 mmol) in 1,4-di Add 4M HCl to the solution of alkane (2 mL) in 1,4-didi The solution was prepared in alkane. The reaction mixture was stirred at room temperature for 1 hour and then concentrated to give 315 mg of ( S )-5-(1-aminoethyl)isobenzofuran-1(3 H )-ketone (97% yield) and then used in the next step.

[0341] Step 3. At 0℃, to ( S )-5-(1-aminoethyl)isobenzofuran-1(3 H )-ketone (2.3 g, 12.99 mmol) was added to a solution of Boc2O (4.47 mL, 19.49 mmol) and NaHCO3 (2.18 g, 25.98 mmol) in THF / H2O (30 / 20 mL), and the reaction mixture was stirred at room temperature for 16 hours. The product was extracted into EtOAc. The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by rapid column chromatography to give 1.9 g of ( S 1-(1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl) tert-butyl carbamate (52% yield) is a white solid.

[0342] Step 4. At 0℃, to ( S 1.9 g (6.85 mol) of tert-butyl carbamate (1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)carbamate was added to a solution of NaOH (412 mg, 10.29 mmol) in THF / H₂O (6 / 24 mL), and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was acidified at 0 °C with 10% HCl solution (pH ~ 5) and extracted with EtOAc. The combined organic layers were dried over Na₂SO₄ and concentrated to give 1.75 g of ( S 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoic acid (88.5% yield) is a white solid.

[0343] Step 5. At -10℃, to ( S TMS-diazomethane (0.912 mL, 16.89 mmol) was added to a solution of 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoic acid (1.0 g, 3.37 mmol) in MeOH / EtOAc (6 / 6 mL). The reaction mixture was stirred for 30 min and quenched with ice water. The product was extracted into EtOAc, dried over Na2SO4, and concentrated to give 1.1 g of ( S Methyl 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoate (crude product). This product can be used in the next step without further purification.

[0344] Step 6. At 0℃, to ( S Methyl 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoate (1.1 g, 3.56 mmol, crude product) was reacted with PPh3 (1.76 g, 5.34 mmol) and CBr4 (1.4 g, 5.34 mmol) in a solution of THF (15 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched with ice water, and the product was extracted into EtOAc. The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by rapid column chromatography to give 610 mg of ( S Methyl benzoate 2-(bromomethyl)-4-(1-((tert-butoxycarbonyl)amino)ethyl)benzoate (36% yield, two steps) is a white solid.

[0345] Step 7. Using the general procedure shown in reaction scheme 5 and method 5 of example above, and with 2-(bromomethyl)-4-[(1 SMethyl benzoate (50.0 mg, 0.134 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (1.200 eq) were used as starting materials for the synthesis of [[(tert-butoxy)carbonyl]amino}ethyl]benzoate (50.0 mg, 0.134 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (1.200 eq). N -[(1 S 1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H-isoindol-5-yl]ethyl] tert-butyl carbamate (67% yield).

[0346] .

[0347] Step 8. Using the general procedure shown in Procedure B of Reaction Scheme 6 and Method 6 of Example 6, and with N 3-{5-[(1-[2-(2,6-dioxopiridin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1H-isoindol-5-yl]methyl} tert-butyl carbamate (30 mg, 0.077 mmol) was synthesized as a starting material. S )-1-aminoethyl]-1-oxo-2,3-dihydro-1 H -isoindol-2-yl}piperidine-2,6-dione (95% yield).

[0348] .

[0349] Example 9: N -[(1 R )-1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1 H -Isoindole- 5-yl]ethyl]carbamate tert-butyl ester (11) and 3-{5-[(1 R )-1-aminoethyl]-1-oxo-2,3-dihydro-1 H -Isoin Synthesis of dol-2-ylpiperidine-2,6-dione (10)

[0350]

[0351] Step 1. At 0°C, to 5-acetylisobenzofuran-1(3 H )-ketone (3.5 g, 19.88 mmol) and ( R Ti(OEt)4 (8.34 mL, 39.90 mmol) was added to a solution of 2-methylpropane-2-sulfinamide (2.65 mmol) in THF (50 mL), and the reaction mixture was stirred at 70 °C for 20 hours. Then, the reaction mixture was added dropwise to a suspension of NaBH4 (3.00 g, 79.5 mmol) in THF at -60 °C, and the mixture was slowly heated to room temperature. The reaction mixture was quenched with MeOH (10 mL), poured into a saline solution, filtered, and diluted with water. The product was extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by rapid column chromatography to give 2.8 g of ( R )-2-methyl- N -(( R)-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)propane-2-sulfinamide (50% yield) is a white solid.

[0352] Step 2. At 10℃, to ( S )-2-methyl- N -(( S )-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)propane-2-sulfinamide (510 mg, 1.815 mmol) in 1,4-di Add 4M HCl to the solution of alkane (2 mL) in 1,4-didi The solution was in alkane. The reaction mixture was stirred at room temperature for 1 hour and concentrated to give 2.1 g of ( R )-5-(1-aminoethyl)isobenzofuran-1(3 H )-ketone (95% yield), is a white solid.

[0353] Step 3. At 0℃, to ( R )-5-(1-aminoethyl)isobenzofuran-1(3 H )-ketone (2.1 g, 11.86 mmol) was added to a solution of Boc2O and NaHCO3 in THF / H2O (20 / 20 mL), and the reaction mixture was stirred at room temperature for 16 hours. The product was extracted into EtOAc. The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by rapid column chromatography to give 2.6 g of ( R 1-(1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl) tert-butyl carbamate (79% yield) is a white solid.

[0354] Step 4. At 0℃, to ( R 1.6 g (5.77 mol) of tert-butyl carbamate (1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)ethyl)carbamate was added to a solution of NaOH (347 mg, 8.66 mmol) in THF / H₂O (6 / 24 mL), and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction mixture was acidified (pH ~ 5) with 10% HCl solution at 0 °C and extracted with EtOAc. The combined organic layers were dried over Na₂SO₄ and concentrated to give 1.51 g of ( R 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoic acid (88.5% yield) is a white solid.

[0355] Step 5. At -10℃, to ( RTMS-diazomethane (12.66 mL, 25.33 mmol) was added to a solution of 1.5 g (5.068 mmol) of 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoic acid in MeOH / EtOAc (8 / 8 mL). The reaction mixture was stirred for 30 min and quenched with ice water. The product was extracted into EtOAc, dried over Na2SO4, and concentrated to give 1.67 g of ( R Methyl 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoate (crude product). The crude product can proceed directly to the next step without purification.

[0356] Step 6. At 0°C, add the stirred ( R Methyl 4-(1-((tert-butoxycarbonyl)amino)ethyl)-2-(hydroxymethyl)benzoate (1.67 g, 5.405 mmol, crude product) was reacted with PPh3 (2.68 g, 8.10 mmol) and CBr4 (2.12 g, 8.10 mmol) in a solution of THF (20 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched with ice water, and the product was extracted into EtOAc. The organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by rapid column chromatography to give 610 mg of ( R Methyl benzoate 2-(bromomethyl)-4-(1-((tert-butoxycarbonyl)amino)ethyl)benzoate (30% yield, two steps) is a white solid.

[0357] Step 7. Using the general procedure shown in reaction scheme 5 and method 5 of Example 5 above, and starting with methyl 2-(bromomethyl)-4-[(1R)-1-{[(tert-butoxy)carbonyl]amino}ethyl]benzoate (50.0 mg, 0.134 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (1.200 eq), synthesize N -[(1 R )-1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1 H -Isoindol-5-yl]ethyl]tert-butyl carbamate (66.8% yield).

[0358] .

[0359] Step 8. Using the general procedure shown in Procedure B of Reaction Scheme 6 and Example Method 6 (95% yield), and with N -[(1 R )-1-[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H -isoindol-5-yl]ethyl]carbamate tert-butyl ester (15.0 mg, 0.039 mmol) was used as a starting material to synthesize 3-{5-[(1 R )-1-aminoethyl]-1-oxo-2,3-dihydro-1 H 2-Isoindol-2-ylpiperidine-2,6-dione hydrochloride.

[0360] .

[0361] Example 10: N -{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1 H -Isoindole-5- Synthesis of [methyl]acetamide (12)

[0362]

[0363] 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1- H [-isoindol-2-yl]piperidine-2,6-dione hydrochloride (20.0 mg, 0.065 mmol) was dissolved in DMF (2.0 mL), and DIPEA (0.034 mL, 0.194 mmol) was added in a single batch. Acetyl chloride (7 μL, 0.093 mmol) was added in a single batch, and the reaction mixture was stirred at room temperature for 24 hours. DMF was removed under reduced pressure, and the residue was purified by preparative HPLC to give 9.6 mg of [a specific product / concentrate]. N -{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1 H -isoindol-5-yl]methyl}acetamide (47% yield).

[0364] .

[0365] Example 11: N -{[2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1-yl] H -Isoindole- Synthesis of 5-yl]methyl}acetamide (13)

[0366]

[0367] Acetic anhydride (3 µL, 0.031 mmol) was added to a mixture of 3-[5-(aminomethyl)-6-fluoro-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione hydrochloride (10.1 mg, 0.031 mmol), DMAP (0.4 mg, 0.003 mmol), DIPEA (11 µL, 0.062 mmol), and DMF (1.0 mL). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was evaporated, and the dried residue was purified by preparative HPLC to give 5.4 mg of [the product / product / concentrate]. N-{[2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl}acetamide (52.1% yield) is a white solid.

[0368] .

[0369] Example 12: N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl] H -isoindol-5-yl] Synthesis of methyl-2-ethoxyacetamide (14)

[0370]

[0371] 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione hydrochloride (20.0 mg, 0.065 mmol) was dissolved in DMF (2.0 mL), and DIPEA (34 µL, 0.194 mmol) was added in a single batch. 2-ethoxyacetyl chloride (11 µL, 0.097 mmol) was added in a single batch, and the reaction mixture was stirred at room temperature for 24 hours. DMF was removed under reduced pressure, and the residue was purified by preparative HPLC to give 9.9 mg of [the product / solution / concentration]. N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H-isoindol-5-yl]methyl}-2-ethoxyacetamide (42% yield).

[0372] .

[0373] Example 13: 3-{5-[(butanoamino)methyl]-1-oxo-2,3-dihydro-1- H -isoindol-2-yl}piperidine-2,6- Synthesis of diketone (15)

[0374]

[0375] To a solution of 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione hydrochloride (60.0 mg, 0.194 mmol) in DMF (1.0 mL), DIPEA (135 µL, 0.775 mmol) was added, followed by iodobutane (24 µL, 0.213 mmol), and the reaction mixture was stirred at room temperature for 18 hours. The solution was concentrated under reduced pressure, and the residue was redissolved in a small amount of water / DMSO and purified by preparative HPLC to give 2.8 mg of 3-{5-[(butamino)methyl]-1-oxo-2,3-dihydro-1H-2-yl]piperidine-2,6-dione hydrochloride. H -Isoindol-2-yl}piperidine-2,6-dione carbamate (3.9% yield).

[0376] .

[0377] Example 14: 3-(5-((dimethylamino)methyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione Synthesis of (16)

[0378]

[0379] 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (70 mg, 0.226 mmol) in water / 1,4-di 0.1 mL of 37% formaldehyde (6 eq) was added to a solution of alkane (1 / 1, 2 mL). The reaction mixture was stirred at room temperature for 6 hours, and NaBH3CN (10 eq) was added. The reaction mixture was then stirred at room temperature for 2 days and concentrated under reduced pressure. The crude product was purified by preparative TLC to give 13.0 mg of 3-(5-((dimethylamino)methyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione (19% yield).

[0380] .

[0381] Example 15: Synthesis of 3-(5-(hydroxymethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione (17)

[0382]

[0383] 3-(5-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (100 mg, 0.31 mmol) was dissolved in DMF (2 mL). (Tributyltinyl)methanol (149.0 mg, 0.46 mmol, 1.5 eq) and Pd(PPh3)4 (35 mg, 0.031 mmol, 0.1 eq) were added, and the reaction mixture was stirred at 90 °C for 18 hours. The crude mixture was purified by preparative HPLC to give 8 mg of 3-(5-(hydroxymethyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (9% yield).

[0384] .

[0385] Example 16: {3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]-2,6-dioxopyranoside} Synthesis of methyl 1-pyridyl 2,2-dimethylpropionate (18)

[0386]

[0387] Step 1. To 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1-oxo-2,3-dihydro-1-oxo-2,3-dihydro-2 ...-oxo-2-dihydro-2-oxo-2-oxo-2-dihydro-2-oxo-2-dihydro-2 H [-isoindol-2-yl]piperidine-2,6-dione hydrochloride (120.0 mg, 0.387 mmol) was added to a suspension in ACN (2.0 mL). N-(benzyloxycarbonyloxy)succinimide (101.4 mg, 0.407 mmol) was added, followed by the addition of DIPEA (0.169 mL, 0.969 mmol), and the reaction mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure, and the crude product was purified by reversed-phase rapid column chromatography to give 135.0 mg. N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl} benzyl carbamate (85% yield).

[0388] Step 2. Put it into the small bottle N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl] H Benzyl 3-[5-({[(benzyloxy)carbonyl]amino}methyl)-1-oxo-2,3-dihydro-1-carbamate (104.0 mg, 0.255 mmol), Cs₂CO₃ (91.5 mg, 0.281 mmol), and tetrabutylammonium iodide (94.3 mg, 0.255 mmol) were added. DMF (2.5 mL) was added, followed by methyl neopentanoate (40 µL, 0.278 mmol), and the reaction mixture was stirred at room temperature for 18 hours. The mixture was filtered through diatomaceous earth, concentrated under reduced pressure, and purified by reversed-phase rapid column chromatography and rapid column chromatography to give 100.0 mg of {3-[5-({[(benzyloxy)carbonyl]amino}methyl)-1-oxo-2,3-dihydro-1-carbamate}. H Methyl isoindol-2-yl]-2,6-dioxopiperidin-1-yl}2,2-dimethylpropionate (75% yield).

[0389] Step 3. To {3-[5-({[(benzyloxy)carbonyl]amino}methyl)-1-oxo-2,3-dihydro-1-methyl] H Methyl methyl 2,2-dimethylpropionate {3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1-yl]-2,6-dioxadiazine-1-yl}2,2-dimethylpropionate (100.0 mg, 0.192 mmol) was added to a solution of methyl 2,2-dimethylpropionate in ethanol (10.0 mL) and the reaction mixture was stirred for 1 hour under a hydrogen atmosphere (1 bar). The reaction mixture was filtered, concentrated under reduced pressure, and purified by preparative HPLC to give 36.0 mg of {3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1-yl]-2,6-dioxadiazine-1-yl}2,2-dimethylpropionate. H Methyl isoindol-2-yl]-2,6-dioxopiperidin-1-yl}2,2-dimethylpropionate (48% yield).

[0390] .

[0391] Example 17: [2-({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl)-1-oxo-2,3-dihydro-1-yl)-1-yl)-1-yl)-2,3-dihydro ...2-yl)-2-yl)-2-yl)-2-yl)-2-yl H -Isoindole-5- Synthesis of methyl[methyl]carbamoyl]phenyl]acetate (19)

[0392]

[0393] Using the general procedure shown in reaction scheme 1 and method 1 of example above, and with 2-(acetoxymethyl)benzoic acid (30.0 mg, 0.154 mmol), 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1 H [2-({[2-(2,6-dioxopiridine-3-yl)-1-oxo-2,3-dihydro-1-yl]piperidine-2,6-dione hydrochloride (1.0 eq) was synthesized using [2-({[2-(2,6-dioxopiridine-3-yl)-1-oxo-2,3-dihydro-1-yl]piperidine-2,6-dione hydrochloride (1.0 eq) as starting material] H 59% yield of methyl isoindole-5-yl]methyl}carbamoyl)phenyl]acetate.

[0394] .

[0395] Example 18: N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl] H -isoindol-5-yl] Synthesis of methyl-2-(hydroxymethyl)benzamide (20)

[0396]

[0397] Using the general procedure shown in reaction scheme 1 and method 1 of example above, and with 2-(hydroxymethyl)benzoic acid (49.1 mg, 0.323 mmol), 3-[5-(aminomethyl)-1-oxo-2,3-dihydro ... H Synthesis using [isoindol-2-yl]piperidine-2,6-dione hydrochloride (1.0 eq) as the starting material N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1H-isoindol-5-yl]methyl}-2-(hydroxymethyl)benzamide (3.4% yield).

[0398] .

[0399] Example 19: 2-[1-({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl) H -Isoindole-5- Synthesis of

[21] methyl[carbamoyl]-2-methylpropyl-2-yl]-3,5-dimethylphenylacetate

[0400]

[0401] Using the general procedure shown in reaction scheme 1 and method 1 of example above, and with 3-(2-acetoxy-4,6-dimethylphenyl)-3-methylbutyric acid (30.0 mg, 0.113 mmol), 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1 H2-[1-({[2-(2,6-dioxopiridine-3-yl)-1-oxo-2,3-dihydro-1-yl]piperidine-2,6-dione hydrochloride (1.2 eq) was synthesized using 2-[1-({[2-(2,6-dioxopiridine-3-yl)-1-oxo-2,3-dihydro-1-yl]piperidine-2,6-dione hydrochloride as the starting material. H -isoindol-5-yl]methyl}carbamoyl)-2-methylpropyl-2-yl]-3,5-dimethylphenylacetate (80% yield).

[0402] .

[0403] Example 20: 4-(((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino Synthesis of formyl)oxy)methyl)phenylacetic acid ester (22)

[0404]

[0405] To a solution of 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione hydrochloride (25 mg, 0.081 mmol) and DIPEA (3 eq) in DMF (1 mL), 4-(acetoxy)benzyl chloroformate (28 mg, 0.121 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. The solvent was removed under vacuum, and the product was purified by preparative HPLC to give 11.0 mg of 4-(((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)carbamoyl)oxy)methyl)phenylacetate (28% yield).

[0406] .

[0407] Example 21: 1-((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino Synthesis of ethyl isobutyrate (23)

[0408]

[0409] DIPEA (0.028 mL, 0.161 mmol) was added to a solution of 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (25 mg, 0.081 mmol) and ethyl 1-(((4-nitrophenoxy)carbonyl)oxy)isobutyrate (26.4 mg, 0.089 mmol) in DMF (1 mL), and the reaction mixture was stirred at room temperature for 18 hours. The product was purified by preparative HPLC to give 15.8 mg of ethyl 1-((((2-(2,6-dioxopiperidine-3-yl)-1-oxoisoindoline-5-yl)methyl)carbamoyl)oxy)isobutyrate (45.4% yield).

[0410] .

[0411] Example 22: (5-methyl-2-oxo-2- H -1,3-dioxacyclopenten-4-yl)methyl N -{[2-(2,6-dioxane) Piperidin-3-yl)-1-oxo-2,3-dihydro-1 H Synthesis of -isoindol-5-yl]methyl}carbamate (24)

[0412]

[0413] In a 10 mL vial, 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidin-2,6-dione hydrochloride (20.0 mg, 0.065 mmol), (5-methyl-2-oxo-1,3-dioxacyclopenten-4-yl)methyl-4-nitrophenyl carbonate (21.0 mg, 0.071 mmol), and DMF (1 mL) were added. DIPEA (0.022 mL, 0.129 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC to give 21.4 mg of (5-methyl-2-oxo-2-dione hydrochloride). H -1,3-dioxacyclopenten-4-yl)methyl N -{[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl] H -Isoindol-5-yl]methyl}carbamate (77% yield).

[0414] .

[0415] Example 23: 2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)- 2-O-ethyl) tert-butyl carbamate (26) and 2-amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoin Synthesis of dolin-5-yl)methyl)acetamide (25)

[0416]

[0417] Step 1. Using the general procedure shown in Reaction Scheme 1 and Method 1 of Example 1 above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (60 mg, 0.194 mmol) and (tert-butoxycarbonyl)glycine (1.200 eq) as starting materials, synthesize tert-butyl carbamate (2-((2-(2,6-dioxopiperidine-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxoethyl)carbamate (86% yield).

[0418] .

[0419] Step 2. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and starting with (2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxoethyl)carbamate tert-butyl ester (51.7 mg, 0.120 mmol) to synthesize 2-amino-N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)acetamide (31% yield).

[0420] .

[0421] Example 24: (( S )-1-(((2-(( S )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl (1)amino)-3-(1) H -imidazol-4-yl)-1-oxopropyl-2-yl)tert-butyl carbamate (30), (( S )-1-(((2-(( R )- 2,6-Dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -imidazol-4-yl)-1-oxo propyl-2-yl) tert-butyl carbamate (29), ( S )-2-amino- N -((2-(( S )-2,6-dioxopiperidin-3-yl)-1-oxo (isoindolin-5-yl)methyl)-3-(1 H -imidazol-4-yl)propanamide (28) and ( S )-2-amino- N -((2-(( R )-2,6-di oxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-(1 H Synthesis of 4-imidazolium-4-yl)propionamide (27)

[0422]

[0423] Step 1. Add 3-[5-(aminomethyl)-1-oxo-2,3-dihydro-1-oxo-2,3-dihydro-1-oxo-2,3-dihydro-2-oxo-2 ... H [-isoindol-2-yl]piperidine-2,6-dione hydrochloride (60.0 mg, 0.194 mmol) and Boc-His-OH (59.3 mg, 0.232 mmol) were dissolved in DMF (6 mL). DIPEA (0.074 mL, 0.426 mmol) was added, followed by HATU (88.4 mg, 0.232 mmol), and the resulting solution was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC to obtain 41 mg of [(...]]. S )-1-(((2-(( S )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -imidazol-4-yl)-1-oxopropyl-2-yl)carbamate tert-butyl ester (41% yield) and 21.0 mg of (( S )-1-(((2-(( R )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -imidazol-4-yl)-1-oxopropyl-2-yl) tert-butyl carbamate (21% yield).

[0424] (( S )-1-(((2-(( S 2,6-Dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1H-imidazol-4-yl)-1-oxopropyl-2-yl)carbamate tert-butyl

[0425] .

[0426] (( S )-1-(((2-((R )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -imidazol-4-yl)-1-oxopropyl-2-yl)tert-butyl carbamate

[0427] .

[0428] Step 2a: Using the general procedure shown in Procedure B of the above reaction scheme 6 and method 6 of Example 6, and with (( S )-1-(((2-(( S The synthesis was carried out using tert-butyl carbamate (10.0 mg, 0.020 mmol) as the starting material. S )-2-amino- N -((2-(( S )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-(1 H -Imidazol-4-yl)propionamide (100% yield).

[0429] .

[0430] Step 2b: Use the general procedure shown in Procedure B of Reaction Scheme 6 and Method 6 of Example 6, and in (( S )-1-(((2-(( R )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H The synthesis was carried out using imidazole-4-yl)-1-oxopropyl-2-yl)carbamate tert-butyl ester (10.0 mg, 0.020 mmol) as the starting material. S )-2-amino- N -((2-(( R )-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-(1H-imidazol-4-yl)propionamide (100% yield).

[0431] .

[0432] Example 25: (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino 3-Methyl-1-oxobut-2-yl)carbamate tert-butyl ester (31) and 2-amino- N -((2-(2,6-dioxopiperidine-3- Synthesis of 1-oxoisoindoline-5-yl)methyl)-3-methylbutyramide (32)

[0433]

[0434] Step 1. Using the general procedure shown in Reaction Scheme 1 and Method 1 of Example 1 above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione hydrochloride (50 mg, 0.161 mmol) and (tert-butoxycarbonyl)valine (1.200 eq) as starting materials, synthesize (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-methyl-1-oxobut-2-yl)carbamate tert-butyl (76% yield).

[0435] .

[0436] Step 2. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and with (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-methyl-1-oxobut-2-yl)carbamate (88.8 mg, 0.188 mmol) as the starting material, synthesize 2-amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-methylbutyramide (92% yield).

[0437] .

[0438] Example 26: (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino 2-Methyl-1-oxopropyl-2-yl)carbamate tert-butyl ester (33) and 2-amino- N -((2-(2,6-dioxopiperidine-3- Synthesis of 5-yl)-1-oxoisoindoline-5-yl)methyl)-2-methylpropionamide (34)

[0439]

[0440] Step 1. Using the general procedure shown in Reaction Scheme 1 and Method 1 of Example 1 above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione hydrochloride (30 mg, 0.097 mmol) and 2-((tert-butoxycarbonyl)amino)-2-methylpropionic acid (1.200 eq) as starting materials, synthesize tert-butyl carbamate (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-methyl-1-oxoprop-2-yl)carbamate (67% yield).

[0441] .

[0442] Step 2. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and with (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-methyl-1-oxopropyl-2-yl)carbamate (26.1 mg, 0.057 mmol) as the starting material, synthesize 2-amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-2-methylpropionamide (83% yield).

[0443] .

[0444] Example 27: (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino (base)-3-(1) H -Indole-3-yl)-1-oxopropyl-2-yl)tert-butyl carbamate (35) and 2-amino- N -((2-(2,6-dioxane) Piperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-(1 H Synthesis of 3-indol-3-yl)propionamide (37)

[0445]

[0446] Step 1. Using the general procedure shown in Reaction Scheme 1 and Method 1 of Example 1 above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione hydrochloride (50 mg, 0.161 mmol) and (tert-butoxycarbonyl)tryptophan (1.200 eq) as starting materials, synthesize (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -Indole-3-yl)-1-oxopropyl-2-yl)tert-butyl carbamate (66% yield).

[0447] .

[0448] Step 2. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and starting with (1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1H-indo-3-yl)-1-oxopropyl-2-yl)carbamate (15.0 mg, 0.027 mmol), synthesize 2-amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-3-(1 H -Indole-3-yl)propionamide (63.3% yield).

[0449] .

[0450] Example 28: N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-3-(1 H - Synthesis of indole-3-yl)acrylamide (36)

[0451]

[0452] Using the general procedure shown in reaction scheme 1 and method 1 of Example 1 above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (40 mg, 0.129 mmol) and 3-indolepropionic acid (26.9 mg, 0.142 mmol) as starting materials, it was synthesized N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-3-(1 H -Indole-3-yl)acrylamide (70% yield).

[0453] .

[0454] Example 29: 2-Amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl Synthesis of 5-guanidinopentanamide (38)

[0455]

[0456] Step 1. Using the general procedure shown in reaction scheme 1 and method 1 of Example above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (40 mg, 0.129 mmol) and ( E )- N 2 ,N ω ,N ω’ Synthesis using tri(tert-butoxycarbonyl)arginine (1,200 eq) as starting material N -(4-{[(Z)-{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}-1-({[2-(2,6-dioxoperidin-3-yl)-1-oxo-2,3-dihydro-1-yl) H -isoindol-5-yl]methyl}carbamoyl)butyl)tert-butyl carbamate (93% yield).

[0457] Step 2. Use the general procedure shown in Procedure A of Reaction Scheme 6 and Method 6 of Example 6, and with N -(4-{[( Z )-{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}-1-({[2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl)-1-oxo-2,3-dihydro-1-yl)-1-amino]-1-amino]-1-amino]-1-amino]-1-amino]-2-(2,6-dioxopiperidin-3-yl)-1-oxo-2,3-dihydro-1-yl ... H 2-Amino-[-isoindol-5-yl]methyl[-carbamoyl]butyl]carbamate (87.6 mg, 0.120 mmol) was used as a starting material to synthesize 2-amino-N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-5-guanidinopentanamide (50% yield).

[0458] .

[0459] Example 30: (2) S ,3 R )-2-amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5- Synthesis of (39)-3-hydroxybutyramide (39)

[0460]

[0461] Step 1. Using the general procedure shown in reaction scheme 1 and method 1 of Example 1 above, and with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (84.8 mg, 0.274 mmol) and (tert-butoxycarbonyl)-L-threonine (1.000 eq) as starting materials, synthesize ((2 S ,3 R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-hydroxy-1-oxobut-2-yl)carbamate tert-butyl ester (69% yield).

[0462] .

[0463] Step 2. Use the general procedure shown in Procedure B of the above reaction scheme 6 and Example 6, and in ((2 S ,3 R The tert-butyl carbamate (70.5 mg, 0.149 mmol) was used as a starting material to synthesize (2) S ,3 R )-2-amino- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-hydroxybutyramide (100% yield).

[0464] .

[0465] Example 31: ((2) S ,3 R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl Synthesis of benzyl (40)-3-hydroxy-1-oxobutyl-2-yl)carbamate (40)

[0466]

[0467] Using the general procedure shown in reaction scheme 1 and method 1 of example above, and with 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione hydrochloride (30 mg, 0.097 mmol) and (2 S ,3 R Synthesized using 2-(((benzyloxy)carbonyl)amino)-3-hydroxybutyric acid (1,200 eq) as starting material (2 S ,3 R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-hydroxy-1-oxobut-2-yl)carbamate (64% yield).

[0468] .

[0469] Example 32: 1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)- Synthesis of 3-(4-hydroxyphenyl)-1-oxopropyl-2-yl)carbamate (41)

[0470]

[0471] The compound was synthesized using the general procedure shown in reaction scheme 1 and method 1 of example above, with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (30 mg, 0.097 mmol) and ((benzyloxy)carbonyl)tyrosine (1.200 eq) as starting materials (52.7% yield).

[0472] .

[0473] Example 33: N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-3-(1 H - Synthesis of indole-3-yl)propionamide (42)

[0474]

[0475] The compound was synthesized using the general procedure shown in reaction scheme 1 and method 1 of example above, with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (40 mg, 0.129 mmol) and 3-(4-chlorophenyl)propionic acid (26.2 mg, 0.142 mmol) as starting materials (yield).

[0476] .

[0477] Example 34: (2) S )- N -((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)pyridine Synthesis of pyrrolidine-2-carboxamide hydrochloride (43)

[0478]

[0479] Step 1. This step was performed using the general procedure shown in reaction scheme 1 and method 1 of example above, with 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione hydrochloride (86.3 mg, 0.279 mmol) and (tert-butoxycarbonyl)-L-proline (50 mg, 0.232 mmol) as starting materials (56.3% yield).

[0480] .

[0481] Step 2. Use the general procedure shown in Procedure B of Reaction Scheme 6 and Method 6 of Example 6, and with (2 S The step was completed using tert-butyl pyrrolidine-1-carboxylic acid (56.9 mg, 0.121 mmol) as the starting material (99.6% yield).

[0482] .

[0483] Example 35: 4-Amino-2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione (44) synthesis

[0484]

[0485] Step 1. To a solution of 30 g (164.7 mmol) of 4-hydroxyphthalic acid in anhydrous MeOH (600 mL), 5 mL of concentrated H₂SO₄ was added, and the mixture was refluxed overnight. After cooling to room temperature, methanol was evaporated, the mixture was diluted with DCM, washed with NaHCO₃ solution, and dried over Na₂SO₄. The mixture was concentrated under reduced pressure to give dimethyl 4-hydroxyphthalate in quantitative yield.

[0486] Step 2. To a solution of 4-hydroxyphthalic acid dimethyl ester (30 g, 142.7 mmol) cooled to -10 °C in concentrated H₂SO₄ (300 mL), 65% HNO₃ (16.5 mL) was added dropwise, and the mixture was stirred at 0 °C for 30 min. The reaction mixture was poured onto ice, and the product was extracted with EtOAc, washed with water, dried over Na₂SO₄, and concentrated under reduced pressure to give a mixture of 4-hydroxy-3-nitrophthalic acid dimethyl ester and 4-hydroxy-5-nitrophthalic acid dimethyl ester, which was separated by column chromatography.

[0487] Step 3. Under an argon atmosphere, Pd / C (5% by weight) was added to a solution of 4-hydroxy-3-nitrophthalic acid ester (5 g, 19.6 mmol) in anhydrous MeOH (100 mL). The flask was filled / emptied with hydrogen, and this process was repeated three times. The solution was stirred at room temperature for 12 hours under a hydrogen atmosphere (1 bar). After the starting material was consumed, the solvent was evaporated to give 3.97 g of dimethyl 3-amino-4-hydroxyphthalate (90% yield).

[0488] Step 4. The mixture of 3-amino-4-hydroxyphthalic acid dimethyl ester (3.97 g, 17.6 mmol) and concentrated HCl aqueous solution (100 mL) was refluxed for 6 hours and evaporated under reduced pressure to give 2.84 g of 3-amino-4-hydroxyphthalic acid salt.

[0489] Step 5. A mixture of 3-amino-4-hydroxyphthalic acid salt (2.84 g, 12.1 mmol), 3-aminopiperidin-2,6-dione hydrochloride (2 g, 18.2 mmol), acetonitrile (26 mL), acetic acid (7 mL), and triethylamine (8.3 mL) was refluxed overnight. The reaction mixture was then cooled to room temperature and poured into water. The precipitated solid was collected and dried to give 1.73 g of 4-amino-2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione (43% yield).

[0490] .

[0491] Example 36: Synthesis of 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)pyrrolidine-2,5-dione (50)

[0492]

[0493] Step 1. Using the general procedure shown in reaction scheme 2 and method 2 of example above, 4-(((tert-butoxycarbonyl)amino)methyl)-2-(hydroxymethyl)benzoic acid (94% yield) was synthesized from tert-butyl carbamate ((1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (500 mg, 1.9 mmol) as the starting material.

[0494] Step 2. Using the general procedure shown in reaction scheme 3 and method 3 of example above, tert-butyl carbamate (3-hydroxy-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (81% yield) was synthesized from 4-(((tert-butoxycarbonyl)amino)methyl)-2-(hydroxymethyl)benzoic acid (430 mg, 1.53 mmol) as the starting material.

[0495] Step 3. Using the general procedure shown in reaction scheme 4 and method 4 of Example 4 above, tert-butyl carbamate ((2-(2,5-dioxopyrrolidine-3-yl)-1-oxoisoindoline-5-yl)methyl)carbamate (50 mg, 0.18 mmol) and 3-aminopyrrolidine-2,5-dione hydrochloride (1 eq) were synthesized as starting materials.

[0496] .

[0497] Step 4. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and starting with ((2-(2,5-dioxopyrrolidine-3-yl)-1-oxoisoindoline-5-yl)methyl)carbamate tert-butyl ester (64.3 mg, 0.18 mmol), synthesize 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)pyrrolidine-2,5-dione (12% yield, two steps).

[0498] .

[0499] Example 37: 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione-5,5-d2 (51)

[0500]

[0501] Step 1. Using the general procedure shown in reaction scheme 4 and method 4 of Example 4 above, tert-butyl carbamate ((2-(2,6-dioxopiridine-3-yl-5,5-d2)-1-oxoisoindoline-5-yl)methyl)carbamate (39% yield) was synthesized from ((3-hydroxy-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (50 mg, 0.18 mmol) and 3-aminopyrrolidine-2,5-dione-3,5,5-d3 (1 eq) as starting materials.

[0502] .

[0503] Step 2. Using the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and starting with ((2-(2,6-dioxopiperidin-3-yl-5,5-d2)-1-oxoisoindoline-5-yl)methyl)carbamate tert-butyl ester (27 mg, 0.073 mmol), synthesize 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione-5,5-d2 (15% yield).

[0504] .

[0505] Example 38: 3-[5-(aminomethyl)-3-methyl-1-oxo-2,3-dihydro-1-yl] H -isoindol-2-yl]piperidine-2, Synthesis of 6-dione (52)

[0506]

[0507] Step 1. To 5-bromo-6-methylisobenzofuran-1(3 H 500 mg (2.21 mmol) of 3-methyl-1-oxo-1,3-dihydroisobenzofuran-5-onitrile was added to a solution of Zn(CN)2 (648.7 mg, 5.52 mmol) in DMF (5 mL), followed by the addition of Pd(PPh3)4 (255 mg, 0.221 mmol). The reaction mixture was heated at 100 °C for 16 hours under an inert atmosphere. The reaction was quenched with ice water, and the product was extracted into EtOAc. The organic layer was dried over Na2SO4, concentrated, and purified by rapid column chromatography to give 314 mg of 3-methyl-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (82% yield).

[0508] Step 2. To a solution of 3-methyl-1-oxo-1,3-dihydroisobenzofuran-5-onitrile (400 mg, 2.30 mmol) in ethanol (5 mL), Boc2O (1.056 mL, 4.598 mmol) was added, followed by Raney nickel (80 mg), and the reaction mixture was stirred under a hydrogen atmosphere (1 bar) for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by rapid column chromatography to give 320 mg of tert-butyl ((3-methyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)methyl)carbamate (50% yield).

[0509] Step 3. Using the general procedure shown in reaction scheme 2 and method 2 of example above, and with N 4-(((tert-butoxycarbonyl)amino)methyl)-2-(1-hydroxyethyl)benzoic acid (99.8% yield) was synthesized from tert-butyl carbamate (32.0 mg, 0.115 mmol) as the starting material.

[0510] Step 4. Using the general procedure shown in reaction scheme 3 and method 3 of Example 3 above, and starting with 4-({[(tert-butoxy)carbonyl]amino}methyl)-2-(1-hydroxyethyl)benzoic acid (33.5 mg, 0.114 mmol), synthesize N -[(3-hydroxy-3-methyl-1-oxo-1,3-dihydro-2-benzofuran-5-yl)methyl] tert-butyl carbamate (80.0% yield).

[0511] Step 5. Using the general procedure shown in reaction scheme 4 and method 4 of example above, and with N Synthesis using tert-butyl carbamate (33.3 mg, 0.091 mmol) as the starting material N -{[2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxo-2,3-dihydro-1H-isoindol-5-yl]methyl} tert-butyl carbamate (4.0% yield).

[0512] Step 6. Using the general procedure shown in Procedure B of Reaction Scheme 6 and Example Method 6 above, and with N -{[2-(2,6-dioxopiperidin-3-yl)-3-methyl-1-oxo-2,3-dihydro-1-yl] H tert-butyl [-isoindol-5-yl]methyl}carbamate (1.4 mg, 0.004 mmol) was used as a starting material to synthesize 3-[5-(aminomethyl)-3-methyl-1-oxo-2,3-dihydro-1-yl]carbamate. H -isoindol-2-yl]piperidine-2,6-dione (100% yield).

[0513] .

[0514] Example 39: 2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1 H -Isoyindole-5-carboxylic acid Synthesis of acid (54)

[0515]

[0516] The following were added: 3-(5-bromo-6-fluoro-1-oxo-2,3-dihydro-1H-isoindol-2-yl)piperidin-2,6-dione (50.0 mg, 0.147 mmol), Mo(CO)6 (0.035 mL, 0.256 mmol), DMAP (35.8 mg, 0.293 mmol), Pd2(dba)3 (13.4 mg, 0.015 mmol), tri-tert-butylphosphine trifluoroborate (8.5 mg, 0.029 mmol), 1,4-di... Alkane (2.0 mL), H₂O (0.200 mL), and DIPEA (51 µL, 0.293 mmol) were placed in a vial. The reaction was carried out in a microwave reactor at 150 °C for 30 min. The crude product was purified by preparative HPLC to give 13.0 mg of 2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxo-2,3-dihydro-1-yl)- ... H -Isoindole-5-carboxylic acid (28% yield) is a white solid.

[0517] .

[0518] Example 40: ( S )-3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)-3-methylpiperidin-2,6-dione Synthesis of (48)

[0519]

[0520] Step 1. To methyl 2-bromomethyl-4-cyanobenzoate (114.0 mg, 0.448 mmol) and ( S 3-Amino-3-methylpiperidin-2,6-dione hydrobromide (100.0 mg, 0.448 mmol) was added to a solution of DIPEA (0.390 mL, 2.242 mmol) in ACN (6 mL), and the reaction mixture was stirred at room temperature for 18 hours. Volatiles were removed under reduced pressure, and the residue was purified by preparative HPLC to give 63.0 mg of (…). S Methyl benzoate 4-cyano-2-(((3-methyl-2,6-dioxopiperidin-3-yl)amino)methyl)benzoate (47% yield).

[0521] Step 2. To ( S Methyl benzoate (67.0 mg, 0.212 mmol) was added to a suspension in anhydrous toluene (6 mL) with bis(trimethylaluminum)-1,4-diazabicyclo[2.2.2]octane adduct (5.4 mg, 0.021 mmol), and the reaction mixture was refluxed for 12 hours. Volatiles were removed under reduced pressure, and the residue was purified by preparative HPLC to give 45 mg of (…). S )-2-(3-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-nitriles (74% yield).

[0522] Step 3. To ( S Raney nickel (33 mg) was added to a solution of 2-(3-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-onitrile (25.0 mg, 0.088 mmol) and Boc2O (38.5 mg, 0.176 mmol) in a mixture of DMF (1.5 mL) and THF (2.5 mL), and the reaction mixture was stirred under hydrogen (1 bar) for 24 hours. The reaction mixture was filtered, concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 18.2 mg of ( S 2-((2-(3-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)tert-butyl carbamate (53% yield).

[0523] Step 4. Use the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and with ( S The synthesis was based on tert-butyl carbamate (18.2 mg, 0.047 mmol) as a starting material. S )-3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)-3-methylpiperidin-2,6-dione (90% yield).

[0524] .

[0525] Example 41: ( R )-3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)-3-methylpiperidin-2,6-dione Synthesis of (49)

[0526]

[0527] Step 1. To methyl 2-bromomethyl-4-cyanobenzoate (57.0 mg, 0.224 mmol) and ( R 3-Amino-3-methylpiperidin-2,6-dione hydrobromide (50.0 mg, 0.224 mmol) was added to a solution of ACN (3 mL) with DIPEA (0.195 mL, 1.121 mmol), and the reaction mixture was stirred at room temperature for 18 hours. Volatiles were removed under reduced pressure, and the residue was purified by preparative HPLC to give 37.0 mg of (…). R Methyl benzoate 4-cyano-2-(((3-methyl-2,6-dioxopiperidin-3-yl)amino)methyl)benzoate (52% yield).

[0528] Step 2. To ( R Methyl benzoate (37.0 mg, 0.117 mmol) was added to a suspension in anhydrous toluene (3 mL) with bis(trimethylaluminum)-1,4-diazabicyclo[2.2.2]octane adduct (3.0 mg, 0.012 mmol), and the reaction mixture was refluxed for 12 hours. Volatiles were removed under reduced pressure, and the residue was purified by preparative HPLC to give 17 mg of (…). R )-2-(3-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-nitriles (51% yield).

[0529] Step 3. To ( RRaney nickel (20 mg) was added to a solution of 2-(3-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-onitrile (15.0 mg, 0.053 mmol) and Boc2O (23.1 mg, 0.106 mmol) in a mixture of DMF (1.0 mL) and THF (1.5 mL), and the reaction mixture was stirred under hydrogen (1 bar) for 24 hours. The reaction mixture was filtered, concentrated under reduced pressure, and the residue was purified by preparative HPLC to give 11.1 mg of ( R 2-((2-(3-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)tert-butyl carbamate (54% yield).

[0530] Step 4. Use the general procedure shown in Procedure A of Reaction Scheme 6 and Example Method 6 above, and with ( R The synthesis was based on tert-butyl carbamate (11.1 mg, 0.029 mmol) as a starting material. R )-3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)-3-methylpiperidine-2,6-dione (72% yield).

[0531] .

[0532] Examples 42-50: Degradation assay, cell viability assay, and cell survival assay

[0533] Table 2. Reference compound IDs and chemical structures.

[0534] .

[0535] Example 42: Fluorescence Polarization (FP) Experiment

[0536] The CRBN-DDB1 protein complex was mixed with Cy5-labeled thalidomide and the test compound (“test compound”). The test solution contained 50 mM Tris pH=7.0, 200 mM NaCl, 0.02% v / v Tween-20, 2 mM MTT, 5 nM Cy5-labeled thalidomide (tracer), 25 nM CRBN-DDB1 protein, and 2% v / v DMSO. This test solution was added to a 384-well plate.

[0537] The plate was spun down (1 minute, 1000 rpm, 22°C) and then shaken for 10 minutes at room temperature (20-25°C) using a VibroTurbulator with the frequency set to level 3. The detection plate containing the protein and tracer was incubated at room temperature (20-25°C) for 60 minutes and then read using a plate reader. Reading was performed using a Cy5 FP Filterset (590nm / 675nm) via a Pherastar plate reader (fluorescence polarization).

[0538] FP experiments were conducted using test compounds at various concentrations to measure K. i value,.

[0539] K, a competitive inhibitor i The values ​​are based on the relationship between compound concentration and measured fluorescence polarization using IC. 50 Values, K of Cy5-T and CRBN / DDB1 complex d The values, as well as the concentrations of protein and tracer in the displacement assay, are calculated using equations (as described in Z. Nikolovska-Coleska et al., Analytical Biochemistry 332 (2004) 261-273).

[0540] Fluorescence polarization (FP) experiment - results

[0541] The compounds are classified according to their affinity for CRBN (defined as Ki). As reported in Table 3 below, the compounds of the present invention interact with the CRBN-DDB1 protein within a similar affinity range as reported in the reference compounds.

[0542] CRBN binding Ki [μM] is represented as follows:

[0543] A < 0.5 μM

[0544] 0.5 μM ≤ B ≤ 1 μM

[0545] Table 3: Fluorescence Polarization (FP) Experiment

[0546]

[0547] .

[0548] Example 43: SALL4 Degradation Assay - Kelly Cell Line

[0549] The effects of various compounds of the present invention and various reference compounds on the degradation of SALL4 in the Kelly cell line were investigated using the following degradation assay protocol.

[0550] Kelly cells were maintained in RPMI-1640 medium supplemented with penicillin / streptomycin and 10% fetal bovine serum (FBS). Cells were seeded into 6-well or 12-well plates, and the desired concentration range of the test compound was added. The final DMSO concentration was 0.25%. After incubation (37°C, 5% CO2) for 24 hours, cells were harvested, washed, and cell lysates were prepared using RIPA lysis buffer. Protein levels were determined by BCA assay, and appropriate amounts were then loaded onto pre-prepared gels for protein separation. After staining with the first and second Abs, the membranes were washed and signals were generated. Densitometry analysis was performed to obtain values ​​used subsequently in protein level assessment.

[0551] The results of treatment with 100 nM compound for 24 hours are shown in Table 4 below.

[0552] Table 4: Percentage reduction in SALL4 protein in Kelly cells after treatment with the compounds of this invention and the reference compound thalidomide. The values ​​shown are the averages from n ≥ 2 experiments.

[0553]

[0554] Thalidomide, lenalidomide, 1, and 44 were also tested for 24 hours at concentrations ranging from 0.01 µM to 1 µM. Figure 1 As shown, the compounds of the present invention induced efficient degradation (>50%) of SALL4 at low concentrations (0.01 M), while lenalidomide and thalidomide exhibited lower activity.

[0555] As shown in Table 4 and Figure 1 As shown, the compounds of the present invention induced the degradation of SALL4 protein in the Kelly (neuroblastoma) cell line at a lower concentration than the reference compound. Therefore, the compounds of the present invention can be used as anticancer drug candidates.

[0556] Example 44: SALL4 Degradation-Time Process in Kelly Cell Line

[0557] The degradation time of SALL4 in the Kelly cell line after treatment with various compounds of the present invention and various reference compounds was also analyzed.

[0558] Kelly cells were maintained in RPMI-1640 medium supplemented with penicillin / streptomycin and 10% fetal bovine serum (FBS). Cells were seeded into 6-well or 12-well plates, and the desired concentration range of the test compound was added. The final DMSO concentration was 0.25%. After incubation (37°C, 5% CO2) for a specified period, cells were harvested, washed, and cell lysates were prepared using RIPA lysis buffer. Protein levels were determined by BCA assay, and appropriate amounts were then loaded onto pre-prepared gels for protein separation. After staining with the first and second Abs, the membranes were washed and signals were generated. Densitometry analysis was performed to obtain values ​​subsequently used in protein level assessment.

[0559] The compounds tested in this experiment were lenalidomide at concentrations of 0.1 µM, 1, and 44, for durations of 3, 6, 12, 24, 48, and 72 h. The results are shown in Figure 2. As shown in the figure, the compounds of the present invention degrade SALL4 more rapidly and efficiently than lenalidomide, indicating that the compounds of the present invention can be administered at lower doses than the reference compounds.

[0560] Example 45: GSPT1 Degradation Assay - Hep3B Cell Line

[0561] The effects of various compounds of the present invention and various reference compounds on the degradation of GSPT1 in the Hep3B cell line were investigated using the following degradation assay protocol.

[0562] Hep3B cells were maintained in EMEM medium supplemented with penicillin / streptomycin and 10% fetal bovine serum (FBS). Cells were seeded into 6-well or 12-well plates, and the desired concentration range of the test compound was added. The final DMSO concentration was 0.25%. After incubation (37 °C, 5% CO2) for 24 hours, cells were harvested, washed, and cell lysates were prepared using RIPA lysis buffer. Protein levels were determined by BCA assay, and appropriate amounts were then loaded onto pre-prepared gels for protein separation. After staining with the first and second Abs, the membranes were washed and signals were generated. Densitometric analysis was performed to obtain values ​​used subsequently in protein level assessment. Densitometric values ​​were normalized to the loading control and calculated as a percentage of the DMSO control.

[0563] The results of treatment with 1 μM and 10 μM compounds for 24 hours are shown in Table 5 below.

[0564] Table 5: Percentage reduction in GSPT1 protein after treatment of HEP3B cells with the compounds of the present invention

[0565]

[0566] A represents a reduction of 80-100% in GSPT1 protein, and B represents a reduction of 50-79% in GSPT1 protein.

[0567] As shown in Table 5 and Figure 3 As shown, the compounds of the present invention induced the degradation of GSPT1 protein in the Hep3B cell line. Therefore, the compounds of the present invention can be used as anticancer drug candidates.

[0568] Example 46: Ikaros Degradation Assay - H929 Cell Line

[0569] Using the above-described degradation assay, the effects of various compounds of the present invention and various reference compounds on the degradation of Ikaros in the H929 cell line were investigated.

[0570] H929 cells were maintained in RPMI-1640 medium supplemented with penicillin / streptomycin and 10% fetal bovine serum (FBS). Cells were seeded into 6-well or 12-well plates, and the desired concentration range of the test compound was added. The final DMSO concentration was 0.25%. After incubation (37 °C, 5% CO2) for 24 hours, cells were harvested, washed, and cell lysates were prepared using RIPA lysis buffer. Protein levels were determined by BCA assay, and appropriate amounts were then loaded onto pre-prepared gels for protein separation. After staining with the first and second Abs, the membranes were washed and signals were generated. Densitometry analysis was performed to obtain values ​​used subsequently in protein level assessment.

[0571] The results of treatment with 10 µM or 20 µM compounds for 24 hours are shown in Table 6 below.

[0572] Table 6: Percentage reduction in IKZF1 protein after treatment of H929 cells with the compounds of the present invention and the reference compounds

[0573]

[0574] C represents a 0-24% reduction in IKZF1 protein.

[0575] Lenalidomide, 1, 44, 28, and 27 were also tested at concentrations of 1 µM and 10 µM for 24 hours. The results are as follows... Figure 4A As shown.

[0576] Compounds 4, 52, 5, 7, and 54 of the present invention were also tested for 24 hours at a concentration of 10 µM, together with reference compound 100, CC-90009, and pomalidomide. The results are as follows: Figure 4B As shown.

[0577] As shown in the examples, the compounds of the present invention are less potent against Ikaros (IKZF1) than the reference compounds.

[0578] Example 47: Aiolos Degradation Assay - H929 Cell Line

[0579] The effects of various compounds of the present invention and various reference compounds on the degradation of Aiolos in the H929 cell line were investigated using the following degradation assay protocol.

[0580] H929 cells were maintained in RPMI-1640 medium supplemented with penicillin / streptomycin and 10% fetal bovine serum (FBS). Cells were seeded into 6-well or 12-well plates, and the desired concentration range of the test compound was added. The final DMSO concentration was 0.25%. After incubation (37°C, 5% CO2) for 24 hours, cells were harvested, washed, and cell lysates were prepared using RIPA lysis buffer. Protein levels were determined by BCA assay, and appropriate amounts were then loaded onto pre-prepared gels for protein separation. After staining with the first and second Abs, the membranes were washed and signals were generated. Densitometry analysis was performed to obtain values ​​used subsequently in protein level assessment.

[0581] The results of treatment with the 20 µM compound for 24 hours are shown in Table 7 below.

[0582] Table 7: Percentage reduction in IKZF3 protein after treatment of H929 cells with the compounds of this invention and the reference compounds

[0583]

[0584] C represents a 0-24% reduction in IKZF1 protein.

[0585] Lenalidomide, 1, 44, 28, and 27 were also tested at concentrations of 1 µM and 10 µM for 24 hours. Densitometric measurements were normalized to the loading control and calculated as a percentage of the DMSO control. Results are as follows: Figure 5 As shown in the examples, the compounds of the present invention are less potent against Aiolos (IKZF3) than the reference compounds.

[0586] The compounds of this invention possess unique degradation properties because they induce the efficient degradation of certain proteins, such as the carcinogenic SALL4 and GSPT1 proteins. Figures 1-3 However, it has no activity or low potency against Ikaros and Aiolos (Figure 4-). Figure 5 ).

[0587] Example 48: Cell viability in Hep3B, Kelly, H929, KG-1 and SNU-398 cell lines

[0588] The effects of various compounds of the present invention and various reference compounds on cell viability in various cell lines were investigated using the cell viability-CTG assay protocol described below.

[0589] Hep3B, Kelly, H929, KG-1, and SNU-398 cells were maintained in their respective cell cultures (see Table 8 below). Cells were seeded into 96-well plates or 384-white plates, and the desired concentration range of the test compound was added. The compound was diluted in DMSO, and a constant 0.25% v / v DMSO concentration was maintained throughout the assay plate. After incubation (37°C, 5% CO2) for 72 hours, CellTiter-Globe cells were analyzed. ® The reagent (Promega / G7570) was added to the wells. The plate was shaken for 4 minutes and incubated in the dark for 8 minutes, then the luminescence (LU) was read using a CLARIOstar Multimode Plate Reader. The signal was proportional to the amount of ATP, which in turn was proportional to the number of cells present in the culture.

[0590] Luminescence (RLU) values ​​were normalized to DMSO control. Dose response was replicated using a nonlinear regression of the mean and IC50 values ​​via a percentage inhibition technique. 50 The calculation is used for evaluation. IC 50 Value reported as absolute IC 50 The value, i.e., the concentration of the test compound at the intersection of the concentration-reaction curve when T / C = 50%. For the average IC50 value... 50 The value is calculated using the geometric mean.

[0591] Table 9 lists the compounds tested in KG-1, Kelly, and Hep3B cell assays. Compounds were tested at concentrations ranging from 0.001 µM to 50 µM for 72 h. Table 9 shows the absolute IC50 values. 50 Values. The dose-response curves of representative compounds 1, 2, 3, 6, 23, 37, and 52 in Hep3B cells are shown below. Figure 6A As shown in Figure 9 and Table 9, the compounds of the present invention exhibited potent anticancer activity in KG-1, Kelly, and Hep3B cells derived from leukemia, neuroblastoma, and hepatocellular carcinoma, respectively.

[0592] Table 10 lists the compounds tested in the H929 cell assay. Compounds were tested in the concentration range of 0.001 µM–50 µM for 72 h. RLU values ​​were normalized to the DMSO control. Table 10 shows the absolute IC50 values. 50 Values. Representative compounds 1, 3, 37, and 52 of this invention, as well as the dose-response curves of reference compound CC-90009 and pomalidomide, are shown below. Figure 6B As shown in the figure, in the H929 cell line, the compounds of the present invention showed no activity or only minimal activity, while the reference compound effectively inhibited cell growth.

[0593] The compounds tested in the SNU-398 cell assay were compound 3 of this invention and the reference clinical-stage compound CC-90009, at concentrations ranging from 0.001 µM to 50 µM for 72 h. The luminescence (RLU) values ​​were normalized to a DMSO control. Results are as follows... Figure 6C As shown in the figure, in the SNU-398 cell line derived from hepatocellular carcinoma, cell growth was effectively inhibited by the compounds of the present invention (IC50). 50 = 82 nM), while CC-90009 showed lower activity (IC50 ...). 50 > 9.9 µM).

[0594] Table 8: Cell lines and culture media

[0595]

[0596] Table 9: Cell viability after treatment with the compounds of the present invention

[0597]

[0598] A represents IC50 ≤ 100 nM, B represents 1 µM ≥ IC50 > 100 nM, and C represents 5 µM ≥ IC50 > 1 µM.

[0599] Table 10: Viability of H929 cells treated with the compounds of the present invention and the reference compounds

[0600]

[0601]

[0602] Inactive means IC50 > 50 µM, and C means 50 µM ≥ IC50 > 5 µM.

[0603] Example 49: Cell viability, other cell lines

[0604] Tumor cells were grown in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum and 50 μg / ml gentamicin in humid air at 37°C and 5% CO2, with up to 20 passages and one or two passages per week.

[0605] Cells were harvested from exponential phase cultures, counted, and seeded into 96-well flat-bottom microtiter plates. Cell density was determined based on the growth rate of cell lines in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum and 50 μg / ml gentamicin (140 μl / well) (4,000 to 20,000 cells / well, depending on the cell line's growth rate; for hematologic malignancies, up to 60,000 cells / well). Cultures were incubated at 37°C under humid conditions with 5% CO2. After 24 hours, 10 μl of the test compound or control medium was added, and the cells were incubated for another 72 hours. Using a Tecan Freedom EVO 200 robotic platform, the compounds were serially diluted in DMSO, transferred to cell culture medium, and added to the assay plates. A constant 0.3% v / v DMSO concentration was maintained throughout the assay plate. Results were obtained via CellTiter-Globe. ® Cell viability was quantified using a cell viability assay (Promega G8462). After cell incubation, 100 μl of CellTiter-Glo was added to each well. ® One Solution Assay reagent. Shake the plate for 2 minutes to induce cell lysis and incubate for 20 minutes, then use EnVision. ® Xcite multi-label reader (Perkin Elmer) reads luminescence (LU).

[0606] For single-agent efficacy assessment, sigmoid concentration-response curves were fitted to the data points (test and control, T / C values) obtained from each tumor model using 4-parameter nonlinear curve fitting (Charles River DRSDatawarehouse Software). IC50 values ​​were reported as absolute IC50 values, i.e., the concentration of the test compound at the intersection of the concentration-response curves at T / C = 50%. The geometric mean was used for calculating the mean IC50 value. Results are presented as heatmaps of all tested tumor models (individual IC50 values ​​relative to the geometric mean IC50 value).

[0607] Table 11: Cell lines sensitive to 1

[0608]

[0609] *Test / Control Activity: A represents test / control activity ≤ 30%, B represents ≤ 60%

[0610] **Absolute IC50:** A represents IC50 ≤ 100 nM, B represents 100 nM < IC50 ≤ 1 μM, C represents IC50 ≥ 30 μM

[0611] Table 12: Cell lines resistant to 1 but sensitive to CC-90009

[0612]

[0613] The compounds of this invention effectively inhibit the growth of the following cancer types: hepatocellular carcinoma (HEP3B, SNU-398), neuroblastoma (Kelly), leukemia (KG-1, KG-1a, UOC-M1, MOLT-3, MOLT-4, MOLM-13, MOLM-1, MOLM-6), prostate cancer (22Rv1), and multiple myeloma (MOLP-2).

[0614] Furthermore, the compounds of this invention did not exhibit activity against H929 and other cell lines listed in Table "Cell lines resistant to 1 but sensitive to CC-90009", demonstrating their distinction from prior art compounds such as the clinical-stage compound CC-90009. This surprising effect corresponds to the clinical appeal of the compounds, which, due to their enhanced selectivity, may correspond to a therapeutic window for specific cancer types such as HCC.

[0615] Example 50: Cell Survival

[0616] The effects of various compounds of this invention and various reference compounds on cell survival in Kelly and Hep3B cell lines were investigated using a cell survival-clonal formation assay protocol.

[0617] To determine the ability of a single cell to form a colony (defined as a colony of at least 50 cells), Kelly and Hep3B cells were kept in RPMI 1640 (Kelly) or EMEM (Hep3B) medium supplemented with penicillin / streptomycin and 10% FBS. Cells were counted at 1 × 10⁶ cells per well. 3 Cells were seeded at a density of 1,000 cells per well in 6-well plates, and the desired concentration of the test compound was added. The cells were then cultured at 37°C / 5% CO2. After colony formation (9–10 days), the cells were washed and treated with a mixture of 6.0% glutaraldehyde and 0.5% crystal violet for 30 minutes, followed by rinsing with water and drying at room temperature (RT).

[0618] The compounds tested in this experiment were lenalidomide at concentrations ranging from 0.1 µM to 10 µM and 1. Crystal violet staining was performed after 9 to 10 days of culture. The results are shown in Figure 7. As shown in the figure, in Kelly and Hep3B cell lines expressing SALL4, the compounds of the present invention inhibited cell survival in most cases, while lenalidomide or other commercially known compounds did not show activity.

[0619] Additional description

[0620] This article also describes compounds for use under the following terms:

[0621] Clause 1. A compound for use in a method of treating cancer, said method comprising administering said compound to a subject in need of it, wherein said compound is:

[0622] (i) A compound of formula (I) or a pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug thereof:

[0623]

[0624] in

[0625] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0626] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0627] R c Selected from NR 1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0628] Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0629] n is 1 or 2;

[0630] X is selected from F, Cl, Br, and I;

[0631] R 1 Selected from H and C1-C4 alkyl groups,

[0632] R 2 Selected from H, C1-C4 alkyl groups and -COR 3 ,

[0633] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0634] R 3 It is an unsubstituted C1-C4 alkyl group; or is substituted by one or more R 4Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 Unsubstituted 5-membered heterocyclic group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group;

[0635] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0636] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2;

[0637] Where R a R b R 1 and R 2 When each is H, then n is 1;

[0638] or

[0639] (ii) Compounds of formula (II) or their pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs:

[0640]

[0641] in:

[0642] Each R 1 Independently selected from H and C1-C4 alkyl groups;

[0643] R 11 Is it OH or OR? 5 ;as well as

[0644] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0645] Clause 2. A pharmaceutical composition for use in a method of treating cancer, said method comprising administering said pharmaceutical composition to a subject in need of it, wherein said pharmaceutical composition comprises:

[0646] (i) A compound of formula (I) or a pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug thereof:

[0647]

[0648] in

[0649] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0650] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0651] R c Selected from NR 1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0652] Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0653] n is 1 or 2;

[0654] X is selected from F, Cl, Br, and I;

[0655] R 1 Selected from H and C1-C4 alkyl groups,

[0656] R 2 Selected from H, C1-C4 alkyl groups and -COR 3 ,

[0657] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0658] R 3 It is an unsubstituted C1-C4 alkyl group; or is substituted by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 Unsubstituted 5-membered heterocyclic group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group;

[0659] R 5It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0660] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2;

[0661] Where R a R b R 1 and R 2 When each is H, then n is 1;

[0662] or

[0663] (ii) Compounds of formula (II) or their pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs:

[0664]

[0665] in:

[0666] Each R 1 Independently selected from H and C1-C4 alkyl groups;

[0667] R 11 Is it OH or OR? 5

[0668] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0669] Clause 3. A compound or composition used for the purposes described in Clause 1 or Clause 2, wherein the compound of formula (I) is

[0670] Compounds of formula (Ia) or their pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs:

[0671] ,

[0672] in;

[0673] R 1 Selected from H and C1-C4 alkyl groups;

[0674] R 2 Selected from H and -COR 3 ;

[0675] R 3It is an unsubstituted C1-C4 alkyl group; or is substituted by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 Unsubstituted 5-membered heterocyclic groups, unsubstituted 6-membered heterocyclic groups, 5-membered heteroaryl groups, and 6-membered heteroaryl groups; and

[0676] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0677] Clause 4. A compound or composition for any of the uses described in any of the preceding clauses, wherein R 11 It is OH.

[0678] Clause 5. A compound or composition for any of the uses described in any of the preceding clauses, wherein NR 1 R 1 It is NH2.

[0679] Clause 6. A compound or composition for any of the uses described in any of the preceding clauses, wherein the cancer is hepatocellular carcinoma, neuroblastoma, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), breast cancer, prostate cancer, bladder cancer, kidney cancer, muscle cancer, ovarian cancer, skin cancer, pancreatic cancer, breast cancer, colon cancer, blood cancer, connective tissue cancer, placental cancer, bone cancer, uterine cancer, cervical cancer, choriocarcinoma, endometrial cancer, gastric cancer, or lung cancer.

[0680] Clause 7. A compound or composition for any of the uses described in any of the preceding clauses, wherein the cancer is hepatocellular carcinoma.

[0681] Clause 8. A compound or composition for any of the uses described in any of Clauses 1-6, wherein the cancer is neuroblastoma.

[0682] Clause 9. A compound or composition for any of the uses described in any of the preceding clauses, wherein the method of treating cancer further includes administering a second cancer therapy to the subject.

[0683] Clause 10. A compound or composition for the purpose described in Clause 9, wherein the second cancer therapy is chemotherapy or radiation therapy.

[0684] Clause 11. A compound or composition for the purpose described in Clause 9 or Clause 10, wherein:

[0685] (a) The cancer is chronic myeloid leukemia (CML), and the second cancer treatment is chemotherapy using imatinib, dasatinib, or nilotinib;

[0686] (b) The cancer is endometrial cancer, and the second cancer treatment is chemotherapy using carboplatin;

[0687] (c) The cancer is glioblastoma, and the second cancer treatment is chemotherapy using temozolomide (TMZ);

[0688] (d) The cancer is lung cancer, and the second cancer treatment is chemotherapy using cisplatin;

[0689] (e) The cancer is lung cancer, and the second cancer treatment is chemotherapy using erlotinib;

[0690] (f) The cancer is lung cancer, and the second cancer treatment is chemotherapy using entinostat;

[0691] (g) The cancer is lung cancer, and the second cancer treatment is chemotherapy using cisplatin, carboplatin, or paclitaxel;

[0692] (h) The cancer is myelodysplastic syndrome (MDS) / acute myeloid leukemia (AML), and the second cancer treatment is chemotherapy using doxorubicin; or

[0693] (i) The cancer is nasopharyngeal carcinoma, and the second cancer treatment is radiotherapy.

[0694] Clause 12. A compound or composition for any of the uses described in any of the preceding clauses, wherein said compound is selected from...

[0695]

[0696] And its pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs.

[0697] Clause 13. Compounds or compositions used for the purposes described in Clause 12, wherein said compound:

[0698] (a) Selected from compounds 12, 14, 30 and 29,

[0699] (b) Selected from compounds 1, 28, 27 and 24,

[0700] (c) Selected from compounds 1, 44, 28, 27 and 24,

[0701] (d) Selected from compounds 44, 28, 27 and 24,

[0702] (e) is selected from compounds 1, 44, 28 and 27.

[0703] (f) Selected from compounds 44 and 1, or

[0704] (g) is compound 1.

[0705] Clause 14. A compound used for a method of modulating target protein levels in a subject, the method comprising administering the compound to the subject, wherein the compound is a compound of formula (III) or a pharmaceutically acceptable salt, ester, optical isomer, racemic mixture, solvate, amino acid conjugate, or prodrug thereof:

[0706] ,

[0707] in;

[0708] R is C=O or CH2;

[0709] R 7 Selected from H, deuterium, X, C1-C4 alkyl and NR 1 R 1 ,

[0710] R 8 Selected from OH, OR 5 and CR a R b R c ,

[0711] in

[0712] When R is C=O and R 8 When it is OH, then R 7 Selected from NR 1 R 1 , deuterium, X and C1-C4 alkyl;

[0713] R 9 and R 10 Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2

[0714] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0715] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0716] R c Selected from NR 1 R2 OH, OR 6 CH2X, CHX2, and CX3;

[0717] n is 1 or 2;

[0718] X is selected from F, Cl, Br, and I;

[0719] R 1 Selected from H and C1-C4 alkyl groups,

[0720] R 2 Selected from H, C1-C4 alkyl groups and -COR 3 ,

[0721] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or when R 8 It is CR a R b R c And R c It is NR 1 R 2 When, then R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0722] R 3 It is unsubstituted C1-C 10 Alkyl; or alkyl; or composed of one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 Unsubstituted 5-membered heterocyclic group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group;

[0723] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0724] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2.

[0725] Clause 15. A pharmaceutical composition for use in a method of modulating target protein levels in a subject, the method comprising administering the pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises a compound of formula (III) or a pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug thereof:

[0726] ,

[0727] in;

[0728] R is C=O or CH2;

[0729] R 7 Selected from H, deuterium, X, C1-C4 alkyl and NR 1 R 1 ,

[0730] R 8 Selected from OH, OR 5 and CR a R b R c ,

[0731] in

[0732] When R is C=O and R 8 When it is OH, then R 7 Selected from NR 1 R 1 , deuterium, X and C1-C4 alkyl;

[0733] R 9 and R 10 Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0734] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0735] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0736] R c Selected from NR 1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0737] n is 1 or 2;

[0738] X is selected from F, Cl, Br, and I;

[0739] R 1 Selected from H and C1-C4 alkyl groups,

[0740] R2 Selected from H, C1-C4 alkyl groups and -COR 3 ,

[0741] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or when R 8 It is CR a R b R c And R c It is NR 1 R 2 When, then R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0742] R 3 It is unsubstituted C1-C 10 Alkyl; or alkyl; or composed of one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 Unsubstituted 5-membered heterocyclic group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group;

[0743] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0744] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2.

[0745] Clause 16. An in vitro method for regulating the level of a target protein in cells, said method comprising administering to said cells a compound of formula (III) or a pharmaceutically acceptable salt, ester, optical isomer, racemic mixture, solvate, amino acid conjugate, or prodrug thereof:

[0746] ,

[0747] in;

[0748] R is C=O or CH2;

[0749] R 7Selected from H, deuterium, X, C1-C4 alkyl and NR 1 R 1 ,

[0750] R 8 Selected from OH, OR 5 and CR a R b R c ,

[0751] in

[0752] When R is C=O and R 8 When it is OH, then R 7 Selected from NR 1 R 1 , deuterium, X and C1-C4 alkyl;

[0753] R 9 and R 10 Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2;

[0754] R a Selected from H, deuterium, and C1-C4 alkyl groups;

[0755] R b Selected from H, deuterium, and C1-C4 alkyl groups;

[0756] R c Selected from NR 1 R 2 OH, OR 6 CH2X, CHX2, and CX3;

[0757] n is 1 or 2;

[0758] X is selected from F, Cl, Br, and I;

[0759] R 1 Selected from H and C1-C4 alkyl groups,

[0760] R 2 Selected from H, C1-C4 alkyl groups and -COR 3 ,

[0761] Alternatively, R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 5-membered or 6-membered heterocycles, wherein the heterocycle is unsubstituted or wherein one or more carbon atoms of the heterocycle form part of a carbonyl group; or when R 8 It is CR a R b R c And R c It is NR 1 R2 When, then R 1 and R a Together with the carbon and nitrogen atoms they are attached to, they form 5-membered or 6-membered heterocycles;

[0762] R 3 It is unsubstituted C1-C 10 Alkyl; or alkyl; or composed of one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 Unsubstituted 5-membered heterocyclic group, unsubstituted 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group;

[0763] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered heteroaryl, and 6-membered heteroaryl groups; and

[0764] R 6 It is an unsubstituted cyclopentyl or cyclohexyl; or a cyclopentyl or cyclohexyl substituted with one or more NH2.

[0765] Clause 17. A compound or composition used for the purposes described in Clause 14 or Clause 15, or a method described in Clause 16, wherein the target protein is SALL4.

[0766] Clause 18. The compound, composition, or method for any of the uses described in Clauses 14-17, wherein, when R a R b R 1 and R 2 When each is H, then n is 1.

[0767] Clause 19. A compound, a composition, or a method for any of the uses described in Clauses 14-18, wherein the compound is a compound of formula (IIIa) or a pharmaceutically acceptable salt, ester, optical isomer, racemate, solvate, amino acid conjugate, or prodrug thereof:

[0768] ,

[0769] in;

[0770] R is C=O or CH2;

[0771] R 7 Selected from H and NR 1 R 1 ,

[0772] R 8 Selected from OH, OR 5 and CH2NR 1 R 2 ,

[0773] in

[0774] When R is C=O and R 8 When it is OH, then R 7 It is NR 1 R 1 ;

[0775] R 1 It is an H or C1-C4 alkyl group;

[0776] R 2 Is it H or -COR? 3 ;

[0777] R 3 It is unsubstituted C1-C 10 Alkyl; or alkyl; or composed of one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 5-membered heterocyclic group, 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group; and

[0778] R 5 It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic groups, 6-membered heterocyclic groups, 5-membered heteroaryl groups and 6-membered heteroaryl groups.

[0779] Clause 20. A compound, a composition or method for any of the uses described in any of Clauses 14-19, wherein R is CH2.

[0780] Clause 21. The compound, composition, or method for the purpose described in Clause 20, wherein R 7 It is H, and R 8 It is CH2NR 1 R 2 .

[0781] Clause 22. The compound or composition for any of the uses described in Clauses 14-19, and the method thereof, wherein R is C=O.

[0782] Clause 23. The compound, composition, or method described in Clause 22 for the purpose described, wherein R 7 It is NR1 R 1 And R 8 Is it OH or OR? 5 .

[0783] Clause 24. The compound, composition, or method for any of the uses described in any of the preceding clauses, wherein R 1 It is H.

[0784] Clause 25. The compound, composition, or method for any of the uses described in any of the preceding clauses, wherein R 2 It is -COR 3 .

[0785] Clause 26. The compound, composition, or method for any of the uses described in any of the preceding clauses, wherein R 3 It is unsubstituted C1-C 10 alkyl.

[0786] Clause 27. The compound, composition, or method described in any one of Clauses 1-25 for the purpose described, wherein R 3 It is by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 NHCOOR 5 OR 5 5-membered heterocyclic group, 6-membered heterocyclic group, 5-membered heteroaryl group and 6-membered heteroaryl group.

[0787] Clause 28. The compound, composition, or method described in Clause 27 for the purpose described, wherein R 3 It is by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2 and NHCOR 5 and NHCOOR 5 .

[0788] Clause 29. The compound, the composition, or the method described in any one of Clauses 14-17 for the purpose described, wherein the compound is selected from...

[0789]

[0790] And its pharmaceutically acceptable salts, esters, optical isomers, racemates, solvates, amino acid conjugates, or prodrugs.

[0791] Clause 30. The compound, composition, or method described in Clause 29 for the purpose described, wherein the compound:

[0792] (a) Selected from compounds 12, 14, 44, 30 and 29,

[0793] (b) Selected from compounds 1, 28, 27 and 24,

[0794] (c) Selected from compounds 1, 44, 28, 27 and 24,

[0795] (d) Selected from compounds 44, 28, 27 and 24,

[0796] (e) is selected from compounds 1, 44, 28 and 27.

[0797] (f) is compound 1.

Claims

1. Compounds of formula (Ia): Or its pharmaceutically acceptable salt. in L can be hydrogen, alkyl, benzyl, -CH2OC(O)Me, or -CH2OC(O). t Bu; Each R 14 Independently selected from deuterium and hydrogen; R 15 Selected from hydrogen, deuterium, and C1-C4 alkyl groups; R g is CR a R b R c , R h Selected from H and C1-C4 alkyl groups; R a and R b Each is H or R a and R b Each is deuterium, or R. a It is H and R b It is methyl; R c Selected from NR 1 R 2 and OH; Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2; n is 1; X is selected from F, Cl, Br, and I; R 1 Selected from H and C1-C3 alkyl groups, R 2 Selected from H, C1-C3 alkyl, -COR 3 and -COOR 3 , R 3 Selected from: Unsubstituted C1-C4 alkyl groups; by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOOR 5 -OH, OCOR 5 substituted or unsubstituted meta-dioxacyclopentenyl, 5-membered heteroaryl, indole, and 6-membered aryl substituted with -OH or -OCO (C1-C4 alkyl); and wherein R 4 Not X; A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and R 5 It is an unsubstituted C1-C6 alkyl group.

2. The compound according to claim 1, wherein when C1-C 10 When alkyl groups are substituted with indole, C1-C 10 The alkyl group is also affected by at least one additional R 4 replace.

3. The compound according to claim 1 or 2, wherein R h It is H.

4. The compound according to claim 1 or 2, wherein R h It is a methyl group.

5. The compound according to claim 1 or 2, wherein R c Selected from NHR 2 .

6. The compound according to claim 1, wherein the compound is selected from: And its pharmaceutically acceptable salts.

7. The compound according to claim 1 or 2, wherein R 2 Selected from H, -COR 3 and -COOR 3 .

8. The compound according to claim 1 or 2, wherein R 3 It is by one or more R 4 Replacement C1-C 10 alkyl.

9. The compound according to claim 1 or 2, wherein each R 4 Independently selected from NH2, OCOR 5 substituted or unsubstituted meta-dioxanepentenyl, indole, and 6-membered aryl groups substituted with one or more -OCO (C1-C4 alkyl) groups; wherein R 4 It's not X.

10. The compound according to claim 1, wherein the compound is selected from compounds 51, 2, 22, 3, 24, 6, 23, 52 and 37: And its pharmaceutically acceptable salts.

11. Compound 43: Or its pharmaceutically acceptable salt.

12. A pharmaceutical composition comprising the compound of claim 1 or 11.

13. Use of the compound in the preparation of a medicament for treating cancer, said treatment comprising administering the compound to a subject in need of it, and said compound being: (i) Compounds of formula (Ia): Or its pharmaceutically acceptable salt. in L can be hydrogen, alkyl, benzyl, -CH2OC(O)Me, or -CH2OC(O). t Bu; Each R 14 Independently selected from deuterium and hydrogen; R 15 Selected from hydrogen, deuterium, and C1-C4 alkyl groups; R g Selected from -COOH and CR a R b R c , R h Selected from H and C1-C4 alkyl groups; R a and R b Each is H, R a and R b Each is deuterium, or R. a It is H and R b It is methyl; R c Selected from NR 1 R 2 and OH; Each R d R e and R f Independently selected from H, deuterium, X, C1-C4 alkyl groups and NH2; n is 1; X is selected from F, Cl, Br, and I; R 1 Selected from H and C1-C4 alkyl groups, R 2 Selected from H, C1-C4 alkyl, -COR 3 and -COOR 3 , R 3 Selected from: Unsubstituted C1-C4 alkyl groups; by one or more R 4 Replacement C1-C 10 Alkyl, wherein each R 4 Independently selected from NH2, NHC(NH)NH2, NHCOOR 5 -OH, OCOR 5 substituted or unsubstituted meta-dioxanepentenyl, 5-membered heteroaryl, indole, and 6-membered aryl substituted with OH or -OCO (C1-C4 alkyl); and wherein R 4 Not X; A 6-membered aryl group substituted with one or more substituents independently selected from CH2-OH or CH2OCO (C1-C4 alkyl); and R 5 It is an unsubstituted C1-C6 alkyl group; or (ii) Compounds of formula (II): Or its pharmaceutically acceptable salt. in: Each R 1 Independently selected from H and C1-C4 alkyl groups; R 11 Is it OH or OR? 5a ;as well as R 5a It is an unsubstituted C1-C6 alkyl group; or a C1-C6 alkyl group substituted by one or more substituents independently selected from 5-membered heterocyclic, 6-membered heterocyclic, 5-membered heteroaryl and 6-membered heteroaryl groups; or (iii) Compound 43: Or its pharmaceutically acceptable salt.

14. The use according to claim 13, wherein when C1-C 10 When alkyl groups are substituted with indole, C1-C 10 The alkyl group is also affected by at least one additional R 4 replace.

15. The use according to claim 13, wherein R 11 It is OH.

16. The use according to claim 13, wherein NR 1 R 1 It is NH2.

17. The use according to claim 13, wherein R h For H.

18. The use according to claim 13, wherein R c Selected from NHR 2 and OH.

19. The use according to claim 13, wherein the compound is selected from: And its pharmaceutically acceptable salts.

20. The use according to claim 13, wherein R c For NHR 2 .

21. The use according to claim 13, wherein R 2 Selected from H, -COR 3 and -COOR 3 .

22. The use according to claim 13, wherein R 3 For one or more R 4 Replacement C1-C 10 alkyl.

23. The use according to claim 13, wherein each R 4 Independently selected from NH2, OCOR 5 Indole and 6-membered aryl groups substituted with one or more -OCO (C1-C4 alkyl); wherein R 4 It's not X.

24. The use according to claim 13, wherein the compound is selected from compounds 51, 2, 22, 3, 24, 6, 23, 52, 37 and 1: 。 25. The use according to claim 13, wherein the cancer is associated with one or more proteins selected from the group consisting of SALL4 or GSPT1.

26. The use according to claim 13, wherein the cancer is hepatocellular carcinoma, neuroblastoma, leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), multiple myeloma, breast cancer, prostate cancer, bladder cancer, kidney cancer, muscle cancer, ovarian cancer, skin cancer, pancreatic cancer, breast cancer, colon cancer, blood cancer, connective tissue cancer, placental cancer, bone cancer, uterine cancer, cervical cancer, choriocarcinoma, endometrial cancer, gastric cancer, or lung cancer.

27. The use according to claim 26, wherein the cancer is hepatocellular carcinoma, neuroblastoma, leukemia, prostate cancer, or multiple myeloma.

28. The use according to claim 27, wherein the cancer is hepatocellular carcinoma.

29. The use according to claim 28, wherein the compound: (a) Selected from compounds 6, 3, 36, 42, 26, 23, 24, 1, 52, 28, 27, 37, 39, 38 and 5; or (b) Selected from compounds 6, 3, 36, 42, 26, 23, 24, 1 and 52.

30. The use according to claim 27, wherein the cancer is neuroblastoma.

31. The use according to claim 30, wherein the compound is selected from compounds 3, 36, 42, 37, 28, 27 and 1.

32. The use according to claim 27, wherein the cancer is leukemia.

33. The use according to claim 32, wherein the compound is selected from compounds 3, 36, 42, 37, 28, 27, 24 and 1.

34. The use according to claim 13, wherein the treatment further comprises administering a second cancer therapy to the subject.

35. The use according to claim 34, wherein the second cancer therapy is chemotherapy, radiotherapy, or immunotherapy.

36. The use according to claim 34, wherein the second agent is selected from therapeutic antibodies that specifically bind to cancer antigens, hematopoietic growth factors, cytokines, anticancer agents, antibiotics, Cox-2 inhibitors, immunomodulators, immunosuppressants, corticosteroids, or their pharmacologically active mutants or derivatives.

37. The use according to claim 13, wherein the treatment comprises orally administering the compound or the pharmaceutical composition to the subject.

38. The compound of claim 1, the pharmaceutical composition of claim 12, or the use of claim 13, wherein L is hydrogen.

39. The compound according to claim 1, the pharmaceutical composition according to claim 12, or the use according to claim 13, wherein each R 14 It is deuterium.

40. The compound of claim 1, the pharmaceutical composition of claim 12, or the use of claim 13, wherein each R 14 It is hydrogen.

41. The compound according to claim 1, the pharmaceutical composition according to claim 12, or the use according to claim 13, wherein R 15 It is deuterium.

42. The compound according to claim 1, the pharmaceutical composition according to claim 12, or the use according to claim 13, wherein R 15 It is hydrogen.

43. The compound according to claim 1, the pharmaceutical composition according to claim 12, or the use according to claim 13, wherein R e It is X.

44. The compound according to claim 1, the pharmaceutical composition according to claim 12, or the use according to claim 13, wherein R 1 Selected from H and methyl.

45. The compound according to claim 1, the pharmaceutical composition according to claim 12, or the use according to claim 13, wherein R 2 Selected from H, methyl, -COR 3 and -COOR 3 .

46. ​​The compound of claim 1 or 11, the pharmaceutical composition of claim 12, or the use of claim 13, wherein administration of the compound or pharmaceutical composition to a subject reduces the level of the target protein in the subject.

47. The compound, pharmaceutical composition, or use according to claim 46, wherein the target protein is selected from SALL-4 or GSPT1.

48. The compound, pharmaceutical composition, or use according to claim 46, wherein administration of the compound or pharmaceutical composition to the subject induces a minimal or substantially no reduction in IKZF1 or IKZF3 protein levels.

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