Pharmaceutical composition for treating adenoid cystic carcinoma and method for treating adenoid cystic carcinoma

Abstract

The present invention relates to: compound (1) or a pharmaceutical composition for treating adenoid cystic carcinoma (ACC), comprising same as an active ingredient; and a method for treating adenoid cystic carcinoma (ACC), the method comprising administering an effective amount of compound (1) or a pharmaceutical composition comprising same as an active ingredient to a subject in need thereof.

Description

Pharmaceutical composition for treating adenocystic carcinoma and method for treating adenocystic carcinoma

[0001] The present invention relates to a pharmaceutical composition for treating adenoid cystic carcinoma (ACC) and a method for treating adenoid cystic carcinoma (ACC).

[0002] Adenocystic carcinoma (ACC) is a slow-growing cancer that typically originates in the salivary glands. ACC is a very rare cancer, with an annual incidence reported to be approximately 4 cases per million people and a prevalence of about 33 cases per million people. This means that in the United States, there are approximately 1,400 new patients and 11,000 existing patients annually. Similar incidence and prevalence rates are reported in Asia and Europe. Although the 5-year survival rate for ACC is relatively high at about 80–90%, it is considered an aggressive “slow-killer” because many patients die from distant metastasis decades after initial diagnosis. While most primary ACC tumors are initially treated with surgical resection and adjuvant radiation therapy, about half of the patients eventually progress to recurrent or metastatic (R / M) disease, requiring systemic treatment.

[0003] However, there are currently no approved drugs for R / M ACC. Although various systemic treatment approaches for R / M ACC have been attempted, clinical success has been limited. Chemotherapy combination therapies such as cyclophosphamide-doxorubicin-cisplatin have been clinically explored, while immune checkpoint inhibitors (e.g., pembrolizumab, nivolumab) have not demonstrated efficacy. This is consistent with the characteristic of ACC being classified as an “immunologically cold tumor” with low PD-L1 expression. Lenvatinib, a multikinase VEGFR inhibitor approved for the treatment of renal cell carcinoma, is used off-label as a first-line treatment for R / M ACC in the United States and has demonstrated clinical efficacy with an Overall Response Rate (ORR) of approximately 15%.

[0004] ACC has a relatively stable genetic profile and a low somatic mutation rate in exon regions. The most common genetic mutations identified in ACC are NOTCH1 mutations (approximately 15% of primary tumors and 40% of relapsed / metastatic (R / M) samples) and MYB-NFIB oncogene fusions, which are caused by the chromosomal translocation t(6;9)(q22-23;p23-24). Meanwhile, the majority of ACC patient tumors (approximately 95%) correspond to the TP53 wild-type. This is ideal for using casein kinase 1 alpha (CK1α) degraders, which are likely to require functional p53 proteins due to their mechanism of action.

[0005] Meanwhile, casein kinase 1 alpha (CK1α) is a serine / threonine protein kinase involved in various cellular pathways and functions, including Wnt signaling, NF-κB signaling, the p53 pathway, autophagy, and the cell cycle. Inhibition of CK1α or a decrease in its expression levels has been shown to induce apoptosis in several types of cancer, such as acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL).

[0006] For example, it has been revealed that inhibition of CK1α selectively eliminates leukemia cells by inducing reduced Rps6 phosphorylation and p53 activation, thereby identifying CK1α as a therapeutic target for AML treatment (Jaras et al., J. Exp. Med. 2014 Vol. 211 No. 4 605-612). Furthermore, there have been reports that inhibition of CK1α, acting through p53 pathway activation, exhibits promising preclinical activity in AML and multiple myeloma (MM) (Janovska et al., Int. J. Mol. Sci. 2020, 21, 9026). In particular, it has been previously established that CK1α controls signaling pathways related to proliferation, survival, and stress in MM (Manni et al., Journal of Hematology & Oncology (2017) 10:157). Furthermore, there have been reports that CK1α is overexpressed in AML patients and acts as a negative factor in the prognosis of AML patients. Consequently, there have been studies suggesting that CK1α inhibits p53 downstream of MDM2-mediated autophagy and apoptosis, and that targeting CK1α and autophagy may provide a therapeutic opportunity to treat AML (Xu et al., ONCOLOGY REPORTS 44: 1895-1904, 2020).

[0007] As such, CK1α has been known in the technical field as a target for preventing or treating blood cancers such as leukemia, lymphoma, and multiple myeloma, but recently, attempts have also been made to extend its application to solid tumors such as colorectal cancer, prostate cancer, and rare cancers. Accordingly, the present invention provides a pharmaceutical composition and a treatment method for treating adenoid cystic carcinoma (ACC) as a novel use of a casein kinase 1 alpha (CK1α) degrader (WO2024 / 003749A).

[0008] The object of the present invention is a pharmaceutical composition for the treatment of adenoid cystic carcinoma (ACC), a rare cancer for which there are no approved drugs, and a method for treating adenoid cystic carcinoma (ACC).

[0009] To achieve the above objective, the present disclosure provides a pharmaceutical composition for treating adenoid cystic carcinoma (ACC) comprising, as an active ingredient, the following compound (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof. Additionally, the present disclosure provides a method for treating adenoid cystic carcinoma (ACC) comprising administering an effective amount of the following compound (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof to a subject.

[0010] (I)

[0011] In chemical formula (I), according to one embodiment, R 1 C1-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C3-C8 cycloalkyl or C5-C 10 Bicycloalkyl, each comprising one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl, and optionally substituted C6-C 10Aryl, C1-C3 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C 10 It is optionally substituted with a bicycloalkyl or an optionally substituted C3-C6 heterocycloalkyl.

[0012] According to one embodiment, X is a -(CH2) that is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl, or C1-C6 alkyl. n - and, C1-C6 alkyl groups can form C3-C6 spiroalkyl rings with bonded atoms, and -(CH2) n - The group may contain 0 to 1 double or triple bond.

[0013] According to one embodiment, n is an integer between 1 and 6.

[0014] According to one embodiment, A is a bond, C4-C8 cycloalkyl, C5-C 10 Bicycloalkyl, C5-C 10 Each is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl, as a heterobicycloalkyl or C3-C8 heterocycloalkyl.

[0015] According to one embodiment, Y is bonded or -(CH2) m -am.

[0016] According to one embodiment, m is an integer of 1 or 2.

[0017] According to one embodiment, Z is H or a halogen.

[0018] According to one embodiment, R 2 is -NR 12 R 5 , -NHC(O)R 6 , -NHC(O)NR 8 (R 9 ), -C(O)NHR 5, a 5-membered or 6-membered heteroaryl, or a C3-C8 heterocycloalkyl or C5-C8 heterocycloalkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl. 10 It is a heterobicycloalkyl ring.

[0019] According to one embodiment, R 5 is H, C1-C5 alkyl, C3-C7 cycloalkyl, 5- or 6-membered heteroaryl, C5-C 10 Bicycloalkyl, (C3-C6 cycloalkyl)-C1-C3 alkyl, (C3-C6 heterocycloalkyl)-C1-C3 alkyl, (C6-C 10 (C1-C3 alkyl, (C1-C5-heteroaryl)-C1-C3 alkyl, or C3-C7 heterocycloalkyl, each comprising 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 It is optionally replaced with ).

[0020] According to one embodiment, R 6 is a C1-C6 alkyl, C3-C6 cycloalkyl, or C3-C6 heterocycloalkyl, each comprising one or more halogens, a C1-C3 haloalkyl, an optionally substituted C3-C6 cycloalkyl, or NR 8 (R 9 It is optionally replaced with ).

[0021] According to one embodiment, R 8 and R 9 Each forms a 4-6 member heterocyclic ring that is independently H, C1-C3 alkyl, or optionally substituted with one or more halogens or C1-C3 haloalkyls together with the nitrogen to which they are bonded.

[0022] According to one embodiment, R 12is H, C1-C5 alkyl, or R 5 and each having one or more halogens, C1-C3 haloalkyl, OH, O-(C1-C3 alkyl), O-(C1-C3 haloalkyl) or CN, together with the nitrogen bonded thereto, (i) a C3-C8 cycloalkyl or heterocycloalkyl ring, or (ii) a C5-C 10 It forms a bicycloalkyl or heterobicycloalkyl ring.

[0023] According to one embodiment, each stereocenter of the compound of formula (I) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers.

[0024] According to one embodiment, each double bond of the compound of formula (I) is independently cis or trans.

[0025] According to one embodiment, the compound can be represented by the chemical formula (Ia).

[0026] (Ia)

[0027] In chemical formula (Ia), according to one embodiment, X is a bond or -(CH2) n -am.

[0028] According to one embodiment, R 1 It is optionally substituted with a C1-C8 alkyl or C3-C8 cycloalkyl, each 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.

[0029] According to one embodiment, A is a C4-C6 cycloalkyl, a C4-C6 heterocycloalkyl, or a C5-C7 bicycloalkyl, each optionally substituted with one or more halogens, OH, a C1-C3 alkyl, or a C1-C3 haloalkyl.

[0030] According to one embodiment, Y is bonded or -CH2-.

[0031] According to one embodiment, 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 comprising 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 It is optionally replaced with ).

[0032] According to one embodiment, R 12 is H, C1-C5 alkyl, or R 5 and together with the nitrogen combined therewith, it forms a C3-C8 heterocycloalkyl ring optionally substituted with one or more halogens or C1-C3 haloalkyls.

[0033] According to one embodiment, the compound can be represented by the chemical formula (Ib).

[0034] (Ib)

[0035] In the chemical formula (Ib), according to one embodiment, X is a bond or -(CH2) n -am.

[0036] According to one embodiment, R 1 is a C3-C6 alkyl selectively substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0037] According to one embodiment, A is a C4-C6 cycloalkyl that is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0038] According to one embodiment, Y is a combination.

[0039] According to one embodiment, R 6 is NR 8 (R 9 It is a C1-C6 alkyl that is selectively substituted with ).

[0040] According to one embodiment, the compound can be represented by the chemical formula (Ic).

[0041] (Ic)

[0042] In the chemical formula (Ic), according to one embodiment, X is bonded or -(CH2) n -am.

[0043] According to one embodiment, R 1 is a C1-C6 alkyl that is optionally substituted with a C3-C8 cycloalkyl or an optionally substituted C3-C6 heterocycloalkyl.

[0044] According to one embodiment, A is bonded to a C4-C6 cycloalkyl, a C4-C6 heterocycloalkyl, or a C5-C7 bicycloalkyl, and is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, or C1-C3 haloalkyl.

[0045] According to one embodiment, Y is a combination.

[0046] According to one embodiment, R 5 is H or C1-C3 alkyl.

[0047] According to one embodiment, compound (I) is selected from [Table A] disclosed in the specific details for carrying out the invention.

[0048] The present disclosure provides a pharmaceutical composition for treating adenoid cystic carcinoma (ACC) comprising the following compound (Id) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof as an active ingredient. Additionally, the present disclosure provides a method for treating adenoid cystic carcinoma (ACC) comprising administering an effective amount of the following compound (Id) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof to a subject.

[0049] (Id)

[0050] In the chemical formula (Id), according to one embodiment, R 1 is a C1-C3 alkyl that is optionally substituted with a C3-C6 cycloalkyl that is optionally substituted.

[0051] According to one embodiment, A is an optionally substituted C4-C6 cycloalkyl.

[0052] According to one embodiment, R 2 is a 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-.

[0053] According to one embodiment, each stereocenter of the compound of formula (Id) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers.

[0054] The present invention provides a pharmaceutical composition for the treatment of adenoid cystic carcinoma (ACC), a rare cancer for which there are no approved drugs, and a method for treating adenoid cystic carcinoma (ACC).

[0055] From the disclosure of this application, various modifications and other embodiments will come to mind to those skilled in the art who have benefit from the teachings of the compositions and methods of the disclosed application presented in the foregoing description and the associated drawings. Accordingly, it should be understood that the content of this disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Those skilled in the art will recognize various variations and adaptations of the aspects disclosed herein. Such variations and adaptations are included in the teachings of this disclosure and are intended to be included in the claims of this application.

[0056] Specific terms are used herein, but are used only in a comprehensive and descriptive sense and are not intended to be restrictive.

[0057] Any method enumerated may be performed in the order of the enumerated events or in any other logically possible order. That is, unless otherwise clearly stated, there is no intention to interpret any method or aspect presented herein as requiring that it be performed in a specific order. Accordingly, unless a method claim specifies that the steps are limited to a specific order, there is no intention to interpret that an order is implied in any way. This applies to non-explicit grounds for interpretation, such as logical details regarding the arrangement of steps or workflow, grammatical constructions or punctuation, or the number or type of aspects described in the specification.

[0058] All publications and patents cited herein are cited to disclose and describe methods and / or materials related to the cited publications. All such publications and patents are incorporated herein by reference as specified, each individual publication or patent specifically and individually by reference. Inclusion by reference is limited to the methods and / or materials described in the explicitly cited publications and patents and does not extend to the dictionary definitions of the cited publications and patents. Any lexical definitions contained in the cited publications and patents that are not explicitly repeated in this application shall not be treated as such definitions and shall not be interpreted as defining any terms appearing in the appended claims. Any citation of a publication is to indicate that it was disclosed prior to the filing date and shall not be interpreted as an acknowledgment that the present disclosure is not entitled to claim a date earlier than that of such publication by prior disclosure. Additionally, the provided publication dates may differ from the actual publication dates, and independent verification may be required.

[0059] The present disclosure describes a pharmaceutical composition for treating adenoid cystic carcinoma (ACC) comprising the following compound (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof as an active ingredient.

[0060] Additionally, the present disclosure describes a method for treating adenoid cystic carcinoma (ACC) comprising administering an effective amount of the following compound (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof to a subject.

[0061] (I)

[0062] In chemical formula (I), according to one embodiment, R 1C1-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C3-C8 cycloalkyl or C5-C 10 Bicycloalkyl, each comprising one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl, and optionally substituted C6-C 10 Aryl, C1-C3 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C 10 It is optionally substituted with a bicycloalkyl or an optionally substituted C3-C6 heterocycloalkyl.

[0063] According to one embodiment, X is a -(CH2) that is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl, or C1-C6 alkyl. n - and, C1-C6 alkyl groups can form C3-C6 spiroalkyl rings with bonded atoms, and -(CH2) n - The group may contain 0 to 1 double or triple bond.

[0064] According to one embodiment, n is an integer between 1 and 6.

[0065] According to one embodiment, A is a bond, C4-C8 cycloalkyl, C5-C 10 Bicycloalkyl, C5-C 10 Each is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl, as a heterobicycloalkyl or C3-C8 heterocycloalkyl.

[0066] According to one embodiment, Y is bonded or -(CH2) m -am.

[0067] According to one embodiment, m is an integer of 1 or 2.

[0068] According to one embodiment, Z is H or a halogen.

[0069] According to one embodiment, R2 is -NR 12 R 5 , -NHC(O)R 6 , -NHC(O)NR 8 (R 9 ), -C(O)NHR 5 , a 5-membered or 6-membered heteroaryl, or a C3-C8 heterocycloalkyl or C5-C8 heterocycloalkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl. 10 It is a heterobicycloalkyl ring.

[0070] According to one embodiment, R 5 is H, C1-C5 alkyl, C3-C7 cycloalkyl, 5- or 6-membered heteroaryl, C5-C 10 Bicycloalkyl, (C3-C6 cycloalkyl)-C1-C3 alkyl, (C3-C6 heterocycloalkyl)-C1-C3 alkyl, (C6-C 10 (C1-C3 alkyl, (C1-C5-heteroaryl)-C1-C3 alkyl, or C3-C7 heterocycloalkyl, each comprising 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 It is optionally replaced with ).

[0071] According to one embodiment, R 6 is a C1-C6 alkyl, C3-C6 cycloalkyl, or C3-C6 heterocycloalkyl, each comprising one or more halogens, a C1-C3 haloalkyl, an optionally substituted C3-C6 cycloalkyl, or NR 8 (R 9 It is optionally replaced with ).

[0072] According to one embodiment, R 8 and R 9Each forms a 4-6 member heterocyclic ring that is independently H, a substituted C1-C3 alkyl, or optionally substituted with one or more halogens or C1-C3 haloalkyls together with the nitrogen to which they are bonded.

[0073] According to one embodiment, R 12 is H, C1-C5 alkyl, or R 5 and each having one or more halogens, C1-C3 haloalkyl, OH, O-(C1-C3 alkyl), O-(C1-C3 haloalkyl) or CN, together with the nitrogen bonded thereto, (i) a C3-C8 cycloalkyl or heterocycloalkyl ring, or (ii) a C5-C 10 It forms a bicycloalkyl or heterobicycloalkyl ring.

[0074] According to one embodiment, each stereocenter of the compound of formula (I) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers.

[0075] According to one embodiment, each double bond of the compound of formula (I) is independently cis or trans.

[0076] According to one embodiment, the compound can be represented by the chemical formula (Ia).

[0077] (Ia)

[0078] In chemical formula (Ia), according to one embodiment, X is a bond or -(CH2) n -am.

[0079] According to one embodiment, R 1It is optionally substituted with a C1-C8 alkyl or C3-C8 cycloalkyl, each 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.

[0080] According to one embodiment, A is a C4-C6 cycloalkyl, a C4-C6 heterocycloalkyl, or a C5-C7 bicycloalkyl, each optionally substituted with one or more halogens, OH, a C1-C3 alkyl, or a C1-C3 haloalkyl.

[0081] According to one embodiment, Y is bonded or -CH2-.

[0082] According to one embodiment, 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 comprising 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 It is optionally replaced with ).

[0083] According to one embodiment, R 12 is H, C1-C5 alkyl, or R 5 and together with the nitrogen combined therewith, it forms a C3-C8 heterocycloalkyl ring optionally substituted with one or more halogens or C1-C3 haloalkyls.

[0084] According to one embodiment, the compound can be represented by the chemical formula (Ib).

[0085] (Ib)

[0086] In the chemical formula (Ib), according to one embodiment, X is a bond or -(CH2) n -am.

[0087] According to one embodiment, R 1 is a C3-C6 alkyl selectively substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0088] According to one embodiment, A is a C4-C6 cycloalkyl that is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl.

[0089] According to one embodiment, Y is a combination.

[0090] According to one embodiment, R 6 is NR 8 (R 9 It is a C1-C6 alkyl that is selectively substituted with ).

[0091] According to one embodiment, the compound can be represented by the chemical formula (Ic).

[0092] (Ic)

[0093] In the chemical formula (Ic), according to one embodiment, X is bonded or -(CH2) n -am.

[0094] According to one embodiment, R 1 is a C1-C6 alkyl that is optionally substituted with a C3-C8 cycloalkyl or an optionally substituted C3-C6 heterocycloalkyl.

[0095] According to one embodiment, A is bonded to a C4-C6 cycloalkyl, a C4-C6 heterocycloalkyl, or a C5-C7 bicycloalkyl, and is optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, or C1-C3 haloalkyl.

[0096] According to one embodiment, Y is a combination.

[0097] According to one embodiment, R 5 is H or C1-C3 alkyl.

[0098] According to one embodiment, the compound can be represented by the chemical formula (Id).

[0099] (Id)

[0100] In the chemical formula (Id), according to one embodiment, R 1 is a C1-C3 alkyl that is optionally substituted with a C3-C6 cycloalkyl. For example, the C3-C6 cycloalkyl may be substituted with one or more halogens, CN, OH, C1-C3 alkyl, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), or C1-C3 haloalkyl.

[0101] According to one embodiment, A is an optionally substituted C4-C6 cycloalkyl. For example, A may 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] According to one embodiment, R 2 is a 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-. For example, R 2 It may be a C1-C3 alkyl optionally substituted with one or more halogens, O-(C1-C3 haloalkyl), O-(C1-C3 alkyl), or C1-C3 haloalkyl.

[0103] In chemical formula (I), according to one embodiment, X and Y are each bonds; A is a C4-C6 cycloalkyl; and R 1 is a C1-C3 alkyl selectively substituted with a C3-C6 cycloalkyl selectively substituted; R 2 is a 5-membered heteroaryl.

[0104] The term “compound” as used herein refers to the specific compound disclosed herein unless otherwise specified, and includes tautomers, positional isomers, geometric isomers, and, where applicable, stereoisomers, optical isomers (enantiomers), and other stereoisomers (diastereomers); and, where applicable, pharmaceutically acceptable salts and derivatives thereof (including prodrug forms) are also included in the context. The term “compound” as used in the context generally means a single compound, but may include other compounds such as stereoisomers, positional isomers, and / or optical isomers (including racemic mixtures), as well as mixtures rich in specific enantiomers or enantiomers of the disclosed compounds. The term may refer to a compound in a modified prodrug form to facilitate delivery and administration to the active site in the context. It should be noted that in describing the compounds of the present disclosure, various factors including numerous related substituents and variables are described. Those skilled in the art will understand that the molecules described herein are stable compounds as generally described below.

[0105] Where the compounds described herein have one or more chiral centers, it should be understood that, unless absolute stereochemistry is explicitly indicated, each center may independently be in an R-configuration or an S-configuration, or a mixture thereof. Accordingly, the compounds provided herein may be enantiomerically pure, enantiomerically abundant, or a mixture of stereoisomers, and include all diastereomers and enantiomer forms. Furthermore, in all compounds described herein having one or more double bonds that produce geometric isomers defined as E or Z, it should be understood that each double bond may independently be E or Z, or a mixture thereof. Stereoisomers are obtained, if desired, through stereoselective synthesis and / or separation of stereoisomers by a chiral chromatography column. Likewise, it should be understood that in all compounds described herein, tautomeric and conformal isomer forms are also intended to be included. A conformal isomer refers to a structure in which the spatial configuration is isomer. Conformational isomerism is a phenomenon in which atoms have the same structural formula but differ in their spatial arrangement based on rotational bonds (conformational isomers).

[0106] As used herein, the “R” group represents a substituent capable of bonding to the indicated atom. The R group may be substituted or may not be substituted. If a group is described as “optionally substituted,” the group may not be substituted or may be substituted with one or more specified substituents. Likewise, if a group is described as “not substituted or substituted,” if it is substituted, the substituent may be selected from one or more of the specified substituents. Unless a substituent is specified, the group indicated as “optionally substituted” or “substituted” means that it may be individually and independently substituted with one or more groups selected individually and independently from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclil, aracleyl, heteroaracleyl, heterocyclil(alkyl), hydroxy, alkoxy, cycloalkoxy, aryloxy, acyl, mercapto, alkylthio, aryltio, cyano, halogen, C-amido, N-amido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, haloalkyl, haloalkoxy, amino (including monosubstituted amino and disubstituted amino), and alkylamino. Where a group is not described as "optionally substituted," "unsubstituted," or "substituted," such group is unsubstituted unless otherwise specified in the definition of the group.

[0107] "C" used herein, where "a" and "b" are integers a to C b " or "C a -C b " refers to the number of carbon atoms in the alkyl, alkenyl, or alkynyl group or the number of carbon atoms in the ring of the cycloalkyl, aryl, heteroaryl, heterocycloalkyl, or heterocyclyl group. That is, the ring of alkyl, alkenyl, alkynyl, or cycloalkyl, the ring of aryl, the ring of heterocycloalkyl, the ring of heteroaryl, or the ring of heterocyclyl may contain carbon atoms of "a" or more and "b" or less.

[0108] As used herein, the term "alkyl" refers to a saturated monovalent chain of carbon atoms that may be optionally branched. In embodiments containing alkyl, the exemplary forms of such embodiments are lower alkyls, e.g., C1-C8, It should be understood that it includes C1-C6, C1-C5, C1-C4, C1-C3 alkyl, methyl, ethyl, propyl, 3-methylpentyl, etc.

[0109] As used herein, the term “alkenyl” refers to a straight-chain or branched hydrocarbon having at least two carbon atoms and at least one double bond. Alkenyls 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. Alkenyl groups may have an appropriate number of double bonds, including, but not limited to, one, two, three, four, five, or more. Examples of the alkenyl group include, but are not limited to, vinyl(ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopenthenyl, 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. The alkenyl group may be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

[0110] As used herein, the term "alkynyl" refers to a straight-chain or branched 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 the alkynyl group include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadininyl, 1-pentinyl, 2-pentinyl, isopentynyl, 1,3-pentadininyl, 1,4-pentadininyl, 1-hexinyl, 2-hexinyl, 3-hexinyl, 1,3-hexadinyl, 1,4-hexadinyl, 1,5-hexadinyl, 2,4-hexadinyl, or 1,3,5-hexatrinyl. The alkynyl group may be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

[0111] 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. The term "C n-m "haloalkyl" or "C n -C m"Haloalkyl" refers to a Cn-m alkyl group having n to m carbon atoms and at least one to {2(n to m)+1} halogen atoms that are identical or different from each other. According to one embodiment, the halogen atom is a fluorine atom. According to one embodiment, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Examples of the haloalkyl group include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5, etc. According to one embodiment, the haloalkyl group may be a fluoroalkyl group.

[0112] The term "cycloalkyl" as used herein refers to a monovalent chain of carbon atoms in which some form a ring. In the examples containing cycloalkyl, it should be understood that the exemplary forms of these examples include lower cycloalkyls, e.g., C3-C8, C3-C7, C3-C6, C3-C5, C4-C8, C4-C7, C4-C6 cycloalkyls, cyclopropyl, cyclohexyl, 3-ethylcyclopentyl, etc.

[0113] The term "bicycloalkyl" as used herein refers to two cycloalkyl groups defined above being linked together to form a bridged, fused, or spiro-type bicyclic compound.

[0114] The term "fused bicycloalkyl" as used herein refers to two cycloalkyl groups that share two adjacent atoms. In other words, the rings are directly connected by a single covalent bond, that is, the so-called bridgehead atoms.

[0115] As used herein, the term “bridged bicycloalkyl” refers to a group of two cycloalkyls in which the moiety shares two or more atoms. That is, the two rings do not share adjacent carbon atoms but share three or more atoms, and separate the two bridgehead atoms with a bridge containing at least one atom. Examples include, but are not limited to, bicyclo[3.2.1]heptyl (“norbornyl”), bicyclo[2.2.2]octyl.

[0116] The term “spirobicycloalkyl” as used herein refers to a bicyclic group in which two rings are joined at a single carbon atom that is a member of each ring. This term includes not only spirobicycloalkyls composed of cycloalkyl rings in which two rings are joined at a single carbon atom that is a member of each ring, but also spirobicycloheteroalkyls in which one ring is a heterocyclil ring and the other is a cycloalkyl ring, or in which both rings are heterocyclil rings in which one carbon atom that is a member of each ring is a heterocyclil ring. Examples of the spirobicyclil group 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.

[0117] As used herein, "heterocycloalkyl" refers to a non-aromatic ring having at least one heteroatom ring member independently selected from nitrogen, sulfur, and oxygen. The heterocycloalkyl group may include monocyclic or polycyclic (e.g., fused, bridged, or spiro) ring systems. The heterocycloalkyl also includes one or more aromatic rings fused to the non-aromatic heterocycloalkyl ring. The ring-forming carbon atoms of the heterocycloalkyl group may be optionally substituted with oxos. The ring-forming heteroatoms of the heterocycloalkyl group may be oxidized to form N-oxide or sulfonyl groups. In the examples comprising heterocycloalkyl, the exemplary forms of such examples should be understood to include lower cycloalkyls, e.g., C3-C8, C3-C7, C3-C6, C3-C5, C4-C8, C4-C7, C4-C6 heterocycloalkyls, etc. Examples of heterocycloalkyl groups include the morpholine ring, pyrrolidine ring, piperazine ring, piperidine ring, tetrahydropyran ring, tetrahydropyridine, azetidine ring, and tetrahydrofuran.

[0118] 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.

[0119] As used herein, the term "aryl" refers to an aromatic carbon ring system having any suitable number of ring atoms and any suitable number of rings. An aryl group may include any suitable number of carbon ring atoms, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, or 6 to 10, 6 to 12, or 6 to 14 ring members. An aryl group may be monocyclic, fused to form a bicyclic or tricyclic group, or linked by bonds to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl having a methylene linkage group. Some aryl groups have 6 to 12 ring members and include phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members and include phenyl or naphthyl. Some other aryl groups have six ring members, including phenyl. The aryl group may be optionally substituted with one or more moiety selected from alkyl, alkenyl, alkynyl, haloalkyl, halogen, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, alkyl carboxylate, amido, nitro, oxo, and cyano.

[0120] As used herein, the term “heteroaryl” refers to a group of substituted and unsubstituted aromatic monocyclic and dicyclic groups having at least one heteroatom (O, S, or N) in at least one ring of “aryl,” wherein the ring containing said heteroatom preferably has any suitable number of heteroatoms independently selected from O, S, and / or N. A dicyclic heteroaryl group must include only an aromatic ring. A heteroaryl ring system may be unsubstituted or may include one or more substituents. Examples of monocyclic heteroaryls include, but are not limited to, thiazolyl, oxazolyl, thiophenyl, furanyl, pyrrolyl, imidazolyl, isosazolyl, pyrazolyl, triazolyl, thiadiazolyl, tetrazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiazolyl, and other similar groups. Examples of bicyclic heteroaryls include, but are not limited to, indolyl, benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisosazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, furinyl, propyridinyl, oxochromene, dioxisoindolin, pyrazolopyridinyl, pyrazolo[1,5-a]pyridinyl, and other similar groups.

[0121] According to one embodiment, the compound is selected from Table A below.

[0122] [Table A]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197] According to one embodiment, compounds (I), (Ia), (Ib), (Ic), and (Id) according to the present disclosure degrade or reduce casein kinase 1 alpha (CK1α). Additionally, according to one embodiment, compounds according to the present disclosure inhibit the activity of casein kinase 1 alpha (CK1α). As used herein, the term "CK1α" refers to casein kinase 1α, a human kinase encoded by the CSNK1A1 gene. CK1α has been found to play an important role in the biology of AML (Jaras M et al, J Exp Med. 2014; 211(4):605–612).

[0198] According to one embodiment, a pharmaceutical composition for treating adenoid cystic carcinoma (ACC) according to the present disclosure optionally comprises a pharmaceutically acceptable excipient or carrier.

[0199] The term "pharmaceuticalally acceptable" describes a substance that does not have biological or other undesirable effects, that is, a substance that does not cause undesirable biological effects at unacceptable levels or interact in a harmful manner.

[0200] The term “pharmaceuticalally acceptable carrier” generally refers to a carrier useful for preparing pharmaceutical compositions suitable for human drug use as well as veterinary use, which is safe, non-toxic, and does not produce biological or other undesirable effects. As used herein in the specification and claims, “pharmaceuticalally acceptable carrier” may include one or more such carriers. The expression “pharmaceuticalally acceptable” means that the carrier must be compatible with other components of the formulation and must not have a harmful effect on the subject receiving it.

[0201] As used herein, the term “pharmaceuticalally acceptable salt” means a salt of an active major agent prepared with an acid or base that is acceptable to the biological system or to the target, or acceptable to both the biological system and the target when administered in a pharmacologically effective amount. If the compounds of the present disclosure contain relatively acidic functional groups, a base addition salt may be obtained by contacting a neutral form of such compounds in a pure state 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, organoamino, magnesium salts, lithium salts, strontium salts, or similar salts. If the compounds of the present disclosure contain relatively basic functional groups, an acid addition salt may be obtained by contacting a neutral form of such compounds in a pure state or in a suitable inert solvent with a sufficient amount of the desired acid. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, salts derived from inorganic acids, e.g., hydrochloric acid, bromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrophosphate, dihydrophosphate, sulfuric acid, monohydrosulfuric acid, hydroiodic acid, or phosphoric acid, and salts derived from relatively non-toxic organic acids, e.g., acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, souveric acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. In addition, salts of amino acids such as arginates and salts of organic acids such as glucuronic acid or galacturonic acid are also included.

[0202] As used herein, the term “effective amount” refers to an amount of activator administered sufficient to degrade CK1α and / or inhibit CK1α activity. According to certain embodiments, the term “effective amount” refers to an amount of activator administered sufficient to treat a CK1α-related disease, preferably adenoid cystic carcinoma (ACC).

[0203] As used herein, the terms "treat," "treatment," or "treating" refer to the administration of a compound or pharmaceutical composition to a subject for the purpose of prevention and / or treatment. The term "preventive treatment" refers to treating a subject who is susceptible to or at risk of a specific disease or condition for other reasons, but who has not yet shown symptoms of such disease or condition, in order to reduce the likelihood of the subject developing the disease or condition. The term "therapeutic treatment" refers to the administration of treatment to a subject who is already suffering from a disease or condition.

[0204] The pharmaceutical compositions described herein may be administered to human patients either as they are or as pharmaceutical compositions mixed with other active ingredients, excipients, or combinations thereof in combination therapy. The appropriate formulation depends on the selected route of administration. The formulations and methods of administration of the compounds described herein are known to those skilled in the art.

[0205] The pharmaceutical compositions disclosed herein may be prepared in a manner previously known, e.g., through conventional mixing, dissolving, granulation, preparation of coated tablets, grinding, emulsifying, encapsulating, encapsulating, or tableting processes. Additionally, the active ingredient is included in an amount effective for achieving the purpose. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts having pharmaceutically compatible counterions.

[0206] Various techniques for administering compounds, salts, and / or compositions exist in the art, including but not limited to oral, rectal, pulmonary, topical, aerosol, injection, infusion, and parenteral administration, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, nasal, and ocular injections. According to one embodiment, the compounds described herein, including the compound of formula (I) or pharmaceutically acceptable salts, enantiomers, stereoisomers, or prodrugs thereof, may be administered orally.

[0207] Additionally, compounds, salts, and / or compositions may be administered topically rather than systemically; for example, the compound may be administered by direct injection or implantation into the affected site, and is often administered in storage or sustained-release formulations. Furthermore, the compound may be administered via a targeted drug delivery system, for example, in liposomes coated with tissue-specific antibodies. The liposomes will be targeted and selectively absorbed by the organ. For example, nasal or pulmonary delivery may be preferred to target respiratory diseases or conditions.

[0208] The composition may be provided as a pack or dispensing device containing one or more unit dose forms containing the active ingredient, as needed. The compound, salt, and / or pharmaceutical composition may be provided to a administering physician or other healthcare professional in the form of a kit. A kit is a package containing a container holding the compound in a suitable pharmaceutical composition and instructions for administering the pharmaceutical composition to a subject.

[0209] As is obvious to those skilled in the art, the useful in vivo dose to be administered and the specific method of administration may vary depending on age, body weight, severity of the disease, mammalian species being treated, the specific compounds used, and the specific uses for which these compounds are used. The determination of effective dose levels, that is, the dose levels required to achieve desired results, can be performed by those skilled in the art using conventional methods such as human clinical trials and in vitro studies.

[0210]

[0211] Examples

[0212] I. Synthesis of Compounds

[0213] Hydrochloride form of Compound 1

[0214] 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0215]

[0216] Step-1: tert-Butyl (4-oxobutyl) carbamate

[0217]

[0218] A solution of oxalyl chloride (5.4 mL, 63.49 mmol, 1.2 equivalents) dissolved in DCM (50 mL) was slowly added to a solution of DMSO (10 mL, 1x) dissolved in DCM (100 mL, 10x) under a nitrogen atmosphere at -78°C for 15 minutes and 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 equivalent) dissolved in DCM (50 mL) was slowly added over 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and N,N-diisopropylethylamine (29.2 mL, 158.7 mmol, 3 equivalents) was slowly added at -78°C. After the addition was completed, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 2:8 / EtOAc: petroleum ether, R f ~ 0.6, confirmed by KMnO4), the reaction mixture was diluted with DCM (200 mL), washed with 10% aqueous citric acid solution (1 × 100 mL), water (1 × 200 mL) and brine (1 × 200 mL), dried with Na2SO4, filtered, and concentrated under vacuum at 30°C or lower to obtain tert-butyl(4-oxobutyl)carbamate (10 g, crude product) as a pale yellow liquid, which was subsequently used without purification.

[0219] 1 H-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).

[0220] Step-2: tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate

[0221]

[0222] Tert-Butyl (4-oxobutyl) carbamate 2 (9.0 g, 0.0484 mol, 1.8 equivalents) and lenalidomide 3 (CAS # 191732-72-6, 7.0 g, 0.0269 mol, 1 equivalent) were dissolved in a mixed solvent of 1,2-dichloroethane (70 mL, 10 times the volume) and DMF (70 mL, 10 times the volume) under a nitrogen atmosphere. Acetic acid (4.8 mL, 0.0807 mol, 3.0 equivalents) was added to this mixture at room temperature. The reaction mixture was stirred at room temperature for 2 hours. At this stage, NaCNBH3 (6.6 g, 0.1076 mol, 4 equivalents) was added in portions at 0°C and stirred at room temperature for 16 hours. Once the reaction is complete (TLC analysis, 8:2 / EtOAc: petroleum ether, R f ~ 0.6 and confirmed by LCMS), the reaction mixture was diluted with DCM (200 mL), washed with water (3 × 200 mL) and brine (200 mL), dried with Na2SO4, filtered, and concentrated. The obtained product was washed with hexane (50 mL), filtered, and dried under reduced pressure to obtain tert-butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (8 g, 69%) as a white solid.

[0223] 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 (MH), Rt (min): 2.552, area% - 97.64.

[0224] Step-3: 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0225]

[0226] HCl (1 M, 8 mL) dissolved in EtOAc was added to a 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 equivalent) dissolved in DCM (10 mL) near freezing point, and 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 freeze-dried to obtain 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (530 mg, 79%) as a yellow solid.

[0227] 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). 13 C-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). Methods: Mobile phase A: 0.1% formic acid in H2O. Mobile phase B: Acetonitrile. Flow rate: 1.5 mL / min. Column: Atlantis dC18 (50 Х 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 x 4.6) mm, 5 μm. R t (min): 7.403; Area% - 98.88.

[0228]

[0229] Hydrochloride form of Compound 2

[0230] 3-(4-((4-Aminobutyl)(methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0231]

[0232] Step-1: tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4- yl)(methyl)amino)butyl)carbamate

[0233]

[0234] Tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (2 g, 4.65 mmol, 1 equivalent) and paraformaldehyde (1.39 g, 46.5 mmol, 10 equivalents) were added together under a nitrogen atmosphere to a mixed solvent of 1,2-dichloroethane (20 mL, 10-fold) and DMF (10 mL, 5-fold). Acetic acid (0.8 mL, 13.95 mmol, 3 equivalents) was added to this mixture, and the reaction mixture was stirred at room temperature for 1 hour. Subsequently, NaCNBH3 (1.15 g, 18.6 mmol, 4 equivalents) was added in portions, and the reaction mixture was stirred at room temperature for an additional 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc R f ~ 0.4, and confirmed by LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3×50 mL) and brine (100 mL), dried with Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 40-45% ethyl acetate in DCM (column size: Biotage R snap cartridge, KP-Sil, 50 g, 230-400 silica gel) to obtain tert-butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(methyl)amino)butyl)carbamate (800 mg, 39%) as a white solid.

[0235] 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 (MH), Rt (min): 2.525, Area% - 99.11.

[0236] Step 2: 3-(4-((4-Aminobutyl)(methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0237]

[0238] HCl (1 M, 8 mL) dissolved in EtOAc was added to a near-freezing solution of tert-butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(methyl)amino)butyl)carbamate 5 (800 mg, 1.80 mmol) dissolved in DCM (10 mL), and 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 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.

[0239] 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). 13 C-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). Methods: Mobile phase A: 0.1% formic acid in H2O. Mobile phase B: Acetonitrile. Flow rate: 1.5 mL / min. Column: Atlantis dC18 (50 Х 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 Х 4.6) mm, 5 μm. Rt (min): 6.313; Area% - 97.81.

[0240]

[0241] Hydrochloride form of Compound 3

[0242] 3-(4-((4-Aminobutyl)(isopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0243]

[0244] Step-1: tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(isopropyl)amino)butyl)carbamate

[0245]

[0246] Tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (1.5 g, 3.4 mmol, 1 equivalent) and 2-Methoxyprofen (1.25 g, 17.5 mmol, 5 equivalents) were added to 1,2-Dichloroethane (30 mL, 20 times the volume), and the suspension was cooled to 0°C. Trifluoroacetic acid (0.22 mL, 3.0 mmol, 0.9 equivalents) was slowly added to this. Subsequently, sodium triacetoxyborohydride (1.07 g, 5.1 mmol, 1.5 equivalents) was added in portions at 0°C under a nitrogen atmosphere, and the reaction mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc R f ~ 0.5 and confirmed by LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (2 × 100 mL) and brine (100 mL), dried with Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by chromatography using 40-45% ethyl acetate in dichloromethane (column size: Biotage R snap cartridge, KP-Sil, 50 g, 230-400 silica gel) 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.

[0247] LCMS: 473.4 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6) 5 μ,

[0248] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.454; area% - 97.90.

[0249] Step 2: 3-(4-((4-Aminobutyl(isopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0250]

[0251] HCl (1 M, 4 mL) dissolved in EtOAc was added to a near-freezing solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(isopropyl)amino)butyl)carbamate 6 (620 mg, 1.313 mmol) dissolved in DCM (4 mL), and the mixture was stirred at room temperature for 12 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum and freeze-dried to obtain the desired compound, 3-(4-((4-Aminobutyl(isopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride (440 mg, 96%), as a light brown solid.

[0252] 1H-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). Methods: Column: Atlantis dC18 (50 χ 4.6) 5 μ,

[0253] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 1.239; area% - 97.330. HPLC: 97.905%, Rt (min): 6.141.

[0254]

[0255] Hydrochloride form of Compound 4

[0256] 3-(4-((4-Aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0257]

[0258] Step-1: tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)carbamate

[0259]

[0260] Tert-butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 4 (1.5 g, 3.4 mmol, 1 equivalent) and propionaldehyde (2.0 g, 3.4 mmol, 10 equivalents) were dissolved in a mixed solvent of 1,2-dichloroethane (20 mL) and DMF (10 mL) under a nitrogen atmosphere. Acetic acid (0.58 mL, 10.2 mmol, 3 equivalents) was added to this mixture, and the reaction mixture was stirred at room temperature for 1 hour. At this point, sodium cyanoborohydride (0.84 g, 1.3 mmol, 4 equivalents) was added in a fractional amount, and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc R f ~ 0.4 and confirmed by LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3×100 mL) and brine (100 mL), dried with Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 40-45% ethyl acetate in DCM (Isolera, column size: Biotage R snap cartridge, KP-Sil, 50 g, 230-400 silica gel) to obtain 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.

[0261] LCMS: 473.4 (M+H). Methods: Column: Atlantis dC18 (50X4.6) 5 μ, Mobile phase: A: 0.1% formic acid in H2O, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.709; Area% - 99.720.

[0262] Step 2: 3-(4-((4-Aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0263]

[0264] HCl (1 M, 5 mL) dissolved in EtOAc was added to a near-freezing solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)carbamate 7 (600 mg, 1.2695 mmol, 1 equivalent) dissolved in DCM (10 mL), and 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 freeze-dried 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.

[0265] 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). 13 C-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). Methods: Column: Atlantis dC18 (50X4.6) 5 μ, Mobile phase: A: 0.1% formic acid in H2O, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.386; Area% - 98.551. HPLC: 98.878 %, Rt (min): 6.95.

[0266]

[0267] Hydrochloride form of Compound 6

[0268] 3-(4-((2-(1-(Aminomethyl)cyclohexyl)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0269]

[0270] Step-1: 2-(1-(((tert-Butoxycarbonyl)amino)methyl)cyclohexyl)acetic acid

[0271]

[0272] NaOH (7.0 g, 175.4 mmol, 1 equivalent) dissolved in water (100 mL, 5 times the volume) and Boc-anhydride (38.2 g, 175.4 mmol, 1 equivalent) were slowly added at 0°C to 2-(1-(Aminomethyl)cyclohexyl)acetic acid 1 (30 g, 175.4 mmol, 1 equivalent) dissolved in THF (300 mL, 10 times the volume). The mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 20% EtOAc in petroleum ether, R f ~ 0.4, and confirmed by LCMS-ELSD), the solvent was evaporated to dry, diluted with water (100 mL), and washed with ethyl acetate (1 × 150 mL). The aqueous layer was separated, acidified to pH 5 to 6 with 1.5N HCl, and then extracted with DCM (2 × 500 mL). The organic layer was washed with brine (250 mL), dried with 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.

[0273] LCMS (ELSD): 270.3 (MH). Methods: Column: Atlantis dC18 (50 × 4.6) 5 μ,

[0274] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.950; area% - 99.502.

[0275] Step 2: Methyl 2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)acetate

[0276]

[0277] Potassium carbonate (20.3 g, 147.6 mmol, 2 equivalents) and methyl iodide (26 mL, 369 mmol, 5 equivalents) were slowly added at 0°C to 2-(1-(((tert-Butoxycarbonyl)amino)methyl)cyclohexyl)acetic acid 2 (20 g, 73.8 mmol, 1 equivalent) dissolved in DMF (200 mL, 10-fold volume). The mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 20% EtOAc in petroleum ether, R f ~ 0.6 and confirmed by LCMS-ELSD), the reaction mixture was diluted with ethyl acetate (200 mL) and washed with H2O (3 × 500 mL) near freezing point. The organic layer was washed with brine (1 × 500 mL), dried with 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.

[0278] LCMS (ELSD): 230.3 (M-tert-Butyl). Methods: Column: Atlantis dC18 (50 × 4.6) 5 μ,

[0279] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 3.374; area% - 99.76.

[0280] Step-3: tert-Butyl 3-hydroxy-2-azaspiro[4.5]decane-2-carboxylate

[0281]

[0282] DIBAL-H (1 M in hexane, 28.07 mL, 28.07 mmol, 1 equivalent) was slowly added to Methyl 2-(1-(((tert-butoxycarbonyl)amino)methyl)cyclohexyl)acetate 3 (8 g, 28.07 mmol, 1 equivalent) dissolved in DCM (150 mL) at -78°C. The mixture was stirred at 0°C for 2 hours.

[0283] Once the reaction is complete (TLC analysis, 20% EtOAc in petroleum ether, R f ~ 0.5 and confirmed by LCMS-ELSD), the reaction mixture was quenched with a 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 with Na2SO4, filtered, and concentrated under vacuum below 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 subsequently used without purification.

[0284] LCMS (ELSD): 202.1 (M-tert-Butyl). Methods: Column: Atlantis dC18 (50 × 4.6) 5 μ,

[0285] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 2.187; area% - 99.976.

[0286] Step-4: tert-Butyl((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethyl)cyclohexyl)methyl)carbamate

[0287]

[0288] Acetic acid (1.82 mL, 30.7 mmol, 4 equivalents) was added under a nitrogen atmosphere to a solution of lenalidomide (CAS # 191732-72-6, 2 g, 7.6 mmol, 1 equivalent) and tert-Butyl 3-hydroxy-2-azaspiro[4.5]decane-2-carboxylate 4 (5.79 g, 2.3 mmol) stirred in a mixture of 1,2-dichloroethane (20 mL) and DMF (20 mL). The reaction mixture was stirred at room temperature for 1 hour. Subsequently, the reaction mixture was cooled to 0°C, sodium cyanoborohydride (1.84 g, 30.7 mmol, 4 equivalents) was added in portions, and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc, R f ~ 0.6 and confirmed by LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3 × 50 mL) and brine (100 mL), dried with Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 40-45% EtOAc in DCM (column size: Biotage R snap 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.

[0289] LCMS: 443.4 (Mt-Bu). Methods: Column: Atlantis dC18 (50 × 4.6) 5 μ,

[0290] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 3.072; area% - 98.042.

[0291] Step-5: 3-(4-((2-(1-(Aminomethyl)cyclohexyl)ethyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0292]

[0293] HCl (1 M, 6 mL) dissolved in EtOAc was added to a near-freezing solution of tert-Butyl((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)ethyl)cyclohexyl)methyl)carbamate 5 (620 mg, 1.2437 mmol, 1 equivalent) dissolved in DCM (10 mL), and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by LCMS, the reaction mixture was concentrated under vacuum to obtain the crude product, distilled with Milli-Q H2O (2 × 10 mL), and dried under vacuum to obtain 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.

[0294] 1H-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). 13 C-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). Methods: Column: Atlantis dC18 (50 × 4.6) 5 μ,

[0295] Mobile phase: A: 0.1% formic acid in H2O, B: ACN, flow rate: 1.5 mL / min. Rt (min): 1.551; area% - 99.47. HPLC: 99.499%, Rt (min): 8.979.

[0296]

[0297] Hydrochloride form of Compound 46

[0298]

[0299] General procedure for the preparation of compound 6b

[0300]

[0301] Acetic acid (1.0 mL) and molecular sieve (1.0 g) were added to compound 4 (1.0 g, 2.32 mmol, 1 equivalent) and compound 5b (336 mg, 4.65 mmol, 2.0 equivalents) dissolved in DCE / DMF (10 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. NaBH3CN (585 mg, 9.3 mmol, 4 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Once the reaction was complete (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 with Na2SO4. The obtained 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.

[0302] TLC: EA = 100%

[0303] R f (Compound 4) = 0.5

[0304] R f (Compound 6b) = 0.6

[0305] LC-MS: 487.3 [M+1] +

[0306] General procedure for the preparation of Compound 46

[0307]

[0308] HCl / EA (2N, 8.73 mmol, 10 equivalents) was added to compound 6b (400 mg, 0.873 mmol, 1 equivalent) dissolved in methanol (1 mL). The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by P-HPLC (acetonitrile / H2O: 30%-40%) to obtain compound 46 (60 mg, 16%) as a white solid.

[0309] TLC: DCM / methanol = 10 / 1

[0310] R f (Compound 6b) = 0.6

[0311] R f (Compound 46) = 0.2

[0312] LC-MS: 387.25 [M+1] +

[0313] 1 H NMR (400 MHz, DO) 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).

[0314]

[0315] Hydrochloride form of Compound 47

[0316] 3-(4-((4-aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0317]

[0318] Step-3: tert-Butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(methyl)amino)butyl)carbamate

[0319] Acetic acid (1.2 mL, 20.91 mmol, 3 equivalents) was added to tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl) carbamate 4 (3 g, 6.97 mmol, 1 equivalent) and pentanal (6.0 g, 69.7 mmol, 10 equivalents) dissolved in a mixed solvent of 1,2-dichloroethane (60 mL, 20 times the volume) and DMF (30 mL, 10 times the volume), and the reaction mixture was stirred at room temperature for 2 hours. NaCNBH3 (1.75 g, 27.9 mmol, 4 equivalents) was added in portions, and the reaction mixture was stirred at room temperature for an additional 16 hours. TLC analysis (70% EtOAc in petroleum ether) confirmed product formation, but the starting material was not completely consumed. Additionally, pentanal (2 × 6.0 g, 69.7 mmol, 10 equivalents), acetic acid (2 × 0.8 mL, 13.94 mmol, 2 equivalents), and NaCNBH3 (0.87 g, 13.94 mmol, 2 equivalents) were added, and the resulting reaction mixture was stirred at room temperature for 36 hours. Upon completion of the reaction (TLC analysis, 70% EtOAc in petroleum ether, R f ~ 0.6 and confirmed by LCMS), the reaction mixture was diluted with DCM (100 mL), washed with water (3 × 100 mL) and brine (300 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 65-70% ethyl acetate in petroleum ether (Biotage R snap cartridge, KP-Sil, 100 g, 230-400 silica gel) to obtain 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.

[0320] 1H-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).

[0321] LC-MS: 501.3 (M+H), Rt (min): 1.853, Area% - 97.842.

[0322] Step 4: 3-(4-((4-aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0323]

[0324] HCl (4M solution dissolved in EtOAc, 12.5 mL) was added to a near-freezing solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)carbamate (2.5 g, 4.99 mmol) dissolved in DCM (25 mL), and the mixture was stirred at room temperature for 2 hours. When the reaction was complete (TLC analysis, 100% EtOAc R f (confirmed as ~0.4), the reaction mixture was concentrated and freeze-dried to obtain 3-(4-((4-aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 47 (2.4 g, 98%) as a milky white solid.

[0325] 1 H-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).

[0326] 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 Х 4.6 mm) 3.5 μm. Rt (min): 1.356; Area%: 96.152.

[0327] 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 χ4.6) mm, 3.5 μm. Rt (min): 2.341; Area%: 99.332.

[0328]

[0329] Hydrochloride form of Compound 48

[0330]

[0331] General procedure for the preparation of compound 6d

[0332]

[0333] Acetic acid (2.0 mL) and a molecular sieve (1.0 g) were added to compound 4 (2.0 g, 4.65 mmol, 1 equivalent) and compound 5d (932 mg, 9.30 mmol, 2.0 equivalents) dissolved in DCE / DMF (20 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. NaBH3CN (1.17 g, 18.6 mmol, 4 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Once the reaction was complete (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 with 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.

[0334] TLC: EA = 100%

[0335] R f (Compound 4) = 0.5

[0336] R f (Compound 6d) = 0.6

[0337] LC-MS: 515.40 [M+1] +

[0338] General procedure for the preparation of Compound 48

[0339]

[0340] HCl / EA (2N, 6.42 mmol, 5 equivalents) was added to compound 6d (660 mg, 1.28 mmol, 1 equivalent) dissolved in DCM (6 mL). The resulting solution was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by P-HPLC (acetonitrile / H2O: 30%-40%) to obtain compound 48 (150.55 mg, 28%) as a white solid.

[0341] TLC: DCM / methanol = 10 / 1

[0342] R f (Compound 6d) = 0.6

[0343] R f (Compound 48) = 0.2

[0344] LC-MS: 415.40 [M+1] +

[0345] 1 H 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).

[0346]

[0347] Hydrochloride form of Compound 49

[0348]

[0349] General procedure for the preparation of compound 2e

[0350]

[0351] Oxalyl chloride (9.5 g, 74.5 mmol, 1.2 equivalents) dissolved in DCM (50 mL) was slowly added to a DMSO (10 mL) solution dissolved in DCM (100 mL) under a nitrogen atmosphere at -78°C for 15 minutes, and stirred at the same temperature for 15 minutes. A solution of compound 1e (10.0 g, 62.1 mmol, 1.0 equivalent) dissolved in DCM (50 mL) was slowly added over 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (18.8 g, 188.2 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (TLC analysis, PE / EA=4 / 1, R f ~0.4, identified as 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 with Na2SO4, filtered, and concentrated under vacuum at 30°C or below to obtain compound 2 (11.4 g, crude product) as a yellow oil, which was subsequently used without purification.

[0352] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0353] R f (1e of compound) = 0.2

[0354] R f (Compound 2e) = 0.4

[0355] LC-MS: Not detected.

[0356] General procedure for the preparation of Compound 5

[0357]

[0358] Acetic acid (0.7 g, 11.6 mmol, 3.0 equivalents) was added to compound 3 (0.2 g, 3.9 mmol, 1.0 equivalents) and compound 4 (1.0 g, 3.9 mmol, 1.0 equivalents) dissolved in DCE / DMF (1 / 1, 20 mL). The reaction mixture was stirred at room temperature for 2 hours. At this stage, NaBH3CN (1.0 g, 15.4 mmol, 4.0 equivalents) was added in a fraction at 0°C and stirred at room temperature for 18 hours. Once the reaction was complete, 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 with Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / methanol: 3:1) to obtain compound 5 (0.75 g, 64%) as a white solid.

[0359] TLC: EA

[0360] R f (Compound 3) = 0.4

[0361] R f (Compound 5) = 0.5

[0362] LC-MS: 302.25 [M+1] + , 300.25 [M-1] -

[0363] 1 H 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).

[0364] 1 H NMR (400 MHz, DMSO-d6): d 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).

[0365] General procedure for the preparation of compound 6e

[0366]

[0367] Acetic acid (20 mL) was added to compound 5 (4.6 g, 15.3 mmol, 1.0 equivalent) and compound 2e (4.4 g, 27.5 mmol, 1.8 equivalent) dissolved in DCE / DMF (80 mL, 1 / 1), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (3.8 g, 61.1 mmol, 4.0 equivalent) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. Once the reaction was complete (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 with 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 6e (0.7 g, 10%) as a white solid.

[0368] TLC: EA = 100%

[0369] R f (Compound 5) = 0.5

[0370] R f (Compound 6e) = 0.6

[0371] LC-MS: 445 [M+1] + , 443 [M-1] -

[0372] 1H 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).

[0373] General procedure for the preparation of Compound 49

[0374]

[0375] HCl / EA (2 mL, 2N, 4.0 mmol, 10.0 equivalents) was added to compound 6e (0.2 g, 0.4 mmol, 1.0 equivalent) dissolved in methanol (2 mL). The resulting solution was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 30%-40%) to obtain compound 49 (147 mg, 95%) as a white solid.

[0376] LC-MS: 345.35 [M+1] +

[0377] 1 H NMR (400 MHz, DO): 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).

[0378]

[0379] Compound 50

[0380] N-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)acetamide

[0381]

[0382] Step-1: N-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(propyl)amino)butyl)acetamide

[0383]

[0384] Acetic anhydride (0.231 mL, 2.445 mmol, 5 equivalents) was added to a solution of 3-(4-((4-aminobutyl)(propyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl compound 4 (200 mg, 0.489 mmol, 1 equivalent) dissolved in pyridine (3 mL) at 0°C, and stirred at room temperature for 12 hours. When the reaction was complete (confirmed by LCMS), the reaction mixture was concentrated under vacuum to obtain the crude product, 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 freeze-dried 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.

[0385] 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).

[0386] LCMS: 415.1 (M+H), Method: Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: ACN, Column: Atlantis dC18 (50×4.6 mm) 5 μm, Flow rate: 1.5 ml / min Rt (min): 1.622, Area %: 98.693.

[0387] HPLC: Method: Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Column: X-Bridge C8(50Х4.6) mm, 3.5 μm Flow rate: 2.0 mL / min, Rt (min): 1.789, Area %: 99.953.

[0388]

[0389] Hydrochloride form of Compound 51

[0390]

[0391] General procedure for the preparation of 2g of compound

[0392]

[0393] DIEA (18.6 g, 144 mmol, 3.0 equivalents) was added to 1 g of the compound (5.0 g, 48 mmol, 1 equivalent) dissolved in DMF (50 mL) and tert-butylchlorodiphenylsilane (13.2 g, 48 mmol, 1.0 equivalent), and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Upon 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 with Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain 2 g of the compound (7.2 g, 44%) as a clear oil.

[0394] TLC: PE / EA = 2 / 1 KMnO4

[0395] R f (1g of compound) = 0.1

[0396] R f (2g of compound) = 0.6

[0397] 1 H 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).

[0398] General procedure for the preparation of 3g of compound

[0399]

[0400] A solution of oxalyl chloride (3.2 g, 25.2 mmol, 1.2 equivalents) dissolved in DCM (40 mL) was slowly added to a solution of DMSO (7.5 mL, 1.0 V) dissolved in DCM (35 mL) under a nitrogen atmosphere at -78°C for 15 minutes and stirred at the same temperature for 15 minutes. A solution of 2 g of the compound (7.2 g, 20.9 mmol, 1.0 equivalents) dissolved in DCM (20 mL) was slowly added at -78°C for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (6.35 g, 62.9 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was completed, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (confirmed by TLC analysis), the reaction mixture was diluted with DCM (50 mL), washed with 10% citric acid (30 mL), water (50 mL), and brine (50 mL), and dried with Na2SO4. The solution was filtered and concentrated under vacuum at 30°C or below to obtain 3 g (7.03 g) of the compound in the form of a clear oil, which was used without further purification.

[0401] TLC: PE / EA = 2 / 1

[0402] R f (2g of compound) = 0.6

[0403] R f (3g of compound) = 0.8

[0404] 1 H 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).

[0405] General procedure for the preparation of 5g of compound

[0406]

[0407] Acetic acid (2.2 mL) and a molecular sieve (7.03 g) were added to compound 4a (2.2 g, 5.1 mmol, 1 equivalent) and compound 3 g (7.03 g, 20.6 mmol, 4.0 equivalents) dissolved in DCE / DMF (10 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. NaBH3CN (1.6 g, 25.6 mmol, 5 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Once the reaction was complete (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 with Na2SO4. The obtained 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 5 g (1.03 g, 27%) of the compound as a white solid.

[0408] TLC: EA = 100%

[0409] R f (Compound 4a) = 0.5

[0410] R f (5g of compound) = 0.7

[0411] LC-MS: 755.60 [M+1] +

[0412] General procedure for the preparation of 6g of compound

[0413]

[0414] THF (13.7 mL, 1 N, 13.7 mmol, 10 equivalents) was added, and the mixture was stirred at room temperature for 4 hours under a nitrogen atmosphere. Once the reaction was complete (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 with 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 6 g (580 mg, 82%) of the compound as a white solid.

[0415] TLC: EA = 100%

[0416] R f (5g of compound) = 0.7

[0417] R f (6g of compound) = 0.4

[0418] LC-MS: 517.30 [M+1] +

[0419] General procedure for the preparation of Compound 51

[0420]

[0421] HCl (2.0 mL, 2N, 4.0 mmol, 10.0 equivalents) was added to 6 g of compound (200 mg, 0.387 mmol, 1 equivalent) dissolved in methanol (1.0 mL). The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was freeze-dried to obtain compound 51 (164.3 mg, 87%) as a white solid.

[0422] TLC: DCM / methanol = 10 / 1

[0423] R f (6g of compound) = 0.4

[0424] R f (Compound 51) = 0.2

[0425] LC-MS: 417.40 [M+1]+

[0426] 1 H NMR (400 MHz, DO): 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).

[0427]

[0428] Hydrochloride form of Compound 52

[0429]

[0430] General procedure for the preparation of compound 2h

[0431]

[0432] A solution of compound 1h (0.5 g, 3.47 mmol, 1.0 equivalent) dissolved in THF (5 mL) was slowly added to LiAlH4 (145 mg, 3.82 mmol, 1.1 equivalent) dissolved in THF (5 mL) at 0°C under a nitrogen atmosphere, and stirred at room temperature for 4 hours. When the reaction was complete (TLC analysis, PE / EA=2 / 1, R f ~0.6, confirmed as KMnO4), the reaction mixture was diluted with MTBE (30 mL) and Na2SO410H2O was added. The mixture was stirred for 10 minutes, filtered, and concentrated to obtain compound 2h (350 mg, 77%) in the form of a clear oil, which was used without further purification.

[0433] TLC: PE / EA = 2:1, KMnO4

[0434] R f (Compound 1h) = 0.2

[0435] R f (Compound 2h) = 0.6

[0436] 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).

[0437] General procedure for the preparation of compound 3h

[0438]

[0439] PCC (1.99 g, 9.22 mmol, 2.0 equivalents) was added to a solution of compound 2h (600 mg, 4.61 mmol, 1 equivalent) dissolved in DCM (30 mL) and stirred at room temperature for 4 hours. When the reaction was complete (confirmed by TLC analysis, PE / EA=3:1), silica gel (4.04 g) was added and stirred for an additional 10 minutes. The reaction mixture was filtered and concentrated under reduced pressure below 30°C to obtain compound 3h (714 mg) in the form of a clear oil, which was used without further purification.

[0440] TLC: PE / EA = 4 / 1 KMnO4

[0441] R f (Compound 2h) = 0.4

[0442] R f (Compound 3h) = 0.7

[0443] 1 H 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).

[0444] General procedure for the preparation of compound 5h

[0445]

[0446] Acetic acid (1 mL) and a molecular sieve (1.0 g) were added to a solution of compound 4a (799 mg, 1.86 mmol, 1 equivalent) and compound 3h (714 mg, 5.57 mmol, 3.0 equivalents) dissolved in DCE / DMF (6 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. NaBH3CN (467 mg, 7.43 mmol, 4 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Once the reaction was complete (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 with 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 5h (275 mg, 27%) as a white solid.

[0447] TLC: EA = 100%

[0448] R f (Compound 4a) = 0.4

[0449] R f (Compound 5h) = 0.6

[0450] LC-MS: 543.55 [M+1] +

[0451] General procedure for the preparation of Compound 52

[0452]

[0453] HCl (1.8 mL, 2N, 3.7 mmol, 10.0 equivalents) was added to a solution of compound 5h (200 mg, 0.368 mmol, 1.0 equivalent) dissolved in methanol (1.0 mL). The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was freeze-dried to obtain compound 52 (170.7 mg, 89%) as a white solid.

[0454] TLC: DCM / methanol = 10 / 1

[0455] R f (Compound 5h) = 0.6

[0456] R f (Compound 52) = 0.1

[0457] LC-MS: 443.45 [M+1] +

[0458] 1 H NMR (400 MHz, DO): 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).

[0459]

[0460] Hydrochloride form of Compound 53

[0461]

[0462] General procedure for the preparation of compound 2i

[0463]

[0464] A solution of oxalyl chloride (0.9 g, 6.9 mmol, 1.2 equivalents) dissolved in DCM (5 mL) was slowly added to a solution of DMSO (1 mL) dissolved in DCM (10 mL) under a nitrogen atmosphere at -78°C for 15 minutes, and stirred at the same temperature for 15 minutes. A solution of compound 1i (1.0 g, 5.7 mmol, 1.0 equivalents) dissolved in DCM (5 mL) was slowly added at -78°C for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (1.7 g, 17.1 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (TLC analysis, PE / EA=4 / 1, R f ~0.4, identified as 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 with Na2SO4, filtered, and concentrated under vacuum at 30°C or below to obtain compound 2i (0.9 g, crude product) in the form of a yellow oil, which was used without further purification.

[0465] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0466] R f (Compound 1i) = 0.2

[0467] R f (Compound 2i) = 0.4

[0468] LC-MS: Not detected.

[0469] 1 H 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).

[0470] General procedure for the preparation of compound 6i

[0471]

[0472] Acetic acid (10 mL) was added to a solution of compound 2i (2.3 g, 13.3 mmol, 2.0 equivalents) and compound 5 (2.0 g, 6.6 mmol, 1.0 equivalents) dissolved in DCE / DMF (1 / 1, 40 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 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. Once the reaction was complete (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 with Na2SO4. The obtained 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.

[0473] TLC: EA

[0474] R f (Compound 2i) = 0.5

[0475] R f (Compound 6i) = 0.6

[0476] LC-MS: 459 [M+1] + , 457.30 [M-1] -

[0477] General procedure for the preparation of Compound 53

[0478]

[0479] TFA (4 mmol, 10.0 equivalents) was added to a solution of compound 6i (0.2 g, 0.4 mmol, 1.0 equivalent) dissolved in DCM / H2O (1 / 1, 2 mL). The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by P-HPLC eluting with acetonitrile / H2O (30%-40%) to obtain compound 53 (105 mg, 65%) as a white solid.

[0480] LC-MS: 359.30 [M+1] + , 357.20 [M-1] -

[0481] 1 H 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).

[0482]

[0483] Hydrochloride form of Compound 55

[0484] 3-(4-((4-aminobutyl)(5-aminopentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[0485]

[0486] Step-1: tert-butyl (5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate

[0487]

[0488] Imidazole (0.837 g, 12.30 mmol, 2.5 equivalents) and TBS-Cl (1.112 g, 7.38 mmol, 1.5 equivalents) were added at 0°C to a solution of tert-butyl (5-hydroxypentyl)carbamate 1 (1.0 g, 4.92 mmol, 1.0 equivalent) dissolved in DCM (20 mL), and the mixture was stirred at room temperature for 2 hours. Upon completion of the reaction (TLC analysis, EtOAc / Petroleum Ether 50%, R f ~ 0.5, confirmed by KMnO4), the reaction mixture was quenched with H2O (20 mL) and extracted with DCM (2 × 20 mL). The organic layer was dried with Na2SO4 and concentrated under vacuum to obtain tert-butyl (5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 2 (1.5 g, 4.70 mmol, 96% yield) as a yellow liquid, which was subsequently used without purification.

[0489] 1 H-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),

[0490] Step-2: tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate

[0491]

[0492] n-BuLi (2.5 M solution in hexane, 2.267 mL, 5.67 mmol, 1.2 equivalents) was added to a solution of tert-butyl (5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 2 (1.5 g, 4.72 mmol, 1.0 equivalent) dissolved in THF (20 mL) while stirring at 0°C, and stirred for 15 minutes at 0°C. At the same temperature, a solution of (Boc)2O (1.316 mL, 5.67 mmol, 1.2 equivalents) dissolved in THF (5 mL) was added to the reaction mixture. The resulting reaction mixture was heated to room temperature and stirred for 1 hour. Upon completion of the reaction (TLC analysis, 10% EtOAc / petroleum ether, R f ~ 0.7, confirmed by KMnO4), the reaction mixture was quenched with water (10 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain 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 subsequently used without purification.

[0493] 1 H-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)

[0494] Step-3: tert-butyl (tert-butoxycarbonyl)(5-hydroxypentyl)carbamate

[0495]

[0496] A solution of tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)pentyl)carbamate 3 (1.9 g, 4.55 mmol) dissolved in THF (20 mL) was stirred at room temperature. The resulting solution was stirred overnight at room temperature. When the reaction was complete (TLC analysis, 20% EtOAc / petroleum ether, R f ~0.2, confirmed by KMnO4), the reaction mixture was quenched with water (10 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain a crude product in the form of a dark yellow liquid. The crude product 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 fraction was 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.

[0497] 1 H-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),

[0498] Step-4: tert-butyl (tert-butoxycarbonyl)(5-oxopentyl)carbamate

[0499]

[0500] DMSO (0.936 ml, 13.18 mmol, 4.0 equivalents) dissolved in CH2Cl2 (5 ml) was added to a solution of oxalyl chloride (0.577 ml, 6.59 mmol, 2.0 equivalents) and CH2Cl2 (20 ml) at -78°C and stirred for 15 minutes at the same temperature. To this, a solution of tert-butyl (tert-butoxycarbonyl)(5-hydroxypentyl)carbamate 4 (1.0 g, 3.30 mmol, 1.0 equivalents) dissolved in CH2Cl2 (5 mL) was added at -78°C. The reaction mixture was stirred at -78°C for 30 minutes, and triethylamine (2.76 ml, 19.78 mmol, 6.0 equivalents) was slowly added at -78°C. Subsequently, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 20% EtOAc / petroleum ether, R f ~ 0.6, confirmed by KMnO4), the reaction mixture was diluted with DCM (40 mL) and washed with 10% aqueous citric acid solution (1 × 40 mL) and water (1 × 40 mL). The mixture was dried with 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 subsequently used without purification.

[0501] 1 H-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),

[0502] 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

[0503]

[0504] Trifluoroacetic acid (0.501 mL, 6.50 mmol, 4.0 equivalents) was added to a mixture of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 6 (0.7 g, 1.626 mmol, 1.0 equivalent) and tert-butyl (tert-butoxycarbonyl)(5-oxopentyl)carbamate 5 (0.980 g, 3.25 mmol, 2.0 equivalents) dissolved in CH2Cl2 (10 mL) and DMF (10 mL), and the mixture was stirred at 0°C for 10 minutes. Sodium triacetoxyborohydride (1.378 g, 6.50 mmol, 4.0 equivalents) was added in a divided manner at 0°C, and the resulting colorless, turbid mixture was stirred at room temperature for 16 hours. When the reaction was complete (TLC analysis, 100% EtOAc, R f(confirmed as ~0.7), the reaction mixture was quenched with water near freezing point (20 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with anhydrous Na2SO4 and concentrated under vacuum to obtain a crude product in the form of a pale yellow liquid. The crude product 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 obtain tert-butyl(tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)carbamate 7 (0.65 g, 0.891 mmol, 54.8% yield) as a milky white solid.

[0505] 1 H-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),

[0506] LCMS: 616.4 (M-Boc). Method: Atlantis dC18 (50 x 4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: ACN, Flow rate: 1.5 ml / min

[0507] HPLC: Method: Mobile Phase A: 0.1% TFA in water, Mobile Phase B: Acetonitrile, Flow Rate: 2.0 ml / min. Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm. RT: 4.972 min, Area: 98.095%

[0508] Step-6: 3-(4-((4-aminobutyl)(5-aminopentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[0509]

[0510] HCl (4 M solution in ethyl acetate, 10 mL) was added at 0°C to a stirred solution of tert-butyl(tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentyl)carbamate 7 (0.35 g, 0.489 mmol, 1.0 equivalent) dissolved in dichloromethane (5 mL), and stirred at room temperature for 3 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum at 40°C or below to obtain a pale yellow solid. The obtained solid was dissolved in water (10 mL) and washed with MTBE (2 × 10 mL). The aqueous layer was freeze-dried to obtain 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.

[0511] 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).

[0512] LCMS: 416.3 (M+H). Methods: 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.

[0513] HPLC: Method: Mobile phase A: 0.1% TFA in water, Mobile phase B: Methanol, Flow rate: 0.7 ml / min. Column: Atlantis dC18 (250 x 4.6) mm, 5 μm. RT: 12.223 min, Area: 98.472%.

[0514]

[0515] Hydrochloride form of Compound 56

[0516]

[0517] General procedure for the preparation of compound 2L

[0518]

[0519] DIEA (32.7 g, 254 mmol, 3.0 equivalents) was added to a solution of tert-butylchlorodiphenylsilane (23.25 g, 85.6 mmol, 1.0 equivalent) and 1 liter of compound (10 g, 85.6 mmol, 1 equivalent) dissolved in DMF (100 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Upon 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 with Na2SO4, and filtered. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain 2 liters (10.27 g, 35%) of compound in the form of a clear oil.

[0520] TLC: PE / EA = 2 / 1 KMnO4

[0521] R f (1 liter of compound) = 0.1

[0522] R f (Compound 2l) = 0.6

[0523] 1 H 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).

[0524] General procedure for the preparation of compound 3L

[0525]

[0526] Oxalyl chloride (2.14 g, 16.8 mmol, 1.2 equivalents) dissolved in DCM (20 mL) was slowly added to a DMSO (5 mL, 1.0 V) solution dissolved in DCM (20 mL) under a nitrogen atmosphere at -78°C for 15 minutes and stirred at the same temperature for 15 minutes. A solution of compound (2 L, 5.0 g, 14.02 mmol, 1.0 equivalent) dissolved in DCM (10 mL) was slowly added at -78°C for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (4.3 g, 42.1 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. Once the reaction was complete (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 with Na2SO4, filtered, and concentrated under vacuum at 30°C or lower to obtain 3 L (4.9 g) of the compound in the form of a clear oil, which was used without further purification.

[0527] TLC: PE / EA = 2 / 1

[0528] R f (Compound 2l) = 0.6

[0529] R f (Compound 3l) = 0.8

[0530] General procedure for the preparation of compound 5l

[0531]

[0532] Acetic acid (1.5 mL) and molecular sieve (5.0 g) were added to a solution of compound 4a (1.5 g, 3.45 mmol, 1 equivalent) and compound 3 L (4.9 g, 13.8 mmol, 4.0 equivalents) dissolved in DCE / DMF (15 mL), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.08 g, 17.2 mmol, 5 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Once the reaction was complete (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 with Na2SO4. The obtained 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 5 L (1.03 g, 29%) of compound as a white solid.

[0533] TLC: EA = 100%

[0534] R f (Compound 4a) = 0.5

[0535] R f (5l of compound) = 0.7

[0536] LC-MS: 769.60 [M+1] +

[0537] General procedure for the preparation of compound 6l

[0538]

[0539] TBAF / THF (13.4 mL, 1 N, 13.4 mmol, 10 equivalents) was added to a solution of 5 L (1.03 g, 1.34 mmol, 1 equivalent) of the compound dissolved in THF, and the mixture was stirred at room temperature for 4 hours under a nitrogen atmosphere. Upon 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 with 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 6 L (620 mg, 87%) of the compound as a white solid.

[0540] TLC: EA = 100%

[0541] R f (5l of compound) = 0.7

[0542] R f (6l of compound) = 0.4

[0543] LC-MS: 531.45 [M+1] +

[0544] General procedure for the preparation of compound 56

[0545]

[0546] HCl (2.8 mL, 2N, 5.6 mmol, 10 equivalents) was added to a solution of compound 6 L (300 mg, 0.566 mmol, 1 equivalent) dissolved in methanol (1 mL). The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was freeze-dried to obtain compound 56 (231 mg, 81%) as a white solid.

[0547] TLC: DCM / methanol = 10 / 1

[0548] R f (Compound 15-A) = 0.4

[0549] R f (Compound 56) = 0.2

[0550] LC-MS: 431.40 [M+1] +

[0551] 1 H NMR (400 MHz, DO): 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).

[0552]

[0553] Hydrochloride form of Compound 57

[0554]

[0555] General procedure for the preparation of compound 2m

[0556]

[0557] A solution of 1 m of compound (14.3 g, 138.6 mmol, 1.0 equivalent) and TEA (42.0 g, 415.8 mmol, 3.0 equivalent) dissolved in DCM (150 mL) was slowly added to a solution of (Boc)2O (36.3 g, 166.3 mmol, 1.2 equivalent), DMAP (1.7 g, 13.9 mmol, 0.1 equivalent) was added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with DCM (300 mL), washed with brine (3 × 50 mL), dried with Na2SO4, and filtered. The mixture was concentrated under vacuum to obtain 2 m of compound (7.1 g, 24%) in the form of a colorless oil, which was subsequently used.

[0558] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0559] R f (1m of compound) = 0.2

[0560] R f (Compound 2m) = 0.4

[0561] LC-MS: Not detected.

[0562] General procedure for the preparation of compound 3m

[0563]

[0564] Oxalyl chloride (5.4 g, 42.4 mmol, 1.2 equivalents) dissolved in DCM (35 mL) was slowly added to a DMSO solution (7 mL, 1.0 V) dissolved in DCM (70 mL) under a nitrogen atmosphere at -78°C for 15 minutes, and stirred at the same temperature for 15 minutes. A solution of compound 2 m (7.1 g, 35.3 mmol, 1.0 equivalent) dissolved in DCM (35 mL) was slowly added at -78°C for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (10.7 g, 105.9 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (TLC analysis, PE / EA=4 / 1, R f ~0.4, identified as 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 with Na2SO4. The mixture was filtered and concentrated under vacuum below 30°C to obtain 3 m (7.4 g) of compound in the form of a yellow oil, which was subsequently used without purification.

[0565] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0566] R f (Compound 2m) = 0.2

[0567] Rf (Compound 3m) = 0.4

[0568] LC-MS: Not detected.

[0569] General procedure for the preparation of compound 5m

[0570]

[0571] Acetic acid (30 mL) was added to a solution of compound 3 m (7.8 g, 41.7 mmol, 1.8 equivalents) and compound 4a (6.0 g, 23.1 mmol, 1.0 equivalent) dissolved in DCE / DMF (1 / 1, 120 mL). The reaction mixture was stirred at room temperature for 2 hours. At this stage, NaBH3CN (5.8 g, 92.6 mmol, 4.0 equivalents) was added in a fraction at 0°C, and the mixture was stirred at room temperature for 18 hours. Once the reaction was complete, 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 with Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 3 / 1) to obtain compound 5 m (2.0 g, 19%) as a white solid.

[0572] TLC: EA

[0573] R f (Compound 3m) = 0.4

[0574] R f (Compound 5m) = 0.6

[0575] LC-MS: 445.35 [M+1] + , 443.30 [M-1] -

[0576] General procedure for the preparation of compound 6m

[0577]

[0578] Acetic acid (10 mL) was added to a solution of compound 5 m (2.0 g, 4.5 mmol, 1.0 equivalent) and compound 6 (2.6 g, 45 mmol, 10.0 equivalent) dissolved in DCE / DMF (1 / 1, 40 mL), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (1.1 g, 18 mmol, 4.0 equivalent) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. Once the reaction was complete (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 with Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / EA: 4 / 1) and P-HPLC (acetonitrile / H2O: 40%-60%) to obtain 6 m (0.87 g, 39%) of compound as a white solid.

[0579] TLC: EA = 100%

[0580] R f (Compound 5m) = 0.5

[0581] R f (Compound 6m) = 0.65

[0582] LC-MS: 487.40 [M+1] + , 485.40 [M-1] -

[0583] General procedure for the preparation of Compound 57

[0584]

[0585] HCl / EA (9 mL, 2 N, 18 mmol, 10.0 equivalents) was added to a solution of compound 6 m (870 mg, 1.8 mmol, 1.0 equivalent) dissolved in methanol (4 mL). The resulting solution was stirred at 30°C for 18 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by P-HPLC (acetonitrile / H2O: 30%-40%) to obtain compound 57 (144 mg, 30%) as a white solid.

[0586] LC-MS: 387.40 [M+1] + , 385.15 [M-1] -

[0587] 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).

[0588]

[0589] Hydrochloride form of Compound 58

[0590]

[0591]

[0592] HCl / EA (2.4 mL, 2 N, 4.8 mmol, 10.0 equivalents) was added to a solution of compound 61 (0.2 g, 0.5 mmol, 1.0 equivalent) dissolved in THF (2 mL), and the mixture was stirred at 30°C for 2 days. The reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 30%-40%) to obtain compound 58 (102 mg, 52%) as a white solid.

[0593] LC-MS: 402.30 [M+1] +, 400.20 [M-1] -

[0594] 1 H 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).

[0595]

[0596] Hydrochloride form of Compound 60

[0597]

[0598]

[0599] General procedure for the preparation of compound 2p

[0600]

[0601] Oxalyl chloride (2.9 g, 22.85 mmol, 1.2 equivalents) dissolved in DCM (10 mL) was slowly added to a DMSO solution (4.2 mL, 1.0 V) dissolved in DCM (20 mL) under a nitrogen atmosphere at -78°C for 15 minutes, and stirred at the same temperature for 15 minutes. A solution of compound 1p (4.2 g, 19.32 mmol, 1.0 equivalent) dissolved in DCM (10 mL) was slowly added at -78°C for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (5.8 g, 57.98 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (TLC analysis, PE / EA=4 / 1, R f~0.6, identified as 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 with Na2SO4, filtered, and concentrated under vacuum at 30°C or below to obtain compound 2p (4.1 g, crude product) in the form of a yellow oil, which was subsequently used without purification.

[0602] TLC: PE / EA = 2:1, KMnO4, ninhydrin

[0603] R f (Compound 1p) = 0.2

[0604] R f (Compound 2p) = 0.6

[0605] LC-MS: Not detected.

[0606] 1 H 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).

[0607] General procedure for the preparation of compound 5p

[0608]

[0609] Acetic acid (2.1 mL) and a molecular sieve (4.1 g) were added to a solution of compound 4a (2.07 g, 4.81 mmol, 1 equivalent) and compound 2p (4.1 g, 19.32 mmol, 4.0 equivalents) dissolved in DCE / DMF (20 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. NaBH3CN (1.51 g, 24.07 mmol, 5 equivalents) was added in portions, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Once the reaction was complete (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 with 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 5p (1.0 g, 34%) as a white solid.

[0610] TLC: EA = 100%

[0611] R f (Compound 4a) = 0.5

[0612] R f (Compound 5p) = 0.6

[0613] LC-MS: 630.60 [M+1] +

[0614] General procedure for the preparation of Compound 60

[0615]

[0616] HCl (1.6 mL, 2N, 3.2 mmol, 10 equivalents) was added to a solution of compound 5p (200 mg, 0.318 mmol, 1 equivalent) dissolved in methanol (1 mL). The resulting solution was stirred at room temperature for 24 hours. The reaction mixture was freeze-dried to obtain compound 60 (130 mg, 76%) as a white solid.

[0617] TLC: DCM / methanol = 10 / 1

[0618] R f (Compound 5p) = 0.6

[0619] R f (Compound 60) = 0.1

[0620] LC-MS: 430.40 [M+1] +

[0621] 1 H NMR (400 MHz, DO): 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).

[0622]

[0623] Compound 61

[0624]

[0625] General procedure for the preparation of compound 2q

[0626]

[0627] Concentrated sulfuric acid (0.58 mL) was added to a solution of compound 1q (5.0 g, 50 mmol, 1.0 equivalent) dissolved in methanol (100 mL), and the mixture was stirred at 90°C for 18 hours. The solution was cooled to 0°C, NaHCO3 (464 mg) was added, and the mixture was stirred for 10 minutes. The reaction mixture was filtered, and the solvent was removed under vacuum. The residue was diluted with DCM (50 mL), dried with Na2SO4, filtered, and concentrated to obtain compound 2q (5.9 g) in the form of a colorless oil, which was subsequently used without purification.

[0628] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0629] R f (Compound 1q) = 0.4

[0630] R f (Compound 2q) = 0.2

[0631] LC-MS: Not detected.

[0632] General procedure for the preparation of compound 3q

[0633]

[0634] Oxalyl chloride (7.6 g, 59.9 mmol, 1.2 equivalents) dissolved in DCM (33 mL) was slowly added to a DMSO (6.6 mL) solution dissolved in DCM (66 mL) under a nitrogen atmosphere at -78°C for 15 minutes, and stirred at the same temperature for 15 minutes. A solution of compound 2q (6.6 g, 49.9 mmol, 1.0 equivalent) dissolved in DCM (33 mL) was slowly added at -78°C for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (15.2 g, 149.8 mmol, 3.0 equivalents) was slowly added at -78°C. After the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (TLC analysis, PE / EA=4 / 1, R f ~0.4, identified as 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 with Na2SO4, filtered, and concentrated under vacuum at 30°C or below to obtain compound 3q (7.45 g, crude product) in the form of a yellow oil, which was subsequently used without purification.

[0635] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0636] R f (Compound 2q) = 0.2

[0637] R f (Compound 3q) = 0.4

[0638] LC-MS: Not detected.

[0639] 1 H 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).

[0640] General procedure for the preparation of Compound 61

[0641]

[0642] Acetic acid (15 mL) was added to a solution of compound 3q (2.5 g, 19.4 mmol, 1.8 equivalents) and compound 5 (3.2 g, 10.8 mmol, 1.0 equivalent) dissolved in DCE / DMF (60 mL, 1 / 1), and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. NaBH3CN (2.7 g, 43 mmol, 4.0 equivalents) was added in portions and stirred overnight at room temperature under a nitrogen atmosphere. Once the reaction was complete (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 with 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 61 (0.4 g, 13%) as a white solid.

[0643] TLC: EA = 100%

[0644] R f (Compound 5) = 0.5

[0645] R f (Compound 61) = 0.6

[0646] LC-MS: 416.35 [M+1] +

[0647] 1 H 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).

[0648]

[0649] Compound 63

[0650]

[0651] HATU (136 mg, 0.4 mmol, 1.2 equivalents) and DIEA (151 mg, 1.5 mmol, 5.0 equivalents) were added to a solution of compound 58 (120 mg, 0.3 mmol, 1.0 equivalent) dissolved in THF (2 mL). The resulting solution was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 30%-40%) to obtain compound 63 (63.4 mg, 50%) as a white solid.

[0652] LC-MS: 415.35 [M+1] + , 413.25 [M-1] -

[0653] 1H 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).

[0654]

[0655] Hydrochloride form of Compound 66

[0656]

[0657] General procedure for the preparation of Compound 2

[0658]

[0659] TEA (2.7 g, 26.3 mmol, 3.0 equivalents) and 9-fluorenylmethyl chloroformate (3.4 g, 13.2 mmol, 1.0 equivalents) were added to a solution of Compound 1 (0.5 g, 8.77 mmol, 1.0 equivalent) dissolved in DCM (10 mL). The mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. Upon 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 with Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain Compound 2 (0.4 g, 16%) as a white solid.

[0660] TLC: PE / EA = 1 / 1, ninhydrin

[0661] Rf (Compound 1) = 0.1

[0662] R f (Compound 2) = 0.8

[0663] 1 H 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).

[0664] General procedure for the preparation of Compound 3

[0665]

[0666] Acrolein (69 mg, 1.2 mmol, 1.1 equivalents) and HG in a solution of Compound 2 (0.3 g, 1.1 mmol, 1.0 equivalent) dissolved in DCM (10 mL). 2nd (34 mg, 0.05 mmol, 0.05 equivalents) was added. The mixture was stirred overnight at 50°C under a nitrogen atmosphere. When the reaction was complete (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 with 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.

[0667] TLC: PE / EA = 3 / 1

[0668] R f (Compound 2) = 0.4

[0669] R f (Compound 3) = 0.2

[0670] 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).

[0671] General procedure for the preparation of Compound 5

[0672]

[0673] Acetic acid (1.2 mL) and a molecular sieve (1.0 g) were added to a solution of Compound 4 (450 mg, 1.7 mmol, 1.0 equivalent) and Compound 3 (1.1 g, 3.5 mmol, 2.0 equivalent) dissolved in DCE / DMF (3 / 1, 8 mL). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere, and NaBH3CN (437 mg, 6.9 mmol, 4.0 equivalent) was added in portions. The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. Once the reaction was complete (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 with Na2SO4. The obtained 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.

[0674] TLC: EA = 100%

[0675] R f (Compound 4) = 0.4

[0676] R f (Compound 5) = 0.5

[0677] LCMS: [C 32 H 30 N4O5] + Calculated value for: 551, Detected value: 551.

[0678] General procedure for the preparation of Compound 6

[0679]

[0680] Butyraldehyde (71.8 mg, 1.0 mmol, 4.0 equivalents), acetic acid (1.0 mL), and a molecular sieve (1.0 g) were added to a solution of compound 5 (137 mg, 0.25 mmol, 1.0 equivalent) dissolved in DCE / DMF (3 / 1, 8 mL). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere, and NaBH3CN (62.6 mg, 1.0 mmol, 4.0 equivalents) was added in portions. The reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. Once the reaction was complete (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 with Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 1 / 1) to obtain compound 6 (41 mg, 27%) as a white solid.

[0681] TLC: EA = 100%

[0682] R f (Compound 5) = 0.5

[0683] R f (Compound 6) = 0.6

[0684] LCMS: [C 36 H 38 N4O5] + Calculated value for: 607, Detected value: 607.

[0685] General procedure for the preparation of Compound 66

[0686]

[0687] Piperidine (66.7 mg, 0.8 mmol, 8.0 equivalents) was added to a solution of compound 6 (47 mg, 0.1 mmol, 1.0 equivalent) dissolved in DCM (4.0 mL). 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.

[0688] TLC: DCM / methanol = 10 / 1

[0689] R f (Compound 6) = 0.8

[0690] R f (Compound 66) = 0.1

[0691] LCMS: [C 21 H 28 N4O3] + Calculated value for: 385, Detected value: 385.

[0692] 1 H NMR (400 MHz, DO): 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).

[0693]

[0694] Hydrochloride form of Compound 67

[0695]

[0696] General procedure for the preparation of Compound 2

[0697]

[0698] TEA (17.6 g, 174.3 mmol, 5.0 equivalents) and DMAP (0.4 g, 3.5 mmol, 0.1 equivalents) were added to a solution of Compound 1 (3.0 g, 34.8 mmol, 1.0 equivalent) dissolved in THF (60 mL) and tert-butylchlorodiphenylsilane (2.9 g, 10.5 mmol, 0.3 equivalents). The mixture was stirred at room temperature under a nitrogen atmosphere for 17 hours. Upon 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 with Na2SO4. The resulting 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%) in the form of a clear oil.

[0699] TLC: PE / EA = 5 / 1

[0700] R f (Compound 1) = 0.1

[0701] R f (Compound 2) = 0.3

[0702] 1 H 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).

[0703] General procedure for the preparation of Compound 3

[0704]

[0705] TEA (2.8 g, 27.8 mmol, 3.0 equivalents) and MsCl (1.59 g, 13.9 mmol, 1.5 equivalents) dissolved in DCM (35 mL) were added to a solution of Compound 2 (3.0 g, 9.3 mmol, 1.0 equivalent) dissolved in DCM (30 mL) under a nitrogen atmosphere at 0°C for 15 minutes. The mixture was stirred at the same temperature for 15 minutes. Subsequently, the reaction mixture was slowly raised to room temperature and stirred for 4 hours. Upon 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 with Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE / EA: 10 / 1) to obtain Compound 3 (2.9 g, 78%) in the form of a clear oil.

[0706] TLC: PE / EA = 2 / 1

[0707] R f (Compound 2) = 0.60

[0708] R f (Compound 3) = 0.63

[0709] LCMS: [C 21 H 26 O4SSi] + Calculated value for Na: 425, Detected value: 425.

[0710] 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).

[0711] General procedure for the preparation of Compound 4

[0712]

[0713] Compound 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. Once the reaction was complete (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.

[0714] TLC: DCM / EA = 1 / 1

[0715] R f (Compound 3) = 0.9

[0716] R f (Compound 4) = 0.1

[0717] LCMS: [C 20 H 25 NOSi] + Calculated value for: 324, Detected value: 324.

[0718] 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).

[0719] General procedure for the preparation of Compound 5

[0720]

[0721] Compound 4 (2.08 g, 6.4 mmol, 1 equivalent) was dissolved in DCM (20 mL), and Fmoc-Osu (2.6 g, 7.7 mmol, 1.2 equivalents) and TEA (1.94 g, 19.3 mmol, 3.0 equivalents) were added. The mixture was stirred at room temperature under a nitrogen atmosphere for 17 hours. Upon 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 with Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (PE / EA: 1 / 1) to obtain Compound 5 (2.6 g, 74%) as a white solid.

[0722] TLC: DCM / EA = 1 / 1

[0723] R f (Compound 4) = 0.1

[0724] R f (Compound 5) = 0.8

[0725] LCMS: [C 35 H 35 Calculated value for NO3Si]+Na: 568, Detected value: 568.

[0726] General procedure for the preparation of Compound 6

[0727]

[0728] TEA-3HF (7.7 g, 47.7 mmol, 10.0 equivalents) was added to Compound 5 (2.6 g, 4.8 mmol, 1.0 equivalent) dissolved in THF (1.0 mL). The resulting solution was stirred overnight at room temperature. Upon 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 with 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.

[0729] TLC: PE / EA = 2 / 1

[0730] R f (Compound 5) = 0.8

[0731] R f (Compound 6) = 0.2

[0732] 1 H 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).

[0733] General procedure for the preparation of Compound 7

[0734]

[0735] MnO2 (4.24 g, 49.0 mmol, 10.0 equivalents) was added to Compound 6 (1.5 g, 4.9 mmol, 1.0 equivalent) dissolved in DCM (20 mL). The mixture was stirred overnight at room temperature. The reaction mixture was filtered and concentrated to obtain Compound 7 (1.15 g, 77%) as a white solid. This was subsequently used without purification.

[0736] TLC: PE / EA = 2 / 1

[0737] R f (Compound 6) = 0.2

[0738] R f (Compound 7) = 0.5

[0739] 1 H 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).

[0740] General procedure for the preparation of Compound 9

[0741]

[0742] Acetic acid (1.5 mL) and a molecular sieve (1.76 g) were added to compound 8 (1.0 g, 3.8 mmol, 1.0 equivalent) and compound 7 (1.2 g, 3.8 mmol, 1.0 equivalent) dissolved in DCE / DMF (6 mL). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Then, NaBH3CN (1.0 g, 15.3 mmol, 4.0 equivalent) was added in portions, and the mixture was stirred for an additional 16 hours at room temperature. Once the reaction was complete (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 with Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 40%-60%) to obtain compound 9 (1.3 g, 63%) as a white solid.

[0743] TLC: EA = 100%

[0744] R f (Compound 8) = 0.4

[0745] R f (Compound 9) = 0.5

[0746] General procedure for the preparation of Compound 10

[0747]

[0748] Acetic acid (1.5 mL) and molecular sieve (1.8 g) were added to compound 9 (1.2 g, 2.2 mmol, 1.0 equivalent) and butyraldehyde (0.6 g, 8.7 mmol, 4.0 equivalent) dissolved in DCE / DMF (3 / 1, 4 mL). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Then, NaBH3CN (546 mg, 8.7 mmol, 4.0 equivalent) was added in portions, and the mixture was stirred for an additional 16 hours at room temperature. Once the reaction was complete (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 with Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by P-HPLC (acetonitrile / H2O: 40%-60%) to obtain compound 10 (315 mg, 24%) as a white solid.

[0749] TLC: EA = 100%

[0750] R f (Compound 9) = 0.5

[0751] R f (Compound 10) = 0.6

[0752] LCMS: [C 36 H 36 N4O5] + Calculated value for: 605, Detected value: 605.

[0753] General procedure for the preparation of Compound 11

[0754]

[0755] Lindler catalyst (60 mg, 10%) and quinoline (12.5 mg, 0.1 mmol, 0.1 equivalent) were added to compound 10 (587 mg, 1.0 mmol, 1.0 equivalent) dissolved in EtOH (5 mL). The mixture was stirred at room temperature for 30 minutes under an N2 / H2 (50%) atmosphere. The reaction mixture was filtered, concentrated under reduced pressure, and purified by P-HPLC (acetonitrile / H2O: 40%-60%) to obtain compound 11 (100 mg, 17%) as a white solid.

[0756] TLC: DCM / EA = 1 / 1

[0757] R f (Compound 10) = 0.7

[0758] R f (Compound 11) = 0.72

[0759] LCMS: [C 36 H 38 N4O5] + Calculated value for: 607, Detected value: 607.

[0760] General procedure for the preparation of Compound 67

[0761]

[0762] Piperidine (280 mg, 3.3 mmol, 20.0 equivalents) was added to Compound 11 (100 mg, 0.17 mmol, 1.0 equivalent) dissolved in DCM (5.0 mL). The mixture was stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 20%-45%) to obtain Compound 67 (14.2 mg, 19%) as a white solid.

[0763] TLC: DCM / methanol = 10 / 1

[0764] R f (Compound 11) = 0.9

[0765] R f (Compound 67) = 0.1

[0766] LCMS: [C 21 H 28 N4O3] + Calculated value for: 385, Detected value: 385.

[0767] 1 H NMR (400 MHz, DO): 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).

[0768]

[0769] Hydrochloride form of compound 68

[0770]

[0771] General procedure for the preparation of Compound 2

[0772]

[0773] HCl / EA (17.6 mL, 3 N, 52.8 mmol, 10.0 equivalents) was added to compound 1 (1.0 g, 5.28 mmol, 1.0 equivalent) dissolved in EA (3 mL), and the mixture was stirred at room temperature for 3 hours under a nitrogen atmosphere. When the reaction was complete (confirmed by TLC analysis), the solution was concentrated under vacuum to obtain compound 2 (1.2 g, crude product) in the form of a yellow oil, which was subsequently used without purification.

[0774] TLC: PE / EA = 1 / 1 KMnO4

[0775] R f (Compound 1) = 0.4

[0776] R f (Compound 2) = 0.1

[0777] General procedure for the preparation of Compound 3

[0778]

[0779] Fmoc-Osu (1.7 g, 5.1 mmol, 1.5 equivalents) and TEA (340 mg, 3.4 mmol, 1.0 equivalents) were added to Compound 2 (0.3 g, 3.4 mmol, 1.0 equivalent) dissolved in THF / H2O (3 / 1, 8 mL). The mixture was stirred overnight at room temperature. Upon 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 with Na2SO4. The resulting mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE / EA: 5 / 1) to obtain Compound 3 (160 mg, 21%) as a white solid.

[0780] TLC: PE / EA = 2 / 1

[0781] R f (Compound 2) = 0.1

[0782] R f (Compound 3) = 0.7

[0783] 1 H 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).

[0784] General procedure for the preparation of Compound 4

[0785]

[0786] A solution of DMSO (0.4 mL, 1.0 V) dissolved in DCM (2 mL) was slowly added to a solution of oxalyl chloride (200 mg, 1.6 mmol, 1.2 equivalents) dissolved in DCM (3 mL) over 15 minutes at -78°C under a nitrogen atmosphere, and stirred for 15 minutes at the same temperature. Subsequently, Compound 3 (410 mg, 1.32 mmol, 1.0 equivalent) dissolved in DCM (3 mL) was slowly added to the previously prepared solution over 15 minutes at -78°C. The mixture was stirred at -78°C for 30 minutes, and TEA (400 mg, 3.96 mmol, 3.0 equivalents) was added at -78°C. Once the addition was complete, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. When the reaction was complete (confirmed by TLC analysis), the reaction mixture was diluted with DCM (20 mL) and quenched with a 10% aqueous citric acid solution (10 mL). The resulting solution was washed with water (20 mL) and brine (20 mL) and dried with Na2SO4. The solvent was removed under vacuum at 30°C or below to obtain compound 4 (640 mg, crude product) in the form of a yellow oil, which was used immediately without further purification.

[0787] TLC: PE / EA = 4:1, KMnO4, ninhydrin

[0788] R f (Compound 3) = 0.4

[0789] R f (Compound 4) = 0.6

[0790] General procedure for the preparation of Compound 6

[0791]

[0792] Acetic acid (1.5 mL) and a molecular sieve (1.0 g) were added to a solution of Compound 5 (410 mg, 1.6 mmol, 1.0 equivalent) and Compound 4 (979 mg, 3.2 mmol, 2.0 equivalent) dissolved in DCM (10 mL). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Then, NaBH3CN (398 mg, 6.4 mmol, 4.0 equivalent) was added in a fraction, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. Once the reaction was complete (confirmed by TLC analysis), the reaction mixture was diluted with DCM (20 mL). The mixture was washed with water (30 mL × 3) and brine (30 mL) and dried with 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.

[0793] TLC: EA = 100%

[0794] R f (Compound 5) = 0.4

[0795] R f (Compound 6) = 0.5

[0796] General procedure for the preparation of Compound 7

[0797]

[0798] A solution of crotonaldehyde (87.6 mg, 1.3 mmol, 1.1 equivalents) in H2SO4 (3.11 mL, 4 N, 12.4 mmol, 10.7 equivalents) was slowly added at 0°C to a solution of Compound 6 (642 mg, 1.2 mmol, 1.0 equivalents) dissolved in THF (3.0 mL). Then, NaBH4 (26.8 mg, 0.7 mmol, 0.6 equivalents) was added at 0°C and stirred for 40 minutes at the same temperature. The reaction mixture was poured into ice water and extracted with DCM (30 mL × 3). 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 obtain Compound 7 (120 mg, 17%) as a white solid.

[0799] TLC: EA = 100%

[0800] R f (Compound 6) = 0.5

[0801] R f (Compound 7) = 0.6

[0802] LCMS: [C 36 H 38 N4O5] + Calculated value for: 607, Detected value: 607.

[0803] General procedure for the preparation of Compound 68

[0804]

[0805] Piperidine (171 mg, 2.0 mmol, 10.0 equivalents) was added to a solution of Compound 7 (120 mg, 0.2 mmol, 1.0 equivalent) dissolved in DCM (4.0 mL). The mixture was stirred at room temperature for 17 hours. It was concentrated under vacuum, and the residue was purified by P-HPLC (acetonitrile / H2O: 20%-40%) to obtain Compound 68 (42.5 mg, 46%) as a white solid.

[0806] TLC: DCM / methanol = 10 / 1

[0807] R f (Compound 7) = 0.5

[0808] R f (Compound 68) = 0.1

[0809] LCMS: [C 21 H 28 N4O3] + Calculated value for: 385, Detected value: 385.

[0810] 1 H NMR (400 MHz, DO): 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).

[0811]

[0812] Hydrochloride form of compound 69

[0813]

[0814] General procedure for the preparation of Compound 2

[0815]

[0816] MnO2 (7.7 g, 88.5 mmol, 10.0 equivalents) and a molecular sieve (3.1 g) were added to a solution of compound 1 (620 mg, 8.8 mmol, 1.0 equivalent) dissolved in DCM (30 mL), and the mixture was stirred at room temperature for 17 hours. When the reaction was complete (confirmed by TLC analysis), the reaction mixture was filtered and concentrated under reduced pressure at 30°C or below to obtain compound 2 (110 mg, 17%) in the form of a clear oil, which was used immediately without further purification.

[0817] TLC: PE / EA = 3 / 1 KMnO4

[0818] R f (Compound 1) = 0.3

[0819] R f (Compound 2) = 0.3

[0820] 1 H NMR (400 MHz, CDCl3): δ 9.13 (s, 1H), 2.05 (s, 3H).

[0821] General procedure for the preparation of Compound 4

[0822]

[0823] Acetic acid (2.0 mL) and a molecular sieve (4.0 g) were added to a solution of Compound 3 (2.0 g, 4.6 mmol, 1.0 equivalent) and Compound 2 (1.62 g, 18.4 mmol, 4.0 equivalent) dissolved in THF (3.0 mL). The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Subsequently, NaBH3CN (1.2 g, 18.4 mmol, 4.0 equivalent) was added in portions, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. Once the reaction was complete (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 with Na2SO4. The obtained mixture was concentrated under reduced pressure and purified by P-HPLC (acetonitrile / H2O: 40%-60%) to obtain compound 4 (110 mg, 5%) as a white solid.

[0824] TLC: EA = 100%

[0825] R f (Compound 3) = 0.4

[0826] R f (Compound 4) = 0.5

[0827] LCMS: [C 26 H 34 N4O5] + Calculated value for: 483, Detected value: 483.

[0828] General procedure for the preparation of Compound 5

[0829]

[0830] Lindla catalyst (10 mg, 10%) and quinoline (1.8 mg, 0.02 mmol, 0.1 equivalent) were added to a solution of compound 4 (100 mg, 0.2 mmol, 1.0 equivalent) dissolved in ethanol (5 mL). The mixture was stirred at room temperature for 30 minutes, filtered, and concentrated under reduced pressure. The residue was purified by P-HPLC (acetonitrile / H2O: 40%-60%) to obtain compound 5 (20 mg, 20%) as a white solid.

[0831] TLC: DCM / methanol = 1 / 1

[0832] R f (Compound 4) = 0.6

[0833] R f (Compound 5) = 0.62

[0834] LCMS: [C 26 H 36 N4O5] + Calculated value for: 485, Detected value: 485.

[0835] General procedure for the preparation of Compound 69

[0836]

[0837] HCl (0.4 mL, 2 N, 0.8 mmol, 11.0 equivalents) was added to a solution of compound 5 (33 mg, 0.07 mmol, 1.0 equivalent) dissolved in MeOH (1 mL). The mixture was stirred at room temperature for 24 hours and concentrated under reduced pressure. The resulting residue was purified with Biotage (C18, acetonitrile / H2O: 30%-60%) to obtain compound 69 (17.8 mg, 57%) as a white solid.

[0838] TLC: DCM / methanol = 10 / 1

[0839] R f (Compound 5) = 0.5

[0840] R f (Compound 69) = 0.1

[0841] LCMS: [C21 H 28 N4O3] + Calculated value for: 385, Detected value: 385.

[0842] 1 H NMR (400 MHz, DO): 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).

[0843]

[0844] Hydrochloride form of Compound 80

[0845] 3-(4-((4-(methylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione.Hydrochloride

[0846]

[0847] Step-1: tert-butyl methyl(4-oxobutyl)carbamate

[0848]

[0849] DMSO (0.419 mL, 5.90 mmol, 2.4 equivalents) was slowly added to a solution of oxalyl chloride (0.25 mL, 2.95 mmol, 1.2 equivalents) dissolved in DCM (5 mL) under a nitrogen atmosphere at -78°C for 5 minutes and stirred for 15 minutes at the same temperature. To this, a solution of tert-butyl (4-hydroxybutyl)(methyl)carbamate 1 (500 mg, 5.90 mmol, 1 equivalent) dissolved in DCM (2.5 mL) was slowly added for 5 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and triethylamine (1.713 mL, 12.30 mmol, 5 equivalents) was slowly added at -78°C. After addition, the reaction mixture was slowly raised to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 50% ethyl acetate / petroleum ether, R ­f ~ 0.4, confirmed by KMnO4), the reaction mixture was diluted with DCM (20 mL), washed with 10% aqueous citric acid solution (20 mL), water (1 × 10 mL), and brine (1 × 10 mL), dried with 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 subsequently used without purification. .

[0850] 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).

[0851] Step-2: tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)(methyl)carbamate

[0852]

[0853] A mixture of tert-butyl methyl(4-oxobutyl)carbamate (279 mg, 1.389 mmol, 1.2 equivalents) and lenalidomide 3 (300 mg, 1.157 mmol, 1 equivalent) was dissolved in a mixture of 1,2-dichloroethane (3 mL, 10-fold) and DMF (3 mL, 10-fold) under a nitrogen atmosphere. To this solution, TFA (0.08 mL, 1.041 mmol, 0.9 equivalents) and Na(OAc)3BH (368 mg, 1.736 mmol, 1.5 equivalents) were added in a fractional manner at 0°C, and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc, R ­f (approximately 0.4 and confirmed by UPLC), the reaction mixture was diluted with DCM (20 mL), washed with water (3 × 20 mL) and brine (20 mL), dried with Na2SO4, filtered, and concentrated to obtain the crude product. This was purified using Isolera (Biotage R snap cartridge, KP-Sil, 25 g, 230-400 mesh silica gel) with 80-85% ethyl acetate in petroleum ether. The pure fraction was collected and concentrated under vacuum to obtain 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 a milky white solid.

[0854] 1H-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).

[0855] LCMS:345.1 (M-Boc), Rt (min): 2.267, Area %: 96.498.

[0856] Step-3: tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)(methyl)carbamate

[0857]

[0858] A 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 equivalent) and fentanal (339 mg, 3.94 mmol, 5 equivalents) 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 equivalents) and STAB (250 mg, 1.181 mmol, 1.5 equivalents) were added in a fraction at 0°C, and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc, R ­f~ 0.6, and confirmed by UPLC), the reaction mixture was diluted with DCM (20 mL), washed with water (3 × 20 mL) and brine (20 mL), dried with anhydrous Na2SO4, filtered, and concentrated to obtain the crude product. The crude product was purified with Isolera (Biotage R snap cartridge, KP-Sil, 25 g, 230-400 mesh silica gel) using 70-80% ethyl acetate in petroleum ether. The pure fraction was 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.

[0859] 1 H-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).

[0860] LCMS: 515.4 (M+H), Rt (min): 1.989, Area %: 95.589.

[0861] Step-4: 3-(4-((4-(methylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0862]

[0863] HCl (4M solution in EtOAc, 0.7 mL) was added to a near-freezing tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate (130 mg, 0.240 mmol, 1 equivalent) suspension dissolved in DCM (1.5 mL), and the mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, and the residue was ground with MTBE (10 mL). The resulting solid was freeze-dried to obtain 3-(4-((4-(methylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione.hydrocholide 80 (70 mg, 63.2%) as a pale yellow solid.

[0864] 1 H-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).

[0865] LCMS: 415.2 (M+H), Method: Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: ACN, Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm, Flow rate: 1.5 ml / min Rt (min): 1.599, Area %: 95.709.

[0866] HPLC: Method: Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm Flow rate: 2.0 mL / min, Rt (min): 2.409, Area %: 97.597.

[0867]

[0868] Formate form of Compound 81

[0869] 3-(4-((4-(dimethylamino)butyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione formic acid salt

[0870]

[0871] AcOH (0.129 mL, 2.247 mmol, 3 equivalents) was added to a solution of 3-(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) dissolved in DCM (6 mL, 20 volumes), 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 portions, and the mixture was stirred at room temperature for an additional 40 hours. When the reaction was complete (TLC analysis, EtOAc, R ­f(confirmed as ~0.4), the reaction mixture was diluted with DCM (10 mL), washed with water (2 × 20 mL) and brine (20 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by preparative HPLC (HCOOH:ACN method) and freeze-dried to obtain 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.

[0872] 1 H-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, 3 H).

[0873] 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 (50X4.6 mm) 3.5 μm. Rt (min): 1.824; Area%: 97.584.

[0874] 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 x 4.6) mm, 3.5 μm. Rt (min): 3.658; Area %: 97.694.

[0875]

[0876] Compound 82

[0877] N-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)acetamide

[0878]

[0879] Acetic anhydride (0.236 mL, 2.497 mmol, 5 equivalents) was slowly added to a solution of 3-(4-((4-aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 47 (200 mg, 0.499 mmol) dissolved in pyridine (3 mL), and the reaction mixture was stirred at room temperature for 13 hours. When the reaction was complete (TLC analysis, 10% MeOH in DCM, R ­f Approximately 0.8 (confirmed by LCMS), the reaction mixture was concentrated under vacuum, diluted with ethyl acetate (10 mL), washed with water (2 × 20 mL) and brine (20 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 3-4% MeOH in DCM (Biotage R snap cartridge, KP-Sil, 25 g, 230-400 silica gel) and freeze-dried to obtain N-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)acetamide 82 (120 mg, 0.267 mmol, 53.5% yield) as a milky white solid.

[0880] 1 H-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).

[0881] 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 Х 4.6 mm) 3.5 μm. Rt (min): 1.414; Area%: 98.439.

[0882] 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 Х 4.6) mm, 3.5 μm. Rt (min): 2.445; Area%: 98.636.

[0883]

[0884] Hydrochloride form of Compound 87

[0885] 3-(4-(((1S,4S)-4-aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0886]

[0887] Step-1: tert-butyl ((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate

[0888]

[0889] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (0.5 g, 1.547 mmol, 1 equivalent), tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate 2 (0.995 g, 4.64 mmol, 3 equivalents), and NaOt-Bu (0.446 g, 4.64 mmol, 3 equivalents) were dissolved in DMF (2 mL) and degassed under nitrogen for 20 minutes. Under a nitrogen atmosphere, Pd-PEPPSI-IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equivalents) was added, and the reaction was heated at 110°C for 4 hours. Upon completion of the reaction (TLC analysis, 100% ethyl acetate, R ­f The reaction mixture was filtered onto a Celite bed, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse-phase column chromatography (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain 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.

[0890] LCMS: 401.1 (M-Boc). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.110; Area% - 94.51.

[0891] 1 H-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).

[0892] Step-2: tert-butyl ((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamate

[0893]

[0894] TFA (0.017 mL, 0.217 mmol, 0.9 equivalents) was added to 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 equivalent) and pentanal 4 (0.128 mL, 1.205 mmol, 5 equivalents) dissolved in DCM (4 mL) and DMF (4 mL) at 0°C, followed by the addition of sodium triacetoxyborohydride (77.0 mg, 0.361 mmol, 1.5 equivalents). The reaction mixture was raised to room temperature and stirred at the same temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% ethyl acetate R ­f ~0.5 and confirmed by LCMS), the reaction mixture was diluted with near-freezing water (15 mL), extracted with ethyl acetate (2 × 15 mL), washed with brine (25 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue 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). The pure fraction from the column was freeze-dried to obtain 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.

[0895] LCMS: 527.3 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.644; Area% - 99.9%.

[0896] 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).

[0897] Step-3: 3-(4-(((1S,4S)-4-aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0898]

[0899] HCl (4 M solution of EtOAc, 4 mL) was added to 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 equivalent) dissolved in DCM (4 mL) near freezing point, and the mixture was stirred at room temperature for 1.5 hours. The progress of the reaction was monitored using LCMS. The reaction mixture was concentrated, washed with MTBE (5 mL × 2), and freeze-dried to obtain 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 a milky white solid.

[0900] LCMS: 427.2 (M-HCl). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.634; Area% - 85.00.

[0901] HPLC: 97.53%, Rt (min): 9.103. Method: Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Flow rate: 1.0 mL / min.

[0902] 1 H-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).

[0903]

[0904] Hydrochloride form of compound 88

[0905] 3-(4-(((1R,4R)-4-aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0906]

[0907] Step-1: tert-butyl ((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate

[0908]

[0909] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (0.5 g, 1.547 mmol, 1 equivalent), tert-butyl ((1R,4R)-4-aminocyclohexyl)carbamate 2 (0.995 g, 4.64 mmol, 3 equivalents), and NaOt-Bu (0.446 g, 4.64 mmol, 3 equivalents) were dissolved in DMF (2 mL) and degassed under nitrogen for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equivalents) was added under a nitrogen atmosphere and reacted at 110 °C for 4 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc R ­f ~ 0.4 and confirmed by 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 (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain 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.

[0910] LCMS: 401.1 (M-Boc). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.188; Area% - 98.18.

[0911] 1 H-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).

[0912] Step-2: tert-butyl ((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)carbamate

[0913]

[0914] TFA (0.012 mL, 0.158 mmol, 1.8 equivalents) and sodium triacetoxyborohydride (56 mg, 0.262 mmol, 3 equivalents) were added to a stirred solution of 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 equivalent) and pentanal (0.076 g, 0.876 mmol, 10 equivalents) dissolved in DCM (1.5 mL) and DMF (1.5 mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc R ­f ~ 0.5 and confirmed by LCMS), the reaction mixture was diluted with near-freezing water (15 mL), extracted with ethyl acetate (2 × 15 mL), washed with brine (25 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue 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 freeze-dried to obtain 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.

[0915] LCMS: 527.3 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.876; Area% - 99.04.

[0916] Step-3: 3-(4-(((1R,4R)-4-aminocyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0917]

[0918] HCl (4M solution dissolved in EtOAc, 0.5 mL) was added to a near-freezing solution of 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, 1 equivalent) dissolved in DCM (1 mL), and the mixture was stirred at room temperature for 3 hours. The reaction progress was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and freeze-dried 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 a milky white solid.

[0919] LCMS: 427.2 (M-HCl). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 1.579; Area% - 98.76.

[0920] HPLC: 99.10%, Rt (min): 2.381. Method: Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Flow rate: 2.0 mL / min.

[0921] 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).

[0922]

[0923] 화합물 89의 염산염 형태

[0924] 3-(4-(((1S,4S)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0925]

[0926] 단계-1: tert-butyl (((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate

[0927]

[0928] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (1.0 g, 3.09 mmol, 1 equivalent), tert-butyl (((1s,4s)-4-aminocyclohexyl)methyl)carbamate 2 (1.060 g, 4.64 mmol, 1.5 equivalents), and NaOt-Bu (0.892 g, 9.28 mmol, 3 equivalents) were dissolved in DMF (5 mL) and degassed under nitrogen for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.151 g, 0.155 mmol, 0.05 equivalents) was added under a nitrogen atmosphere, and the reaction mixture was heated at 110°C for 4 hours. Completion of the reaction was determined by TLC analysis (100% EtOAc, R ­f ~ 0.4) and confirmed by LCMS. The reaction mixture was filtered onto a Celite bed, washed with THF (10 mL), concentrated to a minimum volume (3 mL), and purified by reverse-phase column chromatography (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain 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.

[0929] LCMS: 415.1 (M-Boc). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.325; Area% - 89.09.

[0930] Step-2: tert-butyl (((1s,4s)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamate

[0931]

[0932] TFA (0.012 mL, 0.153 mmol, 0.9 equivalents) was added 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 equivalent) and fentanal 4 (73.2 mg, 0.850 mmol, 5 equivalents) dissolved in DCM (2 mL) and DMF (2 mL) at 0°C, and sodium triacetoxyborohydride (54 mg, 0.255 mmol, 1.5 equivalents) was added. The reaction mixture was raised to room temperature and stirred at the same temperature for 16 hours. After the reaction was complete (TLC analysis, 100% EtOAc R ­f ~ 0.5, and confirmed by LCMS), the reaction mixture was diluted with near-freezing water (15 mL) and extracted with ethyl acetate (2 x 15 mL). The organic layer was washed with brine (25 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue 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 freeze-dried to obtain 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 a milky white solid.

[0933] LCMS: 541.3 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.648; Area% - 93.13.

[0934] Step-3: 3-(4-(((1S,4S)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0935]

[0936] HCl (4M solution in ethyl acetate, 3 mL) was added to a near-freezing solution of tert-butyl (((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamate 5 (35 mg, 0.065 mmol, 1 equivalent) dissolved in DCM (1 mL), and the mixture was stirred at room temperature for 1.5 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and freeze-dried to obtain 3-(4-(((1S,4S)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 89 (22.5 mg, 0.045 mmol, 69% yield) as a milky white solid.

[0937] LCMS: 441.3 (M-HCl). Methods: Column: Atlantis dC18 (50 Х 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.

[0938] HPLC: 94.36 %, Rt (min): 8.083. Method: Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Flow rate: 1.0 mL / min.

[0939] 1 H-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).

[0940]

[0941] Hydrochloride form of Compound 90

[0942] 3-(4-(((1R,4R)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0943]

[0944] Step-1: tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate

[0945]

[0946] Tert-butyl (((1r,4r)-4-aminocyclohexyl)methyl)carbamate 2 (1.060 g, 4.64 mmol, 3 equivalents) and sodium tert-butoxide (0.446 g, 4.64 mmol, 3 equivalents) were added at room temperature to a solution of 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (0.500 g, 1.547 mmol, 1 equivalent) dissolved in DMF (2 mL), and degassed with nitrogen gas for 15 minutes. Pd-PEPPSI-Ihept(Cl) (0.075 g, 0.077 mmol, 0.05 equivalents) was added to this reaction mixture under nitrogen conditions, degassed for an additional 5 minutes, and stirred at 110°C for 4 hours. Upon 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 was purified by reverse-phase chromatography (Grace® column: C18 40 μm, 120 g, product eluted with 44% ACN in 0.1% HCOOH). The pure fraction was freeze-dried 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), a yellow solid.

[0947] LCMS: 415.2 (M-56). Method: Column: Xbridge C8 (50 Х 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% - 55.37 %.

[0948] Step-2: tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamate

[0949]

[0950] TFA (0.033 mL, 0.425 mmol, 2.0 equivalents) was added to 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 equivalent) and pentanal 4 (0.183 g, 2.125 mmol, 10 equivalents) dissolved in DCE (1.5 mL) and DMF (1.5 mL), and the mixture was stirred at 0°C for 10 minutes. Sodium triacetoxyborohydride (0.135 g, 0.638 mmol, 3.0 equivalents) was added to this mixture in portions at 0°C, and the resulting colorless, cloudy mixture was stirred at room temperature for 16 hours (monitored by UPLC). The reaction mixture was quenched with near-freezing water (5 mL) and extracted with dichloromethane (10 mL × 3). The bound organic layer was dried with Na2SO4 and concentrated under vacuum to obtain a crude product in the form of a pale yellow liquid. The crude product was purified by reverse-phase chromatography (elution with 50% ACN in 0.1% HCOOH) to obtain tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4yl)(pentyl)amino)cyclohexyl)methyl)carbamate (0.020 g, 0.037 mmol, 17.18% yield) as a white solid.

[0951] 1H-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).

[0952] LCMS: 541.4 (M+H). Methods: Column: XBridge C8 (50 Х 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 %.

[0953] HPLC: Method: Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm Mobile phase: A: 0.1% TFA in water Mobile phase: B: ACN, Flow rate: 2.0 mL / min, Area% - 98.72 %.

[0954] Step-3: 3-(4-(((1R,4R)-4-(aminomethyl)cyclohexyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[0955]

[0956] HCl (4M solution in dioxane, 0.5 mL) was added at 0°C to a stirred solution of tert-butyl (((1r,4r)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)cyclohexyl)methyl)carbamate 5 (20 mg, 0.037 mmol, 1.0 equivalent) dissolved in dichloromethane (1 mL), and the mixture was stirred at room temperature for 2 hours. The reaction proceeded via TLC (10% MeOH; DCM, R ­f It was confirmed to be ~ 0.0). The reaction mixture was concentrated under vacuum to below 40°C to obtain a milky white solid. The obtained solid was dissolved in water (5 mL), washed with MTBE (2 × 5 mL), and the aqueous layer was freeze-dried to obtain 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 a milky white solid.

[0957] 1 H-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).

[0958] LCMS: 441.3 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: ACN, Flow rate: 1.5 ml / min, Rt- 1.526 min, Area: 97.05 %.

[0959] HPLC: Method: Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Flow rate: 2.0 mL / min, Rt- 2.287 min, Area: 99.27 %.

[0960]

[0961] Hydrochloride form of Compound 91

[0962]

[0963] Step-1: tert-butyl ((1s,4s)-4-formylcyclohexyl)carbamate

[0964]

[0965] To a cold solution of tert-butyl ((1S,4S)-4-(hydroxymethyl)cyclohexyl)carbamate (200 mg, 0.872 mmol, 1 equivalent) dissolved in DCM (5 mL), a solution of DMSO (1.5 mL), DIPEA (438 mg, 3.39 mmol, 3.89 equivalents), and pyridine sulfur trioxide (540 mg, 3.39 mmol, 3.89 equivalents) dissolved in DMSO (1.5 mL) was slowly added, and the mixture was stirred at 0°C for 1 hour. When the reaction was complete, (TLC (40% ethyl acetate in petroleum ether, R ­f(confirmed as : 0.4), the reaction was quenched with 1.5N HCl (20 mL) and extracted with DCM (2 × 20 mL). The bound organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure to obtain tert-butyl ((1S,4S)-4-formylcyclohexyl)carbamate 2A (200 mg, 0.880 mmol, 101% yield) as a colorless liquid crude product.

[0966] Step-2: tert-butyl((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate

[0967]

[0968] TFA (79 mg, 0.694 mmol, 0.9 equivalents) and Na(OAc)3BH (245 mg, 1.157 mmol, 1.5 equivalents) were added at 0°C to a solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (200 mg, 0.771 mmol, 1 equivalent) and tert-butyl ((1S,4S)-4-formylcyclohexyl)carbamate 2A (210 mg, 0.926 mmol, 5 equivalents) dissolved in a mixture of DCE (2 mL) and DMF (0.5 mL), and the mixture was stirred at room temperature for 12 hours. When the reaction was complete (confirmed by UPLC), the reaction mixture was quenched with ice water (25 mL) and extracted with DCM (2 × 10 mL). The combined organic layer was concentrated under reduced pressure to obtain a crude product, which was purified by reverse-phase chromatography (Grace® column: standard C18, 40 g snap, 0.1% formic acid / ACN, 20 mL / min, 70-75% ACN in H2O) to obtain 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 a milky white solid.

[0969] 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).

[0970] LCMS: 470.0(M+H), Rt (min): 2.306, 면적 %: 99.775.

[0971] HPLC: Rt (min): 4.229, 면적 %: 99.789.

[0972] 단계-3: tert-butyl ((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamate

[0973]

[0974] Pentanal (82 mg, 0.956 mmol, 5 equivalents), TFA (19.63 mg, 0.172 mmol, 0.9 equivalents), and Na(OAc)3BH (48.6 mg, 0.230 mmol, 1.5 equivalents) were added at 0°C to a solution of tert-butyl ((1s,4s)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate (90 mg, 0.191 mmol, 1 equivalent) dissolved in DCE (5 mL) and DMF (0.5 mL), and stirred at room temperature for 12 hours. When the reaction was complete (confirmed by LCMS), the reaction was quenched with ice water (25 mL) and extracted with DCM (10 mL × 2). The combined organic layer was concentrated under vacuum to obtain the crude product, which was purified using Isolera (column size: Biotage R snap cartridge, KP-Sil, 10 g, silica gel - 230-400 mesh) with 70-80% ethyl acetate in petroleum ether. The pure fractions were collected individually and concentrated under vacuum to obtain 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, 29.0% yield) as a white solid.

[0975] 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 (J = 16. Hz), J6. Hz, 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, J m. = 1.16. 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 = 1, 7.20 (3H), 1.24 (t, J = 10.00 Hz, 5H), 0.84 (t, J = 6.80 Hz, 3H).

[0976] LCMS: 485.9 (M-56), Rt (min): 2,828, 면적 %: 99,796.

[0977] HPLC: Rt (min): 4,910, 면적 %: 98,887.

[0978] 단계-4: 3-(4-((((1s,4s)-4-aminocyclohexyl)methyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl

[0979]

[0980] HCl (4 M in ethyl acetate, 0.5 mL, 16.46 mmol) was added at 0°C to a near-freezing 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) dissolved in DCM (2 mL), and stirred at room temperature for 6 hours. When the reaction was complete (confirmed by LCMS), the reaction mixture was concentrated and the crude product obtained by washing with MTBE (10 mL) was freeze-dried to obtain 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 a milky white solid.

[0981] 1 H-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).

[0982] LCMS: 442.0 (M+H), Method: Mobile phase: A: 0.1% formic acid in H2O, B: ACN, Column: Atlantis dC18 (50 Х 4.6) 5 μ, Flow rate: 1.5 ml / min; Rt (min): 1.691, Area %: 99.845.

[0983] HPLC: Method: Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: ACN, Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm, Flow rate: 1.5 ml / min, Rt (min): 2.839, Area %: 98.026.

[0984]

[0985] Hydrochloride form of Compound 92

[0986]

[0987] Step-1: tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate

[0988]

[0989] To a cooled solution of tert-butyl ((1r,4r)-4-(hydroxymethyl)cyclohexyl)carbamate (500 mg, 2.180 mmol, 1 equivalent) dissolved in DCM (11 mL), DMSO (3.7 mL), a solution of DIPEA (0.388 mL, 2.180 mmol, 3.89 equivalents), and pyridine sulfur trioxide (347 mg, 2.180 mmol, 3.89 equivalents) dissolved in DMSO (3.7 mL) was slowly added, and the mixture was stirred at 0°C for 1 hour. Upon completion of the reaction (TLC analysis (40% ethyl acetate in petroleum ether, R ­f (confirmed as : 0.4), the reaction was quenched with 1.5N HCl (20 mL) and extracted with DCM (20 mL × 2). The bound organic layer was dried with anhydrous Na2SO4 and concentrated under vacuum to obtain tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (480 mg, 2.112 mmol, 97% yield) as a yellow liquid.

[0990] 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).

[0991] LCMS: 172.4 (M-56), Rt (min): 2.593, 면적 %: 59.513.

[0992] 단계-2: tert-butyl((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)methyl)cyclohexyl)carbamate

[0993]

[0994] TFA (139 mg, 1.215 mmol, 0.9 equivalents) and Na(OAc)3BH (429 mg, 2.025 mmol, 1.5 equivalents) were added at 0°C to a solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (350 mg, 1.350 mmol, 1 equivalent) and tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (368 mg, 1.620 mmol, 5 equivalents) dissolved in a mixture of DCE (1.5 mL) and DMF (0.5 mL), and the mixture was stirred at room temperature for 12 hours. When the reaction was complete (confirmed by TLC), the reaction was quenched with ice water (25 mL) and extracted with DCM (10 mL × 2). The bound organic layer was concentrated under vacuum to obtain a crude product, which was purified by reverse-phase chromatography (Grace®, column: generic C18, 40 g snap, ACN containing 0.1% formic acid, 20 mL / min, 70-75% ACN in H2O). The pure fraction was freeze-dried to obtain 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.

[0995] 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 = 8.60 (2 H6, Hz), J = 2.5 Hz, 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, 2. H-98), 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 = 1.7 Hz (2H), s 1.12 (q, J = 11.60 Hz, 3H), 0.99 (t, J = 12.00 Hz, 2H),

[0996] LCMS: 415.3 (M-56), Rt (min): 2,127, 면적 %: 95,713.

[0997] HPLC: Rt (min): 4,211, 면적 %: 98,834.

[0998] 단계-3: tert-butyl ((1r,4r)-4-((((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamate

[0999]

[1000] Pentanal (229 mg, 2.66 mmol, 5 equivalents), TFA (54.5 mg, 0.478 mmol, 0.9 equivalents), and Na(OAc)3BH (169 mg, 0.797 mmol, 1.5 equivalents) were added at 0°C to 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 equivalent) dissolved in DCE (2.5 mL) and DMF (0.5 mL), and the mixture was stirred at room temperature for 12 hours. When the reaction was complete (confirmed by LCMS), the reaction was quenched with ice water (25 mL) and extracted with DCM (10 mL × 2). The combined organic layer was concentrated under vacuum to obtain a crude product, which was purified using Isolera (column size: Biotage R snap cartridge, KP-Sil, 25 g, silica gel - 230-400 mesh) with 70-80% ethyl acetate in petroleum ether. The pure fractions were collected individually and concentrated under vacuum to obtain 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.

[1001] 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).

[1002] LCMS: 541.3 (M+H), Rt (min): 2.849, 면적 %: 95.954.

[1003] HPLC: Rt (min): 4.907, 면적 %: 95.462.

[1004] 단계-4: 3-(4-((((1r,4r)-4-aminocyclohexyl)methyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl

[1005]

[1006] HCl (4M in ethyl acetate) (0.12 mL, 3.95 mmol) was added at 0°C to a near-freezing solution of tert-butyl ((1r,4r)-4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)methyl)cyclohexyl)carbamate (120 mg, 0.222 mmol) dissolved in DCM (5 mL), and stirred at room temperature for 6 hours. When the reaction was complete (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.

[1007] 1H-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).

[1008] LCMS: 441.3 (M+H), Method: Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: ACN, Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm, Flow rate: 1.5 ml / min, Rt (min): 1.695, Area %: 96.479.

[1009] HPLC: Method: A: 0.1% TFA in H2O, B: ACN, Flow rate: 2.0 mL / min

[1010] Column: Xbridge C8(50 Х 4.6) mm, 3.5 μm, Rt (min): 2.950, Area %: 95.528.

[1011]

[1012] Hydrochloride form of Compound 93

[1013] (E)-3-(4-((4-aminobutyl)(pent-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1014]

[1015] Step-1: (E)-pent-3-enal

[1016]

[1017] DIBAL-H (17.52 mL, 17.52 mmol, 0.8 equivalents) dissolved in hexane was slowly added at -78°C to a stirred solution of methyl (E)-pent-3-enoate 2 (2.5 g, 21.90 mmol, 1.0 equivalent) dissolved in CH2Cl2 (50 mL). Then, the reaction mixture was stirred at 78°C for 5 minutes. When the reaction was complete (TLC analysis, 20% ethyl acetate in petroleum ether, R ­f (confirmed to be about 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 with Na2SO4, filtered, and used in the next step without concentration.

[1018] Step-2: tert-butyl (E)-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-en-1-yl)amino)butyl)carbamate

[1019]

[1020] AcOH (0.332 mL, 5.81 mmol, 5.0 equivalents) was added under a nitrogen atmosphere to a stirred solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 1 (0.5 g, 1.161 mmol, 1.0 equivalent) and (E)-pent-3-enal 3 (1.954 g, 23.23 mmol, 20.0 equivalents) dissolved in a mixture of CH2Cl2 (10 mL) and DMF (5 mL), and stirred for 1 hour. NaCNBH4 (0.292 g, 4.65 mmol, 4.0 equivalents) was added to this mixture at 0°C. The resulting reaction mixture was stirred overnight at room temperature. Upon completion of the reaction (TLC and UPLC analysis, 100% ethyl acetate, R ­f (confirmed as ~0.7), the reaction mixture was diluted with DCM (30 mL), washed with water (2 × 30 mL), 10% LiCl solution (30 mL), and brine (30 mL), dried with Na2SO4, filtered, and concentrated to obtain a pale yellow liquid crude product. The crude product was purified by preparative HPLC (column: X-Bridge-c18 19.1X250, mobile phase: 0.1% aqueous formic acid / acetonitrile, flow rate: 15 mL / min) to obtain tert-butyl (E)-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-en-1-yl)amino)butyl)carbamate 4 (35 mg, 0.068 mmol, 5.87% yield) as a milky white solid.

[1021] 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),

[1022] LCMS: 499.2 (M+H), Rt (min):2.492, 면적-97%.

[1023] 단계-3: (E)-3-(4-((4-aminobutyl)(pent-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1024]

[1025] HCl (4M solution in ethyl acetate, 2 mL) was added at 0°C to a stirred solution of tert-butyl (E)-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-en-1-yl)amino)butyl)carbamate 4 (65 mg, 0.130 mmol, 1.0 equivalent) dissolved in ethyl acetate (1 mL), and the mixture was stirred at room temperature for 3 hours. The progress of the reaction was monitored by TLC (100% EtOAc). The reaction mixture was concentrated under reduced pressure at 40°C or below to obtain a faint semi-solid form. The obtained solid was dissolved in water (2 mL) and washed with MTBE (2 mL), additional water (2 mL) was added and freeze-dried to obtain (E)-3-(4-((4-aminobutyl)(pent-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl (30 mg, 0.067 mmol, 79% yield) as a white solid.

[1026] 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)

[1027] LCMS: 399.2 (M+H). Methods: Column: Atlantis dC18 (50 x 4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: Acetonitrile, Flow rate: 1.5 ml / min

[1028] HPLC: Method: Mobile phase: A: 0.1% FA in water, Mobile phase: B: ACN, Flow rate: 2.0 mL / min, Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, RT: 2.301 min, Area: 97.652 %.

[1029]

[1030] trifluoride form of compound 94

[1031] (Z)-3-(4-((4-aminobutyl)(pent-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[1032]

[1033] Steps 1 and 2: (3-(1-oxo-4-(pent-3-yn-1-ylamino)isoindolin-2-yl)piperidine-2,6-dione

[1034]

[1035] Des-martin periodinan (11.09 g, 26.2 mmol, 1.1 equivalents) and water (0.05 mL) were added to a solution of pent-3-yn-1-ol 2 (2.0 g, 23.78 mmol, 1.0 equivalent) and CH2Cl2 (100 mL) at 0°C, and the resulting white suspension was stirred at room temperature for 1 hour. When the reaction was complete (confirmed by TLC, 20% ethyl acetate in petroleum ether), the reaction mixture was cooled to -78°C, diluted with n-pentane (50 mL), and filtered through a silica gel layer. The filtrate was washed with n-pentane (20 mL), and the filtrate was concentrated under vacuum at 25°C until the volume was reduced to about 50%, and the resulting solution of compound 3 was used immediately in a subsequent step.

[1036] In another round-bottom flask, a solution of Compound 3 was added at room temperature to a solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (4.00 g, 15.43 mmol, 0.649 equivalents) dissolved in DMF (40 mL). Acetic acid (1.753 mL, 30.6 mmol, 1.289 equivalents) was added to the reaction mixture and stirred at room temperature for 2 hours. Sodium cyanoborohydride (1.520 g, 24.18 mmol, 1.017 equivalents) was added to the reaction mixture at 0°C and stirred overnight at room temperature. When the reaction was complete (TLC and UPLC analysis, 100% ethyl acetate, R ­f (confirmed as ~0.7), the reaction mixture was diluted with DCM (100 mL), washed with water (2 × 50 mL), dried with Na2SO4, and filtered to obtain a pale yellow liquid crude product. The crude product was purified by reverse-phase chromatography (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase, eluted with 70% ACN in 0.1% HCO2H) to obtain 3-(1-oxo-4-(pent-3-yn-1-ylamino)isoindolin-2-yl)piperidine-2,6-dione 4 (1.0 g, 3.03 mmol, 12.75% yield) as a pale yellow solid.

[1037] 1H-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),

[1038] LCMS: 326.1 (M+H), Rt (min):1.986, 면적-98.611%.

[1039] 단계-3: tert-butyl (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-yn-1-yl)amino)butyl)carbamate

[1040]

[1041] Trifluoroacetic acid (0.106 mL, 1.383 mmol, 0.9 equivalents) was added to a stirred mixture of 3-(1-oxo-4-(pent-3-yn-1-ylamino)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) dissolved in DMF (15 mL) and CH2Cl2 (15 mL), and stirred at 0°C for 10 minutes. Sodium triacetoxyborohydride (0.49 g, 2.305 mmol, 1.5 equivalents) was added in portions at 0°C, and the resulting colorless, turbid mass was stirred at room temperature for 16 hours. When the reaction was complete (confirmed by UPLC analysis), the reaction mixture was quenched with water near freezing point (50 mL) and extracted with dichloromethane (2 × 50 mL). The bound organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude product in the form of a brown liquid. The crude product was purified by reverse-phase chromatography (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase, eluted with 70% ACN in 0.1% HCO2H) to obtain tert-butyl (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-yn-1-yl)amino)butyl)carbamate 10 (0.4 g, 0.649 mmol, 42.2% yield) as a milky white solid.

[1042] 1H-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),

[1043] LCMS: 597.4 (M+H), Rt (min):2.482, 면적-96.894%.

[1044] 단계-4: tert-butyl (Z)-(tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-en-1-yl)amino)butyl)carbamate

[1045]

[1046] Lindlard catalyst (12 mg, 0.113 mmol, 0.449 equivalents) was added to a stirred solution of tert-butyl (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-yn-1-yl)amino)butyl)carbamate 5 (150 mg, 0.251 mmol, 1.0 equivalent) and quinoline (39.0 mg, 0.302 mmol, 1.2 equivalents) dissolved in DMF (2.0 mL), and stirred at room temperature for 16 hours under a hydrogen atmosphere. When the reaction was complete (confirmed by UPLC analysis), the reaction mixture was filtered through a Celite bed and washed with DMF (2 mL). The filtrate was purified by reverse-phase chromatography (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase, eluted with 70-75% ACN in 0.1% HCOOH) to obtain tert-butyl (Z)-(tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-en-1-yl)amino)butyl)carbamate 6 (120 mg, 0.182 mmol, 72.2% yield) as a pale yellow rubbery solid.

[1047] 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),

[1048] LCMS: 599.3 (M+H). Methods: Column: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile Phase: A: 0.1% FA in H2O, Mobile Phase: B: Acetonitrile, Flow Rate: 1.5 ml / min

[1049] HPLC: Method: Mobile phase: A: 0.1% TFA in water, Mobile phase: B: Methanol, Flow rate: 1.0 mL / min, Column: Atlantis dC18 (250 Х4.6) mm, 5 μm, RT: 16.959 min, Area: 90.666%.

[1050] Step-5: (Z)-3-(4-((4-aminobutyl)(pent-3-en-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[1051]

[1052] TFA (2 mL, 26.0 mmol) was added at 0°C to a stirred solution of tert-butyl (Z)-(tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-en-1-yl)amino)butyl)carbamate 6 (60 mg, 0.100 mmol) dissolved in CH2Cl2 (1 mL), and stirred overnight at room temperature. When the reaction was complete (confirmed by UPLC), the reaction mixture was concentrated under reduced pressure at 40°C to obtain a pale yellow viscous liquid. The crude product was purified by formulation HPLC (Column: Atlantis 19x250mm, Mobile phase: TFA in 0.1% water / ACN, Flow rate: 2mg / mL) to obtain (Z)-3-(4-((4-aminobutyl)(pent-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.

[1053] 1 H-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),

[1054] LCMS: 399.2 (M+H). Methods: Column: Atlantis dC18 (50×4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: Acetonitrile, Flow rate: 1.5 ml / min

[1055] HPLC: Method: Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Flow rate: 1.0 mL / min, Column: Atlantis dC18 (250 χ 4.6) mm, 5 μm, RT: 7.709 min, Area: 94.84%.

[1056]

[1057] Hydrochloride form of Compound 95

[1058] 3-(4-((4-aminobutyl)(pent-3-yn-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl

[1059]

[1060] Steps 1 and 2: tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-yn-1-yl)amino)butyl)carbamate

[1061]

[1062] Des-martin periodinan (11.09 g, 26.2 mmol, 1.1 equivalents) was added to a solution of pent-3-yn-1-ol 3 (2 g, 23.78 mmol, 1 equivalent) dissolved in DCM (40 mL) at 0°C, and the resulting white suspension was stirred at room temperature for 1 hour. The reaction mixture was quenched with a mixture of NaHSO3 (5 g) dissolved in a saturated NaHCO3 (50 mL) solution and extracted with DCM (2 × 15 mL). Tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate (0.500 g, 1.162 mmol, 1 equivalent) and acetic acid (0.714 g, 11.627 mmol) were added to the combined organic layer and stirred for 2 hours. Then, NaCNBH3 (0.463 g, 11.627 mmol, 10 equivalents) was added and stirred at room temperature for 24 hours. When the reaction was complete (confirmed by UPLC), the reaction mixture was quenched with water (40 mL) and extracted with DCM (2 × 40 mL). The combined organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude product, which was purified using Isolera (Biotage R snap cartridge, KP-Sil, 25 g, 230-400 silica mesh) containing 60-70% ethyl acetate in petroleum ether. The pure fraction was concentrated under vacuum to obtain tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-yn-1-yl)amino)butyl)carbamate 3 (150 mg, 0.113 mmol, 0.474% yield) as a milky white solid.

[1063] LCMS: 497.2 (M+H), Rt (min): 2.384, Area %: 37.322.

[1064] Step-3:3-(4-((4-aminobutyl)(pent-3-yn-1-yl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl

[1065]

[1066] HCl (4 M in ethyl acetate, 0.294 mL, 1.178 mmol, 10 equivalents) was added to a solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pent-3-yn-1-yl)amino)butyl)carbamate 3 (150 mg, 0.118 mmol, 1 equivalent) dissolved in DCM (3 mL) at 0°C and stirred at room temperature for 12 hours. When the reaction was complete (confirmed by LCMS), the reaction mixture was concentrated and 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 obtain 3-(4-((4-aminobutyl)(pent-3-yn-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.

[1067] 1H-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).

[1068] 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 Х 4.6 mm) 3.5 μm, Flow rate: 1.5 ml / min, Rt (min): 1.340, Area %: 96.271.

[1069] HPLC: Method: Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Column: X-Bridge C8(50 Х 4.6) mm, 3.5 μm, Flow rate: 2.0 mL / min, Rt (min): 2.314, Area %: 98.115.

[1070]

[1071] Hydrochloride form of Compound 96

[1072] 3-(4-(bis(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1073]

[1074]

[1075] Step-3: di-tert-butyl (((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)azanediyl)bis(butane-4,1-diyl))dicarbamate

[1076]

[1077] Sodium triacetoxyborohydride (492 mg, 2.323 mmol, 4 equivalents) was added to 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 equivalents), and TFA (0.179 mL, 2.323 mmol, 4 equivalents) dissolved in a mixture of dichloromethane (0.5 mL, 20-fold) and DMF (0.25 mL, 10-fold), and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, EtOAc, R ­f ~ 0.6, and confirmed by UPLC), the reaction mixture was diluted with DCM (5 mL), washed with water (3 × 10 mL) and brine (10 mL), dried with Na2SO4, filtered, and concentrated. The resulting residue was purified by chromatography using 75-80% ethyl acetate in petroleum ether (column size: Biotage R snap cartridge, KP-Sil, 25 g, 230-400 mesh silica gel) to obtain 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.

[1078] 1 H-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).

[1079] LC-MS: 702.4 (M+H), Rt (min): 2.83, 면적%: 99.289.

[1080] 단계-4: 3-(4-(bis(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1081]

[1082] HCl (4M solution in dioxane) (3 mL, 12.00 mmol) was added to a near-freezing solution of di-tert-butyl (((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)azanediyl)bis(butane-4,1-diyl))dicarbamate 5 (150 mg, 0.249 mmol) dissolved in DCM (5 mL), and the mixture was stirred at room temperature for 2 hours. Once the reaction was complete (confirmed by TLC analysis as 100% ethyl acetate), the reaction mixture was concentrated and freeze-dried to obtain 3-(4-(bis(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl 96 (82 mg, 67.8% yield) as a pale yellow solid.

[1083] 1 H-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).

[1084] 1 H-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).

[1085] 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 Х 4.6 mm) 3.5 μm. Rt (min): 0.887; Area %: 97.781.

[1086] 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 x 4.6) mm, 3.5 μm. Rt (min): 1.08; Area%: 95.532.

[1087]

[1088] Hydrochloride form of Compound 97

[1089] 3-(4-((4-aminobutyl)(3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1090]

[1091] Step-1: tert-butyl (3-oxopropyl)carbamate

[1092]

[1093] A solution of oxalyl chloride (0.599 mL, 6.85 mmol, 1.2 equivalents) dissolved in DCM (10 mL) was slowly added to a solution of DMSO (0.972 mL, 13.70 mmol, 2.4 equivalents) dissolved in DCM (5 mL) under a nitrogen atmosphere at -78°C for 15 minutes, and stirred for 15 minutes at the same temperature. To this, a solution of tert-butyl (3-hydroxypropyl)carbamate 5 (1 g, 5.71 mmol, 1 equivalent) dissolved in DCM (5 mL) was slowly added for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (3.98 mL, 28.5 mmol, 5.0 equivalents) was added in portions at -78°C. Once the addition was complete, the temperature of the reaction mixture was slowly raised to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 3:7 ethyl acetate:petroleum ether, R ­f ~ 0.4, confirmed by KMnO4), the reaction mixture was diluted with DCM (10 mL), washed with 10% aqueous citric acid solution (10 mL), water (10 mL), and brine (10 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure below 30°C to obtain the crude product tert-butyl (3-oxopropyl)carbamate 6 (0.950 g, 4.90 mmol, 86% yield) as a pale yellow liquid, which was subsequently used without purification.

[1094] LCMS: (No mass peak observed). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.644; Area% - 89.26.

[1095] Step-2: tert-butyl (4-((3-((tert-butoxycarbonyl)amino)propyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate

[1096]

[1097] Tert-butyl (3-oxopropyl)carbamate 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 together under a nitrogen atmosphere to a mixture of 1,2-dichloroethane (10 mL, 10 times the volume) and DMF (5 mL, 5 times the volume). Acetic acid (0.332 mL, 5.81 mmol, 5 equivalents) was added to this mixture, and the reaction mixture was stirred at room temperature for 1 hour. NaCNBH3 (0.292 g, 4.65 mmol, 4 equivalents) was added in portions, and the reaction mixture was stirred at room temperature for an additional 16 hours. Once the reaction is complete (TLC analysis, 100% ethyl acetate, R ­f~ 0.5, confirmed by LCMS), the reaction mixture was diluted with near-freezing water (15 mL), extracted with DCM (2 × 15 mL), washed with brine (25 mL), dried with anhydrous Na2SO4, filtered, and concentrated to obtain a white solid crude product, which was purified with preparative HPLC (Method: Equipment: SC-DC-ARD-05-045; Column Number: XBridge C8 - 250-X-Select-C18-19x150mm; 0.1% TFA in water / ACN; Rt: 9.6 min) and freeze-dried 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, A white solid was obtained (7.89% yield).

[1098] LCMS: 588.4 (M+H). Methods: Column: XBridge C8 (50 Х 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.

[1099] 1 H-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).

[1100] Step-3: 3-(4-((4-aminobutyl)(3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1101]

[1102] HCl (4 M solution in EtOAc, 4 mL) was added to a near-freezing solution of tert-butyl (4-((3-((tert-butoxycarbonyl)amino)propyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 7 (60 mg, 0.102 mmol, 1 equivalent) dissolved in ethyl acetate (2 mL), and the mixture was stirred at room temperature for 1.5 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and freeze-dried to obtain 3-(4-((4-aminobutyl)(3-aminopropyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione 97 (20 mg, 0.051 mmol, 50.1% yield) as a milky white solid.

[1103] LCMS: 388.3 (M-2 ХHCl). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 0.6 mL / min. Rt (min): 2.218; Area% - 99.01.

[1104] HPLC: 93.99%, Rt (min): 6.421. Method: Column: Atlantis dC18 (250 Х 4.6) mm, 5 μm, Mobile phase A: 0.1% TFA in H2O, Mobile phase B: Methanol, Flow rate: 1.0 mL / min.

[1105] 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).

[1106]

[1107] 화합물 102의 염산염 형태

[1108] 3-(4-((4-aminobutyl)((1S,4S)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1109]

[1110] 단계-1: tert-butyl((1S,4S)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate

[1111]

[1112] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (0.5 g, 1.547 mmol, 1 equivalent), tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate 2 (0.995 g, 4.64 mmol, 3 equivalents), and NaOt-Bu (0.446 g, 4.64 mmol, 3 equivalents) were dissolved in DMF (2 mL) and degassed under a nitrogen atmosphere for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equivalents) was added under a nitrogen atmosphere, and the reaction mixture was heated at 110°C for 4 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc, R ­f ~ 0.4 and confirmed by 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 (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain 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.

[1113] LCMS: 456.0 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.223; Area% - 95.48.

[1114] 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).

[1115] Step-2: tert-butyl (tert-butoxycarbonyl)(4-(((1S,4S)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate

[1116]

[1117] TFA (0.074 mL, 1.139 mmol, 4 equivalents) was added to 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 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 4 (0.164 g, 0.569 mmol, 2 equivalents) in DCM (4 mL) and DMF (4 mL) at 0°C, followed by the sequential addition of sodium triacetoxyborohydride (0.241 g, 1.139 mmol, 4 equivalents). The reaction mixture was raised to room temperature and stirred at the same temperature for 16 hours. Once the reaction is complete (TLC analysis, 100% EtOAc, R ­f~ 0.5 and confirmed by LCMS), the reaction mixture was diluted with water near freezing point (15 mL), extracted with ethyl acetate (2 × 15 mL), the organic layer was washed with brine (25 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The obtained residue 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 freeze-dried to obtain tert-butyl (tert-butoxycarbonyl)(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.

[1118] LCMS: 726.7 (MH). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.912; Area% - 99.75.

[1119] 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

[1120] 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).

[1121] Step-3: 3-(4-((4-aminobutyl)((1S,4S)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1122]

[1123] HCl (4M solution in EtOAc, 2.5 mL) was added to a near-freezing solution of tert-butyl (tert-butoxycarbonyl)(4-(((1S,4S)-4-((tert-butoxycarbonyl)amino)cyclohexyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 5 (72 mg, 0.099 mmol, 1 equivalent) dissolved in DCM (1 mL), and the mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by LCMS. The reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and freeze-dried to obtain 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 a milky white solid.

[1124] LCMS: 427.2 (M-HCl). Methods: Column: XBridge C8 (50 Х 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.

[1125] HPLC: 95.86%, Rt (min): 1.363. Method: Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, Mobile phase: A: 0.1% TFA in water, Mobile phase: B: ACN, Flow rate: 2.0 mL / min.

[1126] 1 H-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).

[1127]

[1128] Hydrochloride form of compound 103

[1129] 3-(4-((4-aminobutyl)((1r,4r)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl

[1130]

[1131] Step-1: tert-butyl ((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)carbamate

[1132]

[1133] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (0.5 g, 1.547 mmol, 1 equivalent), tert-butyl ((1R,4R)-4-aminocyclohexyl)carbamate 2 (0.995 g, 4.64 mmol, 3 equivalents), and NaOt-Bu (0.446 g, 4.64 mmol, 3 equivalents) were dissolved in DMF (2 mL) and degassed under nitrogen for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.075 g, 0.077 mmol, 0.05 equivalents) was added under a nitrogen atmosphere and reacted at 110°C for 4 hours. When the reaction was complete (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 (Grace® column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain 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.

[1134] LCMS: 401.1 (M-Boc). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.200, Area% - 99.01

[1135] 1H-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).

[1136] Step-2: 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

[1137]

[1138] TFA (0.047 mL, 0.613 mmol, 4 equivalents) was added to 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 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 4 (88 mg, 0.307 mmol, 2 equivalents) dissolved in DCM (5 mL) and DMF (0.5 mL) at 0°C, followed by the addition of sodium triacetoxyborohydride (130 mg, 0.613 mmol, 4 equivalents). The reaction mixture was stirred at room temperature for 16 hours. Once the reaction is complete (TLC analysis, 100% ethyl acetate R­f ~ 0.5, confirmed by LCMS), the reaction mixture was diluted with water near freezing point (15 mL) and extracted with DCM (2 × 10 mL), the organic layer was washed with brine (25 mL), dried with Na2SO4, filtered, and concentrated. The obtained residue was purified by reverse-phase column chromatography (Grace® column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase), and the pure fraction was freeze-dried to obtain 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.

[1139] LCMS: 728.5 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.913; Area% - 99.75.

[1140] Step-3: 3-(4-((4-aminobutyl)((1R,4R)-4-aminocyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl

[1141]

[1142] HCl (4 M solution in ethyl acetate, 1 mL, 4.0 mmol) was added to a near-freezing solution of 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, 1 equivalent) dissolved in DCM (1 mL), and the mixture was stirred at room temperature for 2 hours. When the reaction was complete (TLC analysis, 10% MeOH / DCM, R ­f (confirmed as ~0.1), the reaction mixture was concentrated, washed with MTBE (2 × 5 mL), and freeze-dried to obtain 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 a milky white solid.

[1143] LCMS: 428.3 (M+H) Method: Column: XBridge C8 (50 Х 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.

[1144] HPLC: 99.63%, Rt (min): 11.860. Methods: Column: Atlantis dC18 (250 x 4.6) mm, 5 μm, Mobile Phase A: 0.1% TFA in water, Mobile Phase B: Methanol, Flow Rate: 0.7 ml / min

[1145] 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).

[1146]

[1147] 화합물 104의 염산염 형태

[1148] 3-(4-((4-aminobutyl)((1s,4s)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1149]

[1150] 단계-1:tert-butyl(((1s,4s)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate

[1151]

[1152] Tert-butyl(((1S,4S)-4-aminocyclohexyl)methyl)carbamate 2 (1.060 g, 4.64 mmol, 1.5 equivalents) and sodium tert-butoxide (0.892 g, 9.28 mmol, 3.0 equivalents) were added at room temperature to a solution of 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (1.0 g, 3.09 mmol, 1.0 equivalents) dissolved in DMF (5 mL, 5-fold volume), and degassed for 15 minutes using nitrogen gas. Pd-PEPPSI-IHept(Cl) (0.151 g, 0.155 mmol, 0.05 equivalents) was added to this reaction mixture under a nitrogen atmosphere and degassed for 5 minutes, and the mixture was stirred overnight at 85°C. Once the reaction is complete (TLC and UPLC analysis, 100% EtOAc, R ­f (confirmed as ~0.5), the reaction mixture was filtered on a Celite bed and washed with THF (50 mL), and the filtrate was concentrated under reduced pressure to obtain a brown viscous liquid. The mixture was purified by reverse-phase chromatography (the product was eluted with 75% ACN dissolved in 0.1% HCO2H), and the pure fraction was freeze-dried 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.

[1153] 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).

[1154] LCMS: 415.8 (M-tBu), Rt (min):2.324, 면적-89.8%.

[1155] HPLC: Rt (min): 5.496; Area - 82.3%.

[1156] 단계-2: tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate

[1157]

[1158] A solution of oxalyl chloride (1.815 mL, 20.73 mmol, 2.0 equivalents) dissolved in DCM (20 mL) was slowly added to a solution of DMSO (2.94 mL, 41.5 mmol, 4.0 equivalents) dissolved in DCM (5 mL) under a nitrogen atmosphere at -78°C for 15 minutes and stirred for 15 minutes at the same temperature. To this, a solution of tert-butyl (tert-butoxycarbonyl)(4-hydroxybutyl)carbamate 4 (3.0 g, 10.37 mmol) dissolved in CH2Cl2 (5 mL) was slowly added for 15 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and triethylamine (8.67 mL, 62.2 mmol, 6.0 equivalents) was added in portions at -78°C. Then, the reaction mixture was slowly raised to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 20% EtOAc / petroleum ether, R ­f ~0.6, confirmed by KMnO4), the reaction mixture was diluted with DCM (40 mL), washed with 10% aqueous citric acid solution (40 mL once) and water (40 mL once), dried with Na2SO4, filtered, and concentrated under reduced pressure (below 30°C) to obtain a pale yellow liquid crude product tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (2.9 g, 9.99 mmol), which was subsequently used without purification.

[1159] 1 H-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),

[1160] Step-3: 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

[1161]

[1162] Trifluoroacetic acid (0.131 mL, 1.700 mmol, 4.0 equivalents) was added to 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 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (0.244 g, 0.850 mmol, 2.0 equivalents) dissolved in CH2Cl2 (10 mL) and DMF (10 mL), and the mixture was stirred at 0°C for 10 minutes. Sodium triacetoxyborohydride (0.360 g, 1.700 mmol, 4.0 equivalents) was added in portions at 0°C, and the resulting colorless, turbid mixture was stirred at room temperature for 16 hours (monitored by UPLC). The reaction mixture was cooled to 0°C, and additionally tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (0.244 g, 0.850 mmol, 2.0 equivalents) and trifluoroacetic acid (0.131 mL, 1.700 mmol, 4.0 equivalents) were added and stirred for 10 minutes. Afterward, sodium triacetoxyborohydride (0.360 g, 1.700 mmol, 4.0 equivalents) was added in portions at 0°C, and the resulting colorless, turbid mixture was stirred at room temperature for 16 hours. Once the reaction is complete (TLC analysis, 8:2 / EtOAc:petroleum ether, R ­f(confirmed as ~0.6), the reaction mixture was quenched with near-freezing water (20 mL) and extracted with dichloromethane (2 × 50 mL). The bound organic layer was dried with anhydrous Na2SO4 and concentrated under vacuum to obtain a pale yellow liquid crude product. The crude product 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 collected fraction was concentrated under reduced pressure to obtain a pale yellow liquid.

[1163] The obtained compound was further purified by reverse-phase chromatography (elution with 75% ACN dissolved in 0.1% HCO2H), and the pure fraction was freeze-dried to obtain 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.

[1164] 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).

[1165] LCMS: 741.4 (M+ ), Rt (min):2.945, Area%- 89.8%.

[1166] HPLC: Rt (min): 5.816; Area% - 99.9%.

[1167] Step-4: 3-(4-((4-aminobutyl)((1s,4s)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochoride

[1168]

[1169] HCl (4 M solution in dioxane, 10 mL) was added at 0°C to a stirred solution of 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 (0.1 g, 0.135 mmol, 1.0 equivalent) dissolved in dichloromethane (10 mL), and the mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum below 40°C to obtain a milky white solid. The obtained solid was dissolved in water (10 mL), washed with MTBE (2 × 10 mL), and freeze-dried 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 a milky white solid.

[1170] 1H-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).

[1171] LCMS: 442.3 (M+H). Methods: Column: XBridge C8 (50 Х 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.

[1172] HPLC: Method: Mobile phase A: 0.1% TFA in water, Mobile phase B: Methanol, Flow rate: 1.0 ml / min. Column: Atlantis dC18 (250 Х 4.6) mm, 5 μm. RT: 6.488 min, Area: 95.427 %.

[1173]

[1174] Hydrochloride form of compound 105

[1175] 3-(4-((4-aminobutyl)((1R,4R)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl

[1176]

[1177] Step-1: tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl)methyl)carbamate

[1178]

[1179] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (1.0 g, 3.09 mmol, 1 equivalent), tert-butyl (((1R,4R)-4-aminocyclohexyl)methyl)carbamate 2 (2.120 g, 9.28 mmol, 3 equivalents) and NaOt-Bu (0.892 g, 9.28 mmol, 3 equivalents) were dissolved in DMF (4 mL) and degassed under nitrogen for 20 minutes. Pd-PEPPSI-IHept(Cl) (0.151 g, 0.155 mmol, 0.05 equivalents) was added under a nitrogen atmosphere, and the reaction mixture was heated at 110°C for 4 hours. Upon 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 (Grace® 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.

[1180] LCMS: 401.1 (M-Boc). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm Mobile phase: A: 0.1% FA in H2O, B: ACN, Flow rate: 1.5 mL / min. Rt (min): 2.262, Area% - 96.25

[1181] 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)

[1182] 단계-2: 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

[1183]

[1184] TFA (0.059 mL, 0.765 mmol, 4 equivalents) and sodium triacetoxyborohydride (162 mg, 0.765 mmol, 4 equivalents) were added to a solution of tert-butyl (((1R,4R)-4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)cyclohexyl) methyl)carbamate 3 (90 mg, 0.191 mmol, 1 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 4 (110 mg, 0.383 mmol, 2 equivalents) dissolved in DCM (5 mL) and DMF (2 mL) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 hours. When the reaction was complete (confirmed by LCMS), the reaction mixture was diluted with cold water (10 mL), extracted with DCM (2 × 10 mL), washed with brine (25 mL), dried with Na2SO4, filtered, and concentrated. The resulting residue was purified by reverse-phase column chromatography (Grace® column: C18 40 μm, 100 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase). The pure fraction obtained from the column was freeze-dried to obtain 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.

[1185] LCMS: 742.5 (M+H). Methods: Column: XBridge C8 (50 Х 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.

[1186] 1 H-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).

[1187] Step-3: 3-(4-((4-aminobutyl)((1R,4R)-4-(aminomethyl)cyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl

[1188]

[1189] HCl (4M solution in EtOAc, 1 mL, 4.0 mmol) was added to a near-freezing solution of 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 (20 mg, 0.027 mmol, 1 equivalent) dissolved in DCM (1 mL), and the mixture was stirred at room temperature for 2 hours. When the reaction was complete (TLC analysis, 10% MeOH:DCM, R ­f (confirmed as ~0.1), the reaction mixture was concentrated and freeze-dried 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.

[1190] LCMS: 428.3 (M+H) Method: Column: XBridge C8 (50 Х 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): 0.8 / 66; Area% - 94.61.

[1191] HPLC: 96.75%, Rt (min): 2.674. Methods: Method Information: A: 0.1% TFA in H2O, B: Methanol, Flow rate: 1.0 ml / min, Column: YMC Hydrosphere C18 (150 x 4.6 mm), 3 μm

[1192] 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).

[1193]

[1194] Compound 106 of the form

[1195] 3-(4-((4-aminobutyl)(((1s,4s)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl

[1196]

[1197] Step-1: tert-butyl ((1s,4s)-4-formylcyclohexyl)carbamate

[1198]

[1199] To a cooled solution of tert-butyl ((1s,4s)-4-(hydroxymethyl)cyclohexyl)carbamate (500 mg, 2.180 mmol, 1 equivalent) dissolved in DCM (11 mL), a solution of DMSO (1.5 mL) containing 3.7 mL, DIPEA (1.510 mL, 8.48 mmol, 3.89 equivalents), and pyridine sulfur trioxide (1.35 g, 8.48 mmol, 3.89 equivalents) was slowly added, and the mixture was stirred at 0°C for 1 hour. Upon completion of the reaction (TLC analysis (40% ethyl acetate / petroleum ether, R ­f (confirmed as : 0.4), the reaction was quenched with 1.5N HCl (20 mL) and extracted with DCM (2 × 20 mL). The bound organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure to obtain tert-butyl ((1s,4s)-4-formylcyclohexyl)carbamate 2A (360 mg, 1.584 mmol, 72.6% yield) as a colorless liquid.

[1200] 1 H-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).

[1201] 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

[1202]

[1203] TFA (47.7 mg, 0.418 mmol, 0.9 equivalents) and Na(OAc)3BH (35.0 mg, 0.557 mmol, 1.5 equivalents) were added at 0°C to a solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate Int-4 (200 mg, 0.465 mmol, 1 equivalent) and tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (528 mg, 2.323 mmol, 5 equivalents) dissolved in a mixture of DCE (2 mL) and DMF (0.5 mL), and the mixture was stirred at room temperature for 12 hours. When the reaction was complete (confirmed by UPLC), the reaction mixture was quenched with ice water (25 mL) and extracted with DCM (2 x 10 mL). The bound organic layer was concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase chromatography (Grace® 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 freeze-dried to obtain 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 a milky white solid.

[1204] 1H-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).

[1205] LCMS: 642.4 (M+H), Rt (min): 2.992, 면적 %: 99.757.

[1206] HPLC: Rt (min): 4.691, 면적 %: 99.938.

[1207] 단계-3: 3-(4-((4-aminobutyl)(((1s,4s)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl

[1208]

[1209] HCl (4M solution in ethyl acetate, 1.3 mL, 5.20 mmol) was added at 0°C to a near-freezing 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 equivalent) dissolved in DCM (2.5 mL), and stirred at room temperature for 2 hours. When the reaction was complete (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.

[1210] 1 H-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).

[1211] 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 Х 4.6 mm) 3.5 μm, Flow rate: 1.5 ml / min, Rt (min): 1.027, Area %: 99.640.

[1212] HPLC: Method: A: 0.1% TFA in H2O, B: ACN, Flow rate: 2.0 ml / min, Column: Xbridge C8 (50 Х 4.6) mm, 3.5 μm Rt (min): 1.655, Area %: 99.741.

[1213]

[1214] Hydrochloride form of compound 107

[1215] 3-(4-((4-aminobutyl)(((1r,4r)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl

[1216]

[1217] Step-1: tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A

[1218]

[1219] DMSO (3.7 mL) and DIPEA (1.510 mL, 8.48 mmol, 3.89 equivalents) were added to a cooled solution of tert-butyl ((1r,4r)-4-(hydroxymethyl)cyclohexyl)carbamate (500 mg, 2.180 mmol, 1 equivalent) dissolved in DCM (11 mL), followed by the gradual addition of a solution of pyridine sulfur trioxide (1.35 g, 8.48 mmol, 3.89 equivalents) dissolved in DMSO (3.7 mL). The mixture was stirred at 0°C for 15 minutes. Upon completion of the reaction (TLC (40% ethyl acetate in petroleum ether, R ­f(confirmed as : 0.4), the reaction mixture was quenched with 1.5N HCl (20 mL) and extracted with DCM (20 mL × 2). The combined organic layer was concentrated under reduced pressure to obtain tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (500 mg, 2.123 mmol, 97% yield) as a yellow solid.

[1220] 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).

[1221] LCMS: 172 (M-56), Rt (min): 2.587, Area %: 96.528.

[1222] 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

[1223]

[1224] TFA (47.7 mg, 0.418 mmol, 0.9 equivalents) and Na(OAc)3BH (35.0 mg, 0.557 mmol, 1.5 equivalents) were added at 0°C 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 equivalent) and tert-butyl ((1r,4r)-4-formylcyclohexyl)carbamate 2A (528 mg, 2.323 mmol, 5 equivalents) dissolved in a mixture of DCM (2 mL) and DMF (0.5 mL), and the mixture was stirred at room temperature for 12 hours. When the reaction was complete (confirmed by UPLC), the reaction mixture was quenched with ice water (25 mL) and extracted with DCM (10 mL × 2). The combined organic layer was concentrated under vacuum to obtain the crude product. The crude product was purified with Isolera (Biotage R snap cartridge, KP-Sil, 25 g, silica gel 230-400 mesh) using 50-60% ethyl acetate in petroleum ether to obtain 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.

[1225] 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), 7.09 (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),

[1226] LCMS: 642.4 (M+H), Rt (min): 2.900 min, 면적 %: 99.216.

[1227] HPLC: Rt (min): 4.591, 면적 %: 97.770.

[1228] 단계-3:3-(4-((4-aminobutyl)(((1r,4r)-4-aminocyclohexyl)methyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl

[1229]

[1230] HCl (4 M solution in ethyl acetate, 0.89 mL, 3.56 mmol, 5 times the volume) was added to a near-freezing solution of 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, 1 equivalent) dissolved in DCM (1.7 mL), and stirred at room temperature for 6 hours. When the reaction was complete (confirmed by UPLC), the reaction mixture was concentrated and washed with MTBE (10 mL) to obtain the crude product, which was freeze-dried to obtain 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.

[1231] 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).

[1232] LCMS: 441.0 (MH), Method: Mobile phase: A: 0.1% FA in H2O; Mobile phase: B: ACN; Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm; Flow rate: 1.5 ml / min; Rt (min): 0.995, Area %: 98.488.

[1233] HPLC: Method: Mobile phase A: 0.1% TFA in water; Mobile phase B: Acetonitrile; Column: Atlantis dC18 (250 Х 4.6) mm, 5 μm; Flow rate: 1.0 ml / min; Rt (min): 6.025, Area %: 97.654.

[1234]

[1235] Hydrochloride forms of compounds 108 and 109

[1236] 3-(4-((4-Aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1237]

[1238] 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

[1239]

[1240] Sodium triacetoxyhydroborate (12.26 g, 57.9 mmol, 5.0 equivalents) was added to a stirred solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione 1 (3 g, 11.57 mmol, 1.0 equivalent) dissolved in TFA (30 mL), and the mixture was stirred at -15 °C for 10 minutes. Then, 4-methylcyclohexan-1-one 2 (10.38 g, 93 mmol, 8.0 equivalents) dissolved in DCM (10 mL) was added, and the reaction mixture was stirred for 16 hours. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase column chromatography (Grace® 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), a cis and trans mixture, as a pale white solid. The cis-trans mixture was separated by preparative HPLC (column: X-Select-C18-19 x 250 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.

[1241] Step-2: tert-butyl (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1s,4s)-4-methylcyclohexyl)amino)butyl)carbamate

[1242]

[1243] TFA (0.189 g, 1.654 mmol, 6.0 equivalents) and sodium triacetoxyhydroborate (0.497 g, 2.344 mmol, 8.5 equivalents) were added at 0 °C 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 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate (0.475 g, 1.654 mmol, 6.0 equivalents) dissolved in DCM (5 mL) and DMF (2 mL). The reaction mixture was heated to room temperature and stirred for 16 hours. When the reaction was complete (confirmed by UPLC), ice water (50 mL) was added to the reaction mixture to quench the reaction, and the mixture was extracted with DCM (30 mL × 2). The bound organic layer was dried with sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase column chromatography (Grace® column: C18 40 μm, 120 g; flow rate: 20 mL / min; 0.1% aqueous HCOOH / ACN mobile phase) to obtain the desired product, tert-butyl (tert-butoxycarbonyl)(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 a milky white solid.

[1244] 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),

[1245] LCMS: 627.4 (M+H), Rt (min): 3.41, 면적% - 97.91. HPLC: Rt (min): 4.88, 면적: 99.57%.

[1246] 단계-3: 3-(4-((4-aminobutyl)((1s,4s)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1247]

[1248] HCl (4 M solution in EtOAc, 1 mL) was added at 0 °C to a near-freezing suspension of tert-butyl (tert-butoxycarbonyl)(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 equivalent) dissolved in DCM (3 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and washed with methyl tert-butyl ether (5 mL). The solvent was transferred and concentrated under reduced pressure to obtain the crude product. The crude product was freeze-dried to obtain 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 a milky white solid.

[1249] 1 H-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).

[1250] 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 Х 4.6 mm) 3.5 μm. Rt (min): 1.340; Area% - 99.13.

[1251] HPLC Method: Mobile Phase A: 0.1% TFA in H2O. Mobile Phase B: Acetonitrile. Flow Rate: 2.0 mL / min. Column: Xbridge C8 (50 x 4.6) mm, 3.5 μm. Rt (min): 2.38; Area % - 98.95.

[1252] Step-2A: tert-butyl (tert-butoxycarbonyl)(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)((1r,4r)-4-methylcyclohexyl)amino)butyl)carbamate

[1253]

[1254] In a solution of 3-(4-(((1R,4R)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 109A (0.1 g, 0.281 mmol, 1 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate (0.162 g, 0.563 mmol, 2 equivalents) dissolved in DMF (5 mL):

[1255] TFA (0.160 g, 1.407 mmol, 4.0 equivalents) was added to DCM (5 mL). After 10 minutes, sodium triacetoxyhydroborate (0.239 g, 1.125 mmol, 4.0 equivalents) was added to this mixture in portions at 0°C. The reaction mixture was stirred at room temperature for 4 hours. The completion of the reaction was confirmed by UPLC analysis, and it was confirmed that 39% of the starting material remained. The reaction mixture was cooled to 0°C, and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate (0.485 g, 1.688 mmol, 6.0 equivalents) was added, followed by the additional addition of TFA (0.160 g, 1.407 mmol, 5.0 equivalents). After 10 minutes, sodium triacetoxyhydroborate (0.507 g, 2.391 mmol, 8.5 equivalents) was added, and the reaction mixture was stirred at room temperature for 16 hours. Completion of the reaction was confirmed by UPLC analysis. The reaction mixture was quenched with ice water (20 mL) and extracted with DCM (20 mL × 3). The bound organic layer was dried with sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash column chromatography (silica gel, 230-400 mesh size) using ethyl acetate / petroleum ether (50%) as the eluent to obtain the desired product as a milky white solid. The product was re-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 obtain 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 a milky white solid.

[1256] 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).

[1257] LCMS: 627.3 (MH), Rt (min): 2.27, Area% - 92.32.

[1258] Step-3A: 3-(4-((4-aminobutyl)((1r,4r)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1259]

[1260] HCl (4M solution of EtOAc, 1 mL) was added at 0°C to a near-freezing solution of 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, 0.064 mmol, 1.0 equivalent) dissolved in DCM (3 mL). The reaction mixture was stirred at room temperature for 2 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc, R ­f~ 0.3 and confirmed by LCMS), the reaction mixture was vacuum distilled to obtain a crude product. The crude product was washed with MTBE (5 mL), the solvent was transferred, dried under reduced pressure, and freeze-dried to obtain the desired product, 3-(4-((4-aminobutyl)((1R,4R)-4-methylcyclohexyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride 109 (0.03 g, 101% yield), as a milky white solid.

[1261] 1 H-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).

[1262] 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 Х 4.6 mm) 3.5 μm. Rt (min): 1.351; Area% - 99.19.

[1263] HPLC Method: Mobile Phase A: 0.1% TFA in H2O. Mobile Phase B: Acetonitrile. Flow Rate: 2.0 mL / min. Column: Xbridge C8 (50 x 4.6) mm, 3.5 μm. Rt (min): 2.418; Area % - 99.64.

[1264]

[1265] Hydrochloride form of Compound 115

[1266]

[1267] Piperidine (338 mg, 4.0 mmol, 20.0 equivalents) was added to a solution of Compound 10 (120 mg, 0.2 mmol, 1.0 equivalent) dissolved in DCM (6.0 mL). The mixture was stirred at room temperature for 17 hours. Afterward, the reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (acetonitrile / H2O: 20%-45%) to obtain Compound 115 (11.4 mg, 13%) as a white solid.

[1268] TLC: DCM / methanol = 10 / 1

[1269] R f (Compound 10) = 0.9

[1270] R f (Compound 115) = 0.1

[1271] LCMS: [C 21 H 26 N4O3] + Calculated value for: 383, Detected value: 383.

[1272] 1 H NMR (400 MHz, DO): 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).

[1273]

[1274] Hydrochloride form of compound 116

[1275]

[1276] HCl (0.2 mL, 2 N, 0.4 mmol, 10.0 equivalents) was added to a solution of Compound 4 (20 mg, 0.04 mmol, 1.0 equivalent) dissolved in MeOH (1 mL). The mixture was stirred at room temperature for 24 hours and concentrated under reduced pressure. The residue was purified with Biotage (C18, acetonitrile / H2O: 30%-60%) to obtain Compound 116 (14.0 mg, 74%) as a white solid.

[1277] TLC: DCM / methanol = 10 / 1

[1278] R f (Compound 4) = 0.5

[1279] R f (Compound 116) = 0.1

[1280] LCMS: [C 21 H 26 N4O3] + Calculated value for: 383, Detected value: 383.

[1281] 1 H NMR (400 MHz, DO): 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).

[1282]

[1283] Hydrochloride form of Compound 123

[1284] 3-(4-((4-amino-3,3-dimethylbutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1285]

[1286] Step-1: 4-Methoxy-2,2-dimethyl-4-oxobutanoic acid

[1287]

[1288] Concentrated H2SO4 (10 mL) was slowly added to a solution of 2,2-dimethylsuccinic acid 1 (100 g, 684 mmol, 1 equivalent) dissolved in methanol (1500 mL, 15-fold volume), and the mixture was stirred at room temperature for 16 hours. When the reaction was complete (TLC analysis, 40% EtOAc in petroleum ether, R ­f ~0.5, confirmed by KMnO4), the reaction mixture was concentrated under vacuum at 30°C. The resulting residue was poured into a saturated NaHCO3 solution (1000 mL) near freezing point and washed with EtOAc (2 × 500 mL). The separated aqueous layer was acidified to pH ~2 with an aqueous HCl (6N) solution and extracted with EtOAc (2 x 800 mL). The combined organic layer was washed with saturated brine (1 x 600 mL), dried with 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.

[1289] 1 H-NMR (400 MHz, DMSO-d6): δ 12.17 (s, 1H), 3.57 (s, 3H), 2.53 (s, 2H), 1.18 (s, 6H).

[1290] Step-2: Methyl 4-amino-3,3-dimethyl-4-oxobutanoate

[1291]

[1292] Thionyl chloride (111 g, 937 mmol, 6 equivalents) was slowly added to a near-freezing solution of 4-Methoxy-2,2-dimethyl-4-oxobutanoic acid 2 (25 g, 156 mmol, 1 equivalent) dissolved in DCM (250 mL, 10-fold volume), and stirred at room temperature for 3 hours. When the reaction was complete (TLC analysis, EtOAc 20% in petroleum ether, R ­f ~ 0.4, identified as methyl ester), the reaction mixture was distilled under vacuum and completely dried under reduced pressure.

[1293] The resulting chloride mixture was dissolved in THF (250 mL) and slowly added to a solution of NH3 (0.5 M solution in THF, 375 mL) pre-cooled to -78°C. The reaction mixture was raised to room temperature and stirred for 30 minutes. Upon completion of the reaction (TLC analysis, 60% EtOAc in petroleum ether, R ­f (confirmed as ~0.2), the reaction mixture was concentrated, and the resulting residue was poured into a saturated NaHCO3 solution (500 mL) near freezing point and extracted with EtOAc (2 × 500 mL). The bound organic layer was washed with saturated brine (500 mL), dried with 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.

[1294] 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)

[1295] Step-3: 4-Amino-3,3-dimethylbutan-1-ol

[1296]

[1297] LiAlH4 (2M in THF, 82 mL, 163 mmol, 2 equivalents) was slowly added over 30 minutes at 0°C to Methyl 4-amino-3,3-dimethyl-4-oxobutanoate 3 (13 g, 82 mmol, 1 equivalent) dissolved in THF (100 mL, 8-fold). After the addition was complete, the reaction mixture was heated to room temperature and then slowly raised to 65°C and stirred for 2 hours. Upon completion of the reaction (confirmed by TLC analysis, 100% EtOAc, R ­f ~ 0.1), the reaction mixture was cooled to 0°C and slowly quenched with a saturated Na2SO4 solution (30 mL). The precipitated inorganic salt was filtered through a Celite pad and washed with EtOAc (1 × 100 mL). The combined filtrate was dried with Na2SO4, filtered, and concentrated to obtain 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.

[1298] 1 H-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).

[1299] Step-4: tert-Butyl (4-hydroxy-2,2-dimethylbutyl)carbamate

[1300]

[1301] Sodium bicarbonate (7.2 g, 86 mmol, 1.5 equivalents) and Boc2O (14 mL, 57.2 mmol, 1 equivalent) were slowly added at room temperature to a solution of 4-Amino-3,3-dimethylbutan-1-ol 4 (7.5 g, 57.2 mmol, 1 equivalent) dissolved in THF (150 mL, 20-fold) and water (40 mL, 5-fold). The reaction mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 100% EtOAc, R ­f (confirmed as ~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 layer was washed with a saline solution (1 × 100 mL), dried with Na2SO4, and concentrated under reduced pressure to obtain a pale brown liquid. The remaining residue was purified by chromatography using 40-50% EtOAc in petroleum ether (column size: Biotage R snap cartridge, KP-Sil, 50 g, 100-200 silica gel) to obtain tert-butyl (4-hydroxy-2,2-dimethylbutyl) carbamate 5 (5.0 g, 22.85 mmol, 40.0% yield) as a milky white crystalline solid.

[1302] 1 H-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).

[1303] Step-5: tert-Butyl 2-hydroxy-4,4-dimethylpyrrolidine-1-carboxylate

[1304]

[1305] DMSO (1 mL, 14.08 mmol) was slowly added at -78°C to a solution of oxalyl chloride (0.5 mL, 5.52 mmol, 1.2 equivalents) dissolved in DCM (5 mL), and stirred at the same temperature for 15 minutes. To this, tert-butyl (4-hydroxy-2,2-dimethylbutyl)carbamate (1.0 g, 4.6 mmol, 1 equivalent) dissolved in DCM (3 mL) was slowly added over 15 minutes, and stirred at -78°C for 30 minutes. At this stage, N,N-diisopropylethylamine (2.4 mL, 13.81 mmol, 3 equivalents) was slowly added at -78°C, and the temperature was slowly raised to 0°C. The mixture was stirred at 0°C for 30 minutes, and the completion of the reaction was confirmed by TLC analysis (20% EtOAc in petroleum ether, R ­f ~ 0.6, KMnO4). The reaction mixture was diluted with DCM (20 mL), washed with 10% aqueous citric acid solution (1 × 50 mL), water (1 × 30 mL) and brine (1 × 30 mL), dried with Na2SO4, filtered, and concentrated under vacuum at 30°C or lower to obtain tert-Butyl 2-hydroxy-4,4-dimethylpyrrolidine-1-carboxylate (0.95 g, crude product) as a pale yellow liquid, which was subsequently used without purification.

[1306] GCMS: Mass measurement; MS: 215.1; Rt (min): 3.005. Method: HP5_SCAN350_MS.amx

[1307] Step-6: tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylbutyl)carbamate

[1308]

[1309] Trifluoroacetic acid (1.189 mL, 15.43 mmol, 4.0 equivalents) was added at 0°C to a stirred solution of 3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1.0 g, 3.86 mmol, 1.0 equivalents) and tert-butyl 2-hydroxy-4,4-dimethylpyrrolidine-1-carboxylate (0.996 g, 4.63 mmol, 1.2 equivalents) dissolved in CH2Cl2 (10 mL) and DMF (10 mL), and stirred for 10 minutes. Sodium triacetoxyborohydride (3.27 g, 15.43 mmol, 4.0 equivalents) was added in portions at 0°C, and the resulting colorless, turbid reaction product was stirred at room temperature for 16 hours. Once the reaction is complete (TLC and LCMS analysis, 100% EtOAc, R ­f (confirmed as ~0.7), the reaction mixture was quenched with near-freezing water (20 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under vacuum to obtain a pale yellow liquid crude product. The crude product was purified by Isolera chromatography using 75-85% ethyl acetate in petroleum ether (column size: Biotage R snap cartridge, KP-Sil, 100 g, 100-200 silica gel). The fraction was collected and concentrated under vacuum to obtain 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.

[1310] 1H-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.

[1311] Step-7: tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)-2,2-dimethylbutyl)carbamate

[1312]

[1313] Trifluoroacetic acid (0.202 mL, 2.62 mmol, 4.0 equivalents) was added at 0°C to a stirred mixture of tert-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 equivalents) and pentanal 9 (0.564 g, 6.54 mmol, 10.0 equivalents) dissolved in CH2Cl2 (5 mL) and DMF (5 mL), and stirred for 10 minutes. Sodium triacetoxyborohydride (0.555 g, 2.62 mmol, 4.0 equivalents) was added in portions at 0°C, and the resulting colorless, turbid reaction mixture was stirred at room temperature for 16 hours. Once the reaction is complete (TLC and LCMS analysis, 100% EtOAc, R­f (confirmed as ~0.8), the reaction mixture was quenched with near-freezing water (20 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under vacuum to obtain a crude product as a pale yellow liquid. The crude product was purified by preparative HPLC (Column: X-Bridge-c18 19.1X250, Mobile phase: 0.1% FA / ACN in water, Flow rate: 15 mL / min) to obtain 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 a milky white solid.

[1314] 1 H-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).

[1315] LCMS: 529.3 (M+H), Rt (min): 2.715, Area% - 99.791.

[1316] Step-8: 3-(4-((4-amino-3,3-dimethylbutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione hydrochloride

[1317]

[1318] HCl (4 M in ethyl acetate, 5 mL, 165 mmol) was added at 0°C to a stirred suspension of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)-2,2-dimethylbutyl)carbamate 10 (80 mg, 0.151 mmol) dissolved in dichloromethane (2 mL), and the mixture was stirred at room temperature for 16 hours. When the reaction was complete (TLC analysis, 100% EtOAc, R ­f (confirmed as ~0.1), the reaction mixture was concentrated under vacuum. The obtained solid was dissolved in water (10 mL) and washed with MTBE (10 mL), and the aqueous layer was freeze-dried to obtain 3-(4-((4-amino-3,3-dimethylbutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl 123 (70 mg, 0.145 mmol, 96% yield) as a light brown solid.

[1319] 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)

[1320] LCMS: 429.3 (M+H). Methods: Column: X-BRIDGE C8 (50 X 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.

[1321] HPLC: Method: Mobile phase: A: Ammonium bicarbonate 10 mM in water, Mobile phase: B: Acetonitrile, Flow rate: 1.0 mL / min, Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm, RT: 5.333 min, Area: 96.027%.

[1322]

[1323] Hydrochloride form of Compound 124

[1324] 3-(4-((4-amino-3,3-dimethylbutyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1325]

[1326] Step-1: tert-butyl (4-((tert-butyldimethylsilyl)oxy)butyl)carbamate

[1327]

[1328] Imidazole (4.5 g, 66.0 mmol, 2.5 equivalents) and TBS-Cl (4.78 g, 31.7 mmol, 1.5 equivalents) were added at 0°C to a solution of tert-butyl (4-hydroxybutyl)carbamate (5.0 g, 26.4 mmol, 1.0 equivalent) dissolved in dichloromethane (100 mL), and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 20% EtOAc / petroleum ether, R ­f~ 0.5, confirmed by KMnO4), the reaction mixture was quenched with H2O (50 mL) and extracted with dichloromethane (1 × 50 mL). The organic layer was dried with Na2SO4 and concentrated under vacuum to obtain the crude product. The crude product was purified by chromatography using 10-15% EtOAc in petroleum ether (column size: Biotage R snap cartridge, KP-Sil, 100 g, 230-400 silica gel) to obtain tert-butyl (4-((tert-butyldimethylsilyl)oxy)butyl)carbamate (8.0 g, 26.3 mmol, 99% yield) as a colorless liquid.

[1329] 1 H-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).

[1330] Step-2: tert-butyl (tert-butoxycarbonyl)(4-((tert-butyldimethylsilyl)oxy)butyl)carbamate

[1331]

[1332] n-BuLi (2.5 M solution in hexane, 12.65 mL, 31.6 mmol, 1.2 equivalents) was added at 0°C to a stirred solution of tert-butyl (4-((tert-butyldimethylsilyl)oxy)butyl)carbamate 2 (8.0 g, 26.4 mmol, 1.0 equivalents) dissolved in THF (80 mL), and stirred at 0°C for 15 minutes. A solution of (Boc)2O (7.34 mL, 31.6 mmol, 1.2 equivalents) dissolved in THF (20 mL) was added to the reaction mixture at the same temperature. The resulting reaction mixture was heated to room temperature and stirred for 1 hour. When the reaction was complete (TLC analysis, 10% EtOAc / petroleum ether, R ­f ~ 0.7, confirmed by KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (2 × 100 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain tert-butyl (tert-butoxycarbonyl)(4-((tert-butyldimethylsilyl)oxy)butyl)carbamate 3 (11.0 g, 27.0 mmol, 102% yield) as a pale yellow liquid, which was subsequently used without purification.

[1333] 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).

[1334] Step-3: tert-butyl (tert-butoxycarbonyl)(4-hydroxybutyl)carbamate

[1335]

[1336] To a stirred solution of tert-butyl (tert-butoxycarbonyl)(4-((tert-butyldimethylsilyl) oxy)butyl)carbamate 3 (10 g, 24.77 mmol, 1.0 equivalent) dissolved in 160 mL of THF at room temperature, TBAF (24.77 mL, 24.77 mmol, 1.5 equivalents) was added, and the mixture was stirred overnight at the same temperature. Upon completion of the reaction (TLC analysis, 20% EtOAc in petroleum ether, R ­f ~ 0.2, confirmed by KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain the mixture as a dark yellow liquid. The mixture 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-butyl (tert-butoxycarbonyl)(4-hydroxybutyl)carbamate 4 (6 g, 20.53 mmol, 83% yield) as a yellow liquid.

[1337] 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)

[1338] Step-4: tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate

[1339]

[1340] DMSO (2.95 mL, 41.5 mmol, 4.0 equivalents) dissolved in CH2Cl2 (5 mL) was added to a solution of oxalyl chloride (1.8 mL, 20.73 mmol, 2.0 equivalents) and CH2Cl2 (30 mL) at -78°C and stirred for 15 minutes at the same temperature. To this mixture, a solution of tert-butyl (tert-butoxycarbonyl)(4-hydroxybutyl)carbamate 4 (3 g, 10.37 mmol, 1.0 equivalent) dissolved in CH2Cl2 (10 mL) was added at -78°C. The reaction mixture was stirred at -78°C for 30 minutes, and triethylamine (9 mL, 62.2 mmol, 6.0 equivalents) was added dropwise at -78°C. Afterward, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 45% EtOAc in petroleum ether, R ­f ~ 0.6, confirmed by 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 with Na2SO4, and concentrated under reduced pressure (below 30°C). The mixture was co-distilled with toluene to obtain tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (3.2 g, 11.14 mmol, 107% yield) as a yellow liquid, which was subsequently used without purification.

[1341] 1 H-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),

[1342] 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

[1343]

[1344] Trifluoroacetic acid (0.202 mL, 2.62 mmol, 4.0 equivalents) was added to a stirred mixture of tert-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 equivalent) and tert-butyl (tert-butoxycarbonyl)(4-oxobutyl)carbamate 5 (0.376 g, 1.308 mmol, 2.0 equivalents) dissolved in CH2Cl2 (5 mL) and DMF (5.00 mL), and the mixture was stirred at 0°C for 10 minutes. Sodium triacetoxyborohydride (0.555 g, 2.62 mmol, 4.0 equivalents) was added to this mixture in portions at 0°C, and the resulting colorless, turbid mass was stirred at room temperature for 16 hours. When the reaction was complete (TLC analysis, 100% EtOAc, R ­f(confirmed as ~0.7), the reaction mixture was quenched with near-freezing water (20 mL) and extracted with DCM (2 × 50 mL). The combined organic layer was dried with Na2SO4 and concentrated under vacuum to obtain the crude product. The crude product was purified by preparative HPLC (Column: X-Bridge-c18 19.1 × 250, Mobile phase: 0.1% FA / ACN in water, Flow rate: 15 mL / min) to obtain 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 9 (75 mg, 0.100 mmol, 15.28% yield) as a white solid.

[1345] 1 H-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).

[1346] LCMS: 730.4 (M+H). Method: Atlantis dC18 (50 Х 4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: Acetonitrile, Flow rate: 1.5 ml / min.

[1347] HPLC: Method: Mobile phase A: 0.1% TFA in water, Mobile phase B: Acetonitrile, Flow rate: 2.0 ml / min. Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm. RT: 5.166 min, Area: 97.332 %

[1348] Step-6: 3-(4-((4-amino-3,3-dimethylbutyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1349]

[1350] HCl (4M solution in dioxane, 5 mL) was added at 0°C to a stirred solution of 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 9 (70 mg, 0.096 mmol, 1.0 equivalent) dissolved in CH2Cl2 (3 mL), and the mixture was stirred at room temperature for 2 hours. The progress of the reaction was monitored by LCMS. The reaction mixture was concentrated under vacuum at 40°C or below to obtain a pale yellow solid. The obtained solid was dissolved in water (10 mL), washed with MTBE (2 × 10 mL), and the aqueous layer was freeze-dried to obtain 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 a milky white solid.

[1351] 1H-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),

[1352] LCMS: 430.2 (M+H). Methods: Column: Atlantis dC18 (50 x 4.6) mm, 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: Acetonitrile, Flow rate: 1.5 ml / min.

[1353] HPLC: Method: Mobile phase: 0.1% TFA in water, Mobile phase B: Acetonitrile, Flow rate: 2.0 ml / min. Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm. RT: 1.407 min, Area: 96.954 %.

[1354]

[1355] Hydrochloride form of Compound 125

[1356] 3-(4-((5-amino-3,3-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1357]

[1358] Step-1: 5-ethoxy-3,3-dimethyl-5-oxopentanoic acid

[1359]

[1360] Sodium ethoxide (0.479 g, 7.03 mmol, 0.1 equivalent) was slowly added to a near-freezing solution of 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione 1 (10 g, 70.3 mmol, 1 equivalent) dissolved in ethanol (50 mL, 5 times the volume) and stirred at 85°C for 16 hours. Once the reaction was complete (confirmed by LCMS analysis), the reaction mixture was distilled under reduced pressure. The resulting residue was poured into a near-freezing saturated NaHCO3 solution (200 mL) and washed with EtOAc (2 × 50 mL). The separated aqueous layer was further acidified with an aqueous hydrochloric acid solution (6N, 40 mL, pH ~2) and extracted with EtOAc (2 × 50 mL). The combined organic extract was washed with brine (200 mL), dried with 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.

[1361] 1 H-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).

[1362] Step-2: ethyl 5-amino-3,3-dimethyl-5-oxopentanoate

[1363]

[1364] Thionyl chloride (15 mL, 206 mmol, 6 equivalents) was slowly added to a near-freezing solution of 5-ethoxy-3,3-dimethyl-5-oxopentanoic acid 2 (6 g, 31.9 mmol, 1 equivalent) dissolved in DCM (60 mL, 10-fold volume), followed by the addition of a catalytic amount of DMF (2 drops) and stirring at room temperature for 3.5 hours. Upon completion of the reaction (TLC analysis, petroleum ether / EtOAc 30%, R ­f ~ 0.8, identified as methyl ester), the reaction mixture was distilled under reduced pressure and dried.

[1365] The resulting chloride mixture was dissolved in THF (30 mL) and slowly added to a solution of ammonia (0.5 M in THF, 90 mL, 4159 mmol) pre-cooled to -78°C. The reaction mixture was heated to room temperature and stirred for 30 minutes. When the reaction was complete (TLC analysis, 30% EtOAc in petroleum ether, R ­f (confirmed as ~0.2), the reaction mixture was concentrated, the residue was poured into a saturated NaHCO3 solution (150 mL) near the freezing point, and extracted with EtOAc (2 × 50 mL). The bound organic layer was washed with brine (150 mL), dried with 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.

[1366] 1 H-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).

[1367] LCMS: 188.1 (M+H), Rt (min): 1.694, Area%: 99.678.

[1368] Step 3: 5-amino-3,3-dimethylpentan-1-ol

[1369]

[1370] LAH (2M in THF, 23.50 mL, 47.0 mmol, 2 equivalents) was slowly added over 5 minutes at 0°C to a solution of ethyl 5-amino-3,3-dimethyl-5-oxopentanoate 3 (4.4 g, 23.50 mmol, 1 equivalent) dissolved in THF (15 mL, 8-fold). After the addition was complete, the reaction mixture was warmed to room temperature, then slowly heated to 65°C and stirred for 2 hours. When the reaction was complete (TLC analysis, EtOAc, R ­f (confirmed as ~0.2), the reaction mixture was cooled to 0°C and slowly quenched with a saturated Na2SO4 solution (60 mL), the precipitated salt was filtered through a Celite pad and washed with EtOAc (50 mL). The combined filtrate was dried with Na2SO4, filtered, and concentrated to obtain 5-amino-3,3-dimethylpentan-1-ol 4 (2 g, 13.30 mmol, 56.6% yield) as a brown liquid.

[1371] 1 H-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).

[1372] LCMS: 132.2 (M+H), Rt (min): 0.603, Area%: 87.229.

[1373] Step-4: tert-butyl (5-hydroxy-3,3-dimethylpentyl)carbamate

[1374]

[1375] Boc-anhydride (3.54 mL, 15.24 mmol, 1 equivalent) was slowly added at room temperature to a stirred suspension of 5-amino-3,3-dimethylpentan-1-ol 4 (2 g, 15.24 mmol, 1 equivalent) and sodium bicarbonate (2.56 g, 30.5 mmol, 2 equivalents) dissolved in a mixture of THF (30 mL, 15-fold) and water (2 mL, 1-fold). The resulting reaction mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (confirmed by LCMS analysis), anhydrous Na2SO4 was added to the reaction mixture, filtered, and washed with EtOAc (20 mL). The filtrate was concentrated under reduced pressure to obtain a yellow liquid crude product. The crude product was purified by column chromatography using 20-25% EtOAc in petroleum ether (Biotage R snap cartridge, KP-Sil, 100 g, 100-200 silica gel) to obtain tert-butyl (5-hydroxy-3,3-dimethylpentyl)carbamate 5 (1.7 g, 7.33 mmol, 48.1% yield) as a yellow liquid.

[1376] 1 H-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).

[1377] LCMS: 132.3 (M-Boc), Rt (min): 2.14, Area%: 99.812.

[1378] Step-5: tert-butyl (5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate

[1379]

[1380] TBS-Cl (1.329 g, 8.82 mmol, 1.2 equivalents) was added at 0°C to a solution of tert-butyl (5-hydroxy-3,3-dimethylpentyl)carbamate 5 (1.7 g, 7.35 mmol, 1.0 equivalent) and imidazole (1.251 g, 18.37 mmol, 2.5 equivalents) dissolved in DCM (20 mL), and the mixture was stirred at room temperature for 13 hours. Upon completion of the reaction (TLC analysis, 30% EtOAc in petroleum ether, R ­f ~ 0.9, confirmed by KMnO4), the reaction was quenched with water (50 mL) and extracted with DCM (1 × 30 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain tert-butyl (5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate 6 (2.5 g, 7.22 mmol, 98% yield) as a yellow liquid.

[1381] 1 H-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).

[1382] LCMS: 246.2 (M-Boc), Rt (min): 3.581, Area%: 99.821.

[1383] Step-6: tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate

[1384]

[1385] n-BuLi (2.5 M in hexane, 3.47 mL, 8.68 mmol, 1.2 equivalents) was added at 0°C to a solution of tert-butyl (5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate 6 (2.5 g, 7.23 mmol) dissolved in THF (25 mL, 10-fold). The reaction mixture was stirred at 0°C for 15 minutes. A solution of (Boc)2O (1.316 mL, 5.67 mmol, 1.2 equivalents) dissolved in THF (10 mL) was added to the reaction mixture at the same temperature. The resulting reaction mixture was raised to room temperature and stirred for 1 hour. Upon completion of the reaction (TLC analysis, 5% EtOAc in petroleum ether, R ­f ~ 0.8, confirmed by KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with DCM (2 × 30 mL). The bound organic layer was dried with 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.

[1386] 1 H-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).

[1387] LCMS: 246.3 (M-200), Rt (min): 4.602, Area%: 98.718.

[1388] Step-7: tert-butyl (tert-butoxycarbonyl)(5-hydroxy-3,3-dimethylpentyl)carbamate

[1389]

[1390] TBAF (11.78 mL, 1 M solution in THF, 11.78 mmol, 1.5 equivalents) was added to a solution of tert-butyl-(tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-3,3-dimethylpentyl)carbamate 7 (3.5 g, 7.85 mmol) dissolved in THF (35 mL, 10-fold volume), and the mixture was stirred at room temperature for 16 hours. Upon completion of the reaction (TLC analysis, 30% EtOAc in petroleum ether, R ­f ~ 0.2, confirmed by KMnO4), the reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 × 30 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain a yellow liquid crude product. The crude product was purified by column chromatography using 12-15% EtOAc in petroleum ether (Biotage R snap cartridge, KP-Sil, 50 g, 60-120 silica gel) to obtain tert-butyl (tert-butoxycarbonyl)(5-hydroxy-3,3-dimethylpentyl)carbamate 8 (1.4 g, 4.22 mmol, 53.8% yield) as a pale yellow liquid.

[1391] 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).

[1392] Step-8: tert-butyl (tert-butoxycarbonyl)(3,3-dimethyl-5-oxopentyl)carbamate

[1393]

[1394] A solution of oxalyl chloride 8 (0.317 mL, 3.62 mmol, 1.2 equivalents) dissolved in DCM (10 mL) was slowly added to a DMSO (0.514 mL, 7.24 mmol, 2.4 equivalents) solution at -78°C under a nitrogen atmosphere and stirred for 15 minutes at the same temperature. To this, a solution of tert-butyl (tert-butoxycarbonyl)(5-hydroxy-3,3-dimethylpentyl)carbamate (1 g, 3.02 mmol, 1 equivalent) dissolved in DCM (10 mL) was slowly added over 5 minutes. The reaction mixture was stirred at -78°C for 30 minutes, and TEA (2.103 mL, 15.09 mmol, 5 equivalents) was slowly added at -78°C. Once the addition was complete, the reaction mixture was slowly raised to 0°C. Once the reaction is complete (TLC analysis, 30% EtOAc in petroleum ether, R ­f ~0.8, confirmed by KMnO4), the reaction mixture was diluted with DCM (10 mL) and washed with 10% aqueous citric acid solution (50 mL), water (50 mL), and brine (50 mL). The bound organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure at 30°C or below 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 subsequently used without purification.

[1395] 1 H-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).

[1396] 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

[1397]

[1398] Sodium triacetoxyborohydride (1.969 g, 9.29 mmol, 4 equivalents) was added to a solution of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate Int-4 (1 g, 2.323 mmol), tert-butyl (tert-butoxycarbonyl)(3,3-dimethyl-5-oxopentyl)carbamate 9 (930 mg, 2.82 mmol, 1.2 equivalents), and TFA (0.716 mL, 9.29 mmol, 4 equivalents) dissolved in a mixture of dichloromethane (10 mL, 10-fold) and DMF (10 mL, 10-fold), and the mixture was stirred at room temperature for 16 hours. TLC analysis (EtOAc, R ­f~ 0.7) and LCMS results showed that although a product was formed, the starting material was not completely consumed. The reaction mixture was diluted with DCM (15 mL), washed with water (2 × 30 mL) and brine (30 mL), dried with anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by preparative HPLC (HCOOH:ACN method) and freeze-dried to obtain 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 10 (70 mg, 0.094 mmol, 4.04% yield) as a white solid.

[1399] 1 H-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).

[1400] LC-MS: 744.5 (M+H), Rt (min): 3.010, Area%: 99.774.

[1401] Step-10: 3-(4-((5-amino-3,3-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1402]

[1403] HCl (4M solution in dioxane, 1 mL, 4.00 mmol) was added at 0°C to a stirred suspension of 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 10 (70 mg, 0.094 mmol) dissolved in dichloromethane (3 mL), and the mixture was stirred at room temperature for 2 hours. When the reaction was complete (TLC analysis, EtOAc, R ­f (confirmed as ~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 the aqueous layer was freeze-dried to obtain 3-(4-((5-amino-3,3-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl 125 (12 mg, 0.023 mmol, 24.65% yield) as a milky white solid.

[1404] 1H-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).

[1405] LCMS: 444.3 (M+H). Method: Mobile phase A: 0.1% formic acid in H2O. Mobile phase B: ACN. Flow rate: 1.5 ml / min. Column: Atlantis dC18 (50 x 4.6) 5 μm. Rt (min): 1.035; Area%: 99.826.

[1406] HPLC: Method: Mobile phase A: 0.1% formic acid in H2O. Mobile phase B: Methanol. Flow rate: 1.0 mL / min. Column: X-Bridge C8 (50 Х 4.6) mm, 3.5 μm. Rt (min): 2.679; Area%: 98.409.

[1407]

[1408] Hydrochloride form of Compound 126

[1409] 3-(4-((5-amino-4,4-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1410]

[1411] Step-1: 5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentanenitrile

[1412]

[1413] A solution of LDA (2M in THF / heptane / ethylbenzene, 5.92 mL, 11.85 mmol, 1.5 equivalents) dissolved in THF (10 mL) was cooled to -78°C, and isobutyronitrile 2 (0.764 g, 11.06 mmol, 1.4 equivalents) was added over 5 minutes. The reaction mixture was heated to -40°C, stirred for 30 minutes, and then cooled back to -78°C. Then, (3-bromopropoxy)(tert-butyl)dimethylsilane 1 (2.0 g, 7.90 mmol, 1.0 equivalent) was slowly added to the reaction mixture, heated to 0°C, and stirred for 1 hour. Upon completion of the reaction (TLC and UPLC analysis, 10% EtOAc in petroleum ether, R ­f ~ 0.6, confirmed by KMnO4), the reaction mixture was quenched with 1 N HCl (1 x 10 mL) and extracted with MTBE (2 × 50 mL). The bound organic layer was washed with saturated NaHCO3 (1 x 20 mL) and brine (1 x 20 mL), dried with Na2SO4, and concentrated under reduced pressure to obtain 5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentanenitrile 3 (2.2 g, 9.11 mmol, 115% yield) as a pale yellow liquid, which was subsequently used without further processing.

[1414] 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)

[1415] Step-2: tert-butyl (5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl)carbamate

[1416]

[1417] A stirred solution of 5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentanenitrile 3 (1.0 g, 4.14 mmol, 1.0 equivalent) dissolved in dry MeOH (30 ml) was cooled to 0 °C, and (Boc)2O (1.923 mL, 8.28 mmol, 2.0 equivalent) and NiCl2.6H2O (0.984 g, 4.14 mmol, 1.0 equivalent) were added. Then, NaBH4 (1.097 g, 29.0 mmol, 7.0 equivalent) was added cautiously in a divided manner. The reaction was exothermic with bubble generation. The reaction mixture was heated to room temperature and stirred overnight. When the reaction was complete (TLC analysis, 5% EtOAc / petroleum ether, R ­f ~ 0.3, confirmed by 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 a saturated NaHCO3 solution (1 x 20 mL), the organic layer was dried with Na2SO4, and concentrated under reduced pressure to obtain the crude product 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 subsequently used without purification.

[1418] 1 H-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).

[1419] Step-3: tert-butyl(tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl) carbamate

[1420]

[1421] n-BuLi (3.47 mL, 8.68 mmol, 1.2 equivalents, 2.5 M solution in hexane) was added to a stirred solution of tert-butyl (5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl)carbamate 4 (2.5 g, 7.23 mmol, 1.0 equivalent) dissolved in 50 mL of THF at 0 °C. The reaction mixture was stirred at 0 °C for 15 minutes. A solution of (Boc)2O (2.015 mL, 8.68 mmol, 1.2 equivalents) dissolved in 10 mL of THF was added to the reaction mixture at the same temperature. The reaction mixture was heated to room temperature and stirred for 1 hour. When the reaction was complete (TLC analysis, 10% EtOAc in petroleum ether, R ­f ~ 0.7, confirmed by KMnO4), the reaction mixture was quenched with water (50 mL) and extracted with DCM (2 × 100 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl)carbamate 5 (3.2 g, 6.82 mmol, 94% yield) as a pale yellow liquid, which was subsequently used without purification.

[1422] 1 H-NMR (400 MHz, DMSO-d6): δ 3.59-3.54 (m, 2H), 2.73-2.76 (m, 2H), 1.42 (s, 18H), 1.24-1.36 (m, 2H), 1.07-1.13 (m, 2H), 0.89 (s, 9H), 0.80 (s, 6H), 0.02 (s, 6H).

[1423] Step-4: tert-butyl (tert-butoxycarbonyl)(5-hydroxy-2,2-dimethylpentyl)carbamate

[1424]

[1425] TBAF (10.8 mL, 10.8 mmol, 1.5 equivalents) was added to a solution of tert-butyl (tert-butoxycarbonyl)(5-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpentyl)carbamate (3.2 g, 7.18 mmol, 1.0 equivalent) dissolved in 60 mL of THF at room temperature, and the reaction mixture was stirred overnight at room temperature. When the reaction was complete (TLC analysis, 20% EtOAc / petroleum ether, R ­f ~0.2, confirmed by KMnO4), the reaction mixture was quenched with water (20 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under reduced pressure to obtain a crude product as a dark yellow liquid. The crude product was purified using Isolera (Biotage R snap cartridge, KP-Sil, 100 g, 100-200 silica gel) containing 15-20% EtOAc in ether. The fractions were collected and concentrated under vacuum to obtain tert-butyl (tert-butoxycarbonyl)(5-hydroxy-2,2-dimethylpentyl)carbamate 6 (1.6 g, 4.82 mmol, 67.1% yield) as a colorless liquid.

[1426] 1 H-NMR (400 MHz, DMSO-d6): δ 4.35 (t, J = 5.20 Hz, 1H), 3.36-3.39 (m, 2H), 3.32-3.35 (m, 2H), 1.47 (s, 18H), 1.36-1.40 (m, 2H), 1.12-1.16 (m, 2H), 0.79 (s, 6H).

[1427] Step-5: tert-butyl (tert-butoxycarbonyl)(5-oxopentyl)carbamate

[1428]

[1429] DMSO (1.370 mL, 19.31 mmol, 4.0 equivalents) dissolved in CH2Cl2 (5 mL) was added to a solution of CH2Cl2 (20 mL) and oxalyl chloride (0.845 mL, 9.65 mmol, 2.0 equivalents) and stirred at -78°C for 15 minutes. To this mixture, a solution of tert-butyl (tert-butoxycarbonyl)(5-hydroxy-2,2-dimethylpentyl)carbamate 6 (1.6 g, 4.83 mmol, 1.0 equivalent) dissolved in CH2Cl2 (5 mL) was added at -78°C. The reaction mixture was stirred at -78°C for 30 minutes, and triethylamine (4.04 mL, 29.0 mmol, 6.0 equivalents) was added dropwise at -78°C. Then, the reaction mixture was slowly heated to 0°C and stirred for 30 minutes. Once the reaction is complete (TLC analysis, 20% EtOAc / petroleum ether, R ­f ~0.6, confirmed by KMnO4), the reaction mixture was diluted with DCM (40 mL), washed with 10% aqueous citric acid solution (1 × 40 mL) and water (1 × 40 mL), dried with Na2SO4, and concentrated under vacuum (at 30°C or below) to obtain tert-butyl (tert-butoxycarbonyl)(2,2-dimethyl-5-oxopentyl)carbamate 7 (1.55 g, 3.42 mmol, 70.8% yield) as a pale yellow liquid, which was subsequently used without purification.

[1430] 1H-NMR (400 MHz, DMSO-d6): δ 9.68 (s, 1H), 3.39 (s, 2H), 2.42-2.50 (m, 2H), 1.44 (m, 2H), 1.42 (s, 18H), 0.79 (s, 6H)

[1431] Step-6: tert-butyl (tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylpentyl)carbamate

[1432]

[1433] Trifluoroacetic acid (0.358 mL, 4.65 mmol, 4.0 equivalents) was added at 0°C to a stirred mixture of tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)butyl)carbamate 8 (0.5 g, 1.161 mmol, 1.0 equivalent) and tert-butyl (tert-butoxycarbonyl)(2,2-dimethyl-5-oxopentyl)carbamate 7 (0.765 g, 2.323 mmol, 2.0 equivalents) dissolved in CH2Cl2 (10 mL) and DMF (10 mL), and stirred for 10 minutes. Sodium triacetoxyborohydride (0.985 g, 4.65 mmol, 4.0 equivalents) was added in portions, and the resulting colorless, turbid mass was stirred at room temperature for 16 hours. When the reaction was complete (TLC analysis, 100% EtOAc, R ­f(confirmed as ~0.7), the reaction mixture was quenched with water near freezing point (1 × 20 mL) and extracted with DCM (2 × 50 mL). The bound organic layer was dried with Na2SO4 and concentrated under vacuum to obtain a pale yellow liquid crude product. The crude product 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 to obtain tert-butyl (tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylpentyl)carbamate 9 (0.25 g, 0.335 mmol, 28.9% yield) as a white solid.

[1434] 1 H-NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 7.35-7.39 (m, 1H), 7.06-7.19 (m, 2H), 6.77-6.79 (m, 1H), 5.09-5.13 (m, 1H), 4.27-4.42 (m, 2H), 3.10-3.21 (m, 4H), 2.89-2.90 (m, 4H), 2.67-2.68 (m, 6H), 1.44 (s, 18H), 1.43 (m, 4H), 1.35 (s, 9H), 1.24 (m, 2H), 0.75 (s, 6H),

[1435] LCMS: 744.5 (M+H). Method: Atlantis dC18 (50 x 4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: Acetonitrile, Flow rate: 1.5 ml / min

[1436] HPLC: Method: Mobile Phase A: 0.1% FA in water, Mobile Phase B: Acetonitrile, Flow Rate: 2.0 ml / min. Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm. RT: 6.191 min, Area: 96.037 %

[1437] Step-7: 3-(4-((5-amino-4,4-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione dihydrochloride

[1438]

[1439] HCl (4M solution in dioxane, 10 ml) was added at 0°C to a solution of tert-butyl (tert-butoxycarbonyl)(5-((4-((tert-butoxycarbonyl)amino)butyl)(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2,2-dimethylpentyl)carbamate 9 (0.15 g, 0.202 mmol, 1.0 equivalent) dissolved in dichloromethane (10 ml), and the mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by LCMS. The reaction mixture was concentrated under vacuum at 40°C or below to obtain a pale yellow solid. The obtained solid was dissolved in water (10 ml) and washed with MTBE (1 × 10 ml), and the aqueous layer was freeze-dried to obtain 3-(4-((5-amino-4,4-dimethylpentyl)(4-aminobutyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 2HCl (80 mg, 0.148 mmol, 73.4% yield) as a pale yellow solid.

[1440] 1H-NMR (400 MHz, DMSO-d6): δ 10.99 (s, 1H), 7.94 (s, 6H), 7.43 (m, 1H), 7.25-7.28 (m, 2H), 5.11-5.15 (m, 1H), 4.12-4.50 (m, 2H), 3.20-3.26 (m, 4H), 2.90-2.98 (m, 1H), 2.76-2.89 (m, 2H), 2.64-2.68 (m, 3H), 2.02-2.05 (m, 1H), 1.54 (m, 4H), 1.39 (m, 2H), 1.24-1.26 (m, 3H), 0.93 (s, 6H),

[1441] LCMS: 444.4 (M+H). Methods: Column: Atlantis dC18 (50 Х 4.6 mm) 5 μm, Mobile phase: A: 0.1% FA in H2O, Mobile phase: B: Acetonitrile, Flow rate: 1.5 ml / min.

[1442] HPLC: Method: Mobile phase A: 0.1% TFA in water, Mobile phase B: Methanol, Flow rate: 1.0 ml / min. Column: X-Bridge C8 (50 x 4.6) mm, 3.5 μm. RT: 3.466 min, Area: 95.684 %.

[1443]

[1444] Compound 143

[1445] 1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)-3-methylurea

[1446]

[1447] TEA (0.077 ml, 0.549 mmol, 1.1 equivalents) was added to a solution of 3-(4-((4-aminobutyl)(pentyl)amino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione, HCl (200 mg, 0.499 mmol, 1 equivalent) and N-methyl-1H-imidazole-1-carboxamide (62.5 mg, 0.499 mmol, 1 equivalent) dissolved in DCM (4 ml), and the reaction mixture was stirred at room temperature for 12 hours. When the reaction was complete (confirmed by UPLC), the reaction mixture was concentrated under vacuum to obtain the crude product, which was purified by preparative HPLC (column: XBridge C8 - 150; method: HCl and ACN, flow rate: 20 ml / min) to obtain 1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)(pentyl)amino)butyl)-3-methylurea (45 mg, 0.098 mmol, 19.56% yield) as a white solid.

[1448] 1 H-NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H), 7.61 (s, 2H), 5.13 (q, J = 4.80 Hz, 1H), 4.74 (s, 2H), 2.96-2.88 (m, 3H), 2.65 (q, J = 17.60) Hz, 1H), 2.43 (q, J = 4.00 Hz, 1H), 2.08 (t, J = 5.60 Hz, 1H), 1.37 (t, J = 6.00 Hz, 5H), 1.22 (d, J = 2.80 Hz, 5H), 0.81 (t, J = 6.80 Hz, 3H).

[1449] LCMS: 458.9 (M+H), Method: Mobile phase: A: 0.1% formic acid in H2O, B: ACN; Column...

Claims

A pharmaceutical composition for treating adenoid cystic carcinoma (ACC) comprising the following compound (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof as an active ingredient. (I) In the above chemical formula (I), R 1 C1-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C3-C8 cycloalkyl or C5-C 10 Bicycloalkyl, each comprising one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl, and optionally substituted C6-C 10 Aryl, C1-C3 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C 10 Optionally substituted with a bicycloalkyl or an optionally substituted C3-C6 heterocycloalkyl; X is a bond or optionally substituted with one or more -(CH2) groups, OH, O-C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl, or C1-C6 alkyl. n - and, C1-C6 alkyl groups can form C3-C6 spiroalkyl rings with bonded atoms, and -(CH2) n - The group may include 0-1 double or triple bonds; n is an integer between 1 and 6; A is a bond, C4-C8 cycloalkyl, C5-C 10 Bicycloalkyl, C5-C 10 Each is a heterobicycloalkyl or C3-C8 heterocycloalkyl, optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl; Y is bonded or -(CH2) m - and; m is an integer of 1 or 2; Z is H or a halogen; R 2 is -NR 12 R 5 , -NHC(O)R 6 , -NHC(O)NR 8 (R 9 ), -C(O)NHR 5 , a 5-membered or 6-membered heteroaryl, or a C3-C8 heterocycloalkyl or C5-C8 heterocycloalkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl. 10 It is a heterobicycloalkyl ring; R 5 is H, C1-C5 alkyl, C3-C7 cycloalkyl, 5- or 6-membered heteroaryl, C5-C 10 Bicycloalkyl, (C3-C6 cycloalkyl)-C1-C3 alkyl, (C3-C6 heterocycloalkyl)-C1-C3 alkyl, (C6-C 10 (C1-C3 alkyl, (C1-C5-heteroaryl)-C1-C3 alkyl, or C3-C7 heterocycloalkyl, each comprising 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 Optionally replaced with ); R 6 is a C1-C6 alkyl, C3-C6 cycloalkyl, or C3-C6 heterocycloalkyl, each comprising one or more halogens, a C1-C3 haloalkyl, an optionally substituted C3-C6 cycloalkyl, or NR 8 (R 9 Optionally replaced with ); R 8 and R 9 Each forms a 4-6 member heterocyclic ring that is independently H, a substituted C1-C3 alkyl, or optionally substituted with one or more halogens or C1-C3 haloalkyls together with the nitrogen to which they are bonded; R 12 is H, C1-C5 alkyl, or R 5 and each having one or more halogens, C1-C3 haloalkyl, OH, O-(C1-C3 alkyl), O-(C1-C3 haloalkyl) or CN, together with the nitrogen bonded thereto, (i) a C3-C8 cycloalkyl or heterocycloalkyl ring, or (ii) a C5-C 10 It forms a bicycloalkyl or heterobicycloalkyl ring. Each stereocenter of the compound of chemical formula (I) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers; Each double bond of the compound of chemical formula (I) is independently cis or trans. A method for treating adenoid cystic carcinoma (ACC), comprising administering an effective amount of the following compound (I) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof to a subject. (I) In the above chemical formula (I), R 1 C1-C8 alkyl, C3-C8 alkenyl, C3-C8 alkynyl, C3-C8 cycloalkyl or C5-C 10 Bicycloalkyl, each comprising one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl, and optionally substituted C6-C 10 Aryl, C1-C3 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C 10 Optionally substituted with a bicycloalkyl or an optionally substituted C3-C6 heterocycloalkyl; X is a bond or optionally substituted with one or more -(CH2) groups, OH, O-C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl, or C1-C6 alkyl. n - and, C1-C6 alkyl groups can form C3-C6 spiroalkyl rings with bonded atoms, and -(CH2) n - The group may include 0-1 double or triple bonds; n is an integer between 1 and 6; A is a bond, C4-C8 cycloalkyl, C5-C 10 Bicycloalkyl, C5-C 10 Each is a heterobicycloalkyl or C3-C8 heterocycloalkyl, optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl; Y is bonded or -(CH2) m - and; m is an integer of 1 or 2; Z is H or a halogen; R 2 is -NR 12 R 5 , -NHC(O)R 6 , -NHC(O)NR 8 (R 9 ), -C(O)NHR 5 , a 5-membered or 6-membered heteroaryl, or a C3-C8 heterocycloalkyl or C5-C8 heterocycloalkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, CN, C1-C3 haloalkyl, C1-C3 alkyl, or C3-C5 cycloalkyl. 10 It is a heterobicycloalkyl ring; R 5 is H, C1-C5 alkyl, C3-C7 cycloalkyl, 5- or 6-membered heteroaryl, C5-C 10 Bicycloalkyl, (C3-C6 cycloalkyl)-C1-C3 alkyl, (C3-C6 heterocycloalkyl)-C1-C3 alkyl, (C6-C 10 (C1-C3 alkyl, (C1-C5-heteroaryl)-C1-C3 alkyl, or C3-C7 heterocycloalkyl, each comprising 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 Optionally replaced with ); R 6 is a C1-C6 alkyl, C3-C6 cycloalkyl, or C3-C6 heterocycloalkyl, each comprising one or more halogens, a C1-C3 haloalkyl, an optionally substituted C3-C6 cycloalkyl, or NR 8 (R 9 Optionally replaced with ); R 8 and R 9 Each forms a 4-6 member heterocyclic ring that is independently H, a substituted C1-C3 alkyl, or optionally substituted with one or more halogens or C1-C3 haloalkyls together with the nitrogen to which they are bonded; R 12 is H, C1-C5 alkyl, or R 5 and each having one or more halogens, C1-C3 haloalkyl, OH, O-(C1-C3 alkyl), O-(C1-C3 haloalkyl) or CN, together with the nitrogen bonded thereto, (i) a C3-C8 cycloalkyl or heterocycloalkyl ring, or (ii) a C5-C 10 It forms a bicycloalkyl or heterobicycloalkyl ring. Each stereocenter of the compound of chemical formula (I) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers; Each double bond of the compound of chemical formula (I) is independently cis or trans. In paragraph 1 or 2, The compound is represented by the chemical formula (Ia), and X is bonded or -(CH2) n - Phosphorus, pharmaceutical composition or method. (Ia) In paragraph 3, R 1 A pharmaceutical composition or method comprising a C1-C8 alkyl or C3-C8 cycloalkyl, each 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. In paragraph 3, A pharmaceutical composition or method in which A is a 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. In paragraph 3, Y is a pharmaceutical composition or method that is bonded or -CH2-. In paragraph 3, 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 comprising 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 A pharmaceutical composition or method that is optionally substituted with ). In paragraph 3, R 12 is H, C1-C5 alkyl, or R 5 A pharmaceutical composition or method forming a C3-C8 heterocycloalkyl ring optionally substituted with one or more halogens or C1-C3 haloalkyls together with nitrogen combined therewith. In paragraph 1 or 2, The compound is represented by the chemical formula (Ib), and X is bonded or -(CH2) n - Phosphorus, pharmaceutical composition or method. (Ib) In Paragraph 9, R 1 A pharmaceutical composition or method wherein is a C3-C6 alkyl optionally substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl. In Paragraph 9, A pharmaceutical composition or method, wherein A is a C4-C6 cycloalkyl selectively substituted with one or more halogens, OH, O-C1-C3 alkyl, NH2, C1-C3 haloalkyl. In Paragraph 9, Y is a pharmaceutical composition or method that is a combination. In paragraph 9 R 6 is NR 8 (R 9 A pharmaceutical composition or method, wherein the composition is a C1-C6 alkyl that is selectively substituted with ). In paragraph 1 or 2 The compound is represented by the chemical formula (Ic), and X is bonded or -(CH2) n - Phosphorus, pharmaceutical composition or method. (Ic) In Paragraph 14, R 1 A pharmaceutical composition or method wherein the alkyl group is a C1-C6 alkyl group optionally substituted with a C3-C8 cycloalkyl group optionally substituted with a C3-C6 heterocycloalkyl group optionally substituted. In Paragraph 14, A pharmaceutical composition or method, wherein 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, or C1-C3 haloalkyl. In Paragraph 14, Y is a pharmaceutical composition or method that is a combination. In Paragraph 14 R 5 A pharmaceutical composition or method that is H or C1-C3 alkyl. In paragraph 1 or 2, A pharmaceutical composition or method in which compound (I) is selected from Table A of the detailed description of the invention. A pharmaceutical composition for treating adenoid cystic carcinoma (ACC) comprising the following compound (Id) or a pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug thereof as an active ingredient. (Id) In the above chemical formula (Id), R 1 is a C1-C3 alkyl selectively substituted with a C3-C6 cycloalkyl; A is an optionally substituted C4-C6 cycloalkyl; R 2 is a 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-; Each stereocenter of the compound of chemical formula (Id) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers. A method for treating adenoid cystic carcinoma (ACC), comprising administering an effective amount of the following compound (Id) or its pharmaceutically acceptable salt, enantiomer, stereoisomer, or prodrug to a subject. (Id) In the above chemical formula (Id), R 1 is a C1-C3 alkyl selectively substituted with a C3-C6 cycloalkyl; A is an optionally substituted C4-C6 cycloalkyl; R 2 is a 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-; Each stereocenter of the compound of chemical formula (Id) is independently an R-enantiomer, an S-enantiomer, or a mixture of R- and S-enantiomers.

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