Compounds and methods for degrading casein kinase 1 alpha

Abstract

A novel compound represented by formula (I) that degrades CK1α (casein kinase 1 alpha), a method for degrading CK1α, or a method for preventing or treating blood cancer by administering the compound to a subject in need thereof is provided.

Description

Background Art

[0001] Casein kinase 1 alpha (CK1α) is a serine / threonine protein kinase involved in various cellular pathways and functions including Wnt signaling, NF- K κB signaling, p53 pathway, autophagy, and the cell cycle. Inhibition of CK1α or a decrease in its expression level has been demonstrated to induce cell death in several types of cancer such as acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL).

[0002] For example, it has been reported that CK1α inhibition causes a decrease in Rps6 phosphorylation and p53 activation, leading to the selective elimination of leukemia cells and identifying CK1α as a therapeutic target for AML treatment (Jaras et al., J. Exp. Med. 2014 Vol. 211 No. 4 605-612). Also, it has been reported that inhibition of CK1α acting through the activation of the p53 pathway showed promising preclinical activity in AML and multiple myeloma (MM) (Janovska et al., Int. J. Mol. Sci. 2020, 21, 9026). In particular, CK1α has previously been found to control signaling pathways involved in proliferation, survival, and stress in MM (Manni et al., Journal of Hematology & Oncology (2017) 10:157). Furthermore, since CK1α is overexpressed in AML patients and has a negative impact on the prognosis of AML patients, it has been reported that there may be a therapeutic opportunity to treat AML by targeting CK1α and autophagy, as CK1α inhibits p53 downstream of MDM2-mediated autophagy and apoptosis (Xu et al., ONCOLOGY REPORTS 44: 1895-1904, 2020). Also, it has previously been found that CK1α governs antigen receptor-induced NF-κB activation and the survival of human lymphoma cells.

[0003] Therefore, CK1α is known as a target for preventing or treating blood cancers such as leukemia, lymphoma, and myeloma. Therefore, there is a need to develop CK1α degrading agents or inhibitors that can be used for the prevention or treatment of blood cancers such as leukemia, lymphoma, and myeloma.

SUMMARY OF THE INVENTION

[0004] The present disclosure provides a novel compound represented by the following formula (I), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof. JPEG2025522795000001.jpg26127

[0005] Such a compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof is useful for reducing the CK1α protein level and treating or preventing blood cancer.

[0006] In formula (I), in some embodiments, R 1 is C1-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C3-C8 cycloalkyl, or C5-C 10 bicycloalkyl, each of which is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl, optionally substituted C6-C 10 aryl, C1-C3 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C 10 bicycloalkyl, or optionally substituted C3-C6 heterocycloalkyl.

[0007] In some embodiments, X is a bond, or -(CH2) n - optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl or C1-C6 alkyl. Here, the C1-C6 alkyl group together with the atom to which it is attached forms a C3-C6 spiroalkyl ring, and the -(CH2) n - group may contain 0 to 1 double bond or triple bond.

[0008] In some embodiments, n is an integer from 1 to 6.

[0009] In some embodiments, A is a bond, or a C4-C8 cycloalkyl, C5-C 10 bicycloalkyl, C5-C 10 heterobicycloalkyl or C3-C8 heterocycloalkyl, each optionally substituted with one or more halogen, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl or C3-C5 cycloalkyl.

[0010] In some embodiments, Y is a bond or -(CH2) m -.

[0011] In some embodiments, m is an integer of 1 or 2.

[0012] In some embodiments, Z is H or halogen.

[0013] In some embodiments, R 2 is -NR 12 R 5 , -NHC(O)R 6 , -NHC(O)NR 8 (R 9 ), -C(O)NHR 5 , a 5- or 6-membered heteroaryl, or a C3-C8 heterocycloalkyl or C5-C 10 heterobicycloalkyl ring, optionally substituted with one or more halogen, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl or C3-C5 cycloalkyl.

[0014] In some embodiments, R 5 is H, C1-C5 alkyl, C3-C7 cycloalkyl, a 5- or 6-membered heteroaryl, C5-C 10Bicycloalkyl, (C3-C6 cycloalkyl)-C1-C3 alkyl, (C3-C6 heterocycloalkyl)-C1-C3 alkyl, (C6-C 10 aryl)-C1-C3 alkyl, (C1-C5-heteroaryl)-C1-C3 alkyl or C3-C7 heterocycloalkyl, each of which is one or more halogen, CN, OH, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), C1-C3 haloalkyl, CH3SO2-, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C7 heterocycloalkyl or C(O)NR 8 (R 9 ) is optionally substituted.

[0015] In some embodiments, R 6 is C1-C6 alkyl, C3-C6 cycloalkyl or C3-C6 heterocycloalkyl, each of which is one or more halogen, C1-C3 haloalkyl, optionally substituted C3-C6 cycloalkyl, or NR 8 (R 9 ) is optionally substituted.

[0016] In some embodiments, R 8 and R 9 are each independently H, substituted C1-C3 alkyl, or together with the nitrogen to which they are attached form a 4- to 6-membered heterocyclic ring optionally substituted with one or more halogen or C1-C3 haloalkyl.

[0017] In some embodiments, R 12 is H, C1-C5 alkyl, or R 5 together with the nitrogen to which it is attached forms (i) a C3-C8 cycloalkyl or heterocycloalkyl ring, or (ii) a C5-C 10 bicycloalkyl or heterobicycloalkyl ring, each of which is optionally substituted with one or more halogen, C1-C3 haloalkyl, OH, O-(C1-C3 alkyl), O-(C1-C3 haloalkyl) or CN.

[0018] In some embodiments, each stereocenter in the compound of formula (I) is independently the R-enantiomer, the S-enantiomer, or a mixture of the R- and S-enantiomers.

[0019] In some embodiments, each double bond in the compound of formula (I) is independently cis or trans.

[0020] In some embodiments, the compound is one represented by formula (I-a), JPEG2025522795000002.jpg24127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof.

[0021] In formula (I-a), in some embodiments, X is a bond or -(CH2) n -.

[0022] In some embodiments, R 1 is C1-C8 alkyl or C3-C8 cycloalkyl, each of which is optionally substituted with one or more halogens, OH, NH2, C1-C3 alkyl, C1-C3 haloalkyl, O-(C1-C3 alkyl), optionally substituted C6 aryl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C6 heterocycloalkyl, or optionally substituted C6-C8 bicycloalkyl.

[0023] In some embodiments, A is C4-C6 cycloalkyl, C4-C6 heterocycloalkyl or C5-C7 bicycloalkyl, each of which is optionally substituted with one or more halogens, OH, C1-C3 alkyl or C1-C3 haloalkyl.

[0024] In some embodiments, Y is a bond or -CH2-.

[0025] In some embodiments, R 5is H, C1-C5 alkyl, C3-C6 cycloalkyl, 6-membered heteroaryl, C5 bicycloalkyl, (C3-C4 cycloalkyl)-C1-C2 alkyl, (C3-C4 heterocycloalkyl)-C1-C2 alkyl, (C6 aryl)-C1-C2 alkyl, (C5 heteroaryl)-C1-C2 alkyl or C3-C4 heterocycloalkyl, each of which is optionally substituted with one or more halogen, CN, OH, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), C1-C3 haloalkyl, CH3SO2- or C(O)NR 8 (R 9 ).

[0026] In some embodiments, R 12 is H, C1-C5 alkyl, or R 5 and the nitrogen to which it is attached together form a C3-C8 heterocycloalkyl ring optionally substituted with one or more halogen or C1-C3 haloalkyl.

[0027] In some embodiments, the compound is one represented by formula (I-b), JPEG2025522795000003.jpg29127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0028] In formula (I-b), in some embodiments, X is a bond or -(CH2) n -.

[0029] In some embodiments, R 1 is C3-C6 alkyl and is optionally substituted with one or more halogen, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0030] In some embodiments, A is a bond or a C4-C6 cycloalkyl optionally substituted with one or more halogen, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0031] In some embodiments, Y is a bond.

[0032] In some embodiments, R 6 is C1-C6 alkyl optionally substituted with NR 8 (R 9 ).

[0033] In some embodiments, the compound is of formula (I-c), JPEG2025522795000004.jpg28127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0034] In formula (I-c), in some embodiments, X is a bond or -(CH2) n -.

[0035] In some embodiments, R 1 is C1-C6 alkyl optionally substituted with optionally substituted C3-C8 cycloalkyl or optionally substituted C3-C6 heterocycloalkyl.

[0036] In some embodiments, A is a bond, C4-C6 cycloalkyl, C4-C6 heterocycloalkyl or C5-C7 bicycloalkyl, each optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0037] In some embodiments, Y is a bond.

[0038] In some embodiments, R 5 is H or C1-C3 alkyl.

[0039] In some embodiments, the compound is of formula (I-d), JPEG2025522795000005.jpg32127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0040] In formula (I-d), in some embodiments, R 1 is C1-C3 alkyl optionally substituted with optionally substituted C3-C6 cycloalkyl.

[0041] In some embodiments, A is optionally substituted C4-C6 cycloalkyl.

[0042] In some embodiments, R 2 is C1-C4 alkyl or (C3-C4 cycloalkyl)-C1-C2 alkyl, each optionally substituted with one or more halogens, CN, OH, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), C1-C3 haloalkyl or CH3SO2-.

[0043] In formula (I), in some embodiments, X and Y are each a bond, A is C4-C6 cycloalkyl, R 1 is C1-C3 alkyl optionally substituted with optionally substituted C3-C6 cycloalkyl, and R 2 is a 5-membered heteroaryl.

[0044] In some embodiments, the compound is selected from Table A below.

[0045] [Table A] JPEG2025522795000006.jpg127122JPEG2025522795000007.jpg12798JPEG2025522795000008.jpg127114JPEG2025522795000009.jpg12787JPEG2025522795000010.jpg12792JPEG2025522795000011.jpg12795JPEG2025522795000012.jpg12797JPEG2025522795000013.jpg12791JPEG2025522795000014.jpg12795JPEG2025522795000015.jpg12788JPEG2025522795000016.jpg12792JPEG2025522795000017.jpg12791JPEG2025522795000018.jpg12793JPEG2025522795000019.jpg12788JPEG2025522795000020.jpg127106JPEG2025522795000021.jpg127103JPEG2025522795000022.jpg12798JPEG2025522795000023.jpg12797JPEG2025522795000024.jpg12796JPEG2025522795000025.jpg12797JPEG2025522795000026.jpg127105JPEG2025522795000027.jpg12788JPEG2025522795000028.jpg12799JPEG2025522795000029.jpg12794JPEG2025522795000030.jpg12798JPEG2025522795000031.jpg12799JPEG2025522795000032.jpg127100JPEG2025522795000033.jpg127106JPEG2025522795000034.jpg12793JPEG2025522795000035.jpg12798JPEG2025522795000036.jpg127101JPEG2025522795000037.jpg12791JPEG2025522795000038.jpg12797JPEG2025522795000039.jpg127104JPEG2025522795000040.jpg12796JPEG2025522795000041.jpg12792JPEG2025522795000042.jpg12798JPEG2025522795000043.jpg12794JPEG2025522795000044.jpg12789JPEG2025522795000045.jpg127105JPEG2025522795000046.jpg127101JPEG2025522795000047.jpg127101JPEG2025522795000048.jpg12798JPEG2025522795000049.jpg12797JPEG2025522795000050.jpg12797JPEG2025522795000051.jpg12799JPEG2025522795000052.jpg12794JPEG2025522795000053.jpg12799JPEG2025522795000054.jpg12792JPEG2025522795000055.jpg127111JPEG2025522795000056.jpg127108JPEG2025522795000057.jpg127106JPEG2025522795000058.jpg127107JPEG2025522795000059.jpg127106JPEG2025522795000060.jpg127107JPEG2025522795000061.jpg127108JPEG2025522795000062.jpg12788JPEG2025522795000063.jpg127105JPEG2025522795000064.jpg127115JPEG2025522795000065.jpg12799JPEG2025522795000066.jpg12793JPEG2025522795000067.jpg12787JPEG2025522795000068.jpg12787JPEG2025522795000069.jpg127103JPEG2025522795000070.jpg127105JPEG2025522795000071.jpg127105JPEG2025522795000072.jpg127104JPEG2025522795000073.jpg45127.

[0046] In some embodiments, the compounds of the present disclosure are for use in the degradation and / or reduction of casein kinase 1 alpha (CK1α).

[0047] In some embodiments, the compounds of the present disclosure are for use in the inhibition of the activity of casein kinase 1 alpha (CK1α).

[0048] In some embodiments, the compounds of the present disclosure are for use in the prevention or treatment of blood cancer.

[0049] In some embodiments, the compounds of the present disclosure are for use in the manufacture of a medicament for the treatment of blood cancer.

[0050] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure (represented by formula (I), (I-a), (I-b) or (I-c)), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, and optionally a pharmaceutically acceptable excipient or carrier.

[0051] In some embodiments, the pharmaceutical composition is for use in the degradation and / or reduction of casein kinase 1 alpha (CK1α).

[0052] In some embodiments, the pharmaceutical composition is for use in the inhibition of the activity of casein kinase 1 alpha (CK1α).

[0053] In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of blood cancer.

[0054] In some embodiments, the blood cancer is leukemia, lymphoma, or myeloma.

[0055] The present disclosure also provides a method for degrading and / or reducing casein kinase 1 alpha (CK1α) in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0056] The present disclosure also provides a method for inhibiting the activity of casein kinase 1 alpha (CK1α) in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0057] The present disclosure also provides a method for preventing or treating blood cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0058] In some embodiments, the blood cancer is leukemia, lymphoma, or myeloma.

[0059] In some embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, chronic eosinophilic leukemia (CEL), and myelodysplastic syndrome (MDS).

[0060] In some embodiments, the lymphoma is selected from the group consisting of Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, and peripheral T-cell lymphoma.

[0061] In some embodiments, the myeloma is selected from the group consisting of multiple myeloma, light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma.

DETAILED DESCRIPTION OF THE INVENTION

[0062] Many modifications and other embodiments of the disclosure will come to mind to those of ordinary skill in the art to which the disclosed compositions and methods pertain, having the benefit of the teachings presented in the foregoing description and the related drawings. Accordingly, the disclosure is not to be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. It will be understood by those of ordinary skill in the art that many variations and adaptations of the described aspects will be recognized. These variations and adaptations are intended to be included within the teachings of this disclosure and within the scope of the appended claims.

[0063] Certain terms are used herein, but they are used in a generic and descriptive sense only and not for purposes of limitation.

[0064] Any of the recited methods can be performed in the order of the recited events or in any other order that is logically possible. That is, unless specifically stated otherwise, the methods or aspects described herein are in no way intended to be construed as requiring that their steps be performed in a particular order. Accordingly, when a method claim does not specifically recite that the steps are limited to a particular order in the claims or the specification, no order should be inferred in any respect. This applies to any possible non-explicit basis for interpretation, including logical matters regarding the arrangement of steps or operations, the plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described in the specification.

[0065] All publications and patents cited in this specification are cited for the purpose of disclosing and describing the methods and / or materials in which the publications are cited. All such publications and patents are hereby incorporated by reference into this specification as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and is not extended to dictionary definitions from the cited publications and patents. Any dictionary definitions in the cited publications and patents that are not expressly repeated in this application should not be so treated and should not be read as defining any terms that appear in the appended claims. The citation of any publication is for disclosures prior to the filing date and should not be construed as an admission that the present disclosure has no right to antedate such publication by virtue of prior disclosure. Further, the provided publication dates may be different from the actual publication dates and may need to be individually verified.

[0066] The present disclosure describes a novel compound represented by the following formula (I), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof. The present disclosure also describes analogs or radical forms of the compound represented by formula (I). JPEG2025522795000074.jpg27127

[0067] In formula (I), in some embodiments, R 1 is C1-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C3-C8 cycloalkyl, or C5-C 10 bicycloalkyl, each of which is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl, optionally substituted C6-C 10 aryl, C1-C3 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C 10 bicycloalkyl, or optionally substituted C3-C6 heterocycloalkyl.

[0068] In some embodiments, X is a bond, or -(CH2) optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl or C1-C6 alkyl. n -, where the C1-C6 alkyl group together with the atom to which it is attached forms a C3-C6 spiroalkyl ring, and the -(CH2) n - group may contain 0 to 1 double bond or triple bond.

[0069] In some embodiments, n is an integer from 1 to 6.

[0070] In some embodiments, A is a bond, or a C4-C8 cycloalkyl, C5-C 10 bicycloalkyl, C5-C 10 heterobicycloalkyl or C3-C8 heterocycloalkyl, each optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl or C3-C5 cycloalkyl.

[0071] In some embodiments, Y is a bond or -(CH2) m -.

[0072] In some embodiments, m is an integer of 1 or 2.

[0073] In some embodiments, Z is H or a halogen.

[0074] In some embodiments, R 2 is -NR 12 R 5 , -NHC(O)R 6 , -NHC(O)NR 8 (R 9 ), -C(O)NHR 5 , a 5- or 6-membered heteroaryl, or a C3-C8 heterocycloalkyl or C5-C 10It is a heterobicycloalkyl ring, optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl or C3-C5 cycloalkyl.

[0075] In some embodiments, R 5 is H, C1-C5 alkyl, C3-C7 cycloalkyl, a 5- or 6-membered heteroaryl, C5-C 10 bicycloalkyl, (C3-C6 cycloalkyl)-C1-C3 alkyl, (C3-C6 heterocycloalkyl)-C1-C3 alkyl, (C6-C 10 aryl)-C1-C3 alkyl, (C1-C5-heteroaryl)-C1-C3 alkyl or C3-C7 heterocycloalkyl, each of which is optionally substituted with one or more halogens, CN, OH, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), C1-C3 haloalkyl, CH3SO2-, optionally substituted C3-C6 cycloalkyl, optionally substituted C3-C7 heterocycloalkyl or C(O)NR 8 (R 9 ) and is optionally substituted.

[0076] In some embodiments, R 6 is C1-C6 alkyl, C3-C6 cycloalkyl or C3-C6 heterocycloalkyl, each of which is optionally substituted with one or more halogens, C1-C3 haloalkyl, optionally substituted C3-C6 cycloalkyl, or NR 8 (R 9 ) and is optionally substituted.

[0077] In some embodiments, R 8 and R 9 are each independently H, substituted C1-C3 alkyl, or together with the nitrogen to which they are attached form a 4- to 6-membered heterocyclic ring optionally substituted with one or more halogens or C1-C3 haloalkyl.

[0078] In some embodiments, R 12 is H, C1-C5 alkyl, or R 5and together with the nitrogen to which it is attached forms (i) a C3-C8 cycloalkyl or heterocycloalkyl ring, or (ii) a C5-C 10 bicycloalkyl or heterobicycloalkyl ring, each of which is optionally substituted with one or more halogens, C1-C3 haloalkyl, OH, O-(C1-C3 alkyl), O-(C1-C3 haloalkyl) or CN.

[0079] In some embodiments, each stereocenter in the compounds of formula (I) is independently the R-enantiomer, the S-enantiomer, or a mixture of the R- and S-enantiomers.

[0080] In some embodiments, each double bond in the compounds of formula (I) is independently cis or trans.

[0081] In some embodiments, the compound is one represented by formula (I-a), JPEG2025522795000075.jpg23127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0082] In formula (I-a), in some embodiments, X is a bond or -(CH2) n -.

[0083] In some embodiments, R 1 is C1-C8 alkyl or C3-C8 cycloalkyl, each of which is optionally substituted with one or more halogens, OH, NH2, C1-C3 alkyl, C1-C3 haloalkyl, O-(C1-C3 alkyl), optionally substituted C6 aryl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C6 heterocycloalkyl or optionally substituted C6-C8 bicycloalkyl.

[0084] In some embodiments, A is C4-C6 cycloalkyl, C4-C6 heterocycloalkyl or C5-C7 bicycloalkyl, each optionally substituted with one or more halogens, OH, C1-C3 alkyl or C1-C3 haloalkyl.

[0085] In some embodiments, Y is a bond or -CH2-.

[0086] In some embodiments, R 5 is H, C1-C5 alkyl, C3-C6 cycloalkyl, 6-membered heteroaryl, C5 bicycloalkyl, (C3-C4 cycloalkyl)-C1-C2 alkyl, (C3-C4 heterocycloalkyl)-C1-C2 alkyl, (C6 aryl)-C1-C2 alkyl, (C5 heteroaryl)-C1-C2 alkyl or C3-C4 heterocycloalkyl, each optionally substituted with one or more halogens, CN, OH, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), C1-C3 haloalkyl, CH3SO2- or C(O)NR 8 (R 9 ) and is optionally substituted.

[0087] In some embodiments, R 12 is H, C1-C5 alkyl, or R 5 together with the nitrogen to which it is attached forms a C3-C8 heterocycloalkyl ring optionally substituted with one or more halogens or C1-C3 haloalkyl.

[0088] In some embodiments, the compound is one represented by formula (I-b), JPEG2025522795000076.jpg30127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0089] In formula (I-b), in some embodiments, X is a bond or -(CH2) n -.

[0090] In some embodiments, R 1is C3-C6 alkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0091] In some embodiments, A is a bond, or C4-C6 cycloalkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0092] In some embodiments, Y is a bond.

[0093] In some embodiments, R 6 is C1-C6 alkyl optionally substituted with NR 8 (R 9 ).

[0094] In some embodiments, the compound is one represented by formula (I-c), JPEG2025522795000077.jpg29127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0095] In formula (I-c), in some embodiments, X is a bond or -(CH2) n -.

[0096] In some embodiments, R 1 is C1-C6 alkyl optionally substituted with optionally substituted C3-C8 cycloalkyl or optionally substituted C3-C6 heterocycloalkyl.

[0097] In some embodiments, A is a bond, C4-C6 cycloalkyl, C4-C6 heterocycloalkyl or C5-C7 bicycloalkyl, each of which is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0098] In some embodiments, Y is a bond.

[0099] In some embodiments, R 5 is H or C1-C3 alkyl.

[0100] In some embodiments, the compound is one represented by formula (I-d), JPEG2025522795000078.jpg33127 or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

[0101] In formula (I-d), in some embodiments, R 1 is C1-C3 alkyl optionally substituted with optionally substituted C3-C6 cycloalkyl. For example, the C3-C6 cycloalkyl can be substituted with one or more halogens, CN, OH, C1-C3 alkyl, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl) or C1-C3 haloalkyl.

[0102] In some embodiments, A is optionally substituted C4-C6 cycloalkyl. For example, A can be substituted with one or more halogens, CN, OH, C1-C3 alkyl, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl) or C1-C3 haloalkyl.

[0103] In some embodiments, R 2 is C1-C4 alkyl or (C3-C4 cycloalkyl)-C1-C2 alkyl, each of which is optionally substituted with one or more halogens, CN, OH, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), C1-C3 haloalkyl or CH3SO2-. For example, R 2 is C1-C3 alkyl optionally substituted with one or more halogens, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl) or C1-C3 haloalkyl.

[0104] In formula (I), in some embodiments, X and Y are each a bond, A is C4-C6 cycloalkyl, and R 1is C1-C3 alkyl optionally substituted with C3-C6 cycloalkyl optionally substituted, and R 2 is a 5-membered heteroaryl.

[0105] This disclosure describes a novel compound represented by the following formula (I), or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof. The disclosure also describes analogs or radical forms of the compound represented by formula (I).

[0106] As used herein, the term "compound" refers to the specific chemical compounds disclosed herein, unless otherwise indicated, and includes, in context, tautomers, positional isomers, geometric isomers, and, where applicable, stereoisomers (including optical isomers (enantiomers) and other stereoisomers (diastereomers)), and, where applicable, pharmaceutically acceptable salts and derivatives thereof (including prodrug forms). In its use in that context, the term "compound" typically refers to a single compound, but may also include other compounds such as stereoisomers, positional isomers, and / or optical isomers (including racemic mixtures), as well as specific enantiomers or mixtures enriched in an enantiomer of the disclosed compound. The term also refers, in context, to prodrug forms of the compound modified to facilitate administration and delivery of the compound to the active site. Note that in the description of the present compound, among other things, a number of substituents and variables associated with the compound are described. Those skilled in the art will understand that the molecules described herein are stable compounds as generally described below.

[0107] In a compound having one or more chiral centers described in this specification, if the absolute stereochemistry is not explicitly indicated, it is understood that each center may independently be in the R configuration, the S configuration, or a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, enantiomerically enriched, or a mixture of stereoisomers, including all diastereomeric and enantiomeric forms. Further, in any compound described herein having one or more double bonds that can give rise to geometric isomers defined as E or Z, it is understood that each double bond may independently be E or Z, or a mixture thereof. Stereoisomers can be obtained, if desired, by methods such as stereoselective synthesis and / or separation of stereoisomers by chiral chromatography columns. Similarly, it is understood that all tautomeric and conformational isomeric forms are also intended to be included in any compound described. Conformational isomers are structures that are stereostructural isomers. Stereostructural isomerism is a phenomenon in which molecules have the same structural formula but different steric structures of atoms with respect to rotatable bonds (conformational isomers).

[0108] As used herein, any "R" group represents a substituent that can be attached to the indicated atom. The R group may or may not be substituted. When a group is described as "optionally substituted", the group may be unsubstituted or substituted with one or more of the indicated substituents. Similarly, when a group is described as "unsubstituted or substituted", if substituted, the substituent can be selected from one or more of the indicated substituents. When no substituent is indicated, an indicated "optionally substituted" or "substituted" group means that the group may be independently substituted with one or more groups independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, aralkyl, heteroaralkyl, heterocyclyl(alkyl), hydroxy, alkoxy, cycloalkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, C-amide, N-amide, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, haloalkyl, haloalkoxy, amino (including mono-substituted amino and di-substituted amino), and alkylamino. When a group is not described as "optionally substituted", "unsubstituted" or "substituted", such a group is unsubstituted unless otherwise specified in the definition of such a group.

[0109] As used herein, "C" a ~C b " or "C" a -C b " ("a" and "b" are integers) refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or the number of carbon atoms in the ring of a cycloalkyl, aryl, heteroaryl, heterocycloalkyl or heterocyclyl group. That is, an alkyl, alkenyl, alkynyl, cycloalkyl ring, aryl ring, heterocycloalkyl ring, heteroaryl ring or heterocyclyl ring can contain "a" to "b" (including both ends) carbon atoms.

[0110] As used herein, the term "alkyl" refers to a saturated monovalent chain of carbon atoms which may be optionally branched. In embodiments that include alkyl, exemplary variations of those embodiments include lower alkyl such as C1-C8, C1-C6, C1-C5, C1-C4, C1-C3 alkyl, methyl, ethyl, propyl, 3-methylpentyl, and the like.

[0111] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon having at least two carbon atoms and at least one double bond. Alkenyl may include any number of carbons, such as C2, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C3, C3-C4, C3-C5, C3-C6, C3-C7, C3-C8, C4, C4-C5, C4-C6, C4-C7, C4-C8, C5, C5-C6, C5-C7, C5-C8, C6, C6-C7, C6-C8, C7, C7-C8, and C8. An alkenyl group can have any suitable number of double bonds, including, but not limited to, one, two, three, four, five or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. An alkenyl group can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amide, nitro, oxo, and cyano.

[0112] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon having at least two carbon atoms and at least one triple bond. Alkynyl may contain any number of carbons, such as C2, C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C3, C3-C4, C3-C5, C3-C6, C3-C7, C3-C8, C4, C4-C5, C4-C6, C4-C7, C4-C8, C5, C5-C6, C5-C7, C5-C8, C6, C6-C7, C6-C8, C7, C7-C8, and C8. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatriynyl. The alkynyl group can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amide, nitro, oxo, and cyano.

[0113] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more halogen atoms. "C n-m -haloalkyl" or "C n -C m -haloalkyl" refers to a C n-m alkyl group having n to m carbon atoms and at least 1 to a maximum of {2(n - m)+1} halogen atoms (which may be the same or different). In some embodiments, the halogen atom is a fluoro atom. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Exemplary haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, etc. In some embodiments, the haloalkyl group is a fluoroalkyl group.

[0114] As used herein, the term "cycloalkyl" refers to a monovalent chain of carbon atoms that forms part of a ring. In embodiments that include cycloalkyl, exemplary variations of those embodiments include C3-C8, C3-C7, C3-C6, C3-C5, C4-C8, C4-C7, C4-C6 cycloalkyl, lower cycloalkyl such as cyclopropyl, cyclohexyl, 3-ethylcyclopentyl, and the like.

[0115] As used herein, the term "bicycloalkyl" refers to a structure in which two cycloalkyl groups as defined above are linked to each other to form a bridged, fused, or spiro bicyclic compound.

[0116] As used herein, the term "fused bicycloalkyl" refers to two cycloalkyl groups that share two adjacent atoms. In other words, they share a covalent bond of one ring, that is, the so-called bridgehead atoms are directly linked.

[0117] As used herein, the term "bridged bicycloalkyl" refers to two cycloalkyl groups that have more than two atoms in common in part. That is, the two rings do not share adjacent carbon atoms, share three or more atoms, and separate the two bridgehead atoms by a bridge containing at least one atom. Examples include, but are not limited to, bicyclo[3.2.1]heptyl ("norbornil"), bicyclo[2.2.2]octyl, and the like.

[0118] As used herein, the term "spirobicycloalkyl" refers to a bicyclic group in which two rings are joined by a single carbon atom that is a member of each of the two rings. This term includes both spirobicycloalkyl and spirobicycloheteroalkyl. In spirobicycloalkyl, the two rings are cycloalkyl rings joined by a single carbon atom that is a member of each of the two rings. In spirobicycloheteroalkyl, one ring is a heterocyclyl ring and the other ring is a cycloalkyl ring joined by a single carbon atom that is a member of each of the two rings, or both rings are heterocyclyl rings joined by a single carbon atom that is a member of each of the two rings. Examples of spirobicyclic groups include spiro[3.3]heptenyl, spiro[3.4]octanyl, azaspiro[3.3]heptanyl, oxazaspiro[3.3]heptanyl, oxa-azaspiro[3.3]heptanyl, and azaspiro[3.4]octanyl.

[0119] As used herein, "heterocycloalkyl" is a non-aromatic ring having at least one heteroatom ring member independently selected from nitrogen, sulfur, and oxygen. Heterocycloalkyl groups include monocyclic or polycyclic (e.g., fused, bridged, or spiro) ring systems. Heterocycloalkyl also includes one or more aromatic rings fused to a non-aromatic heterocycloalkyl ring. The ring-forming carbon atoms of a heterocycloalkyl group can be optionally substituted with oxo. The ring-forming heteroatoms of a heterocycloalkyl group can be oxidized to form an N-oxide or a sulfonyl group. In embodiments containing heterocycloalkyl, exemplary variations of those embodiments include lower cycloalkyl such as C3-C8, C3-C7, C3-C6, C3-C5, C4-C8, C4-C7, C4-C6 heterocycloalkyl. Examples of heterocycloalkyl groups include a morpholine ring, a pyrrolidine ring, a piperazine ring, a piperidine ring, a tetrahydropyran ring, a tetrahydropyridine, an azetidine ring, tetrahydrofuran, and the like.

[0120] As used herein, "heterobicycloalkyl" refers to an unsubstituted or substituted bicycloalkyl structure in which at least one carbon atom is replaced by a heteroatom independently selected from oxygen, nitrogen, and sulfur.

[0121] As used herein, the term "aryl" refers to an aromatic carbocyclic system having any suitable number of ring atoms and any suitable number of rings. An aryl group can include any suitable number of carbocyclic atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, and can include 6-10, 6-12, or 6-14 ring members. An aryl group can be monocyclic, can condense to form a bicyclic or tricyclic group, or can be linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl having a methylene linking group. Some aryl groups have 6-12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6-10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. An aryl group can be optionally substituted with one or more moieties selected from alkyl, alkenyl, alkynyl, haloalkyl, halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, carboxylic acid alkyl, amide, nitro, oxo, and cyano.

[0122] As used herein, the term "heteroaryl" refers to substituted or unsubstituted aromatic 5- or 6-membered monocyclic groups and 9- or 10-membered bicyclic groups having at least one heteroatom (O, S, or N) in at least one ring. The heteroatom-containing ring preferably has 1, 2, or 3 heteroatoms independently selected from O, S, and / or N. Each ring of the heteroaryl group containing a heteroatom can contain 1 or 2 oxygen or sulfur atoms and / or 1 to 4 nitrogen atoms, provided that the total number of heteroatoms in each ring is 4 or less and each ring has at least one carbon atom. The fused rings completing the bicyclic group are aromatic and can contain only carbon atoms. Nitrogen and sulfur atoms can be optionally oxidized and nitrogen atoms can be optionally quaternized. The bicyclic heteroaryl group need only contain aromatic rings. The heteroaryl group can be attached to any available nitrogen or carbon atom of any ring. The heteroaryl ring system can be unsubstituted or can contain one or more substituents. Examples of monocyclic heteroaryl include, but are not limited to, thiazolyl, oxazolyl, thiophenyl, furanyl, pyrrolyl, imidazolyl, isoxazolyl, pyrazolyl, triazolyl, thiadiazolyl, tetrazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, and similar groups. Examples of bicyclic heteroaryl include, but are not limited to, indolyl, benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, purinyl, propyridinyl, oxochromene, dioxoisoindoline, pyrazolopyridinyl, pyrazolo[1,5-a]pyridinyl, and other similar groups.

[0123] In some embodiments, the compound is selected from Table A below.

[0124] [Table A] JPEG2025522795000079.jpg94127JPEG2025522795000080.jpg12796JPEG2025522795000081.jpg12790JPEG2025522795000082.jpg12787JPEG2025522795000083.jpg12793JPEG2025522795000084.jpg12796JPEG2025522795000085.jpg12798JPEG2025522795000086.jpg12789JPEG2025522795000087.jpg12794JPEG2025522795000088.jpg12788JPEG2025522795000089.jpg12792JPEG2025522795000090.jpg12791JPEG2025522795000091.jpg12792JPEG2025522795000092.jpg12787JPEG2025522795000093.jpg127106JPEG2025522795000094.jpg127101JPEG2025522795000095.jpg12797JPEG2025522795000096.jpg12796JPEG2025522795000097.jpg12795JPEG2025522795000098.jpg12796JPEG2025522795000099.jpg127105JPEG2025522795000100.jpg12788JPEG2025522795000101.jpg127100JPEG2025522795000102.jpg12794JPEG2025522795000103.jpg12797JPEG2025522795000104.jpg12799JPEG2025522795000105.jpg12799JPEG2025522795000106.jpg127107JPEG2025522795000107.jpg12793JPEG2025522795000108.jpg12799JPEG2025522795000109.jpg127101JPEG2025522795000110.jpg12790JPEG2025522795000111.jpg12797JPEG2025522795000112.jpg127104JPEG2025522795000113.jpg12796JPEG2025522795000114.jpg12791JPEG2025522795000115.jpg12798JPEG2025522795000116.jpg12795JPEG2025522795000117.jpg12790JPEG2025522795000118.jpg127104JPEG2025522795000119.jpg127101JPEG2025522795000120.jpg127100JPEG2025522795000121.jpg12797JPEG2025522795000122.jpg12798JPEG2025522795000123.jpg12798JPEG2025522795000124.jpg12799JPEG2025522795000125.jpg12794JPEG2025522795000126.jpg12799JPEG2025522795000127.jpg12792JPEG2025522795000128.jpg127111JPEG2025522795000129.jpg127106JPEG2025522795000130.jpg127106JPEG2025522795000131.jpg127107JPEG2025522795000132.jpg127106JPEG2025522795000133.jpg127109JPEG2025522795000134.jpg127106JPEG2025522795000135.jpg12787JPEG2025522795000136.jpg127102JPEG2025522795000137.jpg127115JPEG2025522795000138.jpg12799JPEG2025522795000139.jpg12792JPEG2025522795000140.jpg12787JPEG2025522795000141.jpg12787JPEG2025522795000142.jpg127105JPEG2025522795000143.jpg127105JPEG2025522795000144.jpg127104JPEG2025522795000145.jpg127105JPEG2025522795000146.jpg45127.

[0125] In some embodiments, the compounds of the present disclosure are for use in the degradation and / or reduction of casein kinase 1 alpha (CK1α).

[0126] In some embodiments, the compounds of the present disclosure are for use in the inhibition of the activity of casein kinase 1 alpha (CK1α).

[0127] In some embodiments, the compounds of the present disclosure are for use in the prevention or treatment of blood cancer.

[0128] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure (represented by formula (I), (I-a), (I-b) or (I-c)), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, and optionally a pharmaceutically acceptable excipient or carrier.

[0129] The term "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., that does not cause unacceptable levels of undesirable biological effects or interact in a harmful manner.

[0130] The term "pharmaceutically acceptable carrier" is used herein to generally refer to a carrier that is safe, non-toxic, not biologically or otherwise undesirable, and useful for preparing pharmaceutical compositions that are acceptable for veterinary and human pharmaceutical use. The "pharmaceutically acceptable carrier" as used in the specification and claims can include one or more such carriers. "Pharmaceutically acceptable" means that the carrier is compatible with the other ingredients of the formulation and not harmful to its recipient.

[0131] As used herein, the term "pharmaceutically acceptable salt" means a salt of an active ingredient prepared with an acid or base that is acceptable to, or tolerated by, a biological system, or acceptable to and tolerated by a subject, when administered in a therapeutically effective amount. When the compounds of the present disclosure contain relatively acidic functional groups, the base addition salts can be obtained by contacting the neutral form of such compounds, either as such or in a suitable inert solvent, with a sufficient amount of the desired base. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino, magnesium salts, lithium salts, strontium salts or similar salts. When the compounds of the present disclosure contain relatively basic functional groups, the acid addition salts can be obtained by contacting the neutral form of such compounds, either as such or in a suitable inert solvent, with a sufficient amount of the desired acid. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like, but are not limited thereto. Also included are salts of amino acids such as arginate, and salts of organic acids such as glucuronic acid and galactunoric acid.

[0132] The compound represented by formula (I), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, degrades casein kinase 1 alpha (CK1α).

[0133] In some embodiments, the pharmaceutical composition is for use in the degradation and / or reduction of casein kinase 1 alpha (CK1α).

[0134] In some embodiments, the pharmaceutical composition is for use in suppressing the activity of casein kinase 1 alpha (CK1α).

[0135] In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of blood cancer.

[0136] In some embodiments, the blood cancer is leukemia, lymphoma, or myeloma.

[0137] The present disclosure also provides a method of degrading and / or reducing casein kinase 1 alpha (CK1α) in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0138] The present disclosure also provides a method of inhibiting the activity of casein kinase 1 alpha (CK1α) in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0139] The present disclosure also provides a method of preventing or treating blood cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present disclosure.

[0140] As used herein, the term "blood cancer" refers to cancer that occurs in hematopoietic tissues such as bone marrow or cells of the immune system. In some embodiments, "blood cancer" includes any cancer associated with uncontrolled proliferation of blood cells, particularly white blood cells.

[0141] In some embodiments, the blood cancer is leukemia, lymphoma (Hodgkin lymphoma and non-Hodgkin lymphoma), or myeloma.

[0142] In some embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, chronic eosinophilic leukemia (CEL), and myelodysplastic syndrome (MDS).

[0143] In some embodiments, the lymphoma is selected from the group consisting of Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, and peripheral T-cell lymphoma.

[0144] In some embodiments, the myeloma is selected from the group consisting of multiple myeloma, light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma.

[0145] In some embodiments, the compounds of the present disclosure can bind to cereblon (CRBN), alter the specificity of the complex, and induce the ubiquitination and degradation of CK1α, a transcription factor essential for proliferative diseases such as the proliferation of multiple myeloma.

[0146] In some embodiments, the present disclosure provides compounds and compositions comprising an E3 ligase binding moiety capable of binding to an E3 ligase (e.g., CRBN) and a target protein binding moiety capable of binding to a target protein, resulting in ubiquitination of the target protein and causing degradation of the target protein by the proteasome.

[0147] As used herein, the term "degrade" or "degradation" means the degradation of a target protein mediated by an E3 ligase, such as CRBN, which results in a decrease in the protein level. In one embodiment, the target protein may be a casein kinase 1 (CK1, such as CK1α, CK1β1, CK1γ1, CK1γ2, CK1γ3, CK1δ, CK1ε) protein. In certain embodiments, the target protein may preferably be CK1α.

[0148] As used herein, the term "CK1α" refers to casein kinase lα, a kinase in humans encoded by the CSNK1A1 gene. CK1α has been shown to play an important role in the biology of AML (Jaras M et al, J Exp Med. 2014; 211(4):605 - 612).

[0149] As used herein, the term "effective amount" refers to the dosage of an active agent sufficient to degrade CK1α and / or inhibit CK1α activity. In certain embodiments, the term "effective amount" refers to the dosage of an active agent sufficient to treat a CK1α-related disease, preferably a blood cancer.

[0150] As used herein, the terms "treat", "treatment", or "treating" refer to administering a compound or pharmaceutical composition to a subject for prophylactic and / or therapeutic purposes. The term "prophylactic treatment" refers to treating a subject who does not yet exhibit symptoms of a disease or condition but is susceptible to or at risk of a particular disease or condition, thereby reducing the likelihood that the patient will develop the disease or condition through treatment. The term "therapeutic treatment" refers to treating a subject who is already suffering from a disease or condition.

[0151] As used herein, the term "prevent" refers to delaying the progression of a disease, or delaying the onset or symptoms of a disease. Prevention of a disease or disorder includes halting the onset or symptoms of the disease.

[0152] The pharmaceutical compositions described herein can be administered to a human patient per se or as a pharmaceutical composition mixed with other active ingredients, excipients, or combinations thereof, such as in combination therapy. Suitable formulations depend on the selected route of administration. Techniques for formulating and administering the compounds described herein are known to those skilled in the art.

[0153] The pharmaceutical compositions disclosed herein can be manufactured by methods known per se, for example, by conventional mixing, dissolving, granulating, sugar coating, wet milling, emulsifying, encapsulating, entrapping, or tableting processes. Further, the active ingredient is contained in an amount effective to achieve its intended purpose. Many of the compounds used in the pharmaceutical formulations disclosed herein may be provided as salts with pharmaceutically compatible counterions.

[0154] In the art, there are multiple techniques for administering compounds, salts, and / or compositions, including, but not limited to, oral, rectal, pulmonary, topical, aerosol, injection, infusion, and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injection. In some embodiments, the compounds described herein, including the compounds of formula (I) or pharmaceutically acceptable salts, enantiomers, stereoisomers, or prodrugs thereof, can be administered orally.

[0155] Also, the compounds, salts, and / or compositions can be administered topically rather than systemically, for example, by directly injecting or implanting the compound as a depot or sustained release formulation into the affected area. Further, the compounds can be administered using targeted drug delivery systems, such as liposomes coated with tissue-specific antibodies. Liposomes target and are selectively taken up by organs. For example, nasal or pulmonary delivery may be desirable for targeting respiratory diseases and conditions.

[0156] The compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may have a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals. The notice reflects the approval by the government agency of the drug form for human or veterinary administration. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. Compositions that may include the compounds and / or salts described herein, formulated with a compatible pharmaceutical excipient, may also be prepared, placed in a suitable container, and labeled for the treatment of an indicated condition.

[0157] The compounds, salts and / or pharmaceutical compositions may be provided in the form of a kit to the administering physician or other health care professional. The kit is a package containing a container containing the compound in a suitable pharmaceutical composition and instructions for administering the pharmaceutical composition to a subject. The kit may also optionally include one or more additional therapeutic agents. The kit may also include individual doses of the compound or pharmaceutical composition for sequential or sequential administration. The kit may also optionally include one or more diagnostic tools and instructions for use. The kit may include a suitable delivery device, such as a syringe, along with instructions for administering the compound and any other therapeutic agents. The kit may optionally include instructions for storage, reconstitution (if applicable), and administration of any or all of the included therapeutic agents. The kit may include multiple containers reflecting the number of doses to be given to the subject.

[0158] As will be readily apparent to those skilled in the art, the useful in vivo dosage administered and the particular mode of administration will vary depending on age, body weight, disease severity, mammalian species being treated, the particular compound employed, and the particular use for which these compounds are employed. Determination of effective dosage levels, i.e., the dosage levels necessary to achieve the desired result, can be accomplished by those skilled in the art using routine methods such as human clinical trials and in vitro studies.

[0159] Dosages can vary widely depending on the desired effect and the indication being treated. Alternatively, dosages can also be calculated based on the surface area of the patient, as is understood by those skilled in the art. The exact dosage is determined for each drug, but in most cases, some generalization about dosages can be made. The daily dosage regimen for adult patients can be, for example, from about 0.01 mg to 3,000 mg, preferably from about 1 mg to 700 mg, such as from about 5 to 200 mg, as the oral dosage of each active ingredient. The dosage can be a single or a series of two or more administrations given over a course of one or more days, depending on the needs of the subject. In some embodiments, the compound is administered over a continuous treatment period of, for example, one week or more, or over several months or years.

[0160] When the human dosage of a compound has been established for at least some condition, the same dosage, or a dosage that is about 0.1% to 500% of the established human dosage, more preferably about 25% to 250% of the established human dosage, may be used. When the human dosage has not been established, as in the case of a newly discovered pharmaceutical composition, an appropriate human dosage can be inferred from the ED 50 or Id 50 value, or other appropriate values obtained from in vitro or in vivo studies.

[0161] In the case of administration of a pharmaceutically acceptable salt, the dosage can be calculated as the free base. As will be understood by those skilled in the art, in certain circumstances, particularly for the effective and aggressive treatment of malignant diseases or infections, it may be necessary to administer the compounds disclosed herein in amounts exceeding, or even significantly exceeding, the above - preferred dosage ranges.

[0162] Dosages and intervals may be adjusted individually to provide plasma levels of the active moiety sufficient to maintain the modulating effect, or the minimum effective concentration (MEC). The MEC varies for each compound but can be estimated from in vitro data. The dosage required to achieve the MEC will vary according to the individual characteristics and the route of administration. However, plasma concentrations can be measured using an HPLC assay or a bioassay. The dosing interval can also be determined using the MEC value. The composition should be administered using regimens that maintain plasma levels above the MEC for about 10 - 90% of the time, preferably about 30 - 90%, most preferably about 50 - 90% of the time. In the case of topical administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

[0163] It should be noted that the attending physician knows the methods and times to terminate, interrupt, or adjust administration due to toxicity or organ failure. Conversely, the attending physician also knows to adjust the treatment to a higher level if the clinical response is inadequate (excluding toxicity). The magnitude of the dosage administered in the management of the disease of interest varies with the severity of the disease being treated and the route of administration. The severity of the disease can be partially evaluated, for example, by standard prognostic assessment methods. Further, the dosage and perhaps the dosing frequency also vary according to the age, weight, and response of the individual patient. Programs equivalent to those described above can be used in veterinary medicine.

[0164] The compounds disclosed in this specification can be evaluated for efficacy and toxicity using known methods. For example, the toxicity of a specific compound, or a subset of compounds sharing a specific chemical moiety, can be established by determining the in vitro toxicity against cell lines such as mammalian, preferably human cell lines. The results of such studies often predict toxicity in animals such as mammals, more specifically humans. Alternatively, the toxicity of a specific compound in animal models such as mice, rats, rabbits, or monkeys can be determined using known methods. The efficacy of a specific compound can be established using several recognized methods such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, one of ordinary skill in the art can select an appropriate model, dose, route of administration, and / or regimen with reference to the state of the art. Examples I. Synthesis of Compounds

[0165] Hydrochloride form of Compound 1

[0166] 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000147.jpg35127JPEG2025522795000148.jpg39127

[0167] Step - 1: tert-Butyl (4-oxobutyl)carbamate A solution of oxalyl chloride (5.4 mL, 63.49 mmol, 1.2 equiv) in DCM (50 mL) was added dropwise to a solution of DMSO (10 mL, 1 vol) in DCM (100 mL, 10 vol) at -78 °C under a nitrogen atmosphere over 15 minutes, and the mixture was stirred at the same temperature for 15 minutes. To this, a solution of tert-butyl (4-hydroxybutyl)carbamate 1 (10 g, 52.91 mmol, 1 equiv) in DCM (50 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, then N,N-diisopropylethylamine (29.2 mL, 158.7 mmol, 3 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, 2:8 / EtOAc:pet ether, Rf ~0.6, KMnO4), the reaction mixture was diluted with DCM (200 mL) and washed with 10% aqueous citric acid solution (1×100 mL), water (1×200 mL), and brine (1×200 mL), dried over Na2SO4, filtered, and concentrated under vacuum at a temperature below 30 °C to obtain tert-butyl (4-oxobutyl)carbamate (10 g, crude product) as a pale yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 9.66 (s, 1H), 6.84 (s, 1H), 2.89 - 2.93 (m, 2H), 2.42 - 2.44 (m, 2H), 1.60 - 1.64 (m, 2H), 1.38 (s, 9H).

[0168] Step - 2: tert-Butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate A mixture of tert-butyl (4-oxobutyl)carbamate 2 (9.0 g, 0.0484 mol, 1.8 equiv) and lenolidamide 3 (CAS No. 191732-72-6, 7.0 g, 0.0269 mol, 1 equiv) was dissolved in a mixture of 1,2-dichloroethane (70 ml, 10 vol) and DMF (70 ml, 10 vol) under a nitrogen atmosphere. To this was added acetic acid (4.8 ml, 0.0807 mol, 3.0 equiv) at room temperature. The resulting reaction mixture was stirred at room temperature for 2 h. At this stage, NaCNBH3 (6.6 g, 0.1076 mol, 4 equiv) was added portionwise at 0 °C and the mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 8:2 / EtOAc:petroleum ether, Rf ~0.6, and LCMS), the reaction mixture was diluted with DCM (200 mL), washed with water (3 × 200 mL) and brine (200 mL), dried over Na2SO4, filtered and concentrated. The resulting product was triturated with hexane (50 mL), filtered and dried under suction to give tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (8 g, 69%) as a white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.08 (s, 1H), 7.28 (t, J = 7.60 Hz, 1H), 6.93 (d, J = 7.20 Hz, 1H), 6.82 (t, J = 5.60 Hz, 1H), 6.75 (d, J = 8.00 Hz, 1H), 5.58 (t, J = 5.60 Hz, 1H), 5.10-5.14 (m, 1H), 4.10-4.25 (m, 2H), 3.11-3.14 (m, 2H), 2.90-2.98 (m, 3H), 2.60-2.64 (m, 1H), 2.28-2.32 (m, 1H), 2.04-2.06 (m, 1H), 1.46-1.58 (m, 4H), 1.37 (s, 9H). LC-MS: 429.2 (M-H), Rt (min): 2.552, area%-97.64.

[0169] Step - 3: 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride To an ice-cooled suspension of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (800 mg, 1.86 mmol, 1 equiv) in DCM (10 mL) was added HCl (1 M, 8 mL) in EtOAc, and the mixture was stirred at room temperature for 4 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated and freeze-dried to afford 3-(4-((4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (530 mg, 79%) as a yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 8.07 (s, 3H), 7.34 (t, J = 7.60 Hz, 1H), 7.05 (d, J = 7.60 Hz, 1H), 6.92 (d, J = 8.00 Hz, 1H), 5.10 - 5.15 (m, 1H), 4.19 - 4.37 (m, 2H), 3.19 - 3.20 (m, 2H), 2.90 - 2.95 (m, 1H), 2.80 - 2.89 (m, 2H), 2.51 - 2.52 (m, 1H), 2.29 - 2.33 (m, 1H), 2.03 - 2.06 (m, 1H), 1.66 - 1.67 (m, 4H). 1313C-NMR (100 MHz, DMSO-d6): δ 173.34, 171.52, 168.67, 140.34, 133.14, 129.87, 117.06, 114.76, 52.08, 46.58, 44.46, 31.65, 24.90, 23.23. LCMS: 331.3 (M+H). Method: Mobile phase A: 0.1% formic acid aqueous solution. Mobile phase B: acetonitrile. Flow rate: 1.5 mL / min. Column: Atlantis dC18 (50×4.6) mm, 5 μm. Rt (min): 1.638; Area %-99.32. HPLC method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: acetonitrile. Flow rate: 1.0 mL / min. Column: Atlantis dC18 (250×4.6) mm, 5 μm. Rt (min): 7.403; Area %-98.88.

[0170] Hydrochloride form of Compound 2

[0171] 3-(4-((4-Aminobutyl)(methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000152.jpg32127

[0172] Step-1: tert-Butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(methyl)amino)butyl)carbamate JPEG2025522795000153.jpg 36127 (4 - ((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 4 (2 g, 4.65 mmol, 1 equivalent) and paraformaldehyde (1.39 g, 46.5 mmol, 10 equivalents) were added to a mixture of 1,2-dichloroethane (20 ml, 10 volumes) and DMF (10 ml, 5 volumes) under a nitrogen atmosphere. Acetic acid (0.8 ml, 13.95 mmol, 3 equivalents) was added thereto, and the reaction mixture was stirred at room temperature for 1 hour. NaCNBH3 (1.15 g, 18.6 mmol, 4 equivalents) was added portionwise, and the reaction mixture was stirred at room temperature for an additional 16 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~0.4, and LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3×50 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography (column size: Biotage R snap cartridge, KP-Sil, 50 g, 230 - 400 silica gel) using 40 - 45% ethyl acetate in dichloromethane to obtain (4 - ((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(methyl)amino)butyl)carbamic acid tert-butyl (800 mg, 39%) as a white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 7.37 (t, J = 7.60 Hz, 1H), 7.15 (d, J = 7.20 Hz, 1H), 6.99 (d, J = 8.00 Hz, 1H), 6.80 (t, J = 5.60 Hz, 1H), 5.08 - 5.13 (m, 1H), 4.33 - 4.51 (m, 2H), 2.90 - 2.96 (m, 3H), 2.88 (s, 3H), 2.52 - 2.62 (m, 2H), 2.01 - 2.03 (m, 1H), 1.42 - 1.48 (m, 4H), 1.40 (s, 9H). LC-MS: 445.4 (M-H), Rt (min): 2.525, area% - 99.11.

[0173] Step 2: 3-(4-((4-Aminobutyl)(methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride To a solution of JPEG2025522795000154.jpg33127 (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(methyl)amino)butyl)carbamic acid tert-butyl 5 (800 mg, 1.80 mmol) in DCM (10 mL) cooled in ice, HCl (1 M, 8 mL) in EtOAc was added and stirred at room temperature for 4 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated and freeze-dried to obtain 3-(4-((4-aminobutyl)(methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (550 mg, 88%) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.99 (brs, 3H), 7.37 - 7.51 (m, 3H), 5.10 - 5.15 (m, 1H), 4.44 - 4.63 (m, 2H), 3.30 (s, 2H), 3.01 (s, 3H), 2.92 - 2.98 (m, 2H), 2.60 - 2.78 (m, 3H), 2.45 - 2.50 (m, 1H), 2.06 - 2.08 (m, 1H), 1.66 - 1.67 (m, 4H). 1313C-NMR (100 MHz, DMSO-d6): δ 173.34, 171.30, 167.67, 134.36, 133.03, 130.23, 117.06, 122.84, 55.43, 52.21, 48.02, 42.50, 31.65, 23.02, 22.96. LCMS: 345.1 (M+H). Method: Mobile phase A: 0.1% formic acid aqueous solution. Mobile phase B: acetonitrile. Flow rate: 1.5 mL / min. Column: Atlantis dC18 (50×4.6) mm, 5 μm. Rt (min): 1.252; Area % - 98.76. HPLC method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: acetonitrile. Flow rate: 1.0 mL / min. Column: Atlantis dC18 (250×4.6) mm, 5 μm. Rt (min): 6.313; Area % - 97.81.

[0174] Hydrochloride form of Compound 3

[0175] 3-(4-((4-Aminobutyl)(isopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000155.jpg33127

[0176] Step - 1: tert-Butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(isopropyl)amino)butyl)carbamate JPEG2025522795000156.jpg36127tert-butyl 4((4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate (1.5 g, 3.4 mmol, 1 equivalent) and 2-methoxypropene (1.25 g, 17.5 mmol, 5 equivalents) were added to 1,2-dichloroethane (30 ml, 20 vol), and the suspension was cooled to 0 °C. Trifluoroacetic acid (0.22 ml, 3.0 mmol, 0.9 equivalent) was added dropwise thereto. Next, sodium triacetoxyborohydride (1.07 g, 5.1 mmol, 1.5 equivalents) was added portionwise at 0 °C under a nitrogen atmosphere, and the resulting reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf about 0.5, and LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (2 × 100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by chromatography (column size: Biotage R snap cartridge, KP-Sil, 50 g, 230 - 400 silica gel) using 40 - 45% ethyl acetate in dichloromethane to obtain tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(isopropyl)amino)butyl)carbamate (620 mg, 38%) as a white solid. LCMS: 473.4 (M+H). Method: Column: Atlantis dC18 (50×4.6) 5μ, Mobile phase: A: 0.1% aqueous formic acid, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.454; Area%-97.90.

[0177] Step 2: 3-(4-((4-aminobutyl(isopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000157.jpg31127 620 mg (1.313 mmol) of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(isopropyl)amino)butyl)carbamate was added to a solution of HCl (1 M, 4 mL) in EtOAc in a DCM (4 mL) solution cooled in ice, and the mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum and further lyophilized to obtain 3-(4-((4-aminobutyl(isopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (440 mg, 96%) as a light brown solid, which is the target compound. 1 H-NMR (400 MHz, DMSO-d6): δ 11.05 (s, 1H), 7.98 (s, 3H), 7.72 (s, 2H), 5.12-5.08 (m, 1H), 4.56 (dd, J = 18.00, 56.40 Hz, 2H), 3.82-3.51 (m, 1H), 3.47 (s, 2H), 2.90-2.70 (m, 1H), 2.68-2.65 (m, 3H), 2.44-2.41 (m, 1H), 2.09 (d, J = 5.20 Hz, 1H), 1.53 (t, J = 7.60 Hz, 2H), 1.33 (d, J = 6.80 Hz, 2H), 1.23 (s, 6H). 13 C-NMR (100 MHz, DMSO-d6): 173.37, 171.25, 167.38, 136.43, 134.63, 130.36, 126.85, 123.25, 60.12, 52.33, 49.80, 48.17, 40.49, 39.24, 38.53, 31.55, 24.53, 22.91, 18.92, 18.62. LCMS: 373.2 (M-HCl). Method: Column: Atlantis dC18(50×4.6)5μ, Mobile phase: A: 0.1% aqueous formic acid solution, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.239; Area% - 97.330. HPLC: 97.905%, Rt (min): 6.141.

[0178] Hydrochloride form of Compound 4

[0179] 3-(4-((4-aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000158.jpg32127

[0180] Step - 1: tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(propyl)amino)butyl)carbamate JPEG2025522795000159.jpg35127tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate 4 (1.5 g, 3.4 mmol, 1 equiv) and propionaldehyde (2.0 g, 3.4 mmol, 10 equiv) were dissolved in a mixture of 1,2 - dichloroethane (20 mL) and DMF (10 mL) under a nitrogen atmosphere. Acetic acid (0.58 ml, 10.2 mmol, 3 equiv) was added thereto, and the resulting reaction mixture was stirred at room temperature for 1 hour. At this point, sodium cyanoborohydride (0.84 g, 1.3 mmol, 4 equiv) was added portionwise, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ≈ 0.4, and LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3 × 100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography (Isolera, column size: Biotage R snap cartridge, KP - Sil, 50 g, 230 - 400 silica gel) using 40 - 45% EtOAc in DCM to give tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(propyl)amino)butyl)carbamate 7 (600 mg, 37%) as a white solid. LCMS: 473.4 (M + H). Method: Column: Atlantis dC18(50×4.6)5μ, Mobile phase: A: 0.1% aqueous formic acid solution, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.709; area % - 99.720.

[0181] Step 2: 3-(4-((4-aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000160.jpg34127(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)aminuteso)butyl)carbamic acid tert-butyl 7(600 mg, 1.2695 mmol, 1 equivalent) in DCM (10 mL) was cooled in ice, and HCl (1 M, 5 mL) in EtOAc was added. The mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated and further lyophilized to obtain 3-(4-((4-aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (360 mg, 68%) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.99 (s, 3H), 7.53 (s, 2H), 5.12 (q, J = 4.80 Hz, 1H), 4.49 (d, J = 36.40 Hz, 2H), 3.29 (s, 1H), 2.97 (s, 1H), 2.96 - 2.88 (m, 1H), 2.72 (q, J = 22.80 Hz, 2H), 2.62 (d, J = 16.40 Hz, 1H), 2.44 (s, 1H), 2.06 (t, J = 5.20 Hz, 1H), 2.05 (s, 1H), 1.55 (s, 6H), 0.84 (t, J = 7.20 Hz, 3H). 1313C-NMR (100 MHz, DMSO-d6): δ 173.34, 171.27, 167.49, 162.82, 134.55, 130.45, 125.14, 57.35, 55.02, 52.34, 48.12, 38.58, 36.28, 31.59, 31.26, 24.51, 22.94, 21.57, 19.04, 11.25. LCMS: 373.1 (M-HCl). Method: Column: Atlantis dC18(50×4.6) 5μ, Mobile phase: A: 0.1% aqueous formic acid solution, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.386; Area%-98.551. HPLC: 98.878%, Rt (min): 6.95.

[0182] Hydrochloride form of Compound 6

[0183] 3-(4-((2-(1-(Aminomethyl)cyclohexyl)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000161.jpg21127JPEG2025522795000162.jpg35127JPEG2025522795000163.jpg31127

[0184] Step-1: 2-(1-(((tert-Butoxycarbonyl)amino)methyl)cyclohexyl)acetic acid JPEG2025522795000164.jpg201272 - (1 - (aminomethyl)cyclohexyl)acetic acid 1 (30 g, 175.4 mmol, 1 equiv) in THF (300 ml, 10 vol) solution was added dropwise with an aqueous solution of NaOH (7.0 g, 175.4 mmol, 1 equiv) and boc - anhydride (38.2 g, 175.4 mmol, 1 equiv) at 0 °C. The mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc in petroleum ether, Rf about 0.4, and LCMS - ELSD), the solvent was evaporated to dryness, diluted with water (100 mL), and washed with ethyl acetate (1×150 mL). The aqueous layer was separated, acidified to pH 5 - 6 with 1.5N HCl, and extracted with DCM (2×500 mL). The organic layer was washed with brine (250 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain 2 - (1 - ((((tert - butoxycarbonyl)amino)methyl)cyclohexyl)acetic acid 2 (35 g, 75%) as a white solid. LCMS(ELSD): 270.3(M - H). Method: Column: Atlantis dC18(50×4.6) 5μ, Mobile phase: A: 0.1% aqueous formic acid solution, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.950; Area% - 99.502.

[0185] Step 2: Methyl 2 - (1 - ((((tert - butoxycarbonyl)amino)methyl)cyclohexyl)acetate JPEG2025522795000165.jpg221272-(1-(((tert-Butoxycarbonyl)amino)methyl)cyclohexyl)acetic acid22(20 g, 73.8 mmol, 1 equivalent) in DMF (200 ml, 10 vol) was added dropwise with potassium carbonate (20.3 g, 147.6 mmol, 2 equivalents) and methyl iodide (26 ml, 369 mmol, 5 equivalents) at 0 °C. The mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc in petroleum ether, Rf about 0.6, and LCMS-ELSD), the reaction mixture was diluted with ethyl acetate (200 mL) and washed with ice-cold H2O (3 × 500 mL). The organic layer was washed with brine (1 × 500 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain methyl 2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)acetate 3(15 g, 71%) as a pale yellow liquid. LCMS(ELSD): 230.3(M-tert-Butyl). Method: Column: Atlantis dC18(50×4.6)5μ, Mobile phase: A: 0.1% aqueous formic acid, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 3.374; Area%-99.76.

[0186] Step-3: tert-Butyl 3-hydroxy-2-azaspiro[4.5]decane-2-carboxylate JPEG2025522795000166.jpg201272-Methyl 2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)acetate 3(8 g, 28.07 mmol, 1 equivalent) in DCM (150 mL) was added dropwise with DIBAL-H (1 M in hexane, 28.07 ml, 28.07 mmol, 1 equivalent) at -78 °C. The mixture was stirred at 0 °C for 2 h. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc in petroleum ether, Rf ~0.5, and LCMS-ELSD), the reaction mixture was quenched with saturated NH4Cl solution (50 mL), filtered through a celite bed, and washed with DCM (250 mL). The separated organic layer was washed with brine (250 mL), dried over Na2SO4, filtered, and concentrated under vacuum at <35 °C to obtain tert-butyl 3-hydroxy-2-azaspiro[4.5]decane-2-carboxylate 4 (6 g, crude product) as a light brown liquid, which was used without further purification. LCMS (ELSD): 202.1 (M-tert-Butyl). Method: Column: Atlantis dC18 (50×4.6) 5μ, Mobile phase: A: 0.1% aqueous formic acid, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.187; Area%-99.976.

[0187] Step-4: ((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethyl)cyclohexyl)methyl)carbamic acid tert-butyl JPEG2025522795000167.jpg351271, In a stirred solution of lenalidomide (CAS number 191732-72-6, 2 g, 7.6 mmol, 1 equivalent) and tert-butyl 3-hydroxy-2-azaspiro[4.5]decane-2-carboxylate (5.79 g, 2.3 mmol) in a mixture of dichloroethane (20 mL) and DMF (20 mL), acetic acid (1.82 ml, 30.7 mmol, 4 equivalents) was added under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 1 hour. Next, the reaction mixture was cooled to 0 °C, and sodium cyanoborohydride (1.84 g, 30.7 mmol, 4 equivalents) was added portionwise and stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf about 0.6, and LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3 × 50 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 40 - 45% EtOAc in DCM (column size: Biotage Rsnap cartridge, KP-Sil, 25 g, 230 - 400 silica gel) to obtain tert-butyl ((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethyl)cyclohexyl)methyl)carbamate 5 (620 mg, 16%) as a white solid. LCMS: 443.4 (M - t-Bu). Method: Column: Atlantis dC18 (50 × 4.6) 5μ, Mobile phase: A: 0.1% aqueous formic acid, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 3.072; Area%-98.042.

[0188] Step - 5: 3-(4-((2-(1-(aminomethyl)cyclohexyl)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000168.jpg34127((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethyl)cyclohexyl)methyl)carbamic acid tert-butyl5(620 mg, 1.2437 mmol, 1 equiv) in a ice-cooled solution of DCM (10 mL), HCl (1 M, 6 mL) in EtOAc was added and stirred at room temperature for 12 h. After confirming the completion of the reaction by LCMS, the reaction mixture was concentrated under vacuum to obtain the crude product, which was co-distilled with Milli-Q H2O (2 × 10 mL) and dried under vacuum to give 3-(4-((2-(1-(aminomethyl)cyclohexyl)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (480 mg, 98%) as a pale yellow solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 8.05 (s, 3H), 7.43-7.31 (m, 1H), 7.10 (d, J = 7.20 Hz, 1H), 7.01 (d, J = 7.60 Hz, 1H), 5.12 (q, J = 5.20 Hz, 1H), 4.39 (d, J = 17.20 Hz, 1H), 4.24 (d, J = 17.60 Hz, 1H), 3.18-2.97 (m, 2H), 2.94-2.89 (m, 1H), 2.79 (d, J = 5.60 Hz, 2H), 2.63 (d, J = 17.20 Hz, 1H), 2.31 (q, J = 4.40 Hz, 1H), 2.06-2.04 (m, 1H), 1.77 (t, J = 7.20 Hz, 2H), 1.44 (s, 10H). 1313C-NMR (100 MHz, DMSO-d6): δ 173.32, 171.43, 168.37, 138.77, 133.32, 131.39, 129.94, 119.19, 116.76, 52.15, 46.70, 45.18, 41.16, 40.56, 34.88, 33.03, 31.64, 31.49, 25.84, 23.21, 21.11. LCMS: 399.1 (M-HCl). Method: Column: Atlantis dC18(50×4.6) 5μ, Mobile phase: A: 0.1% aqueous formic acid solution, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.551; Area %-99.47. HPLC: 99.499%, Rt (min): 8.979.

[0189] Hydrochloride form of Compound 46 JPEG2025522795000169.jpg28127JPEG2025522795000170.jpg30127

[0190] General procedure for the preparation of Compound 6b JPEG2025522795000171.jpg33127To a solution of Compound 4 (1.0 g, 2.32 mmol, 1 equiv) and Compound 5b (336 mg, 4.65 mmol, 2.0 equiv) in DCE / DMF (10 mL) were added acetic acid (1.0 mL) and molecular sieves (1.0 g), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (585 mg, 9.3 mmol, 4 equiv) was added portionwise, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA, and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3×20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated and purified by silica gel column chromatography (DCM / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40%~60%) to obtain Compound 6b (400 mg, 35%) as a white solid. TLC: EA = 100% R f (Compound 4)=0.5 R f(Compound 6b) = 0.6 LC-MS: 487.3 [M+1] +

[0191] General procedure for the preparation of Compound 46 To a solution of Compound 6b (400 mg, 0.873 mmol, 1 equiv) in methanol (1 mL) was added HCl / EA (2N, 8.73 mmol, 10 equiv). The resulting solution was stirred at room temperature for 24 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by P-HPLC (acetonitrile / H2O: 30% - 40%) to give Compound 46 (60 mg, 16%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 6b) = 0.6 R f (Compound 46) = 0.2 LC-MS: 387.25 [M+1] + 1 1H NMR (400 MHz, D2O) δ 7.84 (s, 1H), 7.70 (s, 2H), 5.07 (d, J = 8.7 Hz, 1H), 4.55 (s, 3H), 3.51 (s, 4H), 3.18 (s, 1H), 2.73 (d, J = 7.9 Hz, 4H), 2.41 (d, J = 7.5 Hz, 1H), 2.16 (s, 1H), 1.31 (dd, J = 93.7, 45.1 Hz, 8H), 0.65 (s, 3H).

[0192] Hydrochloride form of Compound 47

[0193] 3-(4-((4-Aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000173.jpg36127

[0194] Step - 3: tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(methyl)amino)butyl)carbamate To a solution of tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate 4 (3 g, 6.97 mmol, 1 equiv) and pentanal (6.0 g, 69.7 mmol, 10 equiv) in a mixture of 1,2 - dichloroethane (60 mL, 20 vol) and DMF (30 mL, 10 vol), acetic acid (1.2 mL, 20.91 mmol, 3 equiv) was added and the reaction mixture was stirred at room temperature for 2 h. NaCNBH3 (1.75 g, 27.9 mmol, 4 equiv) was added portionwise and the reaction mixture was stirred at room temperature for a further 16 h. TLC analysis (70% EtOAc in petroleum ether) confirmed the formation of the product but it was found that the starting material was not completely consumed. Additional amounts of pentanal (2 x 6.0 g, 69.7 mmol, 10 equiv), acetic acid (2 x 0.8 mL, 13.94 mmol, 2 equiv) and NaCNBH3 (0.87 g, 13.94 mmol, 2 equiv) were added and the resulting reaction mixture was stirred at room temperature for 36 h. After completion of the reaction (confirmed by TLC analysis, 70% EtOAc in petroleum ether, Rf ~ 0.6, and LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3 x 100 mL), brine (300 mL), dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by chromatography (Biotage R snap cartridge, KP - Sil, 100 g, 230 - 400 silica gel) using 65 - 70% ethyl acetate in petroleum ether to give tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(pentyl)amino)butyl)carbamate 5 (2.5 g, 97%) as a white solid. 11H-NMR (400 MHz, DMSO-d6): δ (s, 1H), 7.37 (t, J = 8.00 Hz, 1H), 7.18 (d, J = 7.20 Hz, 1H), 7.07 (d, J = 8.00 Hz, 1H), 6.78 (t, J = 5.60 Hz, 1H), 5.11 (q, J = 5.20 Hz, 1H), 4.42 - 4.27 (m, 2H), 3.17 (t, J = 8.40 Hz, 4H), 2.96 - 2.90 (m, 3H), 2.68 - 2.61 (m, 2H), 2.03 - 2.00 (m, 1H), 1.43 (s, 15H), 1.38 (t, J = 10.80 Hz, 4H), 0.86 (s, 3H). LC-MS: 501.3 (M+H), Rt (min): 1.853, Area% - 97.842.

[0195] Step 4: 3-(4-((4-Aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride To a ice-cooled solution of tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)carbamate (2.5 g, 4.99 mmol) in DCM (25 mL), HCl (4 M solution in EtOAc, 12.5 mL) was added and stirred at room temperature for 2 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~0.4), the reaction mixture was concentrated and lyophilized to give 3-(4-((4-aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 47 (2.4 g, 98%) as an off-white solid. 1H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.92 (s, 4H), 7.39 (s, 2H), 5.15 - 5.10 (m, 1H), 3.28 (s, 4H), 2.98 - 2.92 (m, 1H), 2.75 (d, J = 5.20 Hz, 2H), 2.68 - 2.64 (m, 2H), 2.08 - 2.04 (m, 1H), 1.54 (s, 6H), 1.26 (d, J = 13.60 Hz, 5H), 0.87 - 0.81 (m, 3H). LCMS: 401.2 (M + H). Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: 0.1% TFA in ACN. Flow rate: 1.5 mL / min. Column: XBridge C8 (50×4.6 mm) 3.5 μm. Rt (min): 1.356; Area %: 96.152. HPLC: Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 2.0 mL / min. Column: X-Bridge C8 (50×4.6) mm, 3.5 μm. Rt (min): 2.341; Area %: 99.332.

[0196] Hydrochloride form of Compound 48 JPEG2025522795000175.jpg28127

[0197] General procedure for the preparation of compound 6d A solution of Compound 4 (2.0 g, 4.65 mmol, 1 equiv) and Compound 5d (932 mg, 9.30 mmol, 2.0 equiv) in DCE / DMF (20 mL) was added with acetic acid (2.0 mL) and molecular sieve (1.0 g), and stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.17 g, 18.6 mmol, 4 equiv) was added portionwise, and stirred at room temperature for 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA, and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 30 mL) and brine (30 mL), and dried over Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain Compound 6 (660 mg, 28%) as a white solid. TLC: EA = 100% R f (Compound 4) = 0.5 R f (Compound 6d) = 0.6 LC-MS: 515.40[M+1] +

[0198] General procedure for the preparation of Compound 48 HCl / EA (2N, 6.42 mmol, 5 equiv) was added to a solution of Compound 6d (660 mg, 1.28 mmol, 1 equiv) in DCM (6 mL). The resulting solution was stirred at 25 °C for 2 hours. The obtained mixture was concentrated under reduced pressure. The residue was purified by P-HPLC (acetonitrile / H2O: 30% - 40%) to obtain Compound 48 (150.55 mg, 28%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 6d) = 0.6 R f (Compound 48) = 0.2 LC-MS: 415.40[M+1] + 11H NMR (400 MHz, CDCl3): δ 10.25 (s, 1H), 7.75 (s, 3H), 7.40 - 7.28 (m, 2H), 6.98 (s, 1H), 5.17 (d, J = 8.9 Hz, 1H), 4.40 (dd, J = 58.4, 16.5 Hz, 2H), 3.13 - 2.54 (m, 9H), 2.03 (d, J = 46.6 Hz, 7H), 1.63 (s, 2H), 1.31 (d, J = 63.7 Hz, 13H), 0.85 (s, 3H).

[0199] Hydrochloride form of Compound 49 JPEG2025522795000178.jpg19127JPEG2025522795000179.jpg25127JPEG2025522795000180.jpg27127

[0200] General procedure for the preparation of compound 2e JPEG2025522795000181.jpg21127A solution of oxalyl chloride (9.5 g, 74.5 mmol, 1.2 equiv) in DCM (50 mL) was added dropwise to a solution of DMSO (10 mL) in DCM (100 mL) at -78 °C under a nitrogen atmosphere over 15 minutes and stirred at the same temperature for 15 minutes. A solution of compound 1e (10.0 g, 62.1 mmol, 1.0 equiv) in DCM (50 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, then TEA (18.8 g, 188.2 mmol, 3.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, PE / EA = 4 / 1, Rf ~0.4, KMnO4 or ninhydrin), the reaction mixture was diluted with DCM (50 mL). The mixture was washed with 10% citric acid (500 mL), water (50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum at less than 30 °C to give compound 2 (11.4 g, crude product) as a yellow oily substance, which was used without further purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin Rf (compound 1e) = 0.2 Rf (Compound 2e) = 0.4 LC-MS: Not found.

[0201] General procedure for the preparation of Compound 5 To a solution of Compound 3 (0.2 g, 3.9 mmol, 1.0 equiv) and Compound 4 (1.0 g, 3.9 mmol, 1.0 equiv) in DCE / DMF (1 / 1, 20 mL) was added acetic acid (0.7 g, 11.6 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 2 h. At this stage, NaBH3CN (1.0 g, 15.4 mmol, 4.0 equiv) was added portionwise at 0 °C and the reaction mixture was stirred at room temperature for 18 h. After completion of the reaction, the reaction mixture was diluted with DCM (100 mL). The mixture was washed with water (2 × 20 mL) and brine (2 × 20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / methanol: 3:1) to give Compound 5 (0.75 g, 64%) as a white solid. TLC: EA R f (Compound 3) = 0.4 R f (Compound 5) = 0.5 LC-MS: 302.25 [M+1] + , 300.25 [M-1] - 1 1H NMR (400 MHz, CDCl3): δ 7.10 (d, J = 5.9 Hz, 1H), 6.70 (d, J = 6.8 Hz, 1H), 5.06 (d, J = 10.9 Hz, 1H), 4.19 (d, J = 15.7 Hz, 1H), 4.07 (d, J = 15.7 Hz, 1H), 3.29 (d, J = 24.2 Hz, 1H), 3.07 (s, 2H), 2.75 (s, 2H), 2.25 (s, 1H), 2.09 (s, 1H), 1.57 (d, J = 6.1 Hz, 2H), 0.91 (s, 3H). 11H NMR (400 MHz, DMSO-d6): δ 9.33 (d, J = 1.8 Hz, 1H), 8.47 - 8.39 (m, 2H), 8.21 (d, J = 7.4 Hz, 1H), and 8.14 (d, J = 8.4 Hz, 1H).

[0202] General procedure for the preparation of compound 6e To a solution of compound 5 (4.6 g, 15.3 mmol, 1.0 equiv) and compound 2e (4.4 g, 27.5 mmol, 1.8 equiv) in DCE / DMF (80 mL, 1 / 1) was added acetic acid (20 mL), and the mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. NaBH3CN (3.8 g, 61.1 mmol, 4.0 equiv) was added portionwise, and the mixture was stirred at room temperature for 18 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS with 100% EA), the reaction mixture was diluted with DCM (400 mL). The mixture was washed with water (2 × 40 mL) and brine (2 × 40 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 2:1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to afford compound 6e (0.7 g, 10%) as a white solid. TLC: EA = 100% R f (Compound 5) = 0.5 R f (Compound 6e) = 0.6 LC-MS: 445 [M + 1] + , 443 [M - 1] - 11H NMR (400 MHz, CDCl3): δ 8.14 (s, 1H), 7.42 (d, J = 31.6 Hz, 2H), 7.12 (s, 1H), 5.22 (d, J = 10.3 Hz, 1H), 4.45 (s, 2H), 3.48 (s, 1H), 3.17 (d, J = 51.0 Hz, 5H), 2.97 - 2.75 (m, 2H), 2.21 (s, 1H), 1.67 (s, 2H), 1.42 (d, J = 17.8 Hz, 14H), 0.87 (s, 3H).

[0203] General procedure for the preparation of Compound 49 To a solution of Compound 6e (0.2 g, 0.4 mmol, 1.0 equiv) in methanol (2 mL) was added HCl / EA (2 mL, 2N, 4.0 mmol, 10.0 equiv). The resulting solution was stirred at room temperature for 18 h. The resulting mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 30% - 40%) to give Compound 49 (147 mg, 95%) as a white solid. LC-MS: 345.35 [M+1] + 1 1H NMR (400 MHz, D2O): δ 7.42 - 7.30 (m, 2H), 7.24 (d, J = 7.5 Hz, 1H), 5.00 (dd, J = 12.9, 4.4 Hz, 1H), 4.43 (q, J = 17.3 Hz, 2H), 3.41 (d, J = 5.4 Hz, 2H), 3.06 - 2.93 (m, 4H), 2.75 (dd, J = 25.3, 11.4 Hz, 2H), 2.41 (dd, J = 12.7, 4.6 Hz, 1H), 2.17 - 2.07 (m, 1H), 1.28 (dd, J = 14.2, 7.0 Hz, 2H), 0.65 (t, J = 7.1 Hz, 3H).

[0204] Compound 50

[0205] N-(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)acetamide JPEG2025522795000185.jpg36127

[0206] Step - 1: N-(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)acetamide JPEG2025522795000186.jpg391273-(4-((4-Aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl compound 4 (200 mg, 0.489 mmol, 1 equivalent) in pyridine (3 mL) was added acetic anhydride (0.231 ml, 2.445 mmol, 5 equivalents) at 0 °C and stirred at room temperature for 12 hours. After completion of the reaction (confirmed by LCMS), the reaction mixture was concentrated under vacuum to obtain the crude product compound, which was purified by reverse-phase column chromatography (Grace column: C18 40 μm, 40 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase), and the pure fraction was lyophilized to obtain N-(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)acetamide (86 mg, 0.207 mmol, 43.0% yield) as a yellow solid. 1H-NMR (400 MHz, DMSO-d6): δ 10.97 (s, 1H), 7.77 (t, J = 5.20 Hz, 1H), 7.38 (t, J = 7.60 Hz, 1H), 7.18 (t, J = 0.40 Hz, 1H), 7.07 (d, J = 7.60 Hz, 1H), 5.10 (q, J = 5.20 Hz, 1H), 4.35 (q, J = 16.80 Hz, 2H), 3.21 - 3.13 (m, 4H), 3.00 (q, J = 6.40 Hz, 4H), 2.57 (s, 1H), 2.02 (q, J = 5.20 Hz, 1H), 1.76 (s, 3H), 1.48 - 1.36 (m, 6H), 0.84 (t, J = 7.60 Hz, 3H). LCMS: 415.1 (M + H), Method: Mobile Phase: A: 0.1% FA aqueous solution, Mobile Phase: B: ACN, Column: Atlantis dC18 (50×4.6 mm) 5μm, Flow Rate: 1.5 mL / min, Rt (min): 1.622, Area %: 98.693. HPLC: Method: Mobile Phase: A: 0.1% TFA aqueous solution, Mobile Phase: B: ACN, Column: X - Bridge C8 (50×4.6) mm, 3.5μm Flow Rate: 2.0 mL / min, Rt (min): 1.789, Area %: 99.953.

[0207] Hydrochloride form of Compound 51 JPEG2025522795000187.jpg22127JPEG2025522795000188.jpg27127

[0208] General procedure for the preparation of Compound 2g A solution of compound 1g (5.0 g, 48 mmol, 1 equiv) and tert-butylchlorodiphenylsilane (13.2 g, 48 mmol, 1.0 equiv) in DMF (50 mL) was added with DIEA (18.6 g, 144 mmol, 3.0 equiv), and stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis PE / EA = 2:1 and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain compound 2g (7.2 g, 44%) as a colorless oily substance. TLC: PE / EA = 2 / 1 KMnO4 R f (Compound 1g) = 0.1 R f (Compound 2g) = 0.6 1 1H NMR (400 MHz, CDCl3): δ 7.66 (d, J = 5.7 Hz, 4H), 7.38 (d, J = 6.8 Hz, 6H), 3.64 (dd, J = 17.5, 4.3 Hz, 4H), 1.63 - 1.50 (m, 5H), 1.43 (d, J = 6.3 Hz, 2H), 1.04 (s, 10H).

[0209] General procedure for the preparation of compound 3g A solution of oxalyl chloride (3.2 g, 25.2 mmol, 1.2 equiv) in DCM (40 mL) was added dropwise to a solution of DMSO (7.5 ml, 1.0 V) in DCM (35 mL) at -78 °C under a nitrogen atmosphere over 15 minutes and stirred at the same temperature for 15 minutes. A solution of Compound 2g (7.2 g, 20.9 mmol, 1.0 equiv) in DCM (20 mL) was added dropwise at -78 °C over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, then TEA (6.35 g, 62.9 mmol, 3.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (50 mL) and washed with 10% citric acid (30 mL), water (50 mL), and brine (50 mL), and dried over Na2SO4. The solution was filtered and concentrated under vacuum at a temperature below 30 °C to obtain Compound 3g (7.03 g) as a clear oily substance, which was used without further purification. TLC: PE / EA = 2 / 1 R f (Compound 2g) = 0.6 R f (Compound 3g) = 0.8 1 1H NMR (400 MHz, CDCl3): δ 9.74 (s, 1H), 7.66 (d, J = 7.3 Hz, 4H), 7.48 - 7.33 (m, 6H), 3.67 (t, J = 6.1 Hz, 2H), 2.41 (t, J = 7.3 Hz, 2H), 1.79 - 1.68 (m, 2H), 1.60 (dd, J = 14.3, 6.6 Hz, 3H), 1.05 (s, 9H).

[0210] General procedure for the preparation of Compound 5g In a solution of compound 4a (2.2 g, 5.1 mmol, 1 equiv) and compound 3g (7.03 g, 20.6 mmol, 4.0 equiv) in DCE / DMF (10 mL), acetic acid (2.2 mL) and molecular sieve (7.03 g) were added, and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.6 g, 25.6 mmol, 5 equiv) was added portionwise, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA, and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain compound 5g (1.03 g, 27%) as a white solid. TLC: EA = 100% R f (Compound 4a) = 0.5 R f (Compound 5g) = 0.7 LC-MS: 755.60 [M+1] +

[0211] General procedure for the preparation of compound 6g To a solution of compound 5g (1.03 g, 1.37 mmol, 1 equiv) in THF, TBAF / THF (13.7 mL, 1 N, 13.7 mmol, 10 equiv) was added, and the mixture was stirred at room temperature for 4 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain compound 6g (580 mg, 82%) as a white solid. TLC: EA = 100% R f (Compound 5g) = 0.7 Rf (Compound 6g) = 0.4 LC-MS: 517.30 [M+1] +

[0212] General procedure for the preparation of Compound 51 To a solution of Compound 6g (200 mg, 0.387 mmol, 1 equiv) in methanol (1.0 ml) was added HCl (2.0 mL, 2 N, 4.0 mmol, 10.0 equiv). The resulting solution was stirred at room temperature for 24 h. The reaction mixture was lyophilized to give Compound 51 (164.3 mg, 87%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 6g) = 0.4 R f (Compound 51) = 0.2 LC-MS: 417.40 [M+1] + 1 1H NMR (400 MHz, D2O): δ 7.88 (d, J = 7.2 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 5.10 - 5.01 (m, 1H), 4.53 (t, J = 13.1 Hz, 3H), 3.56 (d, J = 6.9 Hz, 4H), 3.32 (t, J = 5.7 Hz, 2H), 2.76 (dd, J = 27.9, 6.5 Hz, 4H), 2.47 - 2.32 (m, 1H), 2.16 (s, 1H), 1.54 - 1.07 (m, 11H).

[0213] Hydrochloride form of Compound 52 JPEG2025522795000194.jpg17127JPEG2025522795000195.jpg35127

[0214] General procedure for the preparation of Compound 2h To a solution of compound 1h (0.5 g, 3.47 mmol, 1.0 equiv) in THF (5 mL) was added dropwise a solution of LiAlH4 (145 mg, 3.82 mmol, 1.1 equiv) in THF (5 mL) at 0 °C under a nitrogen atmosphere, and then the mixture was stirred at room temperature for 4 h. After completion of the reaction (confirmed by TLC analysis, PE / EA = 2 / 1, Rf ca. 0.6, KMnO4), the reaction mixture was diluted with MTBE (30 mL), and Na2SO4·10H2O was added. The mixture was stirred for 10 min, filtered, and concentrated to give compound 2h (350 mg, 77%) as a clear oil, which was used without further purification. TLC: PE / EA = 2:1, KMnO4 Rf (compound 1h) = 0.2 Rf (compound 2h) = 0.6 1 H NMR (400 MHz, CDCl3): δ 3.62 (t, J = 5.7 Hz, 2H), 1.59 - 1.44 (m, 3H), 1.28 (d, J = 15.7 Hz, 5H), 1.17 (s, 3H), 0.85 (d, J = 5.6 Hz, 7H).

[0215] General procedure for the preparation of compound 3h To a solution of compound 2h (600 mg, 4.61 mmol, 1 equiv) in DCM (30 mL) was added PCC (1.99 g, 9.22 mmol, 2.0 equiv), and the mixture was stirred at room temperature for 4 h. After completion of the reaction (confirmed by TLC analysis, PE / EA = 3:1), silica gel (4.04 g) was added to the reaction mixture, and the mixture was stirred for an additional 10 min. The reaction mixture was filtered and concentrated under reduced pressure at < 30 °C to give compound 3h (714 mg) as a clear oil, which was used without further purification. TLC: PE / EA = 4 / 1 KMnO4 R f (compound 2h) = 0.4 R f (compound 3h) = 0.7 11H NMR (400 MHz, CDCl3): δ 9.75 (s, 1H), 2.41 (s, 2H), 1.56 (d, J = 29.1 Hz, 3H), 1.24 (d, J = 50.5 Hz, 5H), 0.85 (s, 6H).

[0216] General procedure for the preparation of compound 5h JPEG2025522795000198.jpg41127 To a solution of compound 4a (799 mg, 1.86 mmol, 1 equiv) and compound 3h (714 mg, 5.57 mmol, 3.0 equiv) in DCE / DMF (6 mL) were added acetic acid (1 mL) and molecular sieve (1.0 g). The mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. NaBH3CN (467 mg, 7.43 mmol, 4 equiv) was added portionwise, and the mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA, and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to give compound 5h (275 mg, 27%) as a white solid. TLC: EA = 100% R f (Compound 4a) = 0.4 R f (Compound 5h) = 0.6 LC-MS: 543.55 [M + 1] +

[0217] General procedure for the preparation of compound 52 JPEG2025522795000199.jpg38127 To a solution of compound 5h (200 mg, 0.368 mmol, 1.0 equiv) in methanol (1.0 ml) was added HCl (1.8 mL, 2N, 3.7 mmol, 10.0 equiv). The resulting solution was stirred at room temperature for 24 h. The reaction mixture was lyophilized to give compound 52 (170.7 mg, 89%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 5h) = 0.6 R f (Compound 52) = 0.1 LC-MS: 443.45 [M+1] + 1 1H NMR (400 MHz, D2O): δ 7.84 (d, J = 6.9 Hz, 1H), 7.71 (dd, J = 21.3, 7.2 Hz, 2H), 5.04 (d, J = 9.6 Hz, 1H), 4.51 (t, J = 13.0 Hz, 2H), 3.51 (s, 4H), 2.73 (t, J = 14.1 Hz, 4H), 2.39 (d, J = 9.7 Hz, 1H), 2.14 (s, 1H), 1.31 (dd, J = 76.5, 36.4 Hz, 7H), 1.02 (s, 4H), 0.82 (s, 2H), 0.57 (d, J = 5.5 Hz, 6H).

[0218] Hydrochloride form of Compound 53 JPEG2025522795000200.jpg30127JPEG2025522795000201.jpg28127

[0219] General procedure for the preparation of Compound 2i A solution of oxalyl chloride (0.9 g, 6.9 mmol, 1.2 eq) in DCM (5 mL) was added dropwise to a solution of DMSO (1 mL) in DCM (10 mL) at -78 °C under a nitrogen atmosphere over 15 minutes, and the mixture was stirred at the same temperature for 15 minutes. A solution of compound 1i (1.0 g, 5.7 mmol, 1.0 eq) in DCM (5 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, and then TEA (1.7 g, 17.1 mmol, 3.0 eq) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, PE / EA = 4 / 1, Rf ≈ 0.4, KMnO4 or ninhydrin), the reaction mixture was diluted with DCM (10 mL). The mixture was washed with 10% citric acid (10 mL), water (10 mL), and brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum at a temperature below 30 °C to obtain compound 2i (0.9 g, crude product) as a yellow oily substance, which was used without further purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin Rf (compound 1i) = 0.2 Rf (compound 2i) = 0.4 LC-MS: No found. 1 1H NMR (400 MHz, CDCl3): δ 9.79 (s, 1H), 4.89 (s, 1H), 3.39 (s, 2H), 2.64 (d, J = 35.8 Hz, 2H), 1.40 (s, 10H).

[0220] General procedure for the preparation of compound 6i To a solution of compound 2i (2.3 g, 13.3 mmol, 2.0 equiv) and compound 5 (2.0 g, 6.6 mmol, 1.0 equiv) in DCE / DMF (1 / 1, 40 mL) was added acetic acid (10 mL), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.7 g, 26.6 mmol, 4.0 equiv) was added portionwise, and the mixture was stirred at room temperature for 18 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA and LCMS), the reaction mixture was diluted with DCM (200 mL). The mixture was washed with water (2×20 mL) and brine (2×20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / methanol: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain compound 6i (0.4 g, 26%) as a white solid. TLC: EA R f (Compound 2i) = 0.5 R f (Compound 6i) = 0.6 LC-MS: 459 [M + 1] + , 457.30 [M - 1] -

[0221] General procedure for the preparation of compound 53 To a solution of compound 6i (0.2 g, 0.4 mmol, 1.0 equiv) in DCM / H2O (1 / 1, 2 mL) was added TFA (4 mmol, 10.0 equiv). The resulting solution was stirred at room temperature for 24 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by P-HPLC eluting with acetonitrile / H2O (30% - 40%) to obtain compound 53 (105 mg, 65%) as a white solid. LC-MS: 359.30 [M + 1] + , 357.20 [M - 1] - 11H NMR (400 MHz, CD3OD): δ 7.51 - 7.36 (m, 2H), 7.29 (d, J = 7.5 Hz, 1H), 5.14 (dd, J = 12.9, 4.2 Hz, 1H), 4.46 (s, 2H), 3.31 (d, J = 9.3 Hz, 5H), 3.14 (d, J = 6.5 Hz, 2H), 2.92 (dd, J = 19.2, 11.4 Hz, 3H), 2.78 (d, J = 17.2 Hz, 1H), 2.54 (d, J = 9.8 Hz, 1H), 2.19 (d, J = 5.7 Hz, 1H), 1.82 (s, 2H), 1.57 - 1.44 (m, 2H), 0.88 (t, J = 6.8 Hz, 3H).

[0222] Hydrochloride form of Compound 55

[0223] 3-(4-((4-Aminobutyl)(5-aminopentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000205.jpg15127JPEG2025522795000206.jpg43127JPEG2025522795000207.jpg38127

[0224] Step - 1: (5-((tert-Butyldimethylsilyl)oxy)pentyl)carbamic acid tert-butyl In a solution of tert-butyl (5-hydroxypentyl)carbamate 1 (1.0 g, 4.92 mmol, 1.0 equiv) in DCM (20 mL) at 0 °C, imidazole (0.837 g, 12.30 mmol, 2.5 equiv) and TBS-Cl (1.112 g, 7.38 mmol, 1.5 equiv) were added, and the mixture was stirred at room temperature for 2 h. After completion of the reaction (confirmed by TLC, 50% EtOAc / petroleum ether, Rf ~0.5, KMnO4), the reaction mixture was quenched with H2O (20 mL) and extracted with DCM (2 × 20 mL). The organic layer was dried over Na2SO4 and concentrated under vacuum to give the crude product of tert-butyl (5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 2 (1.5 g, 4.70 mmol, 96% yield) as a yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 6.74 (s, 1H), 3.54 - 3.56 (m, 2H), 2.88 - 2.90 (m, 2H), 1.37 - 1.43 (m, 6H), 1.156 (s, 9H), 0.86 (s, 9H), 0.03 (s, 6H),

[0225] Step - 2: tert-Butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate JPEG2025522795000209.jpgTo a stirred solution of tert-butyl (5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 2 (1.5 g, 4.72 mmol, 1.0 equiv) in THF (20 mL) at 0 °C was added n-BuLi (2.5 M solution in hexanes, 2.267 mL, 5.67 mmol, 1.2 equiv), and the reaction mixture was stirred at 0 °C for 15 minutes. At the same temperature, a solution of (Boc)2O (1.316 mL, 5.67 mmol, 1.2 equiv) in THF (5 mL) was added to the reaction mixture. The resulting reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction (confirmed by TLC analysis, 10% EtOAc / petroleum ether, Rf ~0.7, KMnO4), the reaction mixture was quenched with water (10 mL) and extracted with DCM (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the crude product of tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 3 (2.0 g, 4.62 mmol, 98% yield) as a pale yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 3.55-3.58 (m, 2H), 3.44-3.48 (m, 2H), 1.25-1.43 (m, 6H), 1.12 (s, 18H), 0.88 (s, 9H), 0.03 (s, 6H)

[0226] Step-3: tert-butyl (tert-butoxycarbonyl)(5-hydroxypentyl)carbamate JPEG2025522795000210.jpg12127 At room temperature, tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 3 (1.9 g, 4.55 mmol) was stirred in THF (20 mL) to form a solution. The resulting solution was stirred at room temperature overnight. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc / petroleum ether, Rf ≈ 0.2, KMnO4), the reaction mixture was quenched with water (10 mL) and extracted with DCM (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product as a pale yellow liquid. The crude product compound was purified by Isolera chromatography (Biotage R snap cartridge, KP-Sil, 100 g, 100-200 silica gel) using 15-20% EtOAc in petroleum ether. The fractions were collected and concentrated under vacuum to obtain tert-butyl (tert-butoxycarbonyl)(5-hydroxypentyl)carbamate 4 (1.0 g, 3.27 mmol, 71.9% yield) as a colorless liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 4.36 (t, J = 4.80 Hz, 1H), 3.40-3.47 (m, 2H), 3.37-3.40 (m, 2H), 1.48-1.50 (m, 2H), 1.44 (s, 18H), 1.40-1.42 (m, 2H), 1.25-1.27 (m, 2H),

[0227] Step - 4: tert-butyl (tert-butoxycarbonyl)(5-oxopentyl)carbamate JPEG2025522795000211.jpg A solution of oxalyl chloride (0.577 mL, 6.59 mmol, 2.0 equiv) and CH2Cl2 (20 mL) was added dropwise with DMSO (0.936 mL, 13.18 mmol, 4.0 equiv) in CH2Cl2 (5 mL) at -78 °C, and the mixture was stirred at the same temperature for 15 minutes. To this was added dropwise a solution of tert-butyl (tert-butoxycarbonyl)(5-hydroxypentyl)carbamate 4 (1.0 g, 3.30 mmol, 1.0 equiv) in CH2Cl2 (5 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 30 minutes, and then triethylamine (2.76 mL, 19.78 mmol, 6.0 equiv) was added dropwise at -78 °C. Next, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc / petroleum ether, Rf about 0.6, KMnO4), the reaction mixture was diluted with DCM (40 mL), washed with 10% aqueous citric acid solution (1 x 40 mL) and water (1 x 40 mL), dried over Na2SO4, filtered, and concentrated under vacuum (below 30 °C) to obtain tert-butyl (tert-butoxycarbonyl)(5-oxopentyl)carbamate 5 (0.95 g, 3.08 mmol, 93% yield) as a pale yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 0.00 (s, 1H), 3.47 (m, 2H), 2.42-2.46 (m, 2H), 1.47-1.50 (m, 4H), 1.42 (s, 18H),

[0228] Step-5: tert-butyl (tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)carbamate JPEG2025522795000212.jpg41127(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 6 (0.7 g, 1.626 mmol, 1.0 equiv) and (tert-butoxycarbonyl)(5-oxopentyl)carbamic acid tert-butyl 5 (0.980 g, 3.25 mmol, 2.0 equiv) in CH2Cl2 (10 mL) and DMF (10 mL) were stirred, and trifluoroacetic acid (0.501 mL, 6.50 mmol, 4.0 equiv) was added. The mixture was stirred at 0 °C for 10 min. Sodium triacetoxyborohydride (1.378 g, 6.50 mmol, 4.0 equiv) was added portionwise at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf ca. 0.7), the reaction mixture was quenched with ice-cold water (20 mL) and extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude product as a pale yellow liquid. The crude product compound was purified by Isolera chromatography (Biotage R snap cartridge, KP-Sil, 100 - 200 g, silica gel) using 60 - 70% ethyl acetate in petroleum ether to give (tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)carbamic acid tert-butyl 7 (0.65 g, 0.891 mmol, 54.8% yield) as an off-white solid. 11H-NMR (400 MHz, DMSO-d6): δ 0.00 (s, 1H), 7.35 - 7.39 (m, 1H), 7.18 - 7.20 (m, 1H), 7.06 - 7.08 (m, 1H), 6.75 - 6.78 (m, 1H), 5.08 - 5.13 (m, 1H), 4.27 - 4.41 (m, 2H), 3.45 - 3.46 (m, 2H), 2.89 - 2.92 (m, 4H), 2.74 - 2.74 (m, 4H), 2.51 - 2.52 (m, 2H), 2.50 - 2.50 (m, 1H), 2.00 - 2.03 (m, 1H), 1.45 - 1.49 (m, 15H), 1.42 (s, 18H), 1.20 - 1.26 (m, 2H), LCMS: 616.4 (M - Boc). Method: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: CAN, Flow rate: 1.5 ml / min HPLC: Method: Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: acetonitrile, Flow rate: 2.0 ml / min. Column: X - Bridge C8 (50×4.6) mm, 3.5 μm. RT: 4.972 min, Area: 98.095%

[0229] Step - 6: 3-(4-((4 - aminobutyl)(5 - aminopentyl)amino)-1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione dihydrochloride (tert-Butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)carbamic acid tert-butyl 7 (0.35 g, 0.489 mmol, 1.0 equiv) in dichloromethane (5 mL) was stirred and HCl (4 M ethyl acetate solution, 10 mL) was added at 0 °C. The mixture was then stirred at room temperature for 3 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum at < 40 °C to afford a pale yellow solid. The obtained solid was dissolved in water (10 mL), washed with MTBE (2 x 10 mL), and the aqueous layer was lyophilized to give 3-(4-((4-aminobutyl)(5-aminopentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl (0.2 g, 0.404 mmol, 83% yield) as a pale yellow solid. 1 1H-NMR (400 MHz, CD3OD): δ 8.20 (s, 2H), 7.83 (s, 1H), 5.22 - 5.27 (m, 1H), 4.84 - 4.88 (m, 2H), 3.72 (m, 4H), 2.87 - 2.97 (m, 6H), 2.63 - 2.66 (m, 1H), 2.27 - 2.28 (m, 1H), 1.60 - 1.70 (m, 7H), 1.42 - 1.47 (m, 3H). LCMS: 416.3 (M+H). Method: Column: XBridge C8 (50×4.6 mm) 3.5 μm, Mobile phase: A: 0.1% TFA in H2O, Mobile phase: B: 0.1% TFA in acetonitrile, Flow rate: 1.5 ml / min. HPLC: Method: Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: methanol, Flow rate: 0.7 ml / min. Column: Atlantis dC18 (250×4.6) mm, 5 μm. RT: 12.223 min, Area: 98.472%.

[0230] Hydrochloride form of Compound 56 JPEG2025522795000214.jpg23127JPEG2025522795000215.jpg30127

[0231] General procedure for the preparation of compound 2l JPEG2025522795000216.jpg21127 A solution of compound 1l (10 g, 85.6 mmol, 1 equiv) and tert-butylchlorodiphenylsilane (23.25 g, 85.6 mmol, 1.0 equiv) in DMF (100 mL) was added with DIEA (32.7 g, 254 mmol, 3.0 equiv), and stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis PE / EA = 2:1 and LCMS), the reaction mixture was diluted with DCM (20 mL), washed with water (3 × 20 mL) and brine (20 mL), dried over Na2SO4, and filtered. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to give compound 2l (10.27 g, 35%) as a colorless oil. TLC: PE / EA = 2 / 1 KMnO4 R f (Compound 1l) = 0.1 R f (Compound 2l) = 0.6 1 1H NMR (400 MHz, CDCl3): δ 7.67 (d, J = 5.6 Hz, 4H), 7.39 (d, J = 6.8 Hz, 6H), 3.72 - 3.56 (m, 4H), 1.58 (t, J = 14.0 Hz, 5H), 1.31 (d, J = 37.5 Hz, 6H), 1.05 (s, 9H).

[0232] General procedure for the preparation of compound 3l A solution of oxalyl chloride (2.14 g, 16.8 mmol, 1.2 equiv) in DCM (20 mL) was added dropwise to a solution of DMSO (5 mL, 1.0 V) in DCM (20 mL) at -78 °C under a nitrogen atmosphere over 15 minutes and stirred at the same temperature for 15 minutes. A solution of compound 2l (5.0 g, 14.02 mmol, 1.0 equiv) in DCM (10 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, then TEA (4.3 g, 42.1 mmol, 3.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (50 mL). The mixture was washed with 10% citric acid (30 mL), water (50 mL), and brine (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum at a temperature below 30 °C to obtain compound 3l (4.9 g) as a clear oily substance, which was used without further purification. TLC: PE / EA = 2 / 1 R f (Compound 2l) = 0.6 R f (Compound 3l) = 0.8

[0233] General procedure for the preparation of compound 5l To a solution of compound 4a (1.5 g, 3.45 mmol, 1 equiv) and compound 3l (4.9 g, 13.8 mmol, 4.0 equiv) in DCE / DMF (15 mL) were added acetic acid (1.5 mL) and molecular sieves (5.0 g), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.08 g, 17.2 mmol, 5 equiv) was added portionwise, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA, and LCMS), the reaction was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain compound 5l (1.03 g, 29%) as a white solid. TLC: EA = 100% R f (Compound 4a) = 0.5 R f (Compound 5l) = 0.7 LC-MS: 769.60 [M+1] +

[0234] General procedure for the preparation of Compound 6l JPEG2025522795000219.jpg41127 To a solution of Compound 5l (1.03 g, 1.34 mmol, 1 equiv) in THF was added TBAF / THF (13.4 ml, 1 N, 13.4 mmol, 10 equiv), and the mixture was stirred at room temperature for 4 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain Compound 6l (620 mg, 87%) as a white solid. TLC: EA = 100% R f (Compound 5l) = 0.7 R f (Compound 6l) = 0.4 LC-MS: 531.45 [M+1] +

[0235] General procedure for the preparation of Compound 56 JPEG2025522795000220.jpg37127 To a solution of Compound 6l (300 mg, 0.566 mmol, 1 equiv) in methanol (1 ml) was added HCl (2.8 mL, 2 N, 5.6 mmol, 10 equiv). The resulting solution was stirred at room temperature for 24 h. The reaction mixture was lyophilized to obtain Compound 56 (231 mg, 81%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 15-A) = 0.4 R f (Compound 56) = 0.2 LC-MS: 431.40 [M+1] + 1 H NMR (400 MHz, D2O): δ 7.88 (d, J = 7.0 Hz, 1H), 7.74 (dd, J = 22.2, 7.6 Hz, 2H), 5.07 (d, J = 8.9 Hz, 1H), 4.54 (t, J = 13.1 Hz, 2H), 3.55 (d, J = 5.1 Hz, 4H), 3.34 (s, 2H), 2.87 - 2.67 (m, 4H), 2.41 (d, J = 8.3 Hz, 1H), 2.17 (s, 1H), 1.35 (dd, J = 63.6, 23.7 Hz, 9H), 1.10 (s, 4H).

[0236] Hydrochloride form of Compound 57 JPEG2025522795000221.jpg17127JPEG2025522795000222.jpg32127JPEG2025522795000223.jpg31127

[0237] General procedure for the preparation of compound 2m To a solution of 1m (14.3 g, 138.6 mmol, 1.0 equiv) and TEA (42.0 g, 415.8 mmol, 3.0 equiv) in DCM (150 mL) was added dropwise a solution of (Boc)2O (36.3 g, 166.3 mmol, 1.2 equiv), then DMAP (1.7 g, 13.9 mmol, 0.1 equiv) was added, and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with DCM (300 mL), washed with brine (3 × 50 mL), dried over Na2SO4, filtered, and concentrated under vacuum to afford compound 2m (7.1 g, 24%) as a colorless oil, which was used further without purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin R f (Compound 1m) = 0.2 R f (Compound 2m) = 0.4 LC-MS: No found.

[0238] General procedure for the preparation of compound 3m JPEG2025522795000225.jpg22127A solution of oxalyl chloride (5.4 g, 42.4 mmol, 1.2 equiv) in DCM (35 mL) was added dropwise to a solution of DMSO (7 ml, 1.0 V) in DCM (70 mL) at -78 °C under a nitrogen atmosphere over 15 minutes and stirred at the same temperature for 15 minutes. A solution of compound 2m (7.1 g, 35.3 mmol, 1.0 equiv) in DCM (35 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, then TEA (10.7 g, 105.9 mmol, 3.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, PE / EA = 4 / 1, Rf ca. 0.4, KMnO4 or ninhydrin), the reaction mixture was diluted with DCM (70 mL). The mixture was washed with 10% citric acid solution (35 mL), water (35 mL) and brine (35 mL) and dried over Na2SO4. The resulting mixture was filtered and concentrated under vacuum at less than 30 °C to give compound 3m (7.4 g) as a yellow oily substance, which was used further without purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin R f (Compound 2m) = 0.2 R f (Compound 3m) = 0.4 LC-MS: No found.

[0239] General procedure for the preparation of compound 5m To a solution of Compound 3m (7.8 g, 41.7 mmol, 1.8 equiv) and Compound 4a (6.0 g, 23.1 mmol, 1.0 equiv) in DCE / DMF (1 / 1, 120 mL) was added acetic acid (30 mL). The reaction mixture was stirred at room temperature for 2 h. At this stage, NaBH3CN (5.8 g, 92.6 mmol, 4.0 equiv) was added portionwise at 0 °C and the mixture was stirred at room temperature for 18 h. After completion of the reaction, the reaction mixture was diluted with DCM (600 mL). The mixture was washed with water (2 × 60 mL) and brine (2 × 60 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 3 / 1) to afford Compound 5m (2.0 g, 19%) as a white solid. TLC: EA R f (Compound 3m) = 0.4 R f (Compound 5m) = 0.6 LC-MS: 445.35 [M+1] + , 443.30 [M-1] -

[0240] General procedure for the preparation of Compound 6m To a solution of Compound 5m (2.0 g, 4.5 mmol, 1.0 equiv) and Compound 6 (2.6 g, 45 mmol, 10.0 equiv) in DCE / DMF (40 ml, 1 / 1) was added acetic acid (10 ml). The reaction mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. NaBH3CN (1.1 g, 18 mmol, 4.0 equiv) was added portionwise and the mixture was stirred at room temperature for 18 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA and LCMS), the reaction mixture was diluted with DCM (200 mL). The mixture was washed with water (2 × 40 mL) and brine (2 × 40 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (eluting with DCM / EA: 4:1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to afford Compound 6m (0.87 g, 39%) as a white solid. TLC: EA = 100% Rf (Compound 5m) = 0.5 R f (Compound 6m) = 0.65 LC-MS: 487.40 [M+1] + , 485.40 [M-1] -

[0241] General procedure for the preparation of compound 57 To a solution of compound 6m (870 mg, 1.8 mmol, 1.0 eq) in methanol (4 ml) was added HCl / EA (9 mL, 2N, 18 mmol, 10.0 eq). The resulting solution was stirred at 30 °C for 18 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by P-HPLC (acetonitrile / H2O: 30% - 40%) to give compound 57 (144 mg, 30%) as a white solid. LC-MS: 387.40 [M+1] + , 385.15 [M-1] - 1 H NMR (400 MHz, CD3OD): δ 7.59 (s, 3H), 5.18 (d, J = 10.0 Hz, 1H), 4.57 (s, 2H), 3.47 (s, 3H), 3.00 - 2.74 (m, 5H), 2.55 (d, J = 10.6 Hz, 1H), 2.21 (s, 1H), 1.73 - 1.45 (m, 7H), 1.39 (d, J = 6.4 Hz, 3H), 0.90 (t, J = 6.3 Hz, 4H).

[0242] Hydrochloride form of Compound 58 To a solution of compound 61 (0.2 g, 0.5 mmol, 1.0 eq) in THF (2 mL) was added HCl / EA (2.4 ml, 2N, 4.8 mmol, 10.0 eq), and the mixture was stirred at 30 °C for 2 days. The resulting mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 30% - 40%) to give compound 58 (102 mg, 52%) as a white solid. LC-MS: 402.30 [M+1] + , 400.20 [M-1] - 1 1H NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.25 (s, 1H), 6.90 (d, J = 4.0 Hz, 1H), 6.73 (d, J = 4.0 Hz, 1H), 5.57 (s, 1H), 5.59 (d, J = 12.0 Hz, 1H), 4.26 - 3.85 (m, 2H), 3.05 (s, 2H), 3.85 - 3.75 (m, 1H), 2.70 - 2.50 (m, 1H), 2.31 - 2.20 (m, 1H), 1.99 (s, 1H), 1.55 (s, 2H), 0.89 (s, 3H).

[0243] Hydrochloride form of Compound 60 JPEG2025522795000230.jpg28127JPEG2025522795000231.jpg31127

[0244] General procedure for the preparation of compound 2p A solution of oxalyl chloride (2.9 g, 22.85 mmol, 1.2 equiv) in DCM (10 mL) was added dropwise to a solution of DMSO (4.2 mL, 1.0 V) in DCM (20 mL) at -78 °C under a nitrogen atmosphere over 15 minutes, and the mixture was stirred at the same temperature for 15 minutes. A solution of compound 1p (4.2 g, 19.32 mmol, 1.0 equiv) in DCM (10 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, and then TEA (5.8 g, 57.98 mmol, 3.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, PE / EA = 4 / 1, Rf ~0.6, KMnO4 or ninhydrin), the reaction mixture was diluted with DCM (50 mL). The mixture was washed with 10% citric acid (30 mL), water (50 mL), and brine (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum at a temperature below 30 °C to obtain compound 2p (4.1 g, crude product) as a yellow oily substance, which was used without further purification. TLC: PE / EA = 2:1, KMnO4, ninhydrin Rf (compound 1p) = 0.2 Rf (compound 2p) = 0.6 LC-MS: No found. 1 1H NMR (400 MHz, DMSO-d6): δ 9.63 (s, 1H), 6.75 (s, 1H), 2.86 (d, J = 5.2 Hz, 2H), 2.38 (s, 2H), 1.56 - 1.42 (m, 2H), 1.34 (s, 11H), 1.20 (d, J = 5.4 Hz, 3H).

[0245] General procedure for the preparation of compound 5p In a solution of compound 4a (2.07 g, 4.81 mmol, 1 equiv) and compound 2p (4.1 g, 19.32 mmol, 4.0 equiv) in DCE / DMF (20 mL), acetic acid (2.1 mL) and molecular sieve (4.1 g) were added, and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.51 g, 24.07 mmol, 5 equiv) was added portionwise, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA, and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3×20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain compound 5p (1.0 g, 34%) as a white solid. TLC: EA = 100% R f (Compound 4a) = 0.5 R f (Compound 5p) = 0.6 LC-MS: 630.60[M + 1] +

[0246] General procedure for the preparation of compound 60 To a solution of compound 5p (200 mg, 0.318 mmol, 1 equiv) in methanol (1 ml), HCl (1.6 mL, 2N, 3.2 mmol, 10 equiv) was added. The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was lyophilized to obtain compound 60 (130 mg, 76%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 5p) = 0.6 R f (Compound 60) = 0.1 LC-MS: 430.40[M + 1] + 11H NMR (400 MHz, D2O): δ 7.89 (d, J = 7.1 Hz, 1H), 7.80 - 7.67 (m, 2H), 5.13 - 5.01 (m, 1H), 4.53 (t, J = 13.3 Hz, 2H), 3.56 (s, 4H), 2.72 (d, J = 6.9 Hz, 6H), 2.40 (d, J = 7.9 Hz, 1H), 2.16 (s, 1H), 1.38 (t, J = 36.0 Hz, 8H), 1.12 (s, 4H).

[0247] Compound 61 JPEG2025522795000235.jpg29127

[0248] General procedure for the preparation of compound 2 Concentrated sulfuric acid (0.58 mL) was added to a solution of 1q (5.0 g, 50 mmol, 1.0 equiv) in MeOH (100 mL), and the mixture was stirred at 90 °C for 18 h. The solution was cooled to 0 °C, treated with NaHCO3 (464 mg), and stirred for 10 min. The reaction mixture was filtered, and the solvent was removed under vacuum. The residue was diluted with DCM (50 mL), dried over Na2SO4, filtered, and concentrated to give compound 2q (5.9 g) as a colorless oil, which was used without further purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin Rf (compound 1q) = 0.4 Rf (compound 2q) = 0.2 LC-MS: No found.

[0249] General procedure for the preparation of compound 3 A solution of oxalyl chloride (7.6 g, 59.9 mmol, 1.2 equiv) in DCM (33 mL) was added dropwise to a solution of DMSO (6.6 mL) in DCM (66 mL) at -78 °C under a nitrogen atmosphere over 15 minutes, and the mixture was stirred at the same temperature for 15 minutes. A solution of compound 2q (6.6 g, 49.9 mmol, 1.0 equiv) in DCM (33 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, and then TEA (15.2 g, 149.8 mmol, 3.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, PE / EA = 4 / 1, Rf ≈ 0.4, KMnO4 or ninhydrin), the reaction mixture was diluted with DCM (10 mL). The mixture was washed with 10% citric acid (10 mL), water (10 mL), and brine (10 mL), dried over Na2SO4, filtered, and concentrated under vacuum at a temperature below 30 °C to obtain compound 3q (7.45 g, crude product) as a yellow oily substance, which was used without further purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin Rf (compound 2q) = 0.2 Rf (compound 3q) = 0.4 LC-MS: No found. 1 1H NMR (400 MHz, CDCl3): δ 3.65 - 3.52 (m, 5H), 2.29 (td, J = 7.2, 3.8 Hz, 2H), 1.88 - 1.75 (m, 2H), 1.53 (ddt, J = 13.4, 7.4, 3.4 Hz, 2H).

[0250] General procedure for the preparation of compound 61 To a solution of Compound 3q (2.5 g, 19.4 mmol, 1.8 equiv) and Compound 5 (3.2 g, 10.8 mmol, 1.0 equiv) in DCE / DMF (60 mL, 1 / 1) was added acetic acid (15 mL), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (2.7 g, 43 mmol, 4.0 equiv) was added portionwise, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis, 100% EA and LCMS), the reaction mixture was diluted with DCM (400 mL). The mixture was washed with water (2 × 40 mL) and brine (2 × 40 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 2 / 1) and P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain Compound 61 (0.4 g, 13%) as a white solid. TLC: EA = 100% R f (Compound 5) = 0.5 R f (Compound 61) = 0.6 LC-MS: 416.35 [M + 1] + 1 1H NMR (400 MHz, CD3OD): δ 7.36 (dd, J = 25.2, 7.3 Hz, 2H), 7.17 (d, J = 7.8 Hz, 1H), 5.19 - 5.08 (m, 1H), 4.47 (s, 2H), 3.60 (s, 3H), 3.23 - 3.11 (m, 4H), 2.83 (dd, J = 43.9, 15.0 Hz, 2H), 2.57 (d, J = 13.6 Hz, 1H), 2.31 (t, J = 6.6 Hz, 2H), 2.18 (d, J = 12.4 Hz, 1H), 1.55 (dd, J = 34.0, 6.5 Hz, 6H), 0.88 (t, J = 6.9 Hz, 3H).

[0251] Compound 63 To a solution of compound 58 (120 mg, 0.3 mmol, 1.0 equiv) in THF (2 mL) were added HATU (136 mg, 0.4 mmol, 1.2 equiv) and DIEA (151 mg, 1.5 mmol, 5.0 equiv). The resulting solution was stirred at room temperature for 18 h. The obtained mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 30% - 40%) to give compound 63 (63.4 mg, 50%) as a white solid. LC-MS: 415.35[M+1] + , 413.25[M-1] - 1 H NMR (400 MHz, CD3OD): δ 7.82 (s, 1H), 7.36 (dd, J = 25.5, 6.7 Hz, 2H), 7.18 (d, J = 7.4 Hz, 1H), 5.13 (d, J = 12.6 Hz, 1H), 4.46 (s, 2H), 3.18 (d, J = 23.8 Hz, 5H), 2.83 (dd, J = 40.3, 16.7 Hz, 2H), 2.65 (s, 3H), 2.56 (d, J = 13.2 Hz, 1H), 2.14 (s, 3H), 1.55 (d, J = 37.7 Hz, 6H), 1.32 (d, J = 26.3 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H).

[0252] Hydrochloride form of Compound 66 JPEG2025522795000240.jpg16127JPEG2025522795000241.jpg39127JPEG2025522795000242.jpg35127

[0253] General procedure for the preparation of compound 2 To a solution of compound 1 (0.5 g, 8.77 mmol, 1.0 equiv) in DCM (10 mL), TEA (2.7 g, 26.3 mmol, 3.0 equiv) and 9-fluorenylmethyl chloroformate (3.4 g, 13.2 mmol, 1.0 equiv) were added. The mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to afford compound 2 (0.4 g, 16%) as a white solid. TLC: PE / EA = 1 / 1, ninhydrin R f (Compound 1) = 0.1 R f (Compound 2) = 0.8 1 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 6.4 Hz, 2H), 7.59 (d, J = 6.9 Hz, 2H), 7.45 - 7.39 (m, 2H), 7.39 - 7.27 (m, 2H), 5.84 (brs, 1H), 5.25 - 5.05 (m, 2H), 4.82 (s, 1H), 4.45 - 4.30 (m, 2H), 4.22 (s, 1H), 3.82 (s, 2H).

[0254] General procedure for the preparation of compound 3 To a solution of compound 2 (0.3 g, 1.1 mmol, 1.0 equiv) in DCM (10 mL), acrolein (69 mg, 1.2 mmol, 1.1 equiv) and HG 2nd(34 mg, 0.05 mmol, 0.05 eq) was added. The mixture was stirred at 50 °C overnight under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (10 mL). The mixture was washed with water (10 mL) and brine (10 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 4 / 1) to obtain Compound 3 (0.28 g, 85%) as a yellow solid. TLC: PE / EA = 3 / 1 R f (Compound 2) = 0.4 R f (Compound 3) = 0.2 1 1H NMR (400 MHz, CDCl3): δ 9.56 (d, J = 6.1 Hz, 1H), 7.75 (s, 2H), 7.57 (s, 2H), 7.47 - 7.25 (m, 4H), 6.77 (d, J = 14.8 Hz, 1H), 6.25 - 6.10 (m, 1H), 5.03 (s, 1H), 4.47 (s, 2H), 4.21 (s, 1H), 4.07 (s, 2H).

[0255] General procedure for the preparation of Compound 5 To a solution of Compound 4 (450 mg, 1.7 mmol, 1.0 eq) and Compound 3 (1.1 g, 3.5 mmol, 2.0 eq) in DCE / DMF (3 / 1, 8 mL) were added acetic acid (1.2 mL) and molecular sieves (1.0 g). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere, and then NaBH3CN (437 mg, 6.9 mmol, 4.0 eq) was added portionwise. The reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 2 / 1) to obtain Compound 5 (157 mg, 19%) as a white solid. TLC: EA = 100% R f (Compound 4) = 0.4 R f (Compound 5) = 0.5 LCMS: [C 32 H 30 N4O5] + Calculated value: 551, measured value: 551.

[0256] General procedure for the preparation of Compound 6 JPEG2025522795000246.jpg35127 To a solution of Compound 5 (137 mg, 0.25 mmol, 1.0 equiv) in DCE / DMF (3 / 1, 8 mL) were added butyraldehyde (71.8 mg, 1.0 mmol, 4.0 equiv), acetic acid (1.0 mL), and molecular sieves (1.0 g). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere, and NaBH3CN (62.6 mg, 1.0 mmol, 4.0 equiv) was added portionwise. The reaction was stirred at room temperature overnight under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 1 / 1) to give Compound 6 (41 mg, 27%) as a white solid. TLC: EA = 100% R f (Compound 5) = 0.5 R f (Compound 6) = 0.6 LCMS: [C 36 H 38 N4O5] + Calculated value: 607, measured value: 607.

[0257] General procedure for the preparation of Compound 66 A solution of compound 6 (47 mg, 0.1 mmol, 1.0 equiv) in DCM (4.0 mL) was added with piperidine (66.7 mg, 0.8 mmol, 8.0 equiv). The mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 20% - 40%) to obtain compound 66 (4.4 mg, 13%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 6) = 0.8 R f (Compound 66) = 0.1 LCMS: [C 21 H 28 N4O3] + Calculated value: 385, Measured value: 385. 1 H NMR (400 MHz, D2O): δ 7.79 (d, J = 6.3 Hz, 1H), 7.75 - 7.55 (m, 2H), 5.80 - 5.70 (m, 1H), 5.70 - 5.55 (m, 1H), 5.07 (d, J = 12.5 Hz, 1H), 4.55 - 4.42 (m, 2H), 4.20 - 4.05 (m, 2H), 3.61 - 3.37 (m, 2H), 3.34 (s, 2H), 2.87 - 2.68 (m, 1H), 2.50 - 2.05 (m, 1H), 1.37 - 1.24 (m, 2H), 1.24 - 1.05 (m, 2H), 0.67 (s, 3H).

[0258] Hydrochloride form of Compound 67 JPEG2025522795000248.jpg16127JPEG2025522795000249.jpg19127JPEG2025522795000250.jpg31127JPEG2025522795000251.jpg30127JPEG2025522795000252.jpg28127

[0259] General procedure for the preparation of compound 2 A solution of compound 1 (3.0 g, 34.8 mmol, 1.0 equiv) and tert-butylchlorodiphenylsilane (2.9 g, 10.5 mmol, 0.3 equiv) in THF (60 mL) was added with TEA (17.6 g, 174.3 mmol, 5.0 equiv) and DMAP (0.4 g, 3.5 mmol, 0.1 equiv). The mixture was stirred at room temperature for 17 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (50 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain compound 2 (3.0 g, 88%) as a transparent oily substance. TLC: PE / EA = 5 / 1 R f (Compound 1) = 0.1 R f (Compound 2) = 0.3 1 1H NMR (400 MHz, CDCl3): δ 7.75 - 7.65 (m, 4H), 7.50 - 7.77 (m, 6H), 4.35 (s, 2H), 4.19 (s, 2H), 1.05 (s, 9H).

[0260] General procedure for the preparation of compound 3 A solution of compound 2 (3.0 g, 9.3 mmol, 1.0 equiv) in DCM (30 mL) was added dropwise with a solution of TEA (2.8 g, 27.8 mmol, 3.0 equiv) and MsCl (1.59 g, 13.9 mmol, 1.5 equiv) in DCM (35 mL) over 15 min at 0 °C under a nitrogen atmosphere. The mixture was stirred at the same temperature for 15 min. Next, the reaction mixture was slowly warmed to room temperature and stirred for 4 h. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (50 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain compound 3 (2.9 g, 78%) as a transparent oily substance. TLC: PE / EA = 2 / 1 R f (Compound 2) = 0.60 R f (Compound 3) = 0.63 LCMS: [C 21 H 26 O4SSi] + Calculated value of [C 1 H NMR (400 MHz, CDCl3): δ 7.75 - 7.65 (m, 4H), 7.50 - 7.77 (m, 6H), 4.81 (s, 2H), 4.37 (s, 2H), 3.02 (s, 3H), 1.04 (s, 9H).

[0261] General procedure for the preparation of Compound 4 JPEG2025522795000255.jpg18127Compound 3 (2.9 g, 7.2 mmol, 1 equivalent) was dissolved in NH3 / MeOH (29 mL). The mixture was stirred at room temperature for 17 hours. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / MeOH: 20 / 1) to obtain Compound 5 (1.9 g, 82%) as a white solid. TLC: DCM / EA = 1 / 1 R f (Compound 3) = 0.9 R f (Compound 4) = 0.1 LCMS: [C 20 H 25 NOSi] + Calculated value: 324, Measured value: 324. 1 H NMR (400 MHz, CDCl3): δ 8.26 (s, 1H), 7.75 - 7.65 (m, 4H), 7.50 - 7.77 (m, 6H), 4.29 (s, 2H), 3.52 (s, 2H), 1.03 (s, 9H).

[0262] General procedure for the preparation of Compound 5 A solution of compound 4 (2.08 g, 6.4 mmol, 1 equiv) in DCM (20 mL) was added with Fmoc-Osu (2.6 g, 7.7 mmol, 1.2 equiv) and TEA (1.94 g, 19.3 mmol, 3.0 equiv). The mixture was stirred at room temperature for 17 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL) and washed with water (3 × 20 mL) and brine (20 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA: 1 / 1) to obtain compound 5 (2.6 g, 74%) as a white solid. TLC: DCM / EA = 1 / 1 R f (Compound 4) = 0.1 R f (Compound 5) = 0.8 LCMS: [C 35 H 35 NO3Si]+NA calculated value: 568, measured value: 568.

[0263] General procedure for the preparation of compound 6 A solution of compound 5 (2.6 g, 4.8 mmol, 1.0 equiv) in THF (1.0 ml) was added with TEA-3HF (7.7 g, 47.7 mmol, 10.0 equiv). The resulting solution was stirred at room temperature overnight. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction was quenched with H2O (20 mL). The reaction mixture was extracted with EA (20 mL × 3). The organic layer was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 3 / 1) to obtain compound 6 (1.0 g, 68%) as a white solid. TLC: PE / EA = 2 / 1 R f (Compound 5) = 0.8 R f (Compound 6) = 0.2 11H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.0 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 4.43 (d, J = 6.8 Hz, 2H), 4.27 (s, 2H), 4.22 (s, 1H), 4.03 (s, 2H).

[0264] General procedure for the preparation of Compound 7 MnO2 (4.24 g, 49.0 mmol, 10.0 equiv) was added to a solution of Compound 6 (1.5 g, 4.9 mmol, 1.0 equiv) in DCM (20 mL). The mixture was stirred at room temperature overnight. The reaction mixture was filtered and concentrated to give Compound 7 (1.15 m, 77%) as a white solid. It was used directly in the next step without further purification. TLC: PE / EA = 2 / 1 R f (Compound 6) = 0.2 R f (Compound 7) = 0.5 1 1H NMR (399 MHz, CDCl3): δ 9.18 (s, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 7.4 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 5.02 (s, 1H), 4.45 (d, J = 6.6 Hz, 2H), 4.27 - 4.10 (m, 2H).

[0265] General procedure for the preparation of Compound 9 To a solution of Compound 8 (1.0 g, 3.8 mmol, 1.0 equiv) and Compound 7 (1.2 g, 3.8 mmol, 1.0 equiv) in DCE / DMF (6 mL) were added acetic acid (1.5 mL) and molecular sieve (1.76 g). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Next, NaBH3CN (1.0 g, 15.3 mmol, 4.0 equiv) was added portionwise, and the mixture was stirred at room temperature for an additional 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to give Compound 9 (1.3 g, 63%) as a white solid. TLC: EA = 100% R f (Compound 8) = 0.4 R f (Compound 9) = 0.5

[0266] General procedure for the preparation of Compound 10 To a solution of Compound 9 (1.2 g, 2.2 mmol, 1.0 equiv) and butyraldehyde (0.6 g, 8.7 mmol, 4.0 equiv) in DCE / DMF (3 / 1, 4 mL) were added acetic acid (1.5 mL) and molecular sieve (1.8 g). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Next, NaBH3CN (546 mg, 8.7 mmol, 4.0 equiv) was added portionwise, and the mixture was stirred at room temperature for an additional 16 hours under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to give Compound 10 (315 mg, 24%) as a white solid. TLC: EA = 100% R f (Compound 9) = 0.5 R f(Compound 10) = 0.6 LCMS: [C 36 H 36 N4O5] + Calculated value: 605, measured value: 605.

[0267] General procedure for the preparation of Compound 11 JPEG2025522795000261.jpg39127 To a solution of Compound 10 (587 mg, 1.0 mmol, 1.0 equiv) in EtOH (5 mL) were added Lindlar catalyst (60 mg, 10%) and quinoline (12.5 mg, 0.1 mmol, 0.1 equiv). The mixture was stirred at room temperature for 30 minutes under N2 / H2 (50%). The reaction mixture was filtered and concentrated under reduced pressure, and purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to give Compound 11 (100 mg, 17%) as a white solid. TLC: DCM / EA = 1 / 1 R f (Compound 10) = 0.7 R f (Compound 11) = 0.72 LCMS: [C 36 H 38 N4O5] + Calculated value: 607, measured value: 607.

[0268] General procedure for the preparation of Compound 67 JPEG2025522795000262.jpg33127 To a solution of Compound 11 (100 mg, 0.17 mmol, 1.0 equiv) in DCM (5.0 mL) was added piperidine (280 mg, 3.3 mmol, 20.0 equiv). The mixture was stirred at room temperature for 17 hours. Next, the reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 20% - 45%) to give Compound 67 (14.2 mg, 19%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 11) = 0.9 R f (Compound 67) = 0.1 LCMS: [C 21 H 28N4O3] + Calculated value: 385, measured value: 385. 1 H NMR (400 MHz, D2O): δ 7.84 (dd, J = 15.1, 7.8 Hz, 2H), 7.69 (t, J = 7.8 Hz, 1H), 5.72 - 5.62 (m, 1H), 5.62 - 5.50 (m, 1H), 5.05 (dd, J = 13.3, 5.2 Hz, 1H), 4.51 (q, J = 17.5 Hz, 2H), 4.23 (d, J = 7.4 Hz, 2H), 3.66 - 3.52 (m, 2H), 3.38 - 3.21 (m, 2H), 2.85 - 2.65 (m, 2H), 2.39 (td, J = 12.9, 7.7 Hz, 1H), 2.22 - 2.05 (m, 1H), 1.35 - 1.20 (m, 2H), 1.19 - 1.06 (m, 2H), 0.63 (t, J = 7.3 Hz, 3H).

[0269] Hydrochloride form of Compound 68 JPEG2025522795000263.jpg15127JPEG2025522795000264.jpg17127JPEG2025522795000265.jpg31127JPEG2025522795000266.jpg31127

[0270] General procedure for the preparation of Compound 2 JPEG2025522795000267.jpg18127To a solution of Compound 1 (1.0 g, 5.28 mmol, 1.0 equiv) in EA (3 mL) was added HCl / EA (17.6 ml, 3N, 52.8 mmol, 10.0 equiv), and the mixture was stirred at room temperature for 3 h under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis), the solution was concentrated under vacuum to give Compound 2 (1.2 g, crude product) as a yellow oily substance, which was used directly without further purification. TLC: PE / EA = 1 / 1 KMnO4 R f (Compound 1) = 0.4 R f (Compound 2) = 0.1

[0271] General procedure for the preparation of Compound 3 To a solution of Compound 2 (0.3 g, 3.4 mmol, 1.0 equiv) in THF / H2O (3 / 1, 8 mL) were added Fmoc-Osu (1.7 g, 5.1 mmol, 1.5 equiv) and TEA (340 mg, 3.4 mmol, 1.0 equiv). The mixture was stirred at room temperature overnight. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (10 mL). The mixture was washed with water (10 mL) and brine (5 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 5 / 1) to give Compound 3 (160 mg, 21%) as a white solid. TLC: PE / EA = 2 / 1 R f (Compound 2) = 0.1 R f (Compound 3) = 0.7 1 1H NMR (400 MHz, CDCl3): δ 7.75 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 7.4 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.30 (t, J = 8.5, 2H), 4.50 - 4.4 (m, 2H), 4.25 - 4.20 (m, 1H), 3.65 (s, 2H), 3.21 (s, 2H), 1.75 - 1.37 (m, 4H).

[0272] General procedure for the preparation of Compound 4 In a solution of oxalyl chloride (200 mg, 1.6 mmol, 1.2 eq) in DCM (3 mL), a solution of DMSO (0.4 ml, 1.0 V) in DCM (2 mL) was added dropwise over 15 minutes at -78 °C under a nitrogen atmosphere, and the mixture was stirred at the same temperature for 15 minutes. To the previous solution, a solution of compound 3 (410 mg, 1.32 mmol, 1.0 eq) in DCM (3 mL) was added at -78 °C over 15 minutes. The mixture was stirred at -78 °C for 30 minutes, then TEA (400 mg, 3.96 mmol, 3.0 eq) was added at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (20 mL) and quenched with 10% aqueous citric acid solution (10 mL). The resulting solution was washed with water (20 mL) and brine (20 mL), and dried over Na2SO4. The solvent was removed under vacuum at a temperature below 30 °C to obtain compound 4 (640 mg, crude product) as a yellow oily substance, which was used directly without further purification. TLC: PE / EA = 4:1, KMnO4, ninhydrin R f (Compound 3) = 0.4 R f (Compound 4) = 0.6

[0273] General procedure for the preparation of compound 6 To a solution of compound 5 (410 mg, 1.6 mmol, 1.0 eq) and compound 4 (979 mg, 3.2 mmol, 2.0 eq) in DCE / DMF (10 mL), acetic acid (1.5 mL) and molecular sieve (1.0 g) were added. The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Next, NaBH3CN (398 mg, 6.4 mmol, 4.0 eq) was added portionwise, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 30 mL) and brine (30 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to obtain compound 6 (315 mg, 35%) as a white solid. TLC: EA = 100% R f (Compound 5) = 0.4 R f (Compound 6) = 0.5

[0274] General procedure for the preparation of Compound 7 To a solution of Compound 6 (642 mg, 1.2 mmol, 1.0 eq) in THF (3.0 mL) was added dropwise a solution of crotonaldehyde (87.6 mg, 1.3 mmol, 1.1 eq) in H2SO4 (3.11 ml, 4N, 12.4 mmol, 10.7 eq) at 0 °C. Next, NaBH4 (26.8 mg, 0.7 mmol, 0.6 eq) was added at 0 °C and the mixture was stirred at the same temperature for 40 minutes. The reaction mixture was poured into ice water and extracted with DCM (3 × 30 mL). The organic layer was washed with H2O and brine and concentrated under reduced pressure. The residue was purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to give Compound 7 (120 mg, 17%) as a white solid. TLC: EA = 100% R f (Compound 6) = 0.5 R f (Compound 7) = 0.6 LCMS: [C 36 H 38 N4O5] + Calculated value: 607, Measured value: 607.

[0275] General procedure for the preparation of Compound 68 To a solution of Compound 7 (120 mg, 0.2 mmol, 1.0 eq) in DCM (4.0 mL) was added piperidine (171 mg, 2.0 mmol, 10.0 eq). The mixture was stirred at room temperature for 17 hours. After concentration under vacuum, the residue was purified by P-HPLC (acetonitrile / H2O: 20% - 40%) to give Compound 68 (42.5 mg, 46%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 7) = 0.5 Rf (Compound 68) = 0.1 LCMS: [C 21 H 28 N4O3] + Calculated value: 385, Measured value: 385. 1 H NMR (400 MHz, D2O): δ 7.76 (d, J = 7.4 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 5.75 - 5.55 (m, 1H), 5.16 - 5.04 (m, 1H), 4.98 (dd, J = 13.3, 5.1 Hz, 1H), 4.42 (dd, J = 35.2, 17.5 Hz, 2H), 3.96 (d, J = 7.2 Hz, 2H), 3.62 - 3.41 (m, 2H), 2.75 - 2.55 (m, 4H), 2.32 (qd, J = 13.0, 5.1 Hz, 1H), 2.10 - 2.01 (m, 1H), 1.51 - 1.24 (m, 7H).

[0276] Hydrochloride form of Compound 69 JPEG2025522795000273.jpg15127JPEG2025522795000274.jpg31127JPEG2025522795000275.jpg30127

[0277] General procedure for the preparation of Compound 2 JPEG2025522795000276.jpg18127To a solution of Compound 1 (620 mg, 8.8 mmol, 1.0 equiv) in DCM (30 mL), MnO2 (7.7 g, 88.5 mmol, 10.0 equiv) and molecular sieve (3.1 g) were added, and the mixture was stirred at room temperature for 17 h. After completion of the reaction (confirmed by TLC analysis), the reaction mixture was filtered and concentrated under reduced pressure at a temperature below 30 °C to obtain Compound 2 (110 mg, 17%) as a transparent oily substance, which was used directly without further purification. TLC: PE / EA = 3 / 1 KMnO4 R f (Compound 1) = 0.3 Rf (Compound 2) = 0.3 1 H NMR (400 MHz, CDCl3): δ 9.13 (s, 1H), 2.05 (s, 3H).

[0278] General procedure for the preparation of Compound 4 To a solution of Compound 3 (2.0 g, 4.6 mmol, 1.0 equiv) and Compound 2 (1.62 g, 18.4 mmol, 4.0 equiv) in DCE / DMF (3 / 1, 8 mL) were added acetic acid (2.0 mL) and molecular sieves (4.0 g). The mixture was stirred at room temperature for 1 h under a nitrogen atmosphere. NaBH3CN (1.2 g, 18.4 mmol, 4.0 equiv) was added portionwise, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. After completion of the reaction (confirmed by TLC analysis and LCMS), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (3 × 20 mL) and brine (20 mL), and dried over Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to give Compound 4 (110 mg, 5%) as a white solid. TLC: EA = 100% R f (Compound 3) = 0.4 R f (Compound 4) = 0.5 LCMS: [C 26 H 34 N4O5] + Calculated value: 483, Measured value: 483.

[0279] General procedure for the preparation of Compound 5 To a solution of Compound 4 (100 mg, 0.2 mmol, 1.0 equiv) in EtOH (5 mL) were added Lindlar catalyst (10 mg, 10%) and quinoline (1.8 mg, 0.02 mmol, 0.1 equiv). The mixture was stirred at room temperature for 30 min, filtered, and concentrated under reduced pressure. The residue was purified by P-HPLC (acetonitrile / H2O: 40% - 60%) to give Compound 5 (20 mg, 20%) as a white solid. TLC: DCM / methanol = 1 / 1 R f (Compound 4) = 0.6 R f (Compound 5) = 0.62 LCMS: [C 26 H 36 N4O5] + Calculated value: 485, measured value: 485.

[0280] General procedure for the preparation of Compound 69 To a solution of Compound 5 (33 mg, 0.07 mmol, 1.0 equiv) in MeOH (1 mL) was added HCl (0.4 mL, 2N, 0.8 mmol, 11.0 equiv). The mixture was stirred at room temperature for 24 hours and concentrated under reduced pressure. The residue was purified by Biotage (C18, acetonitrile / H2O: 30% - 60%) to give Compound 69 (17.8 mg, 57%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 5) = 0.5 R f (Compound 69) = 0.1 LCMS: [C 21 H 28 N4O3] + Calculated value: 385, measured value: 385. 1 H NMR (400 MHz, D2O): δ 7.87 - 7.77 (m, 2H), 7.72 - 7.61 (m, 1H), 5.80 - 5.61 (m, 1H), 5.27 - 5.12 (m, 1H), 5.04 (d, J = 10.0 Hz, 1H), 4.50 (q, J = 17.4 Hz, 2H), 4.16 (d, J = 6.2 Hz, 2H), 3.70 - 3.55 (m, 2H), 2.82 - 2.61 (m, 4H), 2.48 - 2.30 (m, 1H), 2.20 - 2.04 (m, 1H), 1.58 - 1.31 (m, 4H), 1.22 (d, J = 5.2 Hz, 3H).

[0281] Hydrochloride form of Compound 80

[0282] 3-(4-((4-(Methylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione. Hydrochloride JPEG2025522795000280.jpg33127JPEG2025522795000281.jpg33127

[0283] Step - 1: tert - Butyl methyl(4 - oxobutyl)carbamate To a solution of oxalyl chloride (0.25 ml, 2.95 mmol, 1.2 equiv) in DCM (5 mL) under a nitrogen atmosphere at -78 °C, DMSO (0.419 ml, 5.90 mmol, 2.4 equiv) was added dropwise over 5 minutes, and the mixture was stirred at the same temperature for 15 minutes. To this, a solution of tert - butyl (4 - hydroxybutyl)(methyl)carbamate 1 (500 mg, 5.90 mmol, 1 equiv) in DCM (2.5 mL) was added dropwise over 5 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, and then triethylamine (1.713 ml, 12.30 mmol, 5 equiv) was added dropwise at -78 °C. After the addition, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, 50% EtOAc / petroleum ether, Rf ~0.4, KMnO4), the reaction mixture was diluted with DCM (20 mL), washed with 10% aqueous citric acid solution (20 mL), water (1 x 10 mL) and brine (1 x 10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum (below 30 °C) to obtain tert - butyl methyl(4 - oxobutyl)carbamate 2 (450 mg, crude product) as a pale yellow liquid, which was used further without purification. 1 H - NMR (400 MHz, DMSO - d6): δ 9.67 (s, 1H), 3.16 (t, J = 7.20 Hz, 2H), 2.69 (s, 3H), 2.43 - 2.38 (m, 2H), 1.71 (t, J = 6.80 Hz, 2H), 1.41 (s, 3H).

[0284] Step - 2: tert - Butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)(methyl)carbamate JPEG2025522795000283.jpg37127 A mixture of tert - butyl methyl(4 - oxobutyl)carbamate (279 mg, 1.389 mmol, 1.2 equiv) and lenolidamide 3 (300 mg, 1.157 mmol, 1 equiv) was dissolved in a mixture of 1,2 - dichloroethane (3 ml, 10 vol) and DMF (3 ml, 10 vol) under a nitrogen atmosphere. To this solution, TFA (0.08 ml, 1.041 mmol, 0.9 equiv) and Na(OAc)3BH (368 mg, 1.736 mmol, 1.5 equiv) were added portionwise at 0 °C, and the mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf ~ 0.4, and UPLC), the reaction mixture was diluted with DCM (20 mL), washed with water (3 × 20 mL), brine (20 mL), dried over Na2SO4, filtered, and concentrated to obtain a crude product, which was purified by Isolera (Biotage R snap cartridge, KP - Sil, 25 g, 230 - 400 mesh silica gel) using 80 - 85% ethyl acetate in petroleum ether. The pure fractions were collected and concentrated under vacuum to give tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)(methyl)carbamate 4 (350 mg, 0.760 mmol, 65% yield) as an off - white solid. 11H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.29 (t, J = 7.60 Hz, 1H), 6.93 (d, J = 6.80 Hz, 1H), 6.75 (d, J = 8.40 Hz, 1H), 5.61 (s, 1H), 5.12 (q, J = 5.20 Hz, 1H), 4.23 (d, 1H), 4.13 (d, J = 17.20 Hz, 1H), 3.21 - 3.14 (m, 3H), 2.88 (q, 2H), 2.72 (t, 3H), 2.58 (d, 2H), 2.46 - 2.28 (m, 2H), 1.55 (s, 3H), 1.37 (s, 9H). LCMS: 345.1 (M - Boc), Rt (min): 2.267, Area %: 96.498.

[0285] Step - 3: tert-Butyl (4 - ((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(pentyl)amino)butyl)(methyl)carbamate JPEG2025522795000284.jpg36127A mixture of tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)(methyl)carbamate 4 (350 mg, 0.787 mmol, 1 equiv) and pentanal (339 mg, 3.94 mmol, 5 equiv) was dissolved in a mixture of CH2Cl2 (3.5 mL, 10 vol) and DMF (3.5 mL, 10 vol) under a nitrogen atmosphere. To this, TFA (0.05 ml, 0.709 mmol, 0.9 equiv) and STAB (250 mg, 1.181 mmol, 1.5 equiv) were added portionwise at 0 °C and stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf ~0.6, and UPLC), the reaction mixture was diluted with DCM (20 mL), washed with water (3 x 20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was purified by Isolera (Biotage R snap cartridge, KP-Sil, 25 g, silica gel 230 - 400 mesh) using 70 - 80% ethyl acetate in petroleum ether. The pure fractions were collected and concentrated under vacuum to obtain the desired product, tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)(methyl)carbamate 5 (130 mg, 0.241 mmol, 30% yield) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.38 (t, J = 8.00 Hz, 1H), 7.19 (d, J = 7.20 Hz, 1H), 7.08 (d, J = 8.40 Hz, 1H), 5.11 (q, J = 5.20 Hz, 1H), 4.34 (d, 1H), 4.29 (d, 1H), 3.22 - 3.11 (m, 6H), 2.92 - 2.87 (m, 2H), 2.70 (q, J = 2.00 Hz, 3H), 2.57 (s, 2H), 2.01 (t, J = 5.20 Hz, 1H), 1.25 - 1.40 (m, 18H), 0.84 (t, J = 7.20 Hz, 3H). LCMS: 515.4 (M+H), Rt (min): 1.989, Area %: 95.589.

[0286] Step - 4: 3-(4-((4-(Methylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione JPEG2025522795000285.jpg 38127 To a ice-cooled suspension of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate (130 mg, 0.240 mmol, 1 equiv) in DCM (1.5 mL), HCl (4 M solution in EtOAc, 0.7 mL) was added and stirred at room temperature for 2 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated and the residue was triturated with MTBE (10 mL). The obtained solid was lyophilized to give 3-(4-((4-(methylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 80 (70 mg, 63.2%) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 8.83 (s, 2H), 7.53 (s, 3H), 5.15 - 5.10 (m, 1H), 4.48 - 4.49 (m, 2H), 3.31 (s, 4H), 2.97 - 2.88 (m, 1H), 2.82 (s, 1H), 2.74 (s, 2H), 2.51 - 2.48 (m, 3H), 1.91 (s, 1H), 1.59 (t, J = 6.40 Hz, 2H), 1.46 (d, J = 17.20 Hz, 3H), 1.24 (s, 4H), 0.82 (t, J = 6.80 Hz, 3H). LCMS: 415.2 (M+H), Method: Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: ACN, Column: Atlantis dC18 (50×4.6 mm) 5 μm, Flow rate: 1.5 ml / min, Rt (min): 1.599, Area %: 95.709. HPLC: Method: Mobile phase: A: 0.1% aqueous TFA solution, Mobile phase: B: ACN, Column: X-Bridge C8 (50×4.6) mm, 3.5 μm Flow rate: 2.0 mL / min, Rt (min): 2.409, Area %: 97.597.

[0287] Formate form of Compound 81

[0288] 3-(4-((4-(Dimethylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione formate JPEG2025522795000286.jpg 421273-(4-((4-Aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 47 (300 mg, 0.749 mmol) and paraformaldehyde (450 mg, 14.98 mmol, 20 equivalents) in DCM (6 ml, 20 vol) solution, AcOH (0.129 ml, 2.247 mmol, 3 equivalents) was added, and the reaction mixture was stirred at room temperature for 2 hours. NaCNBH4 (188 mg, 3.00 mmol, 4 equivalents) was added to the mixture in small portions, and the reaction mixture was stirred at room temperature for an additional 40 hours. After completion of the reaction (confirmed by TLC analysis, EtOAc, Rf approximately 0.4), the reaction mixture was diluted with DCM (10 mL), washed with water (2x20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by preparative HPLC (HCOOH:ACN method) and lyophilized to give 3-(4-((4-(Dimethylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 81 (26 mg, 0.059 mmol, 7.90% yield) as a white solid. 11H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.42 - 7.38 (m, 1H), 7.22 (t, J = 7.20 Hz, 1H), 7.12 (q, J = 3.20 Hz, 1H), 5.23 - 5.18 (m, 1H), 5.14 - 5.05 (m, 1H), 4.40 (d, J = 7.20 Hz, 1H), 4.32 (d, J = 4.40 Hz, 1H), 3.25 - 3.17 (m, 4H), 2.82 - 2.68 (m, 3H), 2.53 - 2.50 (m, 6H), 2.34 (s, 2H), 1.63 (q, J = 3.20 Hz, 2H), 1.44 (d, J = 6.40 Hz, 4H), 1.27 - 1.23 (m, 4H), 0.85 - 0.9 (m, 3H). LCMS: 429.3 (M+H). Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: 0.1% TFA in ACN. Flow rate: 1.5 mL / min. Column: XBridge C8 (50×4.6 mm) 3.5 μm. Rt (min): 1.824; Area %: 97.584. HPLC: Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 2.0 mL / min. Column: X-Bridge C8 (50X4.6) mm, 3.5 μm. Rt (min): 3.658; Area %: 97.694.

[0289] Compound 82

[0290] N-(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)acetamide JPEG2025522795000287.jpg301273 - (4 - ((4 - aminobutyl)(pentyl)amino)-1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione47(200 mg, 0.499 mmol) in pyridine(3 mL) was added dropwise with acetic anhydride(0.236 ml, 2.497 mmol, 5 eq), and the reaction mixture was stirred at room temperature for 13 h. After completion of the reaction(TLC analysis, 10% MeOH in DCM, Rf about 0.8, and confirmed by LCMS), the reaction mixture was concentrated under vacuum, diluted with EtOAc(10 mL), washed with water(2×20 mL), brine(20 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by chromatography(using Biotage R snap cartridge, KP - Sil, 25 g, 230 - 400 silica gel) with 3 - 4% MeOH in DCM, and freeze - dried to obtain N-(4 - ((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(pentyl)amino)butyl)acetamide82(120 mg, 0.267 mmol, 53.5% yield) as an off - white solid. 1 1H - NMR (400 MHz, DMSO - d6): δ 11.00 (s, 1H), 7.78 (t, J = 5.60 Hz, 1H), 7.38 (t, J = 7.60 Hz, 1H), 7.19 (d, J = 7.20 Hz, 1H), 7.07 (d, J = 8.00 Hz, 1H), 5.10 (t, J = 5.20 Hz, 1H), 4.40 (d, J = 16.80 Hz, 1H), 4.29 (d, J = 16.80 Hz, 1H), 3.17 (t, J = 8.40 Hz, 4H), 2.95 (q, J = 1.60 Hz, 3H), 2.62 - 2.53 (m, 1H), 2.48 (s, 1H), 2.02 (d, J = 5.20 Hz, 1H), 1.76 (s, 3H), 1.47 - 1.38 (m, 6H), 1.28 - 1.22 (m, 4H), 0.84 (t, J = 6.80 Hz, 3H). LCMS: 443.3 (M+H). Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: 0.1% TFA in ACN. Flow rate: 1.5 mL / min. Column: XBridge C8 (50×4.6 mm) 3.5 μm. Rt (min): 1.414; Area %: 98.439. HPLC: Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 2.0 mL / min. Column: X-Bridge C8 (50×4.6) mm, 3.5 μm. Rt (min): 2.445; Area %: 98.636.

[0291] Hydrochloride form of Compound 87

[0292] 3-(4-(((1S,4S)-4-Aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000288.jpg34127JPEG2025522795000289.jpg38127

[0293] Step-1: ((1S,4S)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamic acid tert-butyl JPEG2025522795000290.jpg331273 - (4 - Bromo - 1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione 1 (0.5 g, 1.547 mmol, 1 equiv), ((1S,4S) - 4 - Aminocyclohexyl)carbamic acid tert - butyl 2 (0.995 g, 4.64 mmol, 3 equiv), and NaOt - Bu (0.446 g, 4.64 mmol, 3 equiv) were dissolved in DMF (2 mL) and degassed with nitrogen for 20 min. Pd - PEPPSI - IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equiv) was added under a nitrogen atmosphere, and the reaction mixture was heated at 110 °C for 4 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~0.4, and LCMS), the reaction mixture was filtered through a celite bed, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse - phase column chromatography (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to give tert - butyl ((1S,4S) - 4 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)carbamate 3 (110 mg, 0.240 mmol, 15% yield) as a pale yellow solid. LCMS: 401.1 (M - Boc). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.110; Area% - 94.51. 1H-NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 7.28 (t, J = 7.60 Hz, 1H), 6.94 (d, J = 6.80 Hz, 1H), 6.79 (d, J = 8.00 Hz, 1H), 6.61 (m, 1H), 5.14 (q, J = 5.20 Hz, 1H), 5.07 (d, J = 6.40 Hz, 1H), 4.22 (q, J = 17.20 Hz, 2H), 3.47-3.42 (m, 2H), 2.98-2.90 (m, 1H), 2.06-2.04 (m, 1H), 1.66 (m, 6H), 1.58-1.54 (m, 4H), 1.39 (s, 9H).

[0294] Step - 2: ((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamic acid tert-butyl JPEG2025522795000291.jpg36127 A solution of tert-butyl ((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate 3 (110 mg, 0.241 mmol, 1 equiv) and pentanal 4 (0.128 ml, 1.205 mmol, 5 equiv) in DCM (4 mL) and DMF (4 mL) was added with TFA (0.017 ml, 0.217 mmol, 0.9 equiv) at 0 °C, followed by sodium triacetoxyborohydride (77.0 mg, 0.361 mmol, 1.5 equiv). The reaction mixture was warmed to RT and stirred at the same temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf about 0.5, and LCMS), the reaction mixture was diluted with ice-cold water (15 mL), extracted with ethyl acetate (2 x 15 mL), washed with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 40 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fractions obtained from the column were lyophilized to give tert-butyl ((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamate 5 (74 mg, 0.140 mmol, 58% yield) as a white solid. LCMS: 527.3 (M+H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% aqueous FA B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.644; Area%-99.9. 1H-NMR (400 MHz, DMSO-d6): δ 10.96 (s, 1H), 7.41 (t, J = 7.60 Hz, 1H), 7.26 (q, J = 7.60 Hz, 2H), 6.97 (d, J = 6.40 Hz, 1H), 5.08 (q, J = 5.20 Hz, 1H), 4.31 (q, J = 17.20 Hz, 2H), 3.55 (s, 1H), 3.32 - 2.91 (m, 3H), 2.96 - 2.80 (m, 1H), 2.60 - 2.55 (m, 1H), 2.04 - 2.00 (m, 1H), 1.82 - 1.79 (m, 2H), 1.65 - 1.63 (m, 2H), 1.46 - 1.40 (m, 12H), 1.27 (m, 6H), 0.81 (t, J = 7.20 Hz, 3H).

[0295] Step - 3: 3-(4-(((1S,4S)-4-Aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000292.jpg36127 A solution of tert-butyl ((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamate 5 (74 mg, 0.141 mmol, 1 equiv) in DCM (4 mL) was cooled in ice, and HCl (4 M solution in EtOAc, 4 mL) was added. The mixture was stirred at room temperature for 1.5 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and further lyophilized to give 3-(4-(((1S,4S)-4-aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 87 (44 mg, 0.093 mmol, 97% yield) as an off-white solid. LCMS: 427.2 (M - HCl). Method: Column: Atlantis dC18 (50 × 4.6 mm) 5 μm Mobile phase: A: 0.1% aqueous FA solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.634; Area% - 85.00. HPLC: 97.53%, Rt (min): 9.103. Method: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: ACN, Flow rate: 1.0 mL / min. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 10.98 (s, 3H), 7.45 - 7.34 (m, 3H), 5.11 (dd, J = 5.20, 13.20 Hz, 1H), 4.41 - 4.32 (m, 2H), 3.24 (m, 4H), 2.93 - 2.92 (m, 1H), 2.63 - 2.50 (m, 2H), 2.02 (t, J = 6.80 Hz, 1H), 1.91 - 1.68 (m, 4H), 1.65 - 1.58 (m, 4H), 1.58 - 1.21 (m, 6H), 0.80 (t, J = 6.80 Hz, 3H).

[0296] Hydrochloride form of Compound 88

[0297] 3-(4-(((1R,4R)-4-Aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000293.jpg32127JPEG2025522795000294.jpg36127

[0298] Step - 1: ((1R,4R)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamic acid tert-butyl JPEG2025522795000295.jpg 321273 - (4 - Bromo - 1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione 1 (0.5 g, 1.547 mmol, 1 equiv), ((1R,4R) - 4 - Aminocyclohexyl)carbamic acid tert - butyl 2 (0.995 g, 4.64 mmol, 3 equiv) and NaOt - Bu (0.446 g, 4.64 mmol, 3 equiv) were dissolved in DMF (2 mL) and degassed with nitrogen for 20 minutes. Pd - PEPPSI - IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equiv) was added under a nitrogen atmosphere and the reaction mixture was heated at 110 °C for 4 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~ 0.4, and LCMS), the reaction mixture was filtered through celite, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse - phase column chromatography (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to give tert - butyl ((1R,4S) - 4 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)carbamate 3 (0.040 g, 0.086 mmol, 5% yield) as a cream - colored solid. LCMS: 401.1 (M - Boc). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.188; Area% - 98.18. 11H-NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 7.27 (t, J = 7.20 Hz, 1H), 6.91 (d, J = 7.20 Hz, 1H), 6.80 (t, J = 8.00 Hz, 2H), 5.29 (d, J = 6.80 Hz, 1H), 5.12 (q, J = 4.80 Hz, 1H), 4.16 (q, J = 17.20 Hz, 2H), 2.93 - 2.89 (m, 1H), 2.70 - 2.60 (m, 2H), 2.30 - 2.20 (m, 2H), 2.09 - 1.99 (m, 3H), 1.80 (m, 2H), 1.39 (s, 9H), 1.32 - 1.24 (m, 4H).

[0299] Step - 2: ((1R,4R)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamic acid tert-butyl JPEG2025522795000296.jpg35127 tert-Butyl ((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate 3 (0.040 g, 0.086 mmol, 1 equiv) and pentanal (0.076 g, 0.876 mmol, 10 equiv) in DCM (1.5 mL) and DMF (1.5 mL) were stirred, and at 0 °C, TFA (0.012 ml, 0.158 mmol, 1.8 equiv) and sodium triacetoxyborohydride (56 mg, 0.262 mmol, 3 equiv) were added. The reaction mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~0.5, and LCMS), the reaction mixture was diluted with ice-cold water (15 mL), extracted with ethyl acetate (2 × 15 mL), washed with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 40 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fractions obtained from the column were lyophilized to give tert-Butyl ((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamate 5 (20 mg, 0.038 mmol, 43% yield) as a white solid. LCMS: 527.3 (M+H). Method: Column: Atlantis dC18 (50 × 4.6 mm) 5 μm Mobile phase: A: 0.1% aqueous FA B: ACN, flow rate: 1.5 mL / min. Rt (min): 1.876; Area%-99.04.

[0300] Step - 3: 3-(4-(((1R,4R)-4-Aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000297.jpg35127 ((1R,4R)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamic acid tert-butyl 5 (20 mg, 0.038 mmol, 1 equiv) in a ice-cooled solution of DCM (1 mL) was added with HCl (4 M solution in EtOAc, 0.5 mL), and stirred at room temperature for 3 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and lyophilized to obtain 3-(4-(((1R,4R)-4-aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 88 (17 mg, 0.036 mmol, 99% yield) as an off-white solid. LCMS: 427.2 (M-HCl). Method: Column: Atlantis dC18 (50 × 4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.579; Area%-98.76. HPLC: 99.10%, Rt (min): 2.381. Method: Column: X-Bridge C8 (50 × 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: ACN, Flow rate: 2.0 mL / min. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.91 (br s, 3H), 7.44 - 7.32 (m, 3H), 5.13 (dd, J = 5.20, 13.20 Hz, 1H), 4.35 - 4.07 (m, 2H), 3.11 - 2.88 (m, 5H), 2.68 - 2.61 (m, 2H), 2.02 - 1.95 (m, 3H), 1.78 (s, 2H), 1.60 - 1.57 (m, 2H), 1.40 - 1.31 (m, 2H), 1.24 - 1.20 (m, 6H), 0.81 - 0.80 (m, 3H).

[0301] Hydrochloride form of Compound 89

[0302] 3-(4-(((1S,4S)-4-(Aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000298.jpg30127JPEG2025522795000299.jpg37127

[0303] Step - 1: tert-Butyl (((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate JPEG2025522795000300.jpg331273-(4-Bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (1.0 g, 3.09 mmol, 1 equiv), tert-Butyl (((1s,4s)-4-aminocyclohexyl)methyl)carbamate 2 (1.060 g, 4.64 mmol, 1.5 equiv) and NaOt-Bu (0.892 g, 9.28 mmol, 3 equiv) were dissolved in DMF (5 mL) and degassed with nitrogen for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.151 g, 0.155 mmol, 0.05 equiv) was added under a nitrogen atmosphere and the reaction was heated at 110 °C for 4 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~ 0.4, and LCMS), the reaction mixture was filtered through a Celite bed, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to give tert-Butyl (((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate 3 (140 mg, 0.272 mmol, 8.78% yield) as a pale yellow solid. LCMS: 415.1 (M - Boc). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.325; Area%-89.09.

[0304] Step - 2: tert - Butyl (((1s,4s)-4-((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(pentyl)amino)cyclohexyl)methyl)carbamate To a solution of tert - Butyl (((1S,4S)-4-((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)methyl)carbamate 3 (80 mg, 0.170 mmol, 1 equiv) and pentanal 4 (73.2 mg, 0.850 mmol, 5 equiv) in JPEG2025522795000301.jpg38127 DCM (2 mL) and DMF (2 mL) at 0 °C, TFA (0.012 ml, 0.153 mmol, 0.9 equiv) was added, followed by sodium triacetoxyborohydride (54 mg, 0.255 mmol, 1.5 equiv). The reaction mixture was warmed to RT and stirred at the same temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~ 0.5, and LCMS), the reaction mixture was diluted with ice - cold water (15 mL), extracted with ethyl acetate (2 x 15 mL), washed with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by reverse - phase column chromatography (GraceR column: C18 40μm, 40 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fractions obtained from the column were lyophilized to give tert - Butyl (((1S,4S)-4-((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(pentyl)amino)cyclohexyl)methyl)carbamate 5 (35 mg, 0.060 mmol, 35.5% yield) as an off - white solid. LCMS: 541.3 (M + H). Method: Column: Atlantis dC18 (50×4.6 mm) 5μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.648; Area% - 93.13.

[0305] Step - 3: 3-(4-(((1S,4S)-4-(Aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000302.jpg38127((1S,4S)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamic acid tert-butyl5(35 mg, 0.065 mmol, 1 equiv) in a ice-cooled solution of DCM (1 mL) was added HCl (4 M solution in EtOAc, 3 mL), and stirred at room temperature for 1.5 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and lyophilized to give 3-(4-(((1S,4S)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride89(22.5 mg, 0.045 mmol, 69% yield) as an off-white solid. LCMS: 441.3 (M-HCl). Method: Column: Atlantis dC18 (50 × 4.6 mm) 5 μm Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN, Flow rate: 1.5 mL / min. Rt (min): 1.408; Area%-94.03. HPLC: 94.36%, Rt (min): 8.083. Method: Column: X-Bridge C8 (50 × 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: ACN, Flow rate: 1.0 mL / min. 11H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.84 (s, 3H), 7.34 (d, J = 6.40 Hz, 3H), 5.11 (dd, J = 4.80, 12.20 Hz, 1H), 4.38 - 4.24 (m, 2H), 3.19 - 3.10 (m, 3H), 2.92 - 2.84 (m, 3H), 2.69 - 2.68 (m, 2H), 1.95 (s, 1H), 1.75 (s, 1H), 1.60 - 1.50 (m, 4H), 1.49 - 1.30 (m, 4H), 1.24 - 1.20 (m, 6H), 0.80 - 0.78 (m, 3H).

[0306] Hydrochloride form of Compound 90

[0307] 3-(4-(((1R,4R)-4-(Aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000303.jpg29127JPEG2025522795000304.jpg28127

[0308] Step - 1: (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamic acid tert-butyl A solution of 1 (0.500 g, 1.547 mmol, 1 eq) of (4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione in DMF (2 mL) was added with tert-butyl (((1r,4r)-4-aminocyclohexyl)methyl)carbamate 2 (1.060 g, 4.64 mmol, 3 eq) and sodium tert-butoxide (0.446 g, 4.64 mmol, 3 eq) at room temperature, and degassed with nitrogen gas for 15 minutes. To this reaction mixture, Pd-PEPPSI-Ihept(Cl) (0.075 g, 0.077 mmol, 0.05 eq) was added under nitrogen conditions, degassed for 5 minutes, and stirred at 110 °C for 4 hours. After completion of the reaction (confirmed by UPLC analysis), the reaction mixture was filtered through a celite bed, washed with CH3CN (5 mL), and the filtrate was concentrated under reduced pressure to obtain a brown viscous liquid. The crude product compound was purified by reverse-phase chromatography (GraceR column: C18 40 μm, 120 g, eluting the product with 44% ACN in 0.1% HCOOH). The pure fraction was lyophilized to obtain tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate 3 (0.100 g, 0.118 mmol, 7.60% yield) as a yellow solid. LCMS: 415.2 (M-56). Method: Column: Xbridge C8 (50×4.6 mm) 3.5 μm, Mobile phase: A: 0.1% TFA in H2O, Mobile phase: B: 0.1% TFA in CAN, Flow rate: 1.5 mL / min, Area%-55.37%.

[0309] Step-2: tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamate JPEG2025522795000306.jpg A mixture of tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate 3 (0.100 g, 0.213 mmol, 1.0 equiv) and pentanal 4 (0.183 g, 2.125 mmol, 10 equiv) in DCE (1.5 mL) and DMF (1.5 mL) was treated with TFA (0.033 mL, 0.425 mmol, 2.0 equiv) and stirred at 0 °C for 10 min. To this mixture was added sodium triacetoxyborohydride (0.135 g, 0.638 mmol, 3.0 equiv) portionwise at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 h (monitored by UPLC). The reaction mixture was quenched with ice-cold water (5 mL) and extracted with dichloromethane (3 x 10 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo to give the crude product as a pale yellow liquid. The crude product compound was purified by reverse-phase chromatography (eluting with 50% ACN in 0.1% HCOOH) to afford tert-butyl (((1r,4r)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamate (0.020 g, 0.037 mmol, 17.18% yield) as a white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H), 7.81 (t, J = 37.20 Hz, 1H), 7.24 (d, J = 6.00 Hz, 2H), 5.26 (d, J = Hz, 1H), 4.72 (t, J = 6.00 Hz, 2H), 3.35 (d, J = 19.60 Hz, 3H), 2.75 (d, J = 6.40 Hz, 3H), 2.33 (d, J = 1.60 Hz, 2H), 1.71 (d, J = 12.00 Hz, 5H), 1.42-1.37 (m, 14H), 0.79 (s, 3H). LCMS: 541.4 (M+H). Method: Column: XBridge C8 (50×4.6 mm) 3.5 μm Mobile phase: A: 0.1% TFA in H2O Mobile phase: B: 0.1% TFA in ACN Flow rate: 1.5 mL / min Area % - 94.78%. HPLC: Method: Column: X-Bridge C8 (50×4.6) mm, 3.5 μm Mobile phase: A: 0.1% TFA aqueous solution Mobile phase: B: ACN, Flow rate: 2.0 mL / min, Area % - 98.72%.

[0310] Step - 3: 3-(4-(((1R,4R)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000307.jpg 32127 (((1r,4r)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamic acid tert-butyl 5 (20 mg, 0.037 mmol, 1.0 equiv) in dichloromethane (1 mL) stirred solution, HCl (4 M solution in dioxane, 0.5 mL) was added at 0 °C, then stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC (10% MeOH; DCM, Rf ~0.0. The reaction mixture was concentrated under vacuum below 40 °C to give an off-white solid. The solid obtained was dissolved in water (5 mL), washed with MTBE (2×5 mL), and the aqueous layer was lyophilized to give 3-(4-(((1R,4R)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (17 mg, 0.035 mmol, 96% yield) as an off-white solid. 11H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.86 (br s, 3H), 7.43 - 7.29 (m, 3H), 5.12 (d, J = 8.80 Hz, 1H), 4.33 - 4.27 (m, 2H), 3.50 - 3.30 (m, 2H), 3.12 - 3.08 (m, 2H), 2.96 - 2.87 (m, 1H), 2.68 - 2.59 (m, 3H), 2.04 - 2.02 (m, 1H), 1.80 - 1.75 (m, 4H), 1.50 - 1.48 (m, 3H), 1.21 (s, 6H), 1.00 - 0.97 (m, 2H), 0.79 (t, J = 6.40 Hz, 3H). LCMS: 441.3 (M + H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: ACN, Flow rate: 1.5 mL / min, Rt - 1.526 min, Area: 97.05%. HPLC: Method: Column: X - Bridge C8 (50×4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: ACN, Flow rate: 2.0 mL / min, Rt - 2.287 min, Area: 99.27%.

[0311] Hydrochloride form of Compound 91 JPEG2025522795000308.jpg86127

[0312] Step - 1: ((1s,4s) - 4 - formylcyclohexyl)carbamic acid tert - butyl JPEG2025522795000309.jpg14127A solution of tert-butyl ((1S,4S)-4-(hydroxymethyl)cyclohexyl)carbamate (200 mg, 0.872 mmol, 1 equiv) in cooled DCM (5 mL) was added dropwise with a solution of sulfur trioxide pyridine (540 mg, 3.39 mmol, 3.89 equiv) in DMSO (1.5 mL), DIPEA (438 mg, 3.39 mmol, 3.89 equiv), and DMSO (1.5 mL), and stirred at 0 °C for 1 h. After completion of the reaction (confirmed by TLC (40% ethyl acetate in petroleum ether, Rf: 0.4)), the reaction was quenched with 1.5 N HCl (20 mL) and extracted with DCM (2 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product of tert-butyl ((1S,4S)-4-formylcyclohexyl)carbamate 2A (200 mg, 0.880 mmol, 101% yield) as a colorless liquid.

[0313] Step - 2: tert-Butyl ((1S,4S)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate 3-(4-Amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (200 mg, 0.771 mmol, 1 equiv) and tert-butyl ((1s,4s)-4-formylcyclohexyl)carbamate 2A (210 mg, 0.926 mmol, 5 equiv) in a mixture of JPEG2025522795000310.jpg32127DCE (2 mL) and DMF (0.5 mL) were added with TFA (79 mg, 0.694 mmol, 0.9 equiv) and Na(OAc)3BH (245 mg, 1.157 mmol, 1.5 equiv) at 0 °C, and stirred at room temperature for 12 h. After completion of the reaction (confirmed by UPLC), the reaction was quenched with ice water (25 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were concentrated under vacuum to obtain the crude product, which was purified by reverse-phase chromatography (GraceR column: generic C18, 40 g snap, 0.1% formic acid in ACN, 20 mL / min, 70 - 75% ACN in H2O) to give tert-butyl ((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate 3 (105 mg, 0.223 mmol, 28.9% yield) as an off-white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 7.28 (t, J = 8.00 Hz, 1H), 6.91 (d, J = 7.20 Hz, 1H), 6.73 (d, J = 8.00 Hz, 2H), 5.64 (t, J = 5.20 Hz, 1H), 5.12 (q, J = 5.20 Hz, 1H), 4.23 (d, J = 17.20 Hz, 1H), 4.12 (d, J = 17.20 Hz, 1H), 3.49 (s, 1H), 3.03 (t, J = 6.00 Hz, 2H), 2.92 (t, J = 8.00 Hz, 1H), 2.06 - 2.02 (m, 1H), 1.69 (s, 1H), 1.50 (t, J = 13.20 Hz, 8H), 1.39 (s, 9H). LCMS: 470.0 (M+H), Rt (min): 2.306, Area %: 99.775. HPLC: Rt (min): 4.229, Area %: 99.789.

[0314] Step - 3: ((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamic acid tert-butyl JPEG2025522795000311.jpg A solution of ((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamic acid tert-butyl (90 mg, 0.191 mmol, 1 equiv) in 30127DCE (5 mL) and DMF (0.5 mL) was added with pentanal (82 mg, 0.956 mmol, 5 equiv), TFA (19.63 mg, 0.172 mmol, 0.9 equiv) and Na(OAc)3BH (48.6 mg, 0.230 mmol, 1.5 equiv) at 0 °C, and stirred at room temperature for 12 h. After completion of the reaction (confirmed by LCMS), the reaction was quenched with ice water (25 mL) and extracted with DCM (2 × 10 mL). The combined organic layers were concentrated under vacuum to obtain a crude product, which was purified by Isolera (column size: Biotage R snap cartridge, KP-Sil, 10 g, silica gel - 230 - 400 mesh) using 70 - 80% ethyl acetate in petroleum ether. The pure fractions were collected separately and concentrated under vacuum to give ((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamic acid tert-butyl (30 mg, 0.055 mmol, 29.0% yield) as a white solid. 1H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.37 (t, J = 7.60 Hz, 1H), 7.18 (d, J = 7.20 Hz, 1H), 7.10 (d, J = 8.00 Hz, 1H), 6.66 (d, J = 7.20 Hz, 1H), 5.11 (q, J = 4.80 Hz, 1H), 4.38 (d, J = 16.80 Hz, 1H), 4.28 (d, J = 17.20 Hz, 1H), 3.45 (s, 1H), 3.34 - 3.16 (m, 2H), 3.11 (d, J = 7.20 Hz, 2H), 2.90 (d, J = 12.00 Hz, 1H), 2.59 (d, J = 19.60 Hz, 2H), 2.02 (t, J = 5.20 Hz, 1H), 1.46 (t, J = 7.20 Hz, 4H), 1.38 (s, 15H), 1.24 (t, J = 10.00 Hz, 5H), 0.84 (t, J = 6.80 Hz, 3H). LCMS: 485.9 (M - 56), Rt (min): 2.828, Area %: 99.796. HPLC: Rt (min): 4.910, Area %: 98.887.

[0315] Step - 4: 3-(4-((((1s,4s)-4-Aminocyclohexyl)methyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl JPEG2025522795000312.jpg31127 To a solution of tert-butyl ((1S,4S)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamate (30 mg, 0.055 mmol) in DCM (2 mL) at ice-cooled temperature, HCl (4 M in ethyl acetate) (0.5 mL, 16.46 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 6 hours. After completion of the reaction (confirmed by LCMS), the reaction mixture was concentrated, washed with MTBE (10 mL) to obtain the crude product, which was lyophilized to give 3-(4-((((1S,4S)-4-aminocyclohexyl)methyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl (30 mg, 0.063 mmol, 113% yield) as an off-white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.78 (s, 6H), 7.39 (t, J = 8.00 Hz, 1H), 7.22 (d, J = 7.60 Hz, 1H), 7.22 (d, J = 7.60 Hz, 1H), 5.12 (q, J = 5.20 Hz, 1H), 4.33 (dd, J = 16.80, 42.40 Hz, 2H), 3.15 (q, J = 7.60 Hz, 5H), 2.05 - 2.01 (m, 1H), 1.59 (s, 5H), 1.45 (d, J = 5.60 Hz, 6H), 1.25 (d, J = 4.80 Hz, 4H), 0.84 (t, J = 7.20 Hz, 3H). LCMS: 442.0 (M+H), method: mobile phase: A: 0.1% aqueous formic acid solution, B: ACN, column: Atlantis dC18 (50×4.6) 5 μm, flow rate: 1.5 mL / min; Rt (min): 1.691, area %: 99.845. HPLC: method: mobile phase: A: 0.1% FA aqueous solution, mobile phase: B: ACN, column: Atlantis dC18 (50×4.6 mm) 5 μm, flow rate: 1.5 mL / min, Rt (min): 2.839, area %: 98.026.

[0316] Hydrochloride form of Compound 92 JPEG2025522795000313.jpg83127

[0317] Step - 1: tert - Butyl ((1r,4r)-4 - formylcyclohexyl)carbamate JPEG2025522795000314.jpg14127A solution of tert - butyl ((1r,4r)-4 - (hydroxymethyl)cyclohexyl)carbamate (500 mg, 2.180 mmol, 1 equiv) in cooled DCM (11 mL) was added dropwise with a solution of sulfur trioxide pyridine (347 mg, 2.180 mmol, 3.89 equiv) in DMSO (3.7 mL), DIPEA (0.388 ml, 2.180 mmol, 3.89 equiv), and DMSO (3.7 mL), and stirred at 0 °C for 1 h. After completion of the reaction (confirmed by TLC (40% ethyl acetate in petroleum ether, Rf: 0.4)), the reaction was quenched with 1.5 N HCl (20 mL) and extracted with DCM (2 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product of tert - butyl ((1r,4r)-4 - formylcyclohexyl)carbamate 2A (480 mg, 2.112 mmol, 97% yield) as a yellow liquid. 1 1H - NMR (400 MHz, DMSO - d6): δ 9.55 (s, 1H), 3.64 - 3.50 (m, 1H), 3.16 - 3.11 (m, 2H), 2.16 - 2.11 (m, 1H), 1.99 (d, J = 57.60 Hz, 2H), 1.88 (t, J = 24.40 Hz, 3H), 1.38 (s, 10H), 1.18 - 1.26 (m, 11H). LCMS: 172.4 (M - 56), Rt (min): 2.593, area%: 59.513.

[0318] Step - 2: tert - Butyl ((1r,4r)-4 - ((((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)methyl)cyclohexyl)carbamate 3-(4-Amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (350 mg, 1.350 mmol, 1 equiv) and tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (368 mg, 1.620 mmol, 5 equiv) in a solution of JPEG2025522795000315.jpg29127DCE (1.5 mL) and DMF (0.5 mL) were added with TFA (139 mg, 1.215 mmol, 0.9 equiv) and Na(OAc)3BH (429 mg, 2.025 mmol, 1.5 equiv) at 0 °C and stirred at room temperature for 12 h. After completion of the reaction (confirmed by TLC), the reaction was quenched with ice water (25 mL) and extracted with DCM (2 × 10 mL). The combined organic layers were concentrated under vacuum to give the crude product, which was purified by reverse-phase chromatography (GraceR, column: generic C18, 40 g snap, 0.1% formic acid in ACN, 20 mL / min, 70 - 75% ACN in H2O), and the pure fractions were lyophilized to give tert-butyl ((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate 3 (410 mg, 0.834 mmol, 61.8% yield) as a white solid. 1H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.27 (t, J = 7.60 Hz, 1H), 6.91 (d, J = 7.60 Hz, 1H), 6.71 (q, J = 8.00 Hz, 2H), 5.62 (t, J = 5.60 Hz, 1H), 5.12 (q, J = 4.80 Hz, 1H), 4.23 (d, J = 17.20 Hz, 1H), 4.12 (d, J = 17.20 Hz, 1H), 3.19 (d, J = 8.00 Hz, 1H), 2.98 - 2.89 (m, 3H), 2.60 (t, J = 22.00 Hz, 2H), 2.35 - 2.28 (m, 2H), 2.06 - 2.02 (m, 1H), 1.81 (t, J = 20.00 Hz, 4H), 1.50 (t, J = 7.20 Hz, 2H), 1.37 (s, 9H), 1.12 (q, J = 11.60 Hz, 3H), 0.99 (t, J = 12.00 Hz, 2H), LCMS: 415.3 (M - 56), Rt (min): 2.127, Area %: 95.713. HPLC: Rt (min): 4.211, Area %: 98.834.

[0319] Step - 3: ((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamic acid tert-butyl A solution of tert-butyl ((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate (250 mg, 0.531 mmol, 1 equiv) in 35127DCE (2.5 mL) and DMF (0.5 mL) was added with pentanal (229 mg, 2.66 mmol, 5 equiv), TFA (54.5 mg, 0.478 mmol, 0.9 equiv) and Na(OAc)3BH (169 mg, 0.797 mmol, 1.5 equiv) at 0 °C, and stirred at room temperature for 12 h. After completion of the reaction (confirmed by LCMS), the reaction was quenched with ice water (25 mL) and extracted with DCM (2 × 10 mL). The combined organic layers were concentrated under vacuum to obtain a crude product, which was purified by Isolera (column size: Biotage R snap cartridge, KP-Sil, 25 g, silica gel - 230 - 400 mesh) using 70 - 80% ethyl acetate in petroleum ether. The pure fractions were collected separately and concentrated under vacuum to give tert-butyl ((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamate 5 (124 mg, 0.229 mmol, 43.2% yield) as a yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.97 (s, 1H), 7.37 (t, J = 8.00 Hz, 1H), 7.13 (dd, J = 8.00, 35.00 Hz, 2H), 6.63 (d, J = 8.00 Hz, 1H), 5.09 (t, J = 5.20 Hz, 1H), 4.31 (t, J = 16.80 Hz, 2H), 3.31 - 3.15 (m, 3H), 3.05 - 3.03 (m, 2H), 2.00 (s, 1H), 1.72 (d, J = 9.60 Hz, 5H), 1.43 - 1.36 (m, 14H), 1.26 - 1.20 (m, 5H), 0.97 (d, J = 27.20 Hz, 4H), 0.83 (t, J = 6.80 Hz, 3H). LCMS: 541.3 (M+H), Rt (min): 2.849, Area %: 95.954. HPLC: Rt (min): 4.907, Area %: 95.462.

[0320] Step - 4: 3-(4-((((1r,4r)-4-Aminocyclohexyl)methyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl JPEG2025522795000317.jpg35127 To a solution of tert-butyl (120 mg, 0.222 mmol) of ((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamate in DCM (5 mL) cooled in ice at 0 °C was added HCl (4 M in ethyl acetate) (0.12, 3.95 mmol) at 0 °C, and the mixture was stirred at room temperature for 6 h. After completion of the reaction (confirmed by LCMS), the reaction mixture was concentrated, washed with MTBE (10 mL), and dried to obtain 3-(4-((((1r,4r)-4-aminocyclohexyl)methyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl 92 (30 mg, 0.061 mmol, 27.3% yield) as a pale yellow solid. 11H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.83 (s, 3H), 7.39 (t, J = 7.60 Hz, 1H), 7.20 (d, J = 7.20 Hz, 1H), 7.11 (d, J = 8.00 Hz, 1H), 5.11 (q, J = 4.80 Hz, 1H), 4.39 (d, J = 16.80 Hz, 1H), 4.28 (d, J = 16.80 Hz, 1H), 3.18 (d, J = 4.80 Hz, 2H), 3.18 (d, J = 4.80 Hz, 2H), 2.93 - 2.88 (m, 2H), 2.68 (t, J = 2.00 Hz, 1H), 2.62 (d, J = 2.80 Hz, 1H), 2.02 (d, J = 5.60 Hz, 1H), 1.90 (d, J = 10.40 Hz, 2H), 1.77 (d, J = 12.00 Hz, 2H), 1.41 (t, J = 6.80 Hz, 3H), 1.17 - 1.20 (m, 6H), 0.97 (d, J = 12.00 Hz, 2H), 0.83 (t, J = 6.80 Hz, 3H). LCMS: 441.3 (M+H), Method: Mobile Phase: A: 0.1% FA aqueous solution, Mobile Phase: B: ACN, Column: Atlantis dC18 (50×4.6 mm) 5 μm, Flow Rate: 1.5 mL / min, Rt (min): 1.695, Area %: 96.479. HPLC: Method: A: 0.1% TFA in H2O, B: ACN, Flow Rate: 2.0 mL / min Column: Xbridge C8 (50×4.6) mm, 3.5 μm, Rt (min): 2.950, Area %: 95.528.

[0321] Hydrochloride form of Compound 93

[0322] (E)-3-(4-((4-Aminobutyl)(penta-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000318.jpg49127

[0323] Step - 1: (E)-penta-3-enal To a stirred solution of 2(2.5 g, 21.90 mmol, 1.0 equiv) of methyl (E)-penta-3-enoate in CH2Cl2 (50 mL) was added dropwise DIBAL-H (17.52 ml, 17.52 mmol, 0.8 equiv) in hexane at -78 °C. Next, the resulting reaction mixture was stirred at 78 °C for 5 minutes. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc in petroleum ether, Rf ~ 0.5), the reaction mixture was quenched with 1.5 N HCl solution (10 mL) and extracted with DCM (20 mL). The separated organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and used in the next step without concentration.

[0324] Step - 2: (E)-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-en-1-yl)amino)butyl)carbamic acid tert-butyl (4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 1 (0.5 g, 1.161 mmol, 1.0 equiv) and (E)-penta-3-enal 3 (1.954 g, 23.23 mmol, 20.0 equiv) in a stirred solution of a mixture of CH2Cl2 (10 mL) and DMF (5 mL) were added AcOH (0.332 mL, 5.81 mmol, 5.0 equiv) under a nitrogen atmosphere and stirred for 1 h. To this mixture, NaCNBH4 (0.292 g, 4.65 mmol, 4.0 equiv) was added at 0 °C. The resulting reaction mixture was stirred at room temperature overnight. After completion of the reaction (confirmed by TLC and UPLC analysis, 100% EtOAc, Rf ≈ 0.7), the reaction mixture was diluted with DCM (30 mL) and washed with water (2 × 30 mL), 10% LiCl solution (30 mL) and brine (30 mL), dried over Na2SO4, filtered, and concentrated to give the crude product as a pale yellow liquid. The crude product compound was purified by preparative HPLC (column: X-Bridge-c18 19.1X250, mobile phase: 0.1% FA in water / ACN, flow rate: 15 mL / min) to give (E)-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-en-1-yl)amino)butyl)carbamic acid tert-butyl 4 (35 mg, 0.068 mmol, 5.87% yield) as an off-white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.36 - 7.40 (m, 1H), 7.18 - 7.20 (m, 1H), 7.06 - 7.08 (m, 1H), 6.78 (s, 1H), 5.40 - 5.42 (m, 3H), 5.09 - 5.13 (m, 1H), 4.26 - 4.42 (m, 2H), 3.41 - 3.51 (m, 3H), 3.19 - 3.22 (m, 4H), 2.88 - 2.92 (m, 3H), 2.11 - 2.13 (m, 2H), 2.03 (m, 1H), 1.62 - 1.63 (m, 3H), 1.59 - 1.60 (m, 2H), 1.37 (s, 9H), LCMS: 499.2 (M+H), Rt (min): 2.492, Area - 97%.

[0325] Step - 3: (E)-3-(4-((4-Aminobutyl)(penta-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000321.jpg32127 (E)-(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-en-1-yl)amino)butyl)carbamic acid tert-butyl 4 (65 mg, 0.130 mmol, 1.0 equiv) in ethyl acetate (1 mL) was stirred. HCl (4 M solution in EtOAc, 2 mL) was added at 0 °C and the mixture was stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC (100% EtOAc). The reaction mixture was concentrated under vacuum at 40 °C to give an off-white semi-solid. The resulting solid was dissolved in water (2 mL), washed with MTBE (2 mL), additional water (2 mL) was added, and freeze-dried to give (E)-3-(4-((4-aminobutyl)(penta-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl (30 mg, 0.067 mmol, 79% yield) as an off-white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.84 (s, 3H), 7.09 - 7.43 (m, 3H), 5.40 - 5.41 (m, 2H), 5.10 - 5.14 (m, 1H), 4.32 - 4.39 (m, 2H), 3.23 - 3.48 (m, 4H), 2.90 - 2.93 (m, 1H), 2.74 - 2.78 (m, 2H), 2.53 - 2.68 (m, 1H), 2.12 - 2.14 (m, 2H), 2.02 - 2.09 (m, 2H), 1.52 - 1.60 (m, 7H) LCMS: 399.2 (M+H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: Acetonitrile, Flow rate: 1.5 mL / min HPLC: Method: Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: ACN, Flow rate: 2.0 mL / min, Column: X-Bridge C8 (50×4.6) mm, 3.5 μm, RT: 2.301 min, Area: 97.652%.

[0326] Trifluoroacetate form of Compound 94

[0327] (Z)-3-(4-((4-Aminobutyl)(penta-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione JPEG2025522795000322.jpg40127JPEG2025522795000323.jpg33127

[0328] Steps - 1 and 2: (3-(1-Oxo-4-(penta-3-in-1-ylamino)isoindolin-2-yl)piperidine-2,6-dione JPEG2025522795000324.jpg32127 To a solution of penta-3-in-1-ol 2 (2.0 g, 23.78 mmol, 1.0 equiv) and CH2Cl2 (100 mL) at 0 °C, Dess-Martin periodinane (11.09 g, 26.2 mmol, 1.1 equiv) and water (0.05 mL) were added. The resulting white suspension was stirred at room temperature for 1 h. After completion of the reaction (confirmed by TLC, 20% EtOAc in petroleum ether), the reaction mixture was cooled to -78 °C, diluted with n-pentane (50 mL), and filtered through a silica gel bed. The bed was washed with n-pentane (20 mL), and the filtrate was concentrated under vacuum at 25 °C to about 50% of the volume. The resulting solution of 3 was used directly in the next step. In another RB flask, the above solution of 3 was added to a stirred solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (4.00 g, 15.43 mmol, 0.649 eq) in DMF (40 mL) at room temperature. Acetic acid (1.753 ml, 30.6 mmol, 1.289 eq) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. Sodium cyanoborohydride (1.520 g, 24.18 mmol, 1.017 eq) was added to the reaction mixture at 0 °C, and the mixture was stirred at room temperature overnight. After completion of the reaction (confirmed by TLC and UPLC analysis, 100% EtOAc, Rf about 0.7), the reaction mixture was diluted with DCM (100 mL), washed with water (2 × 50 mL), dried over Na2SO4, filtered, and concentrated to obtain the crude product as a pale yellow liquid. The crude product compound was purified by reverse phase (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase, the product was eluted with 70% ACN in 0.1% HCO2H) to obtain 3-(1-oxo-4-(penta-3-ylamino)isoindolin-2-yl)piperidine-2,6-dione 4 (1.0 g, 3.03 mmol, 12.75% yield) as a pale yellow solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.30 (t, J = 8.00 Hz, 1H), 6.96 (d, J = 7.20 Hz, 1H), 6.78 (d, J = 6.00 Hz, 1H), 5.72 (t, J = 6.00 Hz, 1H), 5.09-5.14 (m, 1H), 4.23 (d, J = 16.80 Hz, 1H), 4.12 (d, J = 17.20 Hz, 1H), 3.25-3.33 (m, 2H), 2.93 (m, 1H), 2.50-2.51 (m, 1H), 2.40-2.42 (m, 2H), 2.27-2.38 (m, 1H), 2.02-2.06 (m, 1H), 1.75 (t, J = 2.00 Hz, 3H), LCMS: 326.1 (M+H), Rt (min): 1.986, area - 98.611%.

[0329] Step - 3: tert - Butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(penta - 3 - yl - 1 - yl)amino)butyl)carbamate JPEG2025522795000325.jpg37127In a stirred mixture of 3-(1 - oxo - 4-(penta - 3 - yl - 1 - yl)amino)isoindolin - 2 - yl)piperidine - 2,6 - dione 4 (0.5 g, 1.537 mmol, 1.0 equivalent) and tert - butyl (tert - butoxycarbonyl)(5 - oxopentyl)carbamate 5 (0.88 g, 3.07 mmol, 2.0 equivalents) in CH2Cl2 (15 mL) and DMF (15 mL), trifluoroacetic acid (0.106 ml, 1.383 mmol, 0.9 equivalent) was added and the mixture was stirred at 0 °C for 10 minutes. Sodium triacetoxyborohydride (0.49 g, 2.305 mmol, 1.5 equivalents) was added portionwise at 0 °C and the resulting colorless turbid mass was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by UPLC analysis), the reaction mixture was quenched with ice - cold water (50 mL) and extracted with dichloromethane (2×50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product as a brown liquid. The crude product compound was purified by (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase, the product was eluted with 70% ACN in 0.1% HCO2H) to give tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)(penta - 3 - yl - 1 - yl)amino)butyl)carbamate 10 (0.4 g, 0.649 mmol, 42.2% yield) as an off - white solid. 11H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.39 (t, J = 10.00 Hz, 1H), 7.23 (d, J = 9.60 Hz, 1H), 7.12 (d, J = 10.80 Hz, 1H), 5.08 - 5.14 (m, 1H), 4.41 (d, J = 22.80 Hz, 1H), 4.31 (d, J = 22.40 Hz, 1H), 3.40 - 3.47 (m, 2H), 3.23 - 3.33 (m, 2H), 2.87 - 2.93 (m, 1H), 2.28 - 2.45 (m, 1H), 2.01 - 2.08 (m, 2H), 1.69 (t, J = 2.00 Hz, 3H), 1.49 (s, 18H), 1.27 - 1.44 (m, 8H), LCMS: 597.4 (M+H), Rt (min): 2.482, Area - 96.894%.

[0330] Step - 4: (Z)-(tert-Butoxycarbonyl)(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-en-1-yl)amino)butyl)carbamic acid tert-butyl JPEG2025522795000326.jpg35127 (tert-Butoxycarbonyl)(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-yl-1-yl)amino)butyl)carbamic acid tert-butyl 5 (150 mg, 0.251 mmol, 1.0 eq), quinoline (39.0 mg, 0.302 mmol, 1.2 eq) in DMF (2.0 mL) was stirred. Lindlar catalyst (12 mg, 0.113 mmol, 0.449 eq) was added and the mixture was stirred at room temperature for 16 h under a hydrogen atmosphere. After completion of the reaction (confirmed by UPLC analysis), the reaction mixture was filtered through a celite bed, washed with DMF (2 mL), and the filtrate was purified by reverse-phase chromatography (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase, the product was eluted with 70 - 75% ACN in 0.1% HCOOH) to give (Z)-(tert-Butoxycarbonyl)(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-en-1-yl)amino)butyl)carbamic acid tert-butyl 6 (120 mg, 0.182 mmol, 72.2% yield) as a pale yellow gummy solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.39 (t, J = 3.20 Hz, 1H), 7.21 (d, J = 4.80 Hz, 1H), 7.11 (d, J = 8.00 Hz, 1H), 5.38 - 5.45 (m, 2H), 5.09 - 5.14 (m, 1H), 4.39 (d, J = 16.80 Hz, 1H), 4.28 (d, J = 17.20 Hz, 1H), 3.36 - 3.47 (m, 3H), 3.18 - 3.24 (m, 4H), 3.18 - 3.20 (m, 1H), 2.50 - 2.53 (m, 2H), 2.44 - 2.50 (m, 1H), 2.16 - 2.18 (m, 2H), 2.00 - 2.01 (m, 1H), 1.59 (d, J = 4.80 Hz, 3H), 1.49 (s, 18H), 1.27 - 1.44 (m, 2H), LCMS: 599.3 (M+H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: Acetonitrile, Flow rate: 1.5 mL / min HPLC: Method: Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: Methanol, Flow rate: 1.0 mL / min, Column: Atlantis dC18 (250×4.6) mm, 5 μm, RT: 16.959 min, Area: 90.666%.

[0331] Step - 5: (Z)-3-(4-((4-Aminobutyl)(penta-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione JPEG2025522795000327.jpg32127 (Z)-(tert-Butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-en-1-yl)amino)butyl)carbamic acid tert-butyl 6 (60 mg, 0.100 mmol) in a stirred solution of CH2Cl2 (1 mL) was added TFA (2 ml, 26.0 mmol) at 0 °C, and then stirred at room temperature overnight. After completion of the reaction (confirmed by UPLC), the reaction mixture was concentrated under reduced pressure at 40 °C to give a pale yellow viscous liquid. The crude product compound was purified by preparative HPLC (Column: Atlantis 19x250 mm, Mobile phase: 0.1% TFA in water / ACN, Flow rate: 2 mg / ml) to give (Z)-3-(4-((4-aminobutyl)(penta-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, TFA 94 (12 mg, 0.022 mmol, 22.15% yield) as a pale yellow solid. 11H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.61 (s, 3H), 7.41 (t, J = 7.60 Hz, 1H), 7.24 (d, J = 6.80 Hz, 1H), 7.14 (d, J = 7.60 Hz, 1H), 5.44 - 5.46 (m, 1H), 5.38 - 5.39 (m, 1H), 5.11 - 5.15 (m, 1H), 4.39 (d, J = 16.80 Hz, 1H), 4.28 (d, J = 16.80 Hz, 1H), 3.22 (t, J = 8.40 Hz, 4H), 2.79 - 2.80 (m, 1H), 2.68 - 2.77 (m, 2H), 2.63 - 2.68 (m, 2H), 2.50 - 2.51 (m, 1H), 2.33 - 2.34 (m, 2H), 2.17 - 2.19 (m, 1H), 1.50 (t, J = 2.00 Hz, 3H), 1.48 - 1.49 (m, 3H), LCMS: 399.2 (M+H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: Acetonitrile, Flow rate: 1.5 mL / min HPLC: Method: Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: ACN, Flow rate: 1.0 mL / min, Column: Atlantis dC18 (250×4.6) mm, 5 μm, RT: 7.709 min, Area: 94.84%.

[0332] Hydrochloride form of Compound 95

[0333] 3-(4-((4-Aminobutyl)(penta-3-yl-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl JPEG2025522795000328.jpg50127

[0334] Steps 1 and 2: tert-Butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-yl-1-yl)amino)butyl)carbamate A solution of 3 (2 g, 23.78 mmol, 1 equiv) of penta-3-yn-1-ol in DCM (40 mL) was added with Dess-Martin periodinate (11.09 g, 26.2 mmol, 1.1 equiv) at 0 °C, and the resulting white suspension was stirred at room temperature for 1 h. The reaction mixture was quenched with a mixture of NaHSO3 (5 g) in saturated NaHCO3 (50 mL) solution and extracted with DCM (2 x 15 mL). To the combined organic layers were added (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl (0.500 g, 1.162 mmol, 1 equiv) and acetic acid (0.714 g, 11.627 mmol), and the mixture was stirred for 2 h. Next, NaCNBH3 (0.463 g, 11.627 mmol, 10 equiv) was added, and the mixture was stirred at room temperature for 24 h. After completion of the reaction (confirmed by UPLC), the reaction mixture was quenched with water (40 mL) and extracted with DCM (2 × 40 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to obtain the crude product, which was purified by Isolera (Biotage R snap cartridge, KP-Sil, 25 g, 230 - 400 silica mesh) using 60 - 70% ethyl acetate in petroleum ether. The pure fractions were concentrated under vacuum to obtain (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-yn-1-yl)amino)butyl)carbamic acid tert-butyl 3 (150 mg, 0.113 mmol, 0.474% yield) as an off-white solid. LCMS: 497.2 (M+H), Rt (min): 2.384, area%: 37.322.

[0335] Step - 3: 3-(4-((4-aminobutyl)(penta-3-yn-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl JPEG2025522795000330.jpg35127 tert-Butyl 3((4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(penta-3-yl-1-yl)amino)butyl)carbamate) (150 mg, 0.118 mmol, 1 equivalent) in a ice-cooled solution of DCM (3 mL) was added with HCl (4 M in ethyl acetate, 0.294 ml, 1.178 mmol, 10 equivalents) at 0 °C and stirred at room temperature for 12 h. After completion of the reaction (confirmed by LCMS), the reaction mixture was concentrated, washed with MTBE (10 mL) to obtain the crude product, which was purified by preparative HPLC (column: X-Bridge-c18 19.1X250; method: 0.1% HCL in water / ACN; flow rate: 15 mL / min) to give 3-(4-((4-aminobutyl)(penta-3-yl-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl 95 (7.5 mg, 0.017 mmol, 14.16% yield) as a white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.78 (s, 1H), 7.41 (t, J = 7.60 Hz, 1H), 7.25 (d, J = 7.20 Hz, 1H), 7.15 (d, J = 8.00 Hz, 1H), 5.15 - 5.10 (m, 1H), 4.45 - 4.30 (m, 2H), 3.33 (t, J = 7.20 Hz, 2H), 3.24 (t, J = 6.40 Hz, 3H), 2.93 (s, 1H), 2.77 - 2.68 (m, 2H), 2.33 - 2.28 (m, 2H), 2.04 (t, J = 5.20 Hz, 1H), 1.70 (t, J = 2.40 Hz, 2H), 1.52 (d, J = 4.00 Hz, 4H), 1.24 (t, J = 3.20 Hz, 1H), 0.84 (t, J = 7.20 Hz, 1H). LCMS: 397.3 (M+H), Method: Mobile Phase: A: 0.1% TFA in H2O, Mobile Phase: B: 0.1% TFA in ACN, Column: X Bridge C8 (50×4.6 mm) 3.5 μm, Flow Rate: 1.5 mL / min, Rt (min): 1.340, Area %: 96.271. HPLC: Method: Mobile Phase: A: 0.1% TFA aqueous solution, Mobile Phase: B: ACN, Column: X-Bridge C8 (50×4.6) mm, 3.5 μm, Flow Rate: 2.0 mL / min, Rt (min): 2.314, Area %: 98.115.

[0336] Hydrochloride form of Compound 96

[0337] 3-(4-(Bis(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000331.jpg34127

[0338] Step - 3: (((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)azanediyl)bis(butane-4,1-diyl))dicarbamic acid di-tert-butyl JPEG2025522795000332.jpg32127 A solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (250 mg, 0.581 mmol), tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5A (334 mg, 1.161 mmol, 2 equiv), and TFA (0.179 mL, 2.323 mmol, 4 equiv) in a mixture of dichloromethane (0.5 mL, 20 vol) and DMF (0.25 mL, 10 vol) was added sodium triacetoxyborohydride (492 mg, 2.323 mmol, 4 equiv), and the mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, EtOAc, Rf about 0.6, and UPLC), the reaction mixture was diluted with DCM (5 mL), washed with water (3x10 mL) and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography (column size: Biotage R snap cartridge, KP-Sil, 25 g, 230 - 400 silica gel) using 75 - 80% ethyl acetate in petroleum ether to give tert-butyl (tert-butoxycarbonyl)(4-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 5 (150 mg, 36.5% yield) as a white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.37 (t, J = 7.60 Hz, 1H), 7.20 (d, J = 7.20 Hz, 1H), 7.08 (d, J = 8.00 Hz, 1H), 6.78 (d, J = 5.20 Hz, 1H), 5.11 (q, J = 5.20 Hz, 1H), 4.39 (d, J = 17.20 Hz, 1H), 4.28 (d, J = 16.80 Hz, 1H), 3.34 (s, 2H), 3.18 (d, J = 6.80 Hz, 4H), 2.90 (s, 4H), 2.00 (s, 2H), 1.40 (s, 33H). LC-MS: 702.4 (M+H), Rt (min): 2.83, Area%: 99.289.

[0339] Step - 4: 3-(4-(Bis(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride To a solution of 5 (150 mg, 0.249 mmol) of JPEG2025522795000333.jpg31127(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)azanediyl)bis(butane-4,1-diyl))dicarbamic acid di-tert-butyl in DCM (5 mL) at ice-cooled temperature, HCl (4 M solution in dioxane) (3 ml, 12.00 mmol) was added and stirred at room temperature for 2 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc), the reaction mixture was concentrated and lyophilized to obtain 3-(4-(bis(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl 96 (82 mg, 67.8% yield) (2.4 g, 98%) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 8.07 (s, 6H), 7.52 (s, 3H), 5.12 (q, J = 5.20 Hz, 1H), 4.65 (s, 8H), 3.42 - 3.43 (m 1H), 2.74 (d, J = 5.20 Hz, 4H), 2.62 (d, J = 16.80 Hz, 1H), 2.07 (t, J = 5.60 Hz, 1H), 1.56 (d, J = 4.80 Hz, 8H). 1 1H-NMR (400 MHz, CD3OD): δ 7.93 (s, 2H), 7.80 (s, 1H), 5.24 (q, J = 4.80 Hz, 1H), 4.48 (q, J = 400.80 Hz, 2H), 3.66 (t, J = 26.40 Hz, 4H), 3.00 - 2.92 (m, 6H), 2.68 - 2.61 (m, 1H), 2.28 - 2.24 (m, 1H), 1.69 (s, 1H). LCMS: 402.3 (M+H). Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: 0.1% TFA in ACN. Flow rate: 1.5 mL / min. Column: XBridge C8 (50×4.6 mm) 3.5 μm. Rt (min): 0.887; Area %: 97.781. HPLC: Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 2.0 mL / min. Column: X-Bridge C8 (50×4.6) mm, 3.5 μm. Rt (min): 1.08; Area %: 95.532.

[0340] Hydrochloride form of Compound 97

[0341] 3-(4-((4-Aminobutyl)(3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000334.jpg52127

[0342] Step - 1: tert-Butyl (3-oxopropyl)carbamate A solution of oxalyl chloride (0.599 mL, 6.85 mmol, 1.2 equiv) in DCM (10 mL) was added dropwise to a solution of DMSO (0.972 mL, 13.70 mmol, 2.4 equiv) in DCM (5 mL) at -78 °C under a nitrogen atmosphere over 15 minutes and stirred at the same temperature for 15 minutes. To this solution, a solution of tert-butyl (3-hydroxypropyl)carbamate 5 (1 g, 5.71 mmol, 1 equiv) in DCM (5 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, then TEA (3.98 mL, 28.5 mmol, 5.0 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, 3:7 / EtOAc:petroleum ether, Rf ~0.4, KMnO4), the reaction mixture was diluted with DCM (10 mL) and washed with 10% aqueous citric acid (1x10 mL), water (1x10 mL) and brine (1x10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure at <30 °C to give the crude product tert-butyl (3-oxopropyl)carbamate 6 (0.950 g, 4.90 mmol, 86% yield) as a pale yellow liquid, which was used further without purification. LCMS: (No mass peak was observed). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% aqueous FA B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.644; Area%-89.26.

[0343] Step - 2: (4-((3-((tert-butoxycarbonyl)amino)propyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl JPEG2025522795000336.jpg41127(3 - Oxopropyl)carbamic acid tert - butyl 6 (0.805 g, 4.65 mmol, 4 equivalents) and tert - butyl (4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate 4 (0.5 g, 1.161 mmol, 1 equivalent) were added to a mixture of 1,2 - dichloroethane (10 ml, 10 vol) and DMF (5 ml, 5 vol) under a nitrogen atmosphere. To this, acetic acid (0.332 ml, 5.81 mmol, 5 equivalents) was added and the reaction mixture was stirred at room temperature for 1 hour. NaCNBH3 (0.292 g, 4.65 mmol, 4 equivalents) was added portionwise and the reaction mixture was stirred at room temperature for an additional 16 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~0.5, and LCMS), the reaction mixture was diluted with ice - cold water (15 mL), extracted with DCM (2 x 15 mL), washed with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain the crude product as a white solid, which was purified by preparative HPLC (method: apparatus: SC - DC - ARD - 05 - 045; column number: XBridgeC8 - 250 - X - Select - C18 - 19x150 mm; 0.1% TFA in water / ACN; Rt: 9.6 min), lyophilized to obtain tert - butyl (4 - ((3 - ((tert - butoxycarbonyl)amino)propyl)(2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate 7 (60 mg, 0.092 mmol, 7.89% yield) as a white solid. LCMS: 588.4(M + H). Method: Column: XBridge C8(50×4.6 mm) 3.5 μm Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN, Flow rate: 1.5 mL / min. Rt (min): 1.911; Area% - 89.75. 1H-NMR (400 MHz, DMSO-d6): δ 10.97 (s, 1H), 7.37 (t, J = 7.60 Hz, 1H), 7.14 (d, J = 7.20 Hz, 1H), 7.08 (d, J = 8.00 Hz, 1H), 6.82 - 6.76 (m, 2H), 5.10 (dd, J = 5.20, 13.20 Hz, 1H), 4.35 (q, J = 17.20 Hz, 2H), 3.20 - 3.16 (m, 4H), 2.96 - 2.89 (m, 5H), 2.04 - 2.01 (m, 1H), 1.58 - 1.51 (m, 2H), 1.58 - 1.51 (m, 23H).

[0344] Step - 3: 3-(4-((4-Aminobutyl)(3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride To a solution of 7 (60 mg, 0.102 mmol, 1 equiv) of tert-butyl 4-((3-((tert-butoxycarbonyl)amino)propyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate in ethyl acetate (2 mL) at ice-cooled temperature was added HCl (4 M solution in EtOAc, 4 mL), and the mixture was stirred at room temperature for 1.5 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and lyophilized to obtain 97 (20 mg, 0.051 mmol, 50.1% yield) of 3-(4-((4-aminobutyl)(3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione as an off-white solid. LCMS: 388.3 (M - 2×HCl). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% aqueous FA solution B: ACN, Flow rate: 0.6 mL / min. Rt (min): 2.218; Area% - 99.01. HPLC: 93.99%, Rt (min): 6.421. Method: Column: Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA in H2O, Mobile phase B: methanol, Flow rate: 1.0 mL / min. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.94 (br s, 6H), 7.43 (t, J = 7.60 Hz, 1H), 7.29 - 7.21 (m, 2H), 5.13 (dd, J = 4.80, 13.20 Hz, 1H), 4.45 (d, J = 17.20 Hz, 1H), 4.31 (d, J = 16.80 Hz, 1H), 3.76 - 3.20 (m, 4H), 2.98 - 2.89 (m, 1H), 2.81 - 2.76 (m, 4H), 2.59 (s, 2H), 2.08 - 2.02 (m, 1H), 1.76 - 1.72 (m, 2H), 1.53 (s, 4H).

[0345] Hydrochloride form of Compound 102

[0346] 3-(4-((4-Aminobutyl)((1S,4S)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000338.jpg31127JPEG2025522795000339.jpg38127

[0347] Step - 1: ((1S,4S)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamic acid tert-butyl JPEG2025522795000340.jpg 321273 - (4 - Bromo - 1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione 1 (0.5 g, 1.547 mmol, 1 equiv), ((1S,4S) - 4 - Aminocyclohexyl)carbamic acid tert - butyl 2 (0.995 g, 4.64 mmol, 3 equiv) and NaOt - Bu (0.446 g, 4.64 mmol, 3 equiv) were dissolved in DMF (2 mL) and degassed with nitrogen for 20 minutes. Pd - PEPPSI - IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equiv) was added under a nitrogen atmosphere and the reaction was heated at 110 °C for 4 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~0.4, and LCMS), the reaction mixture was filtered through a Celite bed, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse - phase column chromatography (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to give tert - butyl ((1S,4S) - 4 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)carbamate 3 (138 mg, 0.289 mmol, 18% yield) as a pale yellow solid. LCMS: 456.0 (M + H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.223; Area% - 95.48. 1 1H - NMR (400 MHz, DMSO - d6): δ 11.04 (s, 1H), 7.28 (t, J = 7.60 Hz, 1H), 6.79 (d, J = 8.00 Hz, 1H), 6.63 (br s, 1H), 5.17 - 5.09 (m, 2H), 4.21 (q, J = 17.20 Hz, 2H), 3.47 - 3.42 (m, 2H), 2.98 - 2.90 (m, 1H), 2.68 - 2.65 (m, 1H), 2.08 - 2.03 (m, 1H), 1.66 (m, 6H), 0.54 (m, 3H), 1.39 (m, 10H).

[0348] Step - 2: tert - Butyl (4 - ((((1S,4S) - 4 - ((tert - butoxycarbonyl)amino)cyclohexyl)(2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate JPEG2025522795000341.jpg A solution of tert - butyl ((1S,4S) - 4 - ((2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)carbamate 3 (130 mg, 0.285 mmol, 1 equiv) and tert - butyl (tert - butoxycarbonyl)(4 - oxobutyl)carbamate 4 (0.164 g, 0.569 mmol, 2 equiv) in DCM (4 mL) and DMF (4 mL) was added with TFA (0.074 ml, 1.139 mmol, 4 equiv) at 0 °C, followed by sodium triacetoxyborohydride (0.241 g, 1.139 mmol, 4 equiv). The reaction mixture was warmed to RT and stirred at the same temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~ 0.5, and LCMS), the reaction mixture was diluted with ice - cold water (15 mL), extracted with ethyl acetate (2 x 15 mL), washed with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The obtained residue was purified by reverse - phase column chromatography (GraceR column: C18 40 μm, 40 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fractions obtained from the column were lyophilized to give tert - butyl (4 - ((((1S,4S) - 4 - ((tert - butoxycarbonyl)amino)cyclohexyl)(2 - (2,6 - dioxopiperidin - 3 - yl) - 1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate 5 (74 mg, 0.140 mmol, 58% yield) as a white solid. LCMS: 726.7 (M - H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.912; Area% - 99.75. 1 H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.40 (t, J = 7.60 Hz, 1H), 7.26 (dd, J = 8.00, 16.60 Hz, 2H), 6.97 (d, J = 7.60 Hz, 1H), 6.97 (q, J = 7.60 Hz, 1H), 4.30 (q, J = 17.20 Hz, 2H), 3.56 (m, 1H), 3.36 - 3.16 (m, 2H), 3.08 (m, 1H), 2.91 - 2.88 (m, 1H), 2.01 - 1.99 (m, 1H), 1.785 1.809 (m, 2H), 1.65 - 1.62 (m, 3H), 1.53 (m, 8H), 1.44 - 1.40 (m, 27H), 1.24 - 1.21 (m, 3H).

[0349] Step - 3: 3-(4-((4-Aminobutyl)((1S,4S)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride To a ice-cooled solution of (tert-butoxycarbonyl)(4-(((1S,4S)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 5 (72 mg, 0.099 mmol, 1 equiv) in DCM (1 mL), HCl (4M in EtOAc solution, 2.5 mL) was added and stirred at room temperature for 2 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and lyophilized to give 3-(4-((4-aminobutyl)((1S,4S)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride 102 (49 mg, 0.095 mmol, 96% yield) as an off-white solid. LCMS: 427.2 (M - HCl). Method: Column: XBridge C8 (50×4.6 mm) 3.5 μm; Mobile phase: A: 0.1% TFA in H2O, B: 0.1% TFA in ACN; Flow rate: 1.5 mL / min. Rt (min): 0.932; Area % - 94.26. HPLC: 95.86%, Rt (min): 1.363. Method: Column: X - Bridge C8 (50×4.6) mm, 3.5 μm; Mobile phase: A: 0.1% TFA aqueous solution, Mobile phase: B: ACN; Flow rate: 2.0 mL / min. 1 1H - NMR (400 MHz, DMSO - d6): δ 10.99 (s, 1H), 8.23 (s, 3H), 7.92 (s, 3H), 7.44 (d, J = 7.20 Hz, 3H), 5.10 (dd, J = 5.20, 12.80 Hz, 1H), 4.47 - 4.29 (m, 2H), 3.25 (s, 4H), 2.73 - 2.70 (m, 1H), 2.68 - 2.68 (m, 4H), 2.05 - 2.02 (m, 1H), 1.92 - 1.83 (m, 4H), 1.92 - 1.83 (m, 6H), 1.32 (s, 2H).

[0350] Hydrochloride form of Compound 103

[0351] 3-(4-((4 - aminobutyl)((1r,4r)-4 - aminocyclohexyl)amino)-1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione, 2HCl JPEG2025522795000343.jpg27127JPEG2025522795000344.jpg34127

[0352] Step - 1: ((1R,4R)-4 - ((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)carbamic acid tert - butyl JPEG2025522795000345.jpg 331273 - (4 - Bromo - 1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione 1 (0.5 g, 1.547 mmol, 1 equiv), ((1R,4R)-4 - Aminocyclohexyl)carbamic acid tert - butyl 2 (0.995 g, 4.64 mmol, 3 equiv) and NaOt - Bu (0.446 g, 4.64 mmol, 3 equiv) were dissolved in DMF (2 mL) and degassed with nitrogen for 20 minutes. Pd - PEPPSI - IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equiv) was added under a nitrogen atmosphere and the reaction mixture was heated at 110 °C for 4 hours. After completion of the reaction (confirmed by UPLC analysis), the reaction mixture was filtered through celite, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse - phase column chromatography (GraceR column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to give tert - butyl ((1R,4S)-4 - ((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)cyclohexyl)carbamate 3 (0.070 g, 0.152 mmol, 9.81% yield) as a cream - colored solid. LCMS: 401.1 (M - Boc). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.200, Area% - 99.01 11H-NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 7.27 (t, J = 7.20 Hz, 1H), 6.91 (d, J = 7.20 Hz, 1H), 6.80 (t, J = 8.00 Hz, 2H), 5.29 (d, J = 6.80 Hz, 1H), 5.12 (q, J = 4.80 Hz, 1H), 4.16 (q, J = 17.20 Hz, 2H), 2.93 - 2.89 (m, 1H), 2.70 - 2.60 (m, 2H), 2.30 - 2.20 (m, 2H), 2.09 - 1.99 (m, 3H), 1.80 (m, 2H), 1.39 (s, 9H), 1.32 - 1.24 (m, 4H).

[0353] Step - 2: (tert-Butoxycarbonyl)(4-(((1R,4R)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl JPEG2025522795000346.jpg38127 A solution of tert-butyl ((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate 3 (70 mg, 0.153 mmol, 1 equiv), tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 4 (88 mg, 0.307 mmol, 2 equiv) in DCM (5 ml) and DMF (0.5 ml) was added with TFA (0.047 ml, 0.613 mmol, 4 equiv) at 0 °C, followed by addition of sodium triacetoxyborohydride (130 mg, 0.613 mmol, 4 equiv). The reaction mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf about 0.5, and LCMS), the reaction mixture was diluted with ice-cold water (15 mL), extracted with DCM (2x10 mL), washed with brine (25 mL), dried over Na2SO4, filtered, and concentrated. The obtained residue was purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fractions obtained from the column were lyophilized to give tert-butyl (tert-butoxycarbonyl)(4-(((1R,4R)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 5 (40 mg, 0.055 mmol, 35.8% yield) as a white solid. LCMS: 728.5 (M+H). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% aqueous FA B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.913; Area%-99.75.

[0354] Step-3: 3-(4-((4-aminobutyl)((1R,4R)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl JPEG2025522795000347.jpg37127 (tert-Butoxycarbonyl)(4-(((1r,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 5 (40 mg, 0.055 mmol, 1 equiv) in a ice-cooled solution of DCM (1 mL) was added HCl (4 M solution in EtOAc, 1 mL, 4.0 mmol), and the mixture was stirred at room temperature for 2 h. After completion of the reaction (confirmed by TLC analysis, 10% MeOH / DCM, Rf ~0.1), the reaction mixture was concentrated, washed with MTBE (2×5 mL), and further lyophilized to give 3-(4-((4-aminobutyl)((1r,4r)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl 103 (13.2 mg, 0.026 mmol, 47.8% yield) as an off-white solid. LCMS: 428.3 (M+H) Method: Column: XBridge C8 (50×4.6 mm) 3.5 μm Mobile phase: A: 0.1% TFA in H2O Mobile phase: B: 0.1% TFA in ACN, Flow rate: 1.5 mL / min, Rt (min): 0.906; Area%-95.74. HPLC: 99.63%, Rt (min): 11.860. Method: Column: Atlantis dC18 (250×4.6) mm, 5 μm, Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: methanol, Flow rate: 0.7 mL / min 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.96 (s, 3H), 7.80 (s, 3H), 7.44 (d, J = 7.20 Hz, 1H), 7.34 (t, J = 8.80 Hz, 2H), 5.12 (q, J = 5.20 Hz, 1H), 4.38 - 4.23 (m, 2H), 3.13 (s, 2H), 2.98 - 2.89 (m, 3H), 2.68 (q, J = 1.60 Hz, 2H), 2.61 (d, J = 18.00 Hz, 2H), 2.04 - 1.91 (m, 3H), 1.78 (s, 2H), 1.62 - 1.50 (m, 4H), 1.45 (t, J = 25.60 Hz, 4H).

[0355] Hydrochloride form of Compound 104

[0356] 3-(4-((4-Aminobutyl)((1s,4s)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000348.jpg45127JPEG2025522795000349.jpg26127

[0357] Step - 1: (((1s,4s)-4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamic acid tert-butyl In a solution of 1 (1.0 g, 3.09 mmol, 1.0 equiv) of 271273-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione in DMF (5 ml, 5 vol), tert-butyl (((1s,4s)-4-aminocyclohexyl)methyl)carbamate 2 (1.060 g, 4.64 mmol, 1.5 equiv) and sodium tert-butoxide (0.892 g, 9.28 mmol, 3.0 equiv) were added at room temperature, and the mixture was degassed by bubbling nitrogen gas for 15 minutes. To this reaction mixture, Pd-PEPPSI-IHept(Cl) (0.151 g, 0.155 mmol, 0.05 equiv) was added under a nitrogen atmosphere, degassed for 5 minutes, and the mixture was stirred at 85 °C overnight. After completion of the reaction (confirmed by TLC and UPLC analysis, 100% EtOAc, Rf about 0.5), the reaction mixture was filtered through a Celite bed, washed with THF (50 mL), and the filtrate was concentrated under reduced pressure to obtain a brown viscous liquid. The crude product compound was purified by reverse-phase chromatography (the product was eluted with 75% ACN in 0.1% HCO2H). The pure fractions were lyophilized to obtain tert-butyl (((1s,4s)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate 3 (0.2 g, 0.350 mmol, 11.30% yield) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 7.25 - 7.29 (m, 1H), 6.90 - 6.93 (m, 1H), 6.80 - 6.87 (m, 1H), 6.77 - 6.79 (m, 1H), 5.11 - 5.21 (m, 2H), 4.15 - 4.30 (m, 2H), 3.47 - 3.51 (m, 1H), 2.92 - 2.99 (m, 3H), 2.67 - 2.68 (m, 1H), 2.30 - 2.34 (m, 1H), 2.02 - 2.08 (m, 1H), 1.41 - 1.46 (m, 3H), 1.38 (m, 15H). LCMS: 415.8 (M-tBu), Rt (min): 2.324, area - 89.8%. HPLC: Rt (min): 5.496; Area - 82.3%.

[0358] Step - 2: tert - Butyl (tert - butoxycarbonyl)(4 - oxobutyl)carbamate JPEG2025522795000351.jpg A solution of oxalyl chloride (1.815 ml, 20.73 mmol, 2.0 eq) in DCM (20 mL) was added dropwise to a solution of DMSO (2.94 ml, 41.5 mmol, 4.0 eq) in DCM (5 mL) at - 78 °C over 15 minutes under a nitrogen atmosphere and stirred at the same temperature for 15 minutes. To this, a solution of tert - butyl (tert - butoxycarbonyl)(4 - hydroxybutyl)carbamate 4 (3.0 g, 10.37 mmol) in CH2Cl2 (5 mL) was added dropwise over 15 minutes. The reaction mixture was stirred at - 78 °C for 30 minutes, then triethylamine (8.67 ml, 62.2 mmol, 6.0 eq) was added dropwise at - 78 °C. Next, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc / petroleum ether, Rf ~ 0.6, KMnO4), the reaction mixture was diluted with DCM (40 mL), washed with 10% aqueous citric acid solution (1x40 mL) and water (1x40 mL), dried over Na2SO4, filtered, and concentrated under vacuum (below 30 °C) to obtain tert - butyl (tert - butoxycarbonyl)(4 - oxobutyl)carbamate 5 (2.9 g, 9.99 mmol, crude product) as a pale yellow liquid and used further without purification. 1 1H - NMR (400 MHz, DMSO - d6): δ 9.66 (s, 1H), 3.47 - 3.50 (m, 1H), 2.51 - 2.55 (m, 2H), 2.42 - 2.44 (m, 2H), 1.69 - 1.75 (m, 2H), 1.43 - 1.45 (s, 18H),

[0359] Step-3: (tert-Butoxycarbonyl)(4-(((1s,4s)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl JPEG2025522795000352.jpg37127To a mixture of tert-butyl (((1s,4s)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate 3 (0.2 g, 0.425 mmol, 1.0 equiv) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (0.244 g, 0.850 mmol, 2.0 equiv) in CH2Cl2 (10 mL) and DMF (10.00 mL) was added trifluoroacetic acid (0.131 ml, 1.700 mmol, 4.0 equiv), and the mixture was stirred at 0 °C for 10 minutes. Sodium triacetoxyborohydride (0.360 g, 1.700 mmol, 4.0 equiv) was added portionwise at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 hours (monitored by UPLC). The reaction mixture was cooled to 0 °C, an additional amount of tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (0.244 g, 0.850 mmol, 2.0 equiv) and trifluoroacetic acid (0.131 ml, 1.700 mmol, 4.0 equiv) were added, and the mixture was stirred for 10 minutes. Then, sodium triacetoxyborohydride (0.360 g, 1.700 mmol, 4.0 equiv) was added portionwise at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC analysis, 8:2 / EtOAc:petroleum ether, Rf ~0.6), the reaction mixture was quenched with ice-cold water (20 mL) and extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product as a pale yellow liquid. The crude product compound was purified by Isolera chromatography (Biotage R snap cartridge, KP-Sil, 100 g, 230 - 400 silica gel) using 60 - 70% ethyl acetate in petroleum ether. The fractions were collected and concentrated under vacuum to give a pale yellow liquid. The obtained compound was further purified by reverse-phase chromatography (eluted with 75% ACN in 0.1% HCO2H), and the pure fraction was lyophilized to give tert-butyl (tert-butoxycarbonyl)(4-(((1s,4s)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 6 (0.11 g, 0.146 mmol, 34% yield) as a white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 10.96 (s, 1H), 7.40-7.43 (m, 1H), 7.27-7.33 (m, 2H), 6.78-6.81 (m, 1H), 5.07-5.12 (m, 1H), 4.24-4.36 (m, 2H), 3.33-3.39 (m, 2H), 3.08-3.12 (m, 3H), 2.91-2.95 (m, 3H), 2.60-2.61 (m, 2H), 2.53-2.56 (m, 2H), 1.98-2.01 (m, 1H), 1.62-1.65 (m, 3H), 1.52-1.53 (m, 6H), 1.48 (m, 27H), 1.22-1.24 (m, 2H). LCMS: 741.4 (M + )、Rt (min): 2.945、area%-89.8%. HPLC: Rt (min): 5.816; area%-99.9%.

[0360] Step-4: 3-(4-((4-aminobutyl)((1s,4s)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochoride JPEG2025522795000353.jpg30127 (tert-Butoxycarbonyl)(4-(((1s,4s)-4-(((tert-Butoxycarbonyl)amino)methyl)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 6 (0.1 g, 0.135 mmol, 1.0 equiv) in dichloromethane (10 mL) was stirred. HCl (4 M solution in dioxane, 10 mL) was added at 0 °C, and then the mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum at a temperature below 40 °C to obtain an off-white solid. The obtained solid was dissolved in water (10 mL), washed with MTBE (2 x 10 mL), and the aqueous layer was lyophilized to obtain 3-(4-((4-Aminobutyl)((1s,4s)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl (62.5 mg, 0.116 mmol, 86% yield) as an off-white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.89 - 7.98 (m, 6H), 7.33 - 7.44 (m, 3H), 5.10 - 5.13 (m, 1H), 4.30 - 4.37 (m, 2H), 3.43 - 3.49 (m, 2H), 3.14 - 3.17 (m, 2H), 2.84 - 2.96 (m, 6H), 2.05 (m, 1H), 1.86 (m, 2H), 1.32 - 1.64 (m, 9H), 1.24 (m, 2H). LCMS: 442.3(M+H). Method: Column: XBridge C8(50×4.6mm) 3.5μm, Mobile phase: A: 0.1% TFA in H2O, Mobile phase: B: 0.1% TFA in acetonitrile, Flow rate: 1.5 mL / min. HPLC: Method: Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: methanol, Flow rate: 1.0 mL / min. Column: Atlantis dC18(250×4.6)mm, 5μm. RT: 6.488 min, Area: 95.427%.

[0361] Hydrochloride form of Compound 105

[0362] 3-(4-((4-Aminobutyl)((1R,4R)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl JPEG2025522795000354.jpg27127JPEG2025522795000355.jpg37127

[0363] Step - 1: tert-Butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate JPEG2025522795000356.jpg361273-(4-Bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (1.0 g, 3.09 mmol, 1 eq), tert-Butyl (((1R,4R)-4-aminocyclohexyl)methyl)carbamate 2 (2.120 g, 9.28 mmol, 3 eq) and NaOt-Bu (0.892 g, 9.28 mmol, 3 eq) were dissolved in DMF (4 mL) and degassed with nitrogen for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.151 g, 0.155 mmol, 0.05 eq) was added under a nitrogen atmosphere and the reaction mixture was heated at 110 °C for 4 hours. After completion of the reaction (confirmed by UPLC analysis), the reaction mixture was filtered through celite, washed with EtOAc (5 mL), concentrated to a minimum volume (3 mL), and purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain tert-Butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate 3 (50 mg, 0.102 mmol, 3.30% yield) as a pale yellow solid. LCMS: 401.1 (M - Boc). Method: Column: Atlantis dC18 (50×4.6 mm) 5 μm Mobile phase: A: 0.1% FA aqueous solution B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.262, Area% - 96.25 1 1H - NMR (400 MHz, DMSO - d6): δ 10.97 (s, 1H), 7.26 (t, J = 7.60 Hz, 1H), 6.91 (d, J = 7.60 Hz, 1H), 6.85 - 6.78 (m, 2H), 5.28 (d, J = 8.40 Hz, 1H), 5.12 (dd, J = 5.20, 13.20 Hz, 1H), 4.16 (d, J = 17.2 Hz, 2H), 4.09 (d, J = 17.2 Hz, 1H), 2.66 (t, J = 14.40 Hz, 1H), 2.34 - 2.27 (m, 1H), 2.04 - 1.99 (m, 3H), 1.73 - 1.70 (m, 2H), 1.38 (s, 11H), 1.29 - 1.14 (m, 2H), 1.05 - 1.03 (m, 2H)

[0364] Step - 2: (tert - Butoxycarbonyl)(4 - (((1R,4R)-4 - (((tert - butoxycarbonyl)amino)methyl)cyclohexyl)(2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)carbamic acid tert - butyl JPEG2025522795000357.jpg39127(((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamic acid tert-butyl 3 (90 mg, 0.191 mmol, 1 equiv), tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 4 (110 mg, 0.383 mmol, 2 equiv), a solution of DCM (5 mL) and DMF (2 mL) was added TFA (0.059 ml, 0.765 mmol, 4 equiv) and sodium triacetoxyborohydride (162 mg, 0.765 mmol, 4 equiv) at 0 °C. The resulting reaction mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by LCMS), the reaction mixture was diluted with ice-cold water (10 mL), extracted with DCM (2 x 10 mL), washed with brine (25 mL), dried over Na2SO4, filtered and concentrated. The resulting residue was purified by reverse phase column chromatography (Grace R column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fractions obtained from the column were lyophilized to give tert-butyl (tert-butoxycarbonyl)(4-(((1R,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 5 (20 mg, 0.025 mmol, 14.03% yield) as a white solid. LCMS: 742.5 (M+H). Method: Column: XBridge C8 (50×4.6 mm) 3.5 μm, Mobile phase: A: 0.1% TFA in H2O, Mobile phase: B: 0.1% TFA in ACN, Flow rate: 1.5 mL / min. Rt (min): 2.238; Area%-98.70. 1H-NMR (400 MHz, DMSO-d6): δ 10.97 (s, 1H), 7.40 (t, J = 7.60 Hz, 1H), 7.26 (q, J = 8.00 Hz, 2H), 6.83 - 6.82 (m, 1H), 5.09 (q, J = 4.80 Hz, 1H), 3.12 (t, J = 6.00 Hz, 2H), 3.00 (m, 2H), 2.90 (d, J = 3.60 Hz, 2H), 1.70 (d, J = 9.60 Hz, 4H), 1.69 - 1.38 (m, 4H), 1.24 (s, 33H), 1.19 (d, J = 9.20 Hz, 2H), 0.87 (q, J = 6.00 Hz, 2H).

[0365] Step - 3: 3-(4-((4-Aminobutyl)((1R,4R)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl JPEG2025522795000358.jpg38127To a ice-cooled solution of (tert-butoxycarbonyl)(4-(((1r,4r)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl (20 mg, 0.027 mmol, 1 equiv) in DCM (1 mL), HCl (4 M solution in EtOAc, 1 mL, 4.0 mmol) was added and stirred at room temperature for 2 h. After confirming the completion of the reaction by TLC analysis (10% MeOH:DCM, Rf ~0.1), the reaction mixture was concentrated and lyophilized to obtain 3-(4-((4-aminobutyl)((1R,4R)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl (7.5 mg, 0.014 mmol, 52.3% yield) as a pale yellow solid. LCMS: 428.3 (M+H). Method: Column: XBridge C8 (50×4.6 mm) 3.5 μm, Mobile Phase: A: 0.1% TFA in H2O, Mobile Phase: B: 0.1% TFA in CAN, Flow Rate: 1.5 mL / min, Rt (min): 0.8 / 66; Area % - 94.61. HPLC: 96.75%, Rt (min): 2.674. Method: Method Information: A: 0.1% TFA in H2O, B: Methanol, Flow Rate: 1.0 ml / min, Column: YMC Hydrosphere C18 (150×4.6 mm), 3 μm 1 1H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.87 - 7.86 (m, 6H), 7.44 (s, 1H), 7.33 (s, 2H), 5.13 - 5.09 (m, 1H), 4.31 (q, J = 16.40 Hz, 2H), 3.13 (s, 2H), 2.98 (t, J = 6.00 Hz, 1H), 2.89 (d, J = 5.20 Hz, 1H), 2.71 (s, 2H), 2.68 (d, J = 1.60 Hz, 3H), 2.03 (d, J = 10.80 Hz, 1H), 1.7 - 1.9 (m, 4H), 1.53 (t, J = 7.20 Hz, 5H), 1.24 - 1.25 (m, 2H), 1.12 - 1.0 (m, 2H).

[0366] Hydrochloride form of Compound 106

[0367] 3-(4-((4-Aminobutyl)(((1s,4s)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl JPEG2025522795000359.jpg40127

[0368] Step - 1: ((1s,4s)-4-Formylcyclohexyl)carbamic acid tert-butyl JPEG2025522795000360.jpg14127A solution of tert-butyl ((1S,4S)-4-(hydroxymethyl)cyclohexyl)carbamate (500 mg, 2.180 mmol, 1 equiv) in cooled DCM (11 mL) was added dropwise with a solution of sulfur trioxide pyridine (1.35 g, 8.48 mmol, 3.89 equiv) in DMSO (3.7 mL), DIPEA (1.510 mL, 8.48 mmol, 3.89 equiv), and DMSO (1.5 mL), and stirred at 0 °C for 1 h. After completion of the reaction (confirmed by TLC (40% ethyl acetate in petroleum ether, Rf: 0.4)), the reaction was quenched with 1.5 N HCl (20 mL) and extracted with DCM (2 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to give the crude product of tert-butyl ((1S,4S)-4-formylcyclohexyl)carbamate 2A (360 mg, 1.584 mmol, 72.6% yield) as a colorless liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 9.58 (s, 1H), 8.23 (s, 1H), 6.74 (s, 1H), 3.65 - 3.56 (m, 2H), 2.55 - 2.50 (m, 13H), 1.41 (s, 3H).

[0369] Step - 2: tert-butyl ((1S,4S)-4-(((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate (4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl Int-4 (200 mg, 0.465 mmol, 1 equiv) and tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (528 mg, 2.323 mmol, 5 equiv) in a mixture of JPEG2025522795000361.jpg38127DCE (2 mL) and DMF (0.5 mL) were added TFA (47.7 mg, 0.418 mmol, 0.9 equiv) and Na(OAc)3BH (35.0 mg, 0.557 mmol, 1.5 equiv) at 0 °C, and the mixture was stirred at room temperature for 12 h. After completion of the reaction (confirmed by UPLC), the reaction mixture was quenched with ice water (25 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were concentrated under vacuum to give the crude product. The crude product was purified by reverse-phase chromatography (GraceR column: generic C18, 40 g snap, 0.1% formic acid in ACN, 20 mL / min, 70 - 75% ACN in H2O), and the product fraction was lyophilized to give tert-butyl ((1r,4r)-4-(((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate 2 (130 mg, 0.202 mmol, 43% yield) as an off-white solid. 11H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.37 (t, J = 7.60 Hz, 1H), 7.19 (d, J = 7.20 Hz, 1H), 7.11 (d, J = 8.00 Hz, 1H), 6.78 (t, J = 5.20 Hz, 1H), 6.64 (d, J = 6.80 Hz, 1H), 5.17 - 5.08 (m, 1H), 4.27 (d, J = 17.20 Hz, 1H), 4.38 (d, J = 16.80 Hz, 1H), 3.44 (s, 1H), 3.13 (dd, J = 6.80, 28.00 Hz, 4H), 2.89 (t, J = 6.00 Hz, 3H), 2.04 (q, J = 4.80 Hz, 1H), 1.52 (t, J = 21.20 Hz, 4H), 1.36 (d, J = 9.20 Hz, 28H). LCMS: 642.4 (M+H), Rt (min): 2.992, Area %: 99.757. HPLC: Rt (min): 4.691, Area %: 99.938.

[0370] Step - 3: 3-(4-((4-Aminobutyl)(((1s,4s)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl JPEG2025522795000362.jpg43127 To a solution of tert-butyl ((1S,4S)-4-(((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate (130 mg, 0.203 mmol, 1 equiv) in DCM (2.5 mL) at ice-cooled temperature, HCl (4 M in ethyl acetate, 1.3 mL, 5.20 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction (confirmed by LCMS), the reaction mixture was concentrated, washed with MTBE (10 mL), and dried to obtain 3-(4-((4-aminobutyl)(((1S,4S)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl, 106 (85 mg, 0.165 mmol, 81% yield) as a pale yellow solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 8.10 (d, J = 35.60 Hz, 5H), 7.43 (s, 1H), 7.26 (s, 1H), 5.12 (q, J = 5.20 Hz, 1H), 4.39 (q, J = 16.80 Hz, 2H), 3.16 (d, J = 26.00 Hz, 4H), 2.97 - 2.88 (m, 1H), 2.76 (d, J = 4.80 Hz, 2H), 2.63 (s, 1H), 2.51 (t, J = 1.60 Hz, 1H), 2.05 (t, J = 5.20 Hz, 1H), 1.68 (s, 12H). LCMS: 442.4 (M+H), Method: Mobile phase: A: 0.1% TFA in H2O; Mobile phase: B: 0.1% TFA in ACN, Column: XBridge C8 (50×4.6 mm) 3.5 μm, Flow rate: 1.5 mL / min, Rt (min): 1.027, Area %: 99.640. HPLC: Method: A: 0.1% TFA in H2O, B: ACN, Flow rate: 2.0 ml / min, Column: Xbridge C8 (50×4.6) mm, 3.5 μm Rt (min): 1.655, Area %: 99.741.

[0371] Hydrochloride form of Compound 107

[0372] 3-(4-((4-Aminobutyl)(((1r,4r)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl JPEG2025522795000363.jpg41127

[0373] Step - 1: ((1r,4r)-4-Formylcyclohexyl)carbamic acid tert-butyl 2A JPEG2025522795000364.jpg15127To a cooled solution of ((1r,4r)-4-(hydroxymethyl)cyclohexyl)carbamic acid tert-butyl (500 mg, 2.180 mmol, 1 equivalent) in DCM (11 mL), DMSO (3.7 mL) and DIPEA (1.510 ml, 8.48 mmol, 3.89 equivalents) were added, and a solution of sulfur trioxide pyridine (1.35 g, 8.48 mmol, 3.89 equivalents) in DMSO (3.7 mL) was added dropwise. The mixture was stirred at 0 °C for 15 minutes. After completion of the reaction (confirmed by TLC (40% ethyl acetate in petroleum ether, Rf: 0.4)), the reaction mixture was quenched with 1.5 N HCl (20 mL) and extracted with DCM (2 × 20 mL). The combined organic layers were concentrated under vacuum to give the crude product of ((1r,4r)-4-formylcyclohexyl)carbamic acid tert-butyl 2A (500 mg, 2.123 mmol, 97% yield) as a yellow solid. 1 H-NMR (400 MHz, DMSO-d6): δ 9.55 (d, J = 0.80 Hz, 1H), 6.75 (d, J = 7.60 Hz, 1H), 3.16 (s, 1H), 2.52 (q, J = 2.00 Hz, 3H), 2.09 (d, J = 17.60 Hz, 2H), 1.92 - 1.83 (m, 5H), 1.38 (s, 1H), 1.19 (d, J = 8.80 Hz, 5H). LCMS: 172 (M - 56), Rt (min): 2.587, area%: 96.528.

[0374] Step - 2: tert - Butyl ((1r,4r)-4-(((4-((tert - butoxycarbonyl)amino)butyl)(2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)methyl)cyclohexyl)carbamate To a solution of tert - Butyl (4-((2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)butyl)carbamate Int4 (200 mg, 0.465 mmol, 1 equiv) and tert - Butyl ((1r,4r)-4 - formylcyclohexyl)carbamate 2A (528 mg, 2.323 mmol, 5 equiv) in a mixture of JPEG2025522795000365.jpg36127DCE (2 mL) and DMF (0.5 mL), TFA (47.7 mg, 0.418 mmol, 0.9 equiv) and Na(OAc)3BH (35.0 mg, 0.557 mmol, 1.5 equiv) were added at 0 °C, and the mixture was stirred at room temperature for 12 h. After completion of the reaction (confirmed by UPLC), the reaction mixture was quenched with ice - water (25 mL) and extracted with DCM (2×10 mL). The combined organic layers were concentrated under vacuum to give a crude product. The crude product was purified by Isolera (column size: Biotage R snap cartridge, KP - Sil, 25 g, silica gel 230 - 400 mesh) using 50 - 60% ethyl acetate in petroleum ether to give tert - Butyl ((1r,4r)-4-(((4-((tert - butoxycarbonyl)amino)butyl)(2-(2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)amino)methyl)cyclohexyl)carbamate 2 (60 mg, 0.093 mmol, 19.97% yield) as a pale yellow solid. 11H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.37 (t, J = 7.60 Hz, 1H), 7.18 (d, J = 7.20 Hz, 1H), 7.09 (d, J = 8.00 Hz, 1H), 6.78 (t, J = 5.60 Hz, 1H), 6.65 (d, J = 8.00 Hz, 1H), 5.13 - 5.08 (m, 1H), 4.38 (d, J = 16.80 Hz, 1H), 4.28 (d, J = 16.80 Hz, 1H), 4.03 (q, J = 7.20 Hz, 1H), 3.17 (d, J = 6.80 Hz, 3H), 3.03 (d, J = 6.80 Hz, 2H), 2.93 - 2.87 (m, 3H), 2.56 (t, J = 2.00 Hz, 2H), 2.02 (t, J = 9.20 Hz, 1H), 1.71 (d, J = 8.80 Hz, 4H), 1.36 (d, J = 2.00 Hz, 24H), 1.04 - 0.90 (m, 4H), LCMS: 642.4 (M+H), Rt (min): 2.900 min, Area %: 99.216. HPLC: Rt (min): 4.591, Area %: 97.770.

[0375] Step - 3: 3-(4-((4-Aminobutyl)(((1r,4r)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl JPEG2025522795000366.jpg36127 ((1r,4r)-4-(((4-((tert-Butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamic acid tert-butyl 2 (60 mg, 0.093 mmol, 1 eq) in a ice-cooled solution of DCM (1.7 mL) was added with HCl (4 M solution in ethyl acetate, 0.89 ml, 3.56 mmol, 5 vol), and stirred at room temperature for 6 hours. After completion of the reaction (confirmed by UPLC), the reaction mixture was concentrated, washed with MTBE (10 mL) to obtain the crude product, which was lyophilized to give 3-(4-((4-aminobutyl)(((1r,4r)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl (35 mg, 0.072 mmol, 77% yield) as a pale yellow solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.98 (t, J = 31.60 Hz, 6H), 7.40 (t, J = 7.60 Hz, 1H), 7.20 (t, J = 22.40 Hz, 2H), 5.12 (q, J = 5.20 Hz, 1H), 4.35 (q, J = 17.20 Hz, 2H), 3.20 (s, 2H), 3.06 (d, J = 6.40 Hz, 2H), 2.96 - 2.90 (m, 2H), 2.88 - 2.68 (m, 2H), 2.61 (d, J = 18.40 Hz, 2H), 2.04 (q, J = 5.20 Hz, 1H), 1.21 (q, J = 12.40 Hz, 2H), 0.98 (q, J = 12.40 Hz, 2H). LCMS: 441.0 (M-H), method: mobile phase: A: 0.1% FA aqueous solution; mobile phase: B: ACN; column: Atlantis dC18 (50×4.6 mm) 5 μm; flow rate: 1.5 mL / min; Rt (min): 0.995, area%: 98.488. HPLC: Method: Mobile phase A: 0.1% aqueous TFA solution; Mobile phase B: Acetonitrile; Column: Atlantis dC18 (250×4.6) mm, 5 μm; Flow rate: 1.0 mL / min; Rt (min): 6.025, Area %: 97.654.

[0376] Hydrochloride form of Compounds 108 and 109

[0377] 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000367.jpg32127JPEG2025522795000368.jpg37127JPEG2025522795000369.jpg34127

[0378] Step - 1: 3-(4-(((1s,4s)-4-Methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione and 3-(4-(((1r,4r)-4-Methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione A solution of 1 (3 g, 11.57 mmol, 1.0 eq) of (4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione in 30 mL of TFA was stirred, and sodium triacetoxyhydroborate (12.26 g, 57.9 mmol, 5.0 eq) was added. The mixture was stirred at -15 °C for 10 minutes, and then 2 (10.38 g, 93 mmol, 8.0 eq) of 4-methylcyclohexan-1-one in 10 mL of DCM was added. The reaction mixture was stirred for 16 hours. The solvent was removed under reduced pressure to obtain a crude product. The crude product compound was purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 350 g; flow rate: 60 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The desired fraction was distilled under reduced pressure to obtain 3-(4-((4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (0.56 g, 13.62% yield) as an off-white solid. The cis-trans mixture was separated by preparative HPLC (column: X-Select-C18-19x250 mm, mobile phase: 0.1% TFA in water / ACN, flow rate: 15 mL / min) to obtain 3-(4-(((1s,4s)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 108A (0.29 g, 7.05% yield) as a pale yellow solid and 3-(4-(((1r,4r)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 109A (0.15 g, 3.65% yield) as a pale yellow solid.

[0379] Step - 2: (tert-Butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1s,4s)-4-methylcyclohexyl)amino)butyl)carbamic acid tert-butyl JPEG2025522795000371.jpg37127DCM (5 mL) and DMF (2 mL) were added to a stirred solution of 3-(4-(((1s,4s)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 108A (0.098 g, 0.276 mmol, 1.0 equiv) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate (0.475 g, 1.654 mmol, 6.0 equiv) at 0 °C. TFA (0.189 g, 1.654 mmol, 6.0 equiv) and sodium triacetoxyborohydride (0.497 g, 2.344 mmol, 8.5 equiv) were added. The reaction mixture was warmed to RT and stirred for 16 h. After completion of the reaction (confirmed by UPLC), the reaction mixture was quenched with ice water (50 mL) and extracted with DCM (2 × 30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by reverse-phase column chromatography (GraceR column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to afford the desired product, tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1s,4s)-4-methylcyclohexyl)amino)butyl)carbamate (0.07 g, 39.7% yield) as an off-white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.42 (t, J = 7.60 Hz, 1H), 7.34 - 7.27 (m, 2H), 5.13 - 5.08 (m, 1H), 4.36 - 4.25 (m, 2H), 3.41 - 3.38 (m, 2H), 3.13 - 3.10 (m, 3H), 2.94 - 2.86 (m, 1H), 2.68 - 2.67 (m, 2H), 2.01 - 1.98 (m, 1H), 1.72 - 1.64 (m, 3H), 1.48 - 1.44 (m, 25H), 1.38 - 1.23 (m, 3H), 0.94 - 0.92 (m, 3H), LCMS: 627.4 (M+H), Rt (min): 3.41, Area%-97.91. HPLC: Rt (min): 4.88, Area: 99.57%.

[0380] Step - 3: 3-(4-((4-Aminobutyl)((1s,4s)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000372.jpg33127 A solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1s,4s)-4-methylcyclohexyl)amino)butyl)carbamate 5 (0.07 g, 0.112 mmol, 1.0 equiv) in DCM (3 mL) was cooled to 0 °C in an ice bath, and HCl (4 M solution in EtOAc, 1 mL) was added at 0 °C. The reaction mixture was stirred at RT for 2 h. The reaction mixture was concentrated and washed with methyl tert-butyl ether (5 mL). The solvent was decanted and concentrated under reduced pressure to obtain the crude product. The crude product was lyophilized to give 3-(4-((4-aminobutyl)((1s,4s)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl 108 (0.06 g, 106% yield) as an off-white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.81 (br s, 4H), 7.34 - 7.44 (m, 2H), 5.12 (t, J = 8.40 Hz, 1H), 4.31 - 4.35 (m, 2H), 3.13 (br s, 2H), 2.92 (s, 1H), 2.60 - 2.71 (m, 3H), 2.01 - 2.05 (m, 1H), 1.69 - 1.77 (m, 4H), 1.57 - 1.63 (m, 8H), 1.24 - 1.31 (m, 2H), 0.93 - 0.94 (m, 3H). LCMS: 427.3 (M+H). Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 1.5 mL / min. Column: XBridge C8 (50×4.6 mm) 3.5 μm. Rt (min): 1.340; Area % - 99.13. HPLC method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 2.0 mL / min. Column: Xbridge C8 (50×4.6) mm, 3.5 μm. Rt (min): 2.38; Area % - 98.95.

[0381] Step - 2A: (tert - Butoxycarbonyl)(4 - ((2 - (2,6 - dioxopiperidin - 3 - yl)-1 - oxoisoindolin - 4 - yl)((1r,4r)-4 - methylcyclohexyl)amino)butyl)carbamic acid tert - butyl JPEG2025522795000373.jpg 391273 - (4 - (((1r,4r)-4 - methylcyclohexyl)amino)-1 - oxoisoindolin - 2 - yl)piperidine - 2,6 - dione, 109A (0.1 g, 0.281 mmol, 1.0 equivalent) and (tert - butoxycarbonyl)(4 - oxobutyl)carbamic acid tert - butyl (0.162 g, 0.563 mmol, 2.0 equivalents) in a stirred solution of DMF (5 mL):DCM (5 mL), TFA (0.160 g, 1.407 mmol, 4.0 equivalents) was added. After 10 minutes, sodium triacetoxyborohydride (0.239 g, 1.125 mmol, 4.0 equiv) was added portionwise to this mixture at 0 °C. The reaction mixture was stirred at room temperature for 4 hours. Monitoring the completion of the reaction by UPLC showed that the SM was 39%. The reaction mixture was cooled to 0 °C, tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate (0.485 g, 1.688 mmol, 6.0 equiv) was added, followed by TFA (0.160 g, 1.407 mmol, 5.0 equiv). After 10 minutes, sodium triacetoxyborohydride (0.507 g, 2.391 mmol, 8.5 equiv) was added and the reaction mixture was stirred at room temperature for 16 hours. The completion of the reaction was monitored by UPLC. The reaction mixture was quenched with ice water (20 mL) and extracted with DCM (3 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product compound was purified by flash column chromatography (silica gel, 230 - 400 mesh size) using ethyl acetate / petroleum ether (50%) as the eluent to give the desired product as an off-white solid. The product was further purified by reverse-phase column chromatography (Grace column: C18 40 μm, 50 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to give tert-butyl (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1r,4r)-4-methylcyclohexyl)amino)butyl)carbamate 5A (0.04 g, 20.87% yield) as an off-white solid. 1H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.42 - 7.38 (m, 1H), 7.30 - 7.23 (m, 2H), 5.11 - 5.07 (m, 1H), 4.34 - 4.23 (m, 2H), 3.12 - 3.11 (m, 2H), 2.86 - 3.01 (m, 2H), 2.65 - 2.70 (m, 1H), 2.01 - 1.98 (m, 1H), 1.69 - 1.66 (m, 4H), 1.53 - 1.46 (m, 5H), 1.37 (s, 18H), 1.22 - 1.33 (m, 3H), 0.99 - 0.91 (m, 2H), 0.85 - 0.83 (m, 3H). LCMS: 627.3 (M - H), Rt (min): 2.27, Area% - 92.32.

[0382] Step - 3A: 3-(4-((4-Aminobutyl)((1r,4r)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000374.jpg39127 To a ice-cooled solution of (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1r,4r)-4-methylcyclohexyl)amino)butyl)carbamic acid tert-butyl 5A (0.04 g, 0.064 mmol, 1.0 equiv) in DCM (3 mL) at 0 °C, HCl (4 M solution in EtOAc, 1 mL) was added. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc Rf ~ 0.3, and LCMS), the reaction mixture was evaporated in vacuo to obtain the crude product. The crude product was washed with MTBE (5 mL), the solvent was decanted, and it was dried under reduced pressure and lyophilized to obtain the desired product 109, 3-(4-((4-aminobutyl)((1r,4r)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (0.03 g, 101% yield) as an off-white solid. 1H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 7.99 (brs, 3H), 7.37 - 7.51 (m, 3H), 5.10 - 5.15 (m, 1H), 4.44 - 4.63 (m, 2H), 3.30 (s, 2H), 3.01 (s, 3H), 2.92 - 2.98 (m, 2H), 2.60 - 2.78 (m, 3H), 2.45 - 2.50 (m, 1H), 2.06 - 2.08 (m, 1H), 1.66 - 1.67 (m, 4H). LCMS: 345.1 (M+H). Method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 1.5 mL / min. Column: XBridge C8 (50×4.6 mm) 3.5 μm. Rt (min): 1.351; Area% - 99.19. HPLC method: Mobile phase A: 0.1% TFA in H2O. Mobile phase B: Acetonitrile. Flow rate: 2.0 mL / min. Column: Xbridge C8 (50×4.6) mm, 3.5 μm. Rt (min): 2.418; Area% - 99.64.

[0383] Hydrochloride form of Compound 115 JPEG2025522795000375.jpg To a solution of compound 10 (120 mg, 0.2 mmol, 1.0 equivalent) in DCM (6.0 mL) was added piperidine (338 mg, 4.0 mmol, 20.0 equivalents). The mixture was stirred at room temperature for 17 h. Next, the reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 20% - 45%) to give compound 115 (11.4 mg, 13%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 10) = 0.9 R f (Compound 115) = 0.1 LCMS: [C 21 H 26 N4O3] + Calculated value: 383, Measured value: 383. 11H NMR (400 MHz, D2O): δ 7.72 (d, J = 7.3 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 5.06 (dd, J = 13.3, 4.9 Hz, 1H), 4.53 (q, J = 17.5 Hz, 2H), 4.27 (s, 2H), 3.60 (s, 2H), 3.50 - 3.35 (m, 2H), 2.97 - 2.62 (m, 2H), 2.47 - 2.30 (m, 1H), 2.25 - 2.10 (m, 1H), 1.37 - 1.20 (m, 2H), 1.13 (dd, J = 14.5, 7.2 Hz, 2H), 0.65 (t, J = 7.2 Hz, 3H).

[0384] Hydrochloride form of Compound 116 To a solution of Compound 4 (20 mg, 0.04 mmol, 1.0 equiv) in MeOH (1 mL) was added HCl (0.2 ml, 2N, 0.4 mmol, 10.0 equiv). The mixture was stirred at room temperature for 24 h and concentrated under reduced pressure. The residue was purified by Biotage (C18, acetonitrile / H2O: 30% - 60%) to give Compound 116 (14.0 mg, 74%) as a white solid. TLC: DCM / methanol = 10 / 1 R f (Compound 4) = 0.5 R f (Compound 116) = 0.1 LCMS: [C 21 H 26 N4O3] + Calculated value: 383, Found: 383. 11H NMR (400 MHz, D2O): δ 8.30 - 8.20 (m, 2H), 7.42 - 7.25 (m, 3H), 4.98 (d, J = 12.6 Hz, 1H), 4.38 (q, J = 17.2 Hz, 2H), 3.67 (s, 2H), 3.11 - 2.98 (m, 2H), 2.81 - 2.62 (m, 4H), 2.42 - 2.22 (m, 1H), 2.15 - 2.01 (m, 1H), 1.53 (s, 3H), 1.50 - 1.28 (m, 4H).

[0385] Hydrochloride form of Compound 123

[0386] 3-(4-((4-Amino-3,3-dimethylbutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride JPEG2025522795000377.jpg20127JPEG2025522795000378.jpg34127JPEG2025522795000379.jpg35127

[0387] Step - 1: 4-Methoxy-2,2-dimethyl-4-oxobutanoic acid JPEG2025522795000380.jpg19127To an ice-cooled solution of dimethyl succinate 1 (100 g, 684 mmol, 1 equiv) in methanol (1500 ml, 15 vol), concentrated H2SO4 (10 mL) was added dropwise, and the mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 40% EtOAc in petroleum ether, Rf ~0.5, KMnO4), the reaction mixture was concentrated under vacuum at 30 °C. The resulting residue was poured into an ice-cooled solution of saturated NaHCO3 solution (1000 mL) and washed with EtOAc (2 × 500 mL). The separated aqueous layer was further acidified to pH ~2 with aqueous HCl solution (6N) and extracted with EtOAc (2x800 mL). The combined organic extracts were washed with brine (1x600 mL), dried over Na2SO4, filtered, and concentrated to obtain 4-methoxy-2,2-dimethyl-4-oxobutanoic acid 2 (40 g, 235 mmol, 34.3% yield) as a colorless liquid. 1H-NMR (400 MHz, DMSO-d6): δ 12.17 (s, 1H), 3.57 (s, 3H), 2.53 (s, 2H), 1.18 (s, 6H).

[0388] Step - 2: Methyl 4 - amino - 3,3 - dimethyl - 4 - oxobutanoate To a solution of 2 (25 g, 156 mmol, 1 equiv) of 2 - methoxy - 2,2 - dimethyl - 4 - oxobutanoic acid in DCM (250 mL, 10 vol) cooled in ice, thionyl chloride (111 g, 937 mmol, 6 equiv) was added dropwise, and the mixture was stirred at room temperature for 3 h. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc in petroleum ether, Rf ≈ 0.4, as the methyl ester), the reaction mixture was distilled under vacuum and dried completely under reduced pressure. The resulting acid chloride mixture was dissolved in THF (250 mL) and added dropwise to a pre - cooled NH3 solution (0.5 M solution in THF, 375 mL) at - 78 °C. The reaction mixture was warmed to room temperature and stirred for 30 min. After completion of the reaction (confirmed by TLC analysis, 60% EtOAc in petroleum ether, Rf ≈ 0.2.), the reaction mixture was concentrated, and the resulting residue was poured into a cooled ice solution of saturated NaHCO3 solution (500 mL) and extracted with EtOAc (2×500 mL). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered, and concentrated to obtain methyl 4 - amino - 3,3 - dimethyl - 4 - oxobutanoate 3 (8 g, 50.3 mmol, 32.2% yield) as a pale yellow liquid. 1 H-NMR (400 MHz, DMSO-d6): δ 6.75 - 7.07 (m, 2H), 3.54 (s, 3H), 2.51 (s, 2H), 1.15 (s, 6H)

[0389] Step - 3: 4 - Amino - 3,3 - dimethylbutan - 1 - ol A solution of methyl 3-(13 g, 82 mmol, 1 equiv) of 3-amino-3,3-dimethyl-4-oxobutanoate in THF (100 mL, 8 vol) was added dropwise with LiAlH4 (2 M in THF, 82 mL, 163 mmol, 2 equiv) over 30 minutes at 0 °C. After the addition was complete, the reaction mixture was warmed to room temperature and slowly heated to 65 °C and stirred for 2 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf ~0.1), the reaction mixture was cooled to 0 °C and slowly quenched with saturated Na2SO4 solution (30 mL). The precipitated inorganic salts were removed by filtration through a Celite pad and washed with EtOAc (1×100 mL). The combined filtrates were dried over Na2SO4, filtered, and concentrated to give 4-amino-3,3-dimethylbutan-1-ol 4 (7.8 g, 64.0 mmol, 78% yield) as a light brown liquid, which was used without further purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 3.40 - 3.43 (m, 2H), 2.30 (s, 2H), 1.35 - 1.38 (m, 2H), 0.80 (s, 6H).

[0390] Step - 4: tert-Butyl (4-hydroxy-2,2-dimethylbutyl)carbamate A solution of 4-amino-3,3-dimethylbutan-1-ol 4 (7.5 g, 57.2 mmol, 1 equiv) in 13127THF (150 ml, 20 vol) and water (40 ml, 5 vol) was added dropwise with sodium bicarbonate (7.2 g, 86 mmol, 1.5 equiv) and Boc2O (14 ml, 57.2 mmol, 1 equiv) at room temperature. The resulting reaction mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf ~0.7), the reaction mixture was diluted with EtOAc (1×100 mL), the layers were separated, and extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with aqueous salt solution (1×100 mL), dried over Na2SO4, and concentrated under reduced pressure to give a light brown liquid. The resulting residue was purified by chromatography (column size: Biotage R snap cartridge, KP-Sil, 50 g, 100 - 200 silica gel) using 40 - 50% EtOAc in petroleum ether to give tert-butyl (4-hydroxy-2,2-dimethylbutyl)carbamate 5 (5.0 g, 22.85 mmol, 40.0% yield) as an off-white crystalline solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 6.70 (s, 1H), 4.28 (t, J = 6.40 Hz, 1H), 3.41 - 3.47 (m, 2H), 2.76 (d, J = 8.80 Hz, 2H), 1.43 (s, 9H), 0.83 (s, 6H).

[0391] Step-5: tert-Butyl 2-hydroxy-4,4-dimethylpyrrolidine-1-carboxylate A solution of oxalyl chloride (0.5 mL, 5.52 mmol, 1.2 equiv) in DCM (5 mL) was added dropwise with DMSO (1 mL, 14.08 mmol) at -78 °C and stirred at the same temperature for 15 minutes. To this was added dropwise (4-hydroxy-2,2-dimethylbutyl)carbamic acid tert-butyl (1.0 g, 4.6 mmol, 1 equiv) in DCM (3 mL) over 15 minutes and stirred at -78 °C for 30 minutes. At this point, N,N-diisopropylethylamine (2.4 mL, 13.81 mmol, 3 equiv) was added dropwise to the mixture at -78 °C and slowly warmed to 0 °C. After stirring at 0 °C for 30 minutes, the completion of the reaction was confirmed by TLC analysis (20% EtOAc in petroleum ether, Rf ~0.6, KMnO4). The reaction mixture was diluted with DCM (20 mL) and washed with 10% aqueous citric acid (1×50 mL), water (1×30 mL) and brine (1×30 mL), dried over Na2SO4, filtered and concentrated under vacuum at <30 °C to give tert-butyl 2-hydroxy-4,4-dimethylpyrrolidine-1-carboxylate (0.95 g, crude product) as a pale yellow liquid and used further without purification. GCMS: Mass found; MS: 215.1; Rt (min): 3.005. Method: HP5_SCAN350_MS.amx

[0392] Step - 6: (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylbutyl)carbamic acid tert-butyl JPEG2025522795000385.jpg 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1.0 g, 3.86 mmol, 1.0 equiv) and tert-butyl 2-hydroxy-4,4-dimethylpyrrolidine-1-carboxylate (0.996 g, 4.63 mmol, 1.2 equiv) in CH2Cl2 (10 mL) and DMF (10.00 mL) were stirred, and trifluoroacetic acid (1.189 ml, 15.43 mmol, 4.0 equiv) was added. The mixture was stirred at 0 °C for 10 minutes. Sodium triacetoxyborohydride (3.27 g, 15.43 mmol, 4.0 equiv) was added portionwise to this mixture at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC and LCMS analysis, 100% EtOAc, Rf ~0.7), the reaction mixture was quenched with ice-cold water (20 mL) and extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum to give the crude product as a pale yellow liquid. The crude product compound was purified by Isolera chromatography (column size: Biotage R snap cartridge, KP-Sil, 100 g, 100 - 200 silica gel) using 75 - 85% ethyl acetate in petroleum ether. The fractions were collected and concentrated under vacuum to give tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylbutyl)carbamate 8 (0.6 g, 1.049 mmol, 27.2% yield) as a pale yellow semi-solid. 11H-NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 7.29 (t, J = 10.40 Hz, 1H), 6.93 (d, J = 10.00 Hz, 1H), 6.82 - 6.86 (m, 1H), 6.75 (d, J = 10.80 Hz, 1H), 5.49 (s, 1H), 5.08 - 5.15 (m, 1H), 4.08 - 4.25 (m, 2H), 3.13 - 3.14 (m, 2H), 2.73 - 2.91 (m, 3H), 2.65 (m, 1H), 2.30 - 2.36 (m, 1H), 2.01 - 2.08 (m, 1H), 1.45 - 1.50 (m, 2H), 1.38 (s, 9H), 0.94 (s, 6H). LC-MS: 403.1 (M - tBu), Rt (min): 2.302, Area% - 80.229.

[0393] Step - 7: tert-Butyl (4 - ((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)-2,2-dimethylbutyl)carbamate JPEG2025522795000386.jpg37127tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylbutyl)carbamate 8 (0.3 g, 0.654 mmol, 1.0 equiv) and pentanal 9 (0.564 g, 6.54 mmol, 10.0 equiv) in CH2Cl2 (5 mL) and DMF (5.00 mL) were stirred, and trifluoroacetic acid (0.202 ml, 2.62 mmol, 4.0 equiv) was added. The mixture was stirred at 0 °C for 10 minutes. Sodium triacetoxyborohydride (0.555 g, 2.62 mmol, 4.0 equiv) was added portionwise at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC and LCMS analysis, 100% EtOAc, Rf ~0.8), the reaction mixture was quenched with ice-cold water (20 mL) and extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum to give the crude product as a pale yellow liquid. The crude product compound was purified by preparative HPLC (column: X-Bridge-c18 19.1X250, mobile phase: 0.1% FA in water / ACN, flow rate: 15 mL / min) to give tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)-2,2-dimethylbutyl)carbamate 10 (80 mg, 0.145 mmol, 22.21% yield) as an off-white solid. 1 1H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.34 - 7.39 (m, 1H), 7.16 - 7.18 (m, 1H), 6.82 - 7.05 (m, 2H), 5.09 - 5.13 (m, 1H), 4.25 - 4.42 (m, 2H), 3.16 - 3.34 (m, 4H), 2.87 - 2.92 (m, 1H), 2.75 - 2.77 (m, 3H), 2.02 (m, 1H), 1.36 (m, 9H), 1.25 (s, 9H), 0.80 - 0.83 (m, 9H). LCMS: 529.3 (M+H), Rt (min): 2.715, area%-99.791.

[0394] Step - 8: 3-(4-((4-Amino-3,3-dimethylbutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride To a stirred suspension of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)-2,2-dimethylbutyl)carbamate (80 mg, 0.151 mmol) in dichloromethane (2 mL) at 0 °C was added HCl (4 M in ethyl acetate, 5 mL, 165 mmol), and then the mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf ~ 0.1), the reaction mixture was concentrated under vacuum. The resulting solid was dissolved in water (10 mL), washed with MTBE (10 mL), and the aqueous layer was lyophilized to give 3-(4-((4-amino-3,3-dimethylbutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl (70 mg, 0.145 mmol, 96% yield) as a light brown solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.87 (s, 3H), 7.16 - 7.43 (m, 3H), 5.11 (s, 1H), 4.33 - 4.35 (m, 2H), 3.22 (m, 4H), 2.90 (m, 1H), 2.63 (m, 3H), 2.50 (m, 3H), 2.10 (m, 1H), 1.43 (m, 3H), 1.24 (m, 4H), 0.94 (m, 5H), 0.83 (m, 3H) LCMS: 429.3 (M + H) 10 mM aqueous ammonium bicarbonate solution. Method: Column: X-BRIDGE C8 (50×4.6) 3.5 μm, Mobile phase: A: 0.1% TFA in H2O, B: 0.1% TFA in acetonitrile, Flow rate: 1.5 mL / min. HPLC: Method: Mobile Phase: A:, Mobile Phase: B: Acetonitrile, Flow Rate: 1.0 mL / min, Column: X-Bridge C8 (50×4.6) mm, 3.5 μm, RT: 5.333 min, Area: 96.027%.

[0395] Hydrochloride form of Compound 124

[0396] 3-(4-((4-Amino-3,3-dimethylbutyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000388.jpg19127JPEG2025522795000389.jpg43127JPEG2025522795000390.jpg40127

[0397] Step - 1: tert-Butyl (4-((tert-butyldimethylsilyl)oxy)butyl)carbamate JPEG2025522795000391.jpg12127To a solution of tert-butyl (4-hydroxybutyl)carbamate (5.0 g, 26.4 mmol, 1.0 eq) in dichloromethane (100 ml) at 0 °C, imidazole (4.5 g, 66.0 mmol, 2.5 eq) and TBS-Cl (4.78 g, 31.7 mmol, 1.5 eq) were added, and the mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC, 20% EtOAc / petroleum ether, Rf ca. 0.5, KMnO4), the reaction mixture was quenched with H2O (50 mL) and extracted with dichloromethane (1×50 mL). The organic layer was dried over Na2SO4 and concentrated under vacuum to give the crude product compound. The crude product compound was purified by chromatography (column size: Biotage R snap cartridge, KP-Sil, 100 g, 230 - 400 silica gel) using 10 - 15% EtOAc in petroleum ether to give tert-Butyl (4-((tert-butyldimethylsilyl)oxy)butyl)carbamate (8.0 g, 26.3 mmol, 99% yield) as a colorless liquid. 1H-NMR (400 MHz, DMSO-d6): δ 6.77 (s, 1H), 3.56 (t, 2H), 2.90 (t, 2H), 1.16-1.20 (m, 4H), 1.07 (s, 9H), 0.86 (s, 9H), 0.03 (s, 6H).

[0398] Step - 2: (tert-Butoxycarbonyl)(4-((tert-butyldimethylsilyl)oxy)butyl)carbamic acid tert-butyl JPEG2025522795000392.jpg13127n-BuLi (2.5 M solution in hexane, 12.65 ml, 31.6 mmol, 1.2 equiv) was added to a stirred solution of tert-butyl (4-((tert-butyldimethylsilyl)oxy)butyl)carbamate 2 (8.0 g, 26.4 mmol, 1.0 equiv) in THF (80 mL) at 0 °C and stirred for 15 minutes at 0 °C. A solution of (Boc)2O (7.34 ml, 31.6 mmol, 1.2 equiv) in THF (20 mL) was added to the reaction mixture at the same temperature. The resulting reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction (confirmed by TLC analysis, 10% EtOAc / petroleum ether, Rf ~0.7, KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (2 × 100 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give the crude product of (tert-butoxycarbonyl)(4-((tert-butyldimethylsilyl)oxy)butyl)carbamic acid tert-butyl 3 (11.0 g, 27.0 mmol, 102% yield) as a pale yellow liquid, which was used further without purification. 1 H-NMR (400 MHz, DMSO-d6): δ 3.58 (t, 2H), 3.48 (t, 2H), 1.16-1.20 (m, 4H), 1.07 (s, 18H), 0.86 (s, 9H), 0.03 (s, 6H).

[0399] Step - 3: (tert-Butoxycarbonyl)(4-hydroxybutyl)carbamic acid tert-butyl JPEG2025522795000393.jpg11127(tert-Butoxycarbonyl)(4-((tert-butyldimethylsilyl)oxy)butyl)carbamic acid tert-butyl 3 (10 g, 24.77 mmol, 1.0 equiv) was added to a stirred solution of THF (160 mL) at room temperature with TBAF (1 M solution in THF, 24.77 mL, 24.77 mmol, 1.5 equiv), and the mixture was stirred overnight at the same temperature. After completion of the reaction (confirmed by TLC analysis, 20% EtOAc / petroleum ether, Rf about 0.2, KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with DCM (2×50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product as a pale yellow liquid. The crude product compound was purified by Isolera chromatography (Biotage R snap cartridge, KP-Sil, 100 g, 230 - 400 silica gel) using 15 - 20% EtOAc in petroleum ether to obtain (tert-butoxycarbonyl)(4-hydroxybutyl)carbamic acid tert-butyl 4 (6 g, 20.53 mmol, 83% yield) as a yellow liquid. 1 H-NMR (400 MHz, DMSO-d6): δ 4.40 (t, J = 6.80 Hz, 1H), 3.41 - 3.48 (m, 2H), 3.33 - 3.39 (m, 2H), 1.44 (m, 4H), 1.27 (s, 18H)

[0400] Step - 4: (tert-Butoxycarbonyl)(4-oxobutyl)carbamic acid tert-butyl JPEG2025522795000394.jpgA solution of oxalyl chloride (1.8 mL, 20.73 mmol, 2.0 equiv) and CH2Cl2 (30 mL) was added dropwise with DMSO (2.95 mL, 41.5 mmol, 4.0 equiv) in CH2Cl2 (5 mL) at -78 °C, and the mixture was stirred at the same temperature for 15 minutes. To this mixture was added dropwise a solution of tert-butyl (tert-butoxycarbonyl)(4-hydroxybutyl)carbamate 4 (3 g, 10.37 mmol, 1.0 equiv) in CH2Cl2 (10 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 30 minutes, and then triethylamine (9 mL, 62.2 mmol, 6.0 equiv) was added dropwise at -78 °C. Next, the reaction mixture was slowly warmed to 0 °C and stirred for 30 minutes. After completion of the reaction (confirmed by TLC analysis, 45% EtOAc / petroleum ether, Rf ≈ 0.6, KMnO4), the reaction mixture was diluted with DCM (20 mL), washed with 10% aqueous citric acid solution (1×100 mL) and water (1×100 mL), dried over Na2SO4, and concentrated under reduced pressure (below 30 °C). The crude product was co-distilled with toluene to give tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (3.2 g, 11.14 mmol, 107% yield) as a yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 9.65 (s, 1H), 3.48 (t, J = 9.20 Hz, 2H), 2.41-2.50 (m, 2H), 1.70-2.30 (m, 2H), 1.44 (s, 18H),

[0401] Step-5: tert-butyl (tert-butoxycarbonyl)(4-((4-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate JPEG2025522795000395.jpg38127(4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylbutyl)carbamic acid tert-butyl 8 (0.3 g, 0.654 mmol, 1.0 equiv) and (tert-butoxycarbonyl)(4-oxobutyl)carbamic acid tert-butyl 5 (0.376 g, 1.308 mmol, 2.0 equiv) in CH2Cl2 (5 mL) and DMF (5.00 mL) were stirred, and trifluoroacetic acid (0.202 ml, 2.62 mmol, 4.0 equiv) was added, and the mixture was stirred at 0 °C for 10 minutes. To this mixture, sodium triacetoxyborohydride (0.555 g, 2.62 mmol, 4.0 equiv) was added portionwise at 0 °C, and the resulting colorless turbid mass was stirred at room temperature for 16 hours. After completion of the reaction (confirmed by TLC analysis, 100% EtOAc, Rf about 0.7), the reaction mixture was quenched with ice-cold water (20 mL) and extracted with DCM (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum to obtain the crude product compound. The crude product compound was purified by preparative HPLC (column: X-Bridge-c18 19.1×250, mobile phase: 0.1% FA in water / ACN, flow rate: 15 mL / min) to give (tert-butoxycarbonyl)(4-((4-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 9 (75 mg, 0.100 mmol, 15.28% yield) as a white solid. 11H-NMR (400 MHz, DMSO-d6): δ 10.95 (s, 1H), 7.35 - 7.39 (m, 1H), 7.19 - 7.21 (m, 1H), 7.05 - 7.07 (m, 1H), 6.79 - 6.81 (m, 1H), 5.10 - 5.14 (m, 1H), 4.26 - 4.41 (m, 2H), 3.43 - 3.46 (m, 2H), 3.17 - 3.19 (m, 4H), 2.89 - 2.93 (m, 1H), 2.68 - 2.75 (m, 2H), 2.00 - 2.02 (m, 2H), 1.29 - 1.49 (m, 16H), 1.24 (s, 18H), 0.80 (s, 6H). LCMS: 730.4 (M + H). Method: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: Acetonitrile, Flow rate: 1.5 mL / min. HPLC: Method: Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: Acetonitrile, Flow rate: 2.0 mL / min. Column: X - Bridge C8 (50×4.6) mm, 3.5 μm. RT: 5.166 min, Area: 97.332%

[0402] Step - 6: 3-(4-((4-Amino-3,3-dimethylbutyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochlorideJPEG2025522795000396.jpg35127 (tert-Butoxycarbonyl)(4-((4-((tert-Butoxycarbonyl)amino)-3,3-dimethylbutyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl 9 (70 mg, 0.096 mmol, 1.0 eq) in a stirred solution of CH2Cl2 (3 mL) was added HCl (4 M solution in dioxane, 5 mL) at 0 °C, and then stirred at room temperature for 2 h. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum below 40 °C to give a pale yellow solid. The obtained solid was dissolved in water (10 mL), washed with MTBE (2 x 10 mL), and the aqueous layer was lyophilized to give 3-(4-((4-Amino-3,3-dimethylbutyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl 124 (40 mg, 0.077 mmol, 80% yield) as an off-white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 7.93 (s, 6H), 7.34 - 7.44 (m, 1H), 7.16 - 7.28 (m, 2H), 5.11 - 5.16 (m, 1H), 4.30 - 4.45 (m, 2H), 3.23 (m, 4H), 2.90 - 2.98 (m, 1H), 2.76 - 2.77 (m, 2H), 2.64 - 2.68 (m, 3H), 2.02 - 2.05 (m, 1H), 1.52 (m, 7H), 0.95 - 0.96 (m, 6H), LCMS: 430.2 (M+H). Method: Column: Atlantis dC18 (50×4.6) mm, 5 μm, Mobile phase: A: 0.1% FA aqueous solution, Mobile phase: B: Acetonitrile, Flow rate: 1.5 mL / min. HPLC: Method: Mobile phase: 0.1% TFA aqueous solution, Mobile phase B: Acetonitrile, Flow rate: 2.0 mL / min. Column: X-Bridge C8 (50×4.6) mm, 3.5 μm. RT: 1.407 min, Area: 96.954%.

[0403] Hydrochloride form of Compound 125

[0404] 3-(4-((5-Amino-3,3-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000397.jpg21127JPEG2025522795000398.jpg20127JPEG2025522795000399.jpg36127

[0405] Step - 1: 5-Ethoxy-3,3-dimethyl-5-oxopentanoic acid To a ice-cooled solution of 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione 1 (10 g, 70.3 mmol, 1 equivalent) in ethanol (50 ml, 5 vol), sodium ethoxide (0.479 g, 7.03 mmol, 0.1 equivalent) was added dropwise and stirred at 85 °C for 16 hours. After completion of the reaction (confirmed by LCMS analysis), the reaction mixture was distilled under reduced pressure. The obtained residue was poured into an ice-cooled solution of saturated NaHCO3 solution (200 mL) and washed with EtOAc (2 × 50 mL). The separated aqueous layer was further acidified with aqueous HCl solution (6N, 40 ml, pH ~ 2) and extracted with EtOAc (2 × 50 mL). The combined organic extracts were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated to obtain 5-ethoxy-3,3-dimethyl-5-oxopentanoic acid 2 (6 g, 31.9 mmol, 45.3% yield) as a colorless liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 12.03 (s, 1H), 4.04 (q, J = 7.20 Hz, 2H), 2.37 (s, 2H), 2.29 (d, J = 7.20 Hz, 2H), 1.18 (t, J = 7.20 Hz, 3H), 1.05 (s, 6H).

[0406] Step - 2: Ethyl 5-amino-3,3-dimethyl-5-oxopentanoate To an ice-cooled solution of 2-(6g, 31.9 mmol, 1 equiv) of 2-ethoxy-3,3-dimethyl-5-oxopentanoic acid in DCM (60 ml, 10 vol), thionyl chloride (15 ml, 206 mmol, 6 equiv) was added dropwise, followed by the dropwise addition of a catalytic amount of DMF (2 drops), and the mixture was stirred at room temperature for 3.5 h. After completion of the reaction (confirmed by TLC analysis, 30% EtOAc in petroleum ether, Rf ~0.8, as the methyl ester), the reaction mixture was distilled under reduced pressure and dried. The resulting acid chloride mixture was dissolved in THF (30 mL) and added dropwise to a pre-cooled ammonia solution (0.5 M in THF, 90 ml, 4159 mmol) at -78 °C. The reaction mixture was warmed to room temperature and stirred for 30 min. After completion of the reaction (confirmed by TLC analysis, 30% EtOAc in petroleum ether, Rf ~0.2.), the reaction mixture was concentrated, and the resulting residue was poured into an ice-cooled solution of saturated NaHCO3 solution (150 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered, and concentrated to obtain ethyl 5-amino-3,3-dimethyl-5-oxopentanoate 3 (4.4 g, 159 mmol, 50% yield) as a yellow liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 7.21 (s, 1H), 6.73 (s, 1H), 4.04 (q, J = 7.20 Hz, 2H), 2.36 (s, 2H), 2.08 (s, 2H), 1.18 (t, J = 7.20 Hz, 3H), 1.03 (s, 6H). LCMS: 188.1 (M+H), Rt (min): 1.694, area %: 99.678.

[0407] Step - 3: 5-Amino-3,3-dimethylpentan-1-ol A solution of ethyl 3-(4.4 g, 23.50 mmol, 1 eq) of 121275-amino-3,3-dimethyl-5-oxopentanoate in THF (15 ml, 8 vol) was added dropwise with LAH (2 M in THF, 23.50 ml, 47.0 mmol, 2 eq) over 5 minutes at 0 °C. After the addition was complete, the reaction mixture was warmed to room temperature and then slowly heated to 65 °C and stirred for 2 hours. After completion of the reaction (confirmed by TLC analysis, EtOAc, Rf about 0.2), the reaction mixture was cooled to 0 °C and quenched slowly with saturated Na2SO4 solution (60 mL). The precipitated salt was removed by filtration through a Celite pad and washed with EtOAc (50 mL). The combined filtrates were dried over Na2SO4, filtered, and concentrated to give 5-amino-3,3-dimethylpentan-1-ol 4 (2 g, 13.30 mmol, 56.6% yield) as a brown liquid. 1 1H-NMR (400 MHz, CDCl3): δ 3.70 (t, J = 7.60 Hz, 3H), 2.73 (q, J = 8.00 Hz, 2H), 1.54 (t, J = 7.60 Hz, 2H), 1.44-1.40 (m, 2H), 1.02 (s, 1H), 0.93 (s, 9H). LCMS: 132.2 (M+H), Rt (min): 0.603, area%: 87.229.

[0408] Step - 4: tert-Butyl (5-hydroxy-3,3-dimethylpentyl)carbamate JPEG2025522795000403.jpg A stirred suspension of 5-amino-3,3-dimethylpentan-1-ol 4 (2 g, 15.24 mmol, 1 equiv) and sodium bicarbonate (2.56 g, 30.5 mmol, 2 equiv) in a mixture of 13127THF (30 ml, 15 vol) and water (2 ml, 1 vol) was added dropwise with Boc-anhydride (3.54 ml, 15.24 mmol, 1 equiv) at room temperature. The resulting reaction mixture was stirred at room temperature for 16 h. After completion of the reaction (confirmed by LCMS analysis), anhydrous Na2SO4 was added to the reaction mixture, filtered, and washed with EtOAc (20 mL). The obtained filtrate was concentrated under reduced pressure to give the crude product as a yellow liquid. The crude product was purified by column chromatography (Biotage R snap cartridge, KP-Sil, 100 g, 100 - 200 silica gel) using 20 - 25% EtOAc in petroleum ether to give tert-butyl (5-hydroxy-3,3-dimethylpentyl)carbamate 5 (1.7 g, 7.33 mmol, 48.1% yield) as a yellow liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 6.70 (s, 1H), 3.43 (d, J = 4.40 Hz, 2H), 2.94 - 2.87 (m, 2H), 1.44 - 1.20 (m, 16H), 0.78 (s, 6H). LCMS: 132.3 (M - Boc), Rt (min): 2.14, area%: 99.812.

[0409] Step - 5: tert-Butyl (5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate JPEG2025522795000404.jpg14127A solution of tert-butyl (5-hydroxy-3,3-dimethylpentyl)carbamate 5 (1.7 g, 7.35 mmol, 1.0 equiv) and imidazole (1.251 g, 18.37 mmol, 2.5 equiv) in DCM (20 mL) was added with TBS-Cl (1.329 g, 8.82 mmol, 1.2 equiv) at 0 °C and stirred at room temperature for 13 h. After completion of the reaction (confirmed by TLC, 30% EtOAc in petroleum ether, Rf ~0.9, KMnO4), the reaction was quenched with water (50 mL) and extracted with DCM (1x30 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give tert-butyl (5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate 6 (2.5 g, 7.22 mmol, 98% yield) as a yellow liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 6.69 (t, J = 5.20 Hz, 1H), 3.63 (t, J = 7.60 Hz, 2H), 2.90 (q, J = 5.60 Hz, 2H), 1.40 (s, 10H), 1.37 (s, 2H), 0.85 (t, J = 3.20 Hz, 14H), 0.03 (q, J = 2.80 Hz, 6H). LCMS: 246.2 (M - Boc), Rt (min): 3.581, area%: 99.821.

[0410] Step - 6: tert-Butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate JPEG2025522795000405.jpg15127A solution of tert-butyl (5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate 6 (2.5 g, 7.23 mmol) in THF (25 ml, 10 vol) was added with n-BuLi (2.5 M in hexane, 3.47 ml, 8.68 mmol, 1.2 eq) at 0 °C. The reaction mixture was stirred at 0 °C for 15 minutes. At the same temperature, a solution of (Boc)2O (1.316 ml, 5.67 mmol, 1.2 eq) in THF (10 mL) was added to the reaction mixture. The resulting reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction (confirmed by TLC analysis, 5% EtOAc in petroleum ether, Rf about 0.8, KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with DCM (2 × 30 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate 7 (3.5 g, 7.75 mmol, 107% yield) as a yellow liquid. 1 1H-NMR (400 MHz, DMSO-d6): δ 1.47 (s, 9H), 1.44 - 1.41 (m, 4H), 0.91 (s, 4H), 0.86 (t, J = 4.40 Hz, 6H), 0.03 (d, J = 4.80 Hz, 3H). LCMS: 246.3 (M - 200), Rt (min): 4.602, area%: 98.718.

[0411] Step - 7: tert-butyl (5-hydroxy-3,3-dimethylpentyl)carbamate JPEG2025522795000406.jpg15127 (tert-Butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamic acid tert-butyl 7 (3.5 g, 7.85 mmol) in THF (35 ml, 10 vol) solution was added with TBAF (11.78 ml, 1 M solution in THF, 11.78 mmol, 1.5 equiv), and stirred at room temperature for 16 h. After completion of the reaction (confirmed by TLC analysis, 30% EtOAc in petroleum ether, Rf about 0.2, KMnO4), the reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 × 30 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product as a yellow liquid. The crude product compound was purified by column chromatography (Biotage R snap cartridge, KP-Sil, 50 g, 60-120 silica gel) using 12-15% EtOAc in petroleum ether to obtain (tert-butoxycarbonyl)(5-hydroxy-3,3-dimethylpentyl)carbamic acid tert-butyl 8 (1.4 g, 4.22 mmol, 53.8% yield) as a pale yellow liquid. 1 H-NMR (400 MHz, DMSO-d6): δ 4.26 (t, J = 4.80 Hz, 1H), 3.50-3.44 (m, 4H), 1.45 (s, 16H), 1.38 (q, J = 7.60 Hz, 5H), 0.88 (s, 6H).

[0412] Step - 8: (tert-Butoxycarbonyl)(3,3-dimethyl-5-oxopentyl)carbamic acid tert-butyl A solution of oxalyl chloride (0.317 mL, 3.62 mmol, 1.2 equiv) in DCM (10 mL) was added dropwise to a solution of DMSO (0.514 mL, 7.24 mmol, 2.4 equiv) at -78 °C under a nitrogen atmosphere and stirred at the same temperature for 15 minutes. To this, a solution of tert-butyl (tert-butoxycarbonyl)(5-hydroxy-3,3-dimethylpentyl)carbamate (1 g, 3.02 mmol, 1 equiv) in DCM (10 mL) was added dropwise over 5 minutes. The reaction mixture was stirred at -78 °C for 30 minutes, and then TEA (2.103 mL, 15.09 mmol, 5 equiv) was added dropwise at -78 °C. After the addition was complete, the reaction mixture was slowly warmed to 0 °C. After completion of the reaction (confirmed by TLC analysis, 30% EtOAc in petroleum ether, Rf ~0.8, KMnO4), the reaction mixture was diluted with DCM (10 mL) and washed with 10% aqueous citric acid (50 mL), water (50 mL), and brine (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum at a temperature below 30 °C to obtain tert-butyl (tert-butoxycarbonyl)(3,3-dimethyl-5-oxopentyl)carbamate 9 (930 mg, 2.82 mmol, 94% yield) as a yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 9.76 (q, J = 2.40 Hz, 1H), 3.49 - 3.44 (m, 2H), 2.29 (d, J = 2.80 Hz, 1H), 1.52 (d, J = 8.00 Hz, 1H), 1.49 (s, 12H), 1.44 (s, 1H), 1.02 (s, 3H), 0.88 (s, 1H).

[0413] Step - 9: tert-Butyl (tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3,3-dimethylpentyl)carbamate JPEG2025522795000408.jpg39127 (4-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamic acid tert-butyl Int-4 (1 g, 2.323 mmol) in a mixture of dichloromethane (10 ml, 10 vol) and DMF (10 ml, 10 vol), (tert-Butoxycarbonyl)(3,3-dimethyl-5-oxopentyl)carbamic acid tert-butyl 9 (930 mg, 2.82 mmol, 1.2 eq), and trifluoroacetic acid (0.716 ml, 9.29 mmol, 4 eq), sodium triacetoxyborohydride (1.969 g, 9.29 mmol, 4 eq) was added, and the mixture was stirred at room temperature for 16 h. Formation of the product was confirmed by TLC analysis (EtOAc, Rf ~0.7) and LCMSn, but the starting material was not completely consumed. The reaction mixture was diluted with DCM (15 mL), washed with water (2 × 30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by preparative HPLC (HCOOH:ACN method) and lyophilized to give (tert-Butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3,3-dimethylpentyl)carbamic acid tert-butyl 10 (70 mg, 0.094 mmol, 4.04% yield) as a white solid. 11H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.38 (t, J = 7.60 Hz, 1H), 7.19 (d, J = 7.20 Hz, 1H), 7.08 (d, J = 7.60 Hz, 1H), 6.77 (s, 1H), 5.12 (q, J = 5.20 Hz, 1H), 4.40 (d, J = 16.80 Hz, 1H), 4.29 (d, J = 17.20 Hz, 1H), 3.46 (t, J = 9.20 Hz, 2H), 3.25 (d, J = 28.00 Hz, 4H), 2.93 (t, J = 7.20 Hz, 3H), 2.04 (t, J = 7.60 Hz, 1H), 1.44 (t, J = 8.40 Hz, 20H), 1.37 (d, J = 14.00 Hz, 12H), 1.02 (s, 6H). LC-MS: 744.5 (M+H), Rt (min): 3.010, Area %: 99.774.

[0414] Step - 10: 3-(4-((5-Amino-3,3-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000409.jpg39127(tert-Butoxycarbonyl)(5-((4-((tert-Butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-3,3-dimethylpentyl)carbamic acid tert-butyl10(70 mg, 0.094 mmol) in dichloromethane (3 mL) was stirred and suspended, and HCl (4 M solution in dioxane, 1 mL, 4.00 mmol) was added at 0 °C, and then the mixture was stirred at room temperature for 2 hours. After completion of the reaction (confirmed by TLC analysis, EtOAc, Rf about 0.1), the reaction mixture was concentrated under reduced pressure. The obtained solid was dissolved in water (30 mL), washed with MTBE (2 × 30 mL), and then the aqueous layer was lyophilized to obtain 3-(4-((5-amino-3,3-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl125(12 mg, 0.023 mmol, 24.65% yield) as an off-white solid. 1 H-NMR (400 MHz, DMSO-d6): δ 11.01 (s, 1H), 8.00 (s, 6H), 7.54 (s, 2H), 5.17-5.12 (m, 1H), 4.49 (d, J = 31.20 Hz, 2H), 3.35 (s, 4H), 2.92 (d, J = 12.40 Hz, 1H), 2.75-2.61 (m, 5H), 2.07 (t, J = 5.20 Hz, 1H), 1.51 (q, J = 8.40 Hz, 6H), 1.39-1.30 (m, 2H), 0.87 (s, 6H). LCMS: 444.3 (M+H). Method: Mobile phase A: 0.1% formic acid aqueous solution. Mobile phase B: ACN. Flow rate: 1.5 mL / min. Column: Atlantis dC18 (50x4.6) 5μm. Rt (min): 1.035; Area %: 99.826. HPLC: Method: Mobile phase A: 0.1% formic acid aqueous solution. Mobile phase B: Methanol. Flow rate: 1.0 mL / min. Column: X-Bridge C8 (50×4.6) mm, 3.5μm. Rt (min): 2.679; Area %: 98.409.

[0415] Hydrochloride form of Compound 126

[0416] 3-(4-((5-Amino-4,4-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride JPEG2025522795000410.jpg23127JPEG2025522795000411.jpg21127JPEG2025522795000412.jpg46127

[0417] Step - 1: 5-((tert-Butyldimethylsilyl)oxy)-2,2-dimethylpentanenitrile A solution of LDA (2 M in THF / heptane / ethylbenzene, 5.92 mL, 11.85 mmol, 1.5 equiv) in THF (10 mL) was cooled to -78 °C, and isobutyronitrile 2 (0.764 g, 11.06 mmol, 1.4 equiv) was added over 5 minutes. The reaction mixture was warmed to -40 °C, stirred for 30 minutes, and cooled again to -78 °C. Next, (3-bromopropoxy)(tert-butyl)dimethylsilane 1 (2.0 g, 7.90 mmol, 1.0 equiv) was added dropwise to the reaction mixture, warmed to 0 °C, and stirred for 1 hour. After completion of the reaction (confirmed by TLC and UPLC analysis, 10% EtOAc / petroleum ether, Rf ca. 0.6, KMnO4), the reaction mixture was quenched with 1 N HCl (1 x 10 mL) and extracted with MTBE (2 x 50 mL). The combined organic layers were washed with saturated NaHCO3 (1 x 20 mL), brine (1 x 20 mL), dried over Na2SO4, and concentrated under reduced pressure to give 5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentanenitrile 3 (2.2 g, 9.11 mmol, 115% yield) as a pale yellow liquid, which was used further without purification. 1 H-NMR (400 MHz, DMSO-d6): δ 3.66 - 3.69 (m, 2H), 1.69 - 1.73 (m, 2H), 1.59 - 1.63 (m, 2H), 1.38 (s, 6H), 0.91 (s, 9H), 0.08 (s, 6H)

[0418] Step - 2: tert-Butyl (5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl)carbamate To a stirred solution of 5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentanenitrile 3 (1.0 g, 4.14 mmol, 1.0 eq) in dry MeOH (30 mL) cooled to 0 °C, (Boc)2O (1.923 ml, 8.28 mmol, 2.0 eq) and NiCl2·6H2O (0.984 g, 4.14 mmol, 1.0 eq) were added, and then NaBH4 (1.097 g, 29.0 mmol, 7.0 eq) was carefully added portionwise. The reaction was exothermic and foamed. The resulting reaction mixture was warmed to room temperature and stirred overnight. After completion of the reaction (confirmed by TLC analysis, 5% EtOAc / petroleum ether, Rf ~0.3, KMnO4), the reaction mixture was diluted with DCM (20 mL), filtered through a celite bed, and washed with DCM (20 mL). The filtrate was washed with saturated NaHCO3 solution (1x20 mL), the organic layer was dried over Na2SO4, and concentrated under reduced pressure to give the crude product of tert-Butyl (5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl)carbamate 4 (1.2 g, 3.39 mmol, 82% yield) as a pale yellow liquid, which was used further without purification. 1 1H-NMR (400 MHz, DMSO-d6): δ 6.71 (t, J = 6.40 Hz, 1H), 3.54 (m, 2H), 2.73 - 2.76 (m, 2H), 1.44 (s, 9H), 1.22 - 1.38 (m, 2H), 1.07 - 1.13 (m, 2H), 0.89 (s, 9H), 0.80 (s, 6H), 0.02 (s, 6H).

[0419] Step - 3: tert-Bu...

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof. Wherein, R 1 is C 1 -C 8 alkyl, C 3 -C 8 alkenyl, C 3 -C 8 alkynyl, C 3 -C 8 cycloalkyl or C 5 -C 10 bicycloalkyl, each of which is one or more halogens, OH, O-C 1 -C 3 alkyl, NH 2 , C 1 -C 3 haloalkyl, optionally substituted C 6 -C 10 aryl, C 1 -C 3 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 5 -C 10 bicycloalkyl or optionally substituted C 3 -C 6 optionally substituted with heterocycloalkyl, X is a bond, or one or more halogens, OH, O-C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 3 -C 6 cycloalkyl or C 1 -C 6 -(CH optionally substituted with alkyl 2 ) n -, and the C 1 -C 6 alkyl group may together with the atom to which it is attached form a C 3 -C 6 spiroalkyl ring, and the -(CH 2 ) n - group may contain 0 to 1 double bond or triple bond, n is an integer from 1 to 6, A is a bond, or each is one or more halogens, OH, O-C 1 -C 3 alkyl, CN, C 1 -C 3 haloalkyl, C 1 -C 3 alkyl or C 3 -C 5 C optionally substituted with cycloalkyl 4 -C 8 cycloalkyl, C 5 -C 10 bicycloalkyl, C 5 -C 10 heterobicycloalkyl or C 3 -C 8 heterocycloalkyl, and Y is a bond or -(CH 2 ) m - and m is an integer of 1 or 2, Z is H or halogen, R 2 is -NR 12 R 5 、 -NHC(O)R 6 、 -NHC(O)NR 8 (R 9 )、 -C(O)NHR 5 、 a 5 - or 6 - membered heteroaryl, or C 3 -C 8 heterocycloalkyl or C 5 -C 10 heterobicycloalkyl ring, optionally substituted with one or more halogen, OH, O - C 1 -C 3 alkyl, CN, C 1 -C 3 haloalkyl, C 1 -C 3 alkyl or C 3 -C 5 cycloalkyl, R 5 is H, C 1 -C 5 alkyl, C 3 -C 7 cycloalkyl, 5- or 6-membered heteroaryl, C 5 -C 10 bicycloalkyl, (C 3 -C 6 cycloalkyl)-C 1 -C 3 alkyl, (C 3 -C 6 heterocycloalkyl)-C 1 -C 3 alkyl, (C 6 -C 10 aryl)-C 1 -C 3 alkyl, (C 1 -C 5 -heteroaryl)-C 1 -C 3 alkyl or C 3 -C 7 heterocycloalkyl, each of which is optionally substituted with one or more halogen, CN, OH, O-(C 1 -C 3 haloalkyl), O-(C 1 -C 3 alkyl), C 1 -C 3 haloalkyl, CH 3 SO 2 -, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 7 heterocycloalkyl or C(O)NR 8 (R 9 ) and is optionally substituted, R 6 is C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or C 3 -C 6 heterocycloalkyl, each of which is optionally substituted with one or more halogens, C 1 -C 3 haloalkyl, optionally substituted C 3 -C 6 cycloalkyl, or NR 8 (R 9 ) and is optionally substituted, R 8 and R 9 each independently is H, substituted C 1 -C 3 alkyl, or together with the nitrogen to which they are attached forms a 4- to 6-membered heterocyclic ring optionally substituted with one or more halogens or C 1 -C 3 haloalkyl, R 12 is H, C 1 -C 5 is alkyl, or R 5 together with the nitrogen to which it is attached forms (i) a C 3 -C 8 cycloalkyl or heterocycloalkyl ring, or (ii) a C 5 -C 10 bicycloalkyl or heterobicycloalkyl ring, each of which is optionally substituted with one or more halogens, C 1 -C 3 haloalkyl, OH, O-(C 1 -C 3 alkyl), O-(C 1 -C 3 haloalkyl) or CN, Each stereocenter in the compound of formula (I) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers, and Each double bond in the compound of formula (I) is independently cis or trans.

2. The compound is represented by formula (I-a), X is a bond or -(CH 2 ) n -, and the compound according to claim 1, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

3. R 1 is C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl, each of which is one or more halogens, OH, NH 2 , C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, O-(C 1 -C 3 alkyl), optionally substituted C 6 aryl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 3 -C 6 heterocycloalkyl or optionally substituted C 6 -C 8 bicycloalkyl, optionally substituted, the compound according to claim 2, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

4. A is C 4 -C 6 cycloalkyl, C 4 -C 6 heterocycloalkyl or C 5 -C 7 bicycloalkyl, each of which is optionally substituted with one or more halogens, OH, C 1 -C 3 alkyl or C 1 -C 3 haloalkyl, and the compound according to claim 2, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

5. Y is a bond or -CH 2 - and the compound according to claim 2, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

6. R 5 is H, C 1 -C 5 alkyl, C 3 -C 6 cycloalkyl, 6-membered heteroaryl, C 5 bicycloalkyl, (C 3 -C 4 cycloalkyl)-C 1 -C 2 alkyl, (C 3 -C 4 heterocycloalkyl)-C 1 -C 2 alkyl, (C 6 aryl)-C 1 -C 2 alkyl, (C 5 heteroaryl)-C 1 -C 2 alkyl or C 3 -C 4 heterocycloalkyl, each of which is optionally substituted with one or more halogen, CN, OH, O-(C 1 -C 3 haloalkyl), O-(C 1 -C 3 alkyl), C 1 -C 3 haloalkyl, CH 3 SO 2 - or C(O)NR 8 (R 9 ) and is the compound according to claim 2, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

7. R 12 is H, C 1 -C 5 alkyl, or R 5 together with the nitrogen to which it is attached, is one or more halogens or C 1 -C 3 optionally substituted with haloalkyl C 3 -C 8 forming a heterocycloalkyl ring, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, of the compound according to claim 2.

8. The compound is represented by formula (I-b), X is a bond or -(CH 2 ) n -, the compound according to claim 1, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

9. R 1 is one or more halogens, OH, O-C 1 -C 3 alkyl, NH 2 , C 1 -C 3 C optionally substituted with haloalkyl 3 -C 6 alkyl, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, of the compound according to claim 8.

10. A is a bond, or one or more halogens, OH, O-C 1 -C 3 alkyl, NH 2 、C 1 -C 3 C optionally substituted with haloalkyl 4 -C 6 cycloalkyl, the compound according to claim 8, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

11. Y is a bond, The compound according to claim 8, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

12. R 6 is NR 8 (R 9 ) optionally substituted C 1 -C 6 alkyl, the compound according to claim 8, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

13. The compound is represented by formula (I-c), X is a bond or -(CH 2 ) n -, and the compound according to claim 1, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

14. R 1 is C 1 -C 6 alkyl, optionally substituted C 3 -C 8 -cycloalkyl or optionally substituted C 3 -C 6 optionally substituted with heterocycloalkyl, a compound according to claim 13, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

15. A is a bond, or each is one or more halogens, OH, O-C 1 -C 3 alkyl, NH 2 、C 1 -C 3 C optionally substituted with haloalkyl 4 -C 6 cycloalkyl, C 4 -C 6 heterocycloalkyl or C 5 -C 7 bicycloalkyl, and the compound according to claim 13, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

16. Y is a bond, The compound according to claim 13, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

17. R 5 is H or C 1 -C 3 The compound according to claim 13, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, characterized in that it is alkyl.

18. X and Y are each a bond, A is C 4 -C 6 is cycloalkyl, R 1 is optionally substituted C 3 -C 6 optionally substituted with cycloalkyl C 1 -C 3 is alkyl, and R 2 The compound according to claim 1, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, wherein R is a 5-membered heteroaryl.

19. A compound of formula (I-d), or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof. Wherein, R 1 is optionally substituted C 3 -C 6 optionally substituted with cycloalkyl C 1 -C 3 is alkyl, A is an optionally substituted C 4 -C 6 cycloalkyl, and R 2 is C 1 -C 4 alkyl or (C 3 -C 4 cycloalkyl)-C 1 -C 2 alkyl, each of which is optionally substituted with one or more halogen, CN, OH, O-(C 1 -C 3 haloalkyl), O-(C 1 -C 3 alkyl), C 1 -C 3 haloalkyl or CH 3 SO 2 -, and Each stereocenter in the compound of formula (I-d) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers.

20. R 2 is one or more halogens, O-(C 1 -C 3 -haloalkyl), O-(C 1 -C 3 -alkyl) or C 1 -C 3 -haloalkyl optionally substituted C 1 -C 3 -alkyl, a compound according to claim 19, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof.

21. The compound is for use in the degradation or reduction of casein kinase 1 alpha (CK1α), The compound according to claim 1 or 19.

22. The compound is for use in inhibiting the activity of casein kinase 1 alpha (CK1α), The compound according to claim 1 or 19.

23. The compound is for use in the prevention or treatment of blood cancer, The compound according to claim 1 or 19.

24. The compound according to claim 1 or 19, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, and Optionally, a pharmaceutically acceptable excipient or carrier, and A pharmaceutical composition comprising.

25. The pharmaceutical composition according to claim 24, characterized in that it is for use in the degradation or reduction of casein kinase 1 alpha (CK1α).

26. The pharmaceutical composition according to claim 24, characterized in that it is for use in inhibiting the activity of casein kinase 1 alpha (CK1α).

27. The pharmaceutical composition according to claim 24, characterized in that it is for use in the prevention or treatment of blood cancer.

28. The pharmaceutical composition according to claim 27, characterized in that the blood cancer is leukemia, lymphoma or myeloma.

29. The pharmaceutical composition according to claim 28, characterized in that the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocyte leukemia, adult T-cell leukemia, chronic eosinophilic leukemia (CEL) and myelodysplastic syndrome (MDS).

30. The pharmaceutical composition according to claim 28, characterized in that the lymphoma is selected from the group consisting of Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocyte lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, and peripheral T-cell lymphoma.

31. The pharmaceutical composition according to claim 28, characterized in that the myeloma is selected from the group consisting of multiple myeloma, light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma.

32. A method for degrading or reducing casein kinase 1 alpha (CK1α) in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 24.

33. A method of inhibiting the activity of casein kinase 1 alpha (CK1α) in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 24.

34. A method of preventing or treating blood cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 24.

35. The method according to claim 34, wherein the blood cancer is leukemia, lymphoma or myeloma.

36. The method according to claim 35, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, chronic eosinophilic leukemia (CEL) and myelodysplastic syndrome (MDS).

37. The method according to claim 35, wherein the lymphoma is selected from the group consisting of Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, and peripheral T-cell lymphoma.

38. The method according to claim 35, wherein the myeloma is selected from the group consisting of multiple myeloma, light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma.

39. The compound of Table A, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof. [Table A]

40. The compound of claim 39, or a pharmaceutically acceptable salt, enantiomer, stereoisomer or prodrug thereof, and optionally, a pharmaceutically acceptable excipient or carrier, and A pharmaceutical composition comprising the same.

41. The pharmaceutical composition according to claim 40, characterized in that it is for use in the degradation or reduction of casein kinase 1 alpha (CK1α).

42. The pharmaceutical composition according to claim 40, characterized in that it is for use in inhibiting the activity of casein kinase 1 alpha (CK1α).

43. The pharmaceutical composition according to claim 40, characterized in that it is for use in the prevention or treatment of blood cancer.

44. The pharmaceutical composition according to claim 40, characterized in that the blood cancer is leukemia, lymphoma or myeloma.

45. The pharmaceutical composition according to claim 44, characterized in that the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia or acute lymphocytic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, chronic eosinophilic leukemia (CEL) and myelodysplastic syndrome (MDS).

46. The pharmaceutical composition according to claim 44, characterized in that the lymphoma is selected from the group consisting of Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, and peripheral T-cell lymphoma.

47. The pharmaceutical composition according to claim 44, characterized in that the myeloma is selected from the group consisting of multiple myeloma, light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma.

48. A method for degrading or reducing casein kinase 1 alpha (CK1α) in a subject in need thereof, comprising administering to the subject an effective amount of a compound comprising the compound according to claim 39.

49. A method of inhibiting the activity of casein kinase 1 alpha (CK1α) in a subject in need thereof, comprising administering to the subject an effective amount of a compound comprising the compound according to claim 39. **Claim 50** A method of preventing or treating blood cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound comprising the compound according to claim 39.

Images