Protein degradation targeting chimera and uses thereof

Abstract

The present application provides a protein degradation targeting chimera having a structure shown in formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof, a pharmaceutical composition and application thereof. The protein degradation targeting chimera provided by the present application can effectively degrade RXRs protein, thereby effectively down-regulating the expression of RXRs in tumor cells, and has a good inhibitory effect on various tumor cells (especially various small cell lung cancer cells), and can be used as a potential drug for anti-tumor (especially small cell lung cancer) treatment.

Description

Technical Field

[0001] This invention relates to the field of chemical and pharmaceutical technology, specifically to a protein degradation-targeting chimera and its applications. Background Technology

[0002] Lung cancer is the most common malignant tumor worldwide and the leading cause of cancer death in my country. Based on pathological type, it can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Currently, immunotherapy and targeted therapy for NSCLC have made groundbreaking progress. Drugs targeting driver genes such as EGFR and ALK, such as gefitinib and crizotinib, are widely used as first-line treatments for NSCLC. In recent years, immunotherapy has also achieved milestone progress in the treatment of NSCLC, with several immune checkpoint inhibitors approved by the US FDA for first-line treatment of NSCLC.

[0003] However, compared to the tremendous success achieved in targeted therapy and immunotherapy for NSCLC, there has been no significant improvement in the detection, treatment, and prognosis of SCLC over the past thirty years. SCLC, referred to as "refractory cancer" by the National Cancer Institute, is a neuroendocrine tumor accounting for approximately 15% of primary lung cancers. It is characterized by high malignancy, aggressiveness, rapid disease progression, high recurrence rate, and drug resistance. About two-thirds of patients are diagnosed with SCLC at an advanced stage (ES-SCLC), no longer eligible for surgery. Only 2%–5% of SCLC patients are suitable for surgical treatment. Most patients initially receive chemotherapy (e.g., cisplatin plus etoposide, i.e., the EP regimen) and concurrent thoracic radiotherapy, with significant efficacy and high response rates (60%–70%), making chemotherapy still the first-line treatment for SCLC. Although SCLC is initially sensitive to chemotherapy drugs and has a high response rate, it is extremely prone to drug resistance and recurrence, resulting in a very poor prognosis, with a 5-year survival rate of less than 7%. Research on targeted therapies for SCLC has progressed slowly, with only anlotinib currently approved by the my country Food and Drug Administration for clinical treatment of SCLC. Drugs targeting EGFR and VEGFR that have been successfully used to treat NSCLC have all failed in clinical trials for SCLC. In recent years, immunotherapy for SCLC has made some progress, but it faces challenges such as a lack of effective biomarkers, a small applicable population, and easy drug resistance. Therefore, the clinical treatment of SCLC currently faces a "drug shortage," urgently requiring a deeper understanding of the mechanisms of SCLC tumor progression, the discovery of new targets with good drug-like properties that drive SCLC progression, and the overcoming of chemotherapy resistance in SCLC, thus providing new strategies for SCLC treatment.

[0004] The nuclear receptor family, consisting of 48 members, is a ligand-dependent, widely distributed family of important transcription factors involved in various biological processes, including human development, metabolism, and reproductive health. Nuclear receptors share common structural features, typically comprising four functional regions: the A / B region (N-terminal activation domain, AF-1), the C region (DNA-binding domain, DBD), the D region (hinge region), and the E / F region (ligand-binding domain, LBD). Small molecule ligands (natural or synthetic) can specifically bind to the LBD region, thereby regulating nuclear receptor activity and the expression of its target genes, thus exerting physiological functions. Abnormal nuclear receptor function is closely related to various pathological processes, such as rheumatoid arthritis, diabetes, asthma, and cancer. Based on this, related small molecule agonists or inhibitors have gradually become a hot topic in new drug development, making nuclear receptors ideal drug targets for targeted cancer therapy. To date, small molecule drugs targeting nuclear receptors approved by the US FDA account for 16% of the 48 members. For example, enzalutamide, an AR antagonist for treating castration-resistant prostate cancer (CPRC), and tamoxifen, an estrogen-like drug for treating breast cancer, have both achieved success in clinical treatment, fully demonstrating the feasibility of using nuclear receptors as drug targets to treat human diseases.

[0005] Retinoid X receptors (RXRs) are members of the ligand-dependent nuclear receptor superfamily, including three subtypes: RXRα, RXRβ, and RXRγ. In the body, RXRs act as central nuclear receptors, typically functioning as homodimers or heterodimers to regulate downstream gene signaling pathways and exert physiological functions. Abnormal expression or function of RXRs is closely related to cancer, metabolic diseases, and cardiovascular diseases, and they are considered important drug targets.

[0006] The three RXR subtypes exhibit different expression distributions and functions in vivo. RXRα is highly expressed in visceral tissues such as the liver, kidneys, and intestines; knockout of RXRα leads to myocardial dysplasia and embryonic lethality in mice. However, RXRα protein is lowly expressed in various cancers, including prostate cancer, gastric cancer, and breast ductal carcinoma; RXRα activators can effectively inhibit tumor growth. RXRβ is widely expressed in various tissues; knockout of RXRβ only causes intrauterine lethality in about 50% of mice, with the remaining mice remaining largely normal (except for male infertility). RXRβ mutations can cause abnormal lipid metabolism in supporting cells. In normal organisms, RXRγ is mainly expressed in myogenic cells and the central nervous system; RXRγ knockout mice are phenotypically normal and fertile. However, RXRγ shows a significant histiocytogenic dependence in tumors; RXRγ expression is absent in ovarian and breast cancer, but significantly overexpressed in thyroid cancer, and its high expression is significantly positively correlated with extrathyroidal invasion and metastasis. A recent study published in Cell has also confirmed that in treated melanoma minimal residual disease (MRD), the nuclear receptor RXRγ can effectively drive the transcription of neural crest stem cells (NCSCs), leading to tumor drug resistance. RXR antagonists can significantly reduce the accumulation of NCSCs in melanoma, delay the development of tumor drug resistance, and prevent melanoma recurrence. Our research indicates that RXRγ plays a crucial role in the development and progression of small cell lung cancer; therefore, developing new drugs targeting RXRs, especially RXRγ, is of great significance.

[0007] Protein degradation targeting chimaeras (PROTACs) are small molecule compounds that specifically recognize and induce the degradation of target proteins. Their structure mainly consists of three parts: a ligand that binds to the target protein, a ligand that binds to E3 ubiquitin ligase, and a linker connecting the two parts. PROTAC molecules link the target protein ligand and the E3 ubiquitin ligase ligand together via the linker. In vivo, they recognize both the target protein and the E3 ubiquitin ligase separately, inducing the recruitment of the E3 ubiquitin ligase to the target protein surface, triggering polyubiquitination and thus inducing target protein degradation. Compared to traditional small molecules, PROTAC molecules have the following advantages: achieving high degradation efficiency with lower doses, targeting undrug-resistant proteins, high selectivity for homologous targets, overcoming drug resistance, and prolonging drug action time. Degradative agents for target proteins such as ER and AR have already been developed and are in clinical development. Summary of the Invention

[0008] Based on this, the present invention provides a protein degradation targeting chimera that can degrade RXRs proteins, which can effectively downregulate the expression of RXRs in tumor cells and has a good inhibitory effect on various small cell lung cancer cells.

[0009] This invention includes the following technical solutions:

[0010] Protein degradation-targeting chimeras having the structure shown in formula (I), or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or prodrug molecules thereof:

[0011]

[0012] Wherein, Y is selected from: -CH2-, -C(O)-;

[0013] L is -X-L1-Z-, where X is attached to a phenyl group in a diazapyridine ring and Z is attached to a phenyl group in a benzo[5]-membered ring;

[0014] X is selected from: 3-12 membered cycloalkyl groups, 3-12 membered heterocyclic groups having 1-4 heteroatoms, -NHC(O)-, -C(O)NH-, -C(O)O-, -OC(O)-;

[0015] Z is selected from: 3-12 membered cycloalkyl groups, 3-12 membered heterocyclic groups having 1-4 heteroatoms, -CH2-, -NH-, -O-, -NHC(O)-, -C(O)NH-, -C≡C-, -CH=CH-, -C(O)O-, -OC(O)-;

[0016] L1 is selected from: 3-12 membered cycloalkyl groups, 3-12 membered heterocyclic groups having 1-4 heteroatoms, and -(CH2). m -、-(CH2-L2-CH2) n -、、-(CH2) m1 -L2-(CH2) m1 -、-(CH2-L2) p -CH2-, -CH2-(CH2-L2-CH2) q -CH2-, -(CH2) m1 -L2-C(O)-(CH2) a -;

[0017] L2 is selected from: 3-12 membered cycloalkyl groups, 3-12 membered heterocyclic groups with 1-4 heteroatoms, -O-, -NH-, -NHC(O)-, -C(O)NH-, -C≡C-, -CH=CH-, -C(O)O-, -OC(O)-;

[0018] m is a positive integer from 1 to 20; n is a positive integer from 1 to 10; each m1 is independently selected from a positive integer from 1 to 10; p is a positive integer from 1 to 10; q is a positive integer from 1 to 10; a is a positive integer from 1 to 20;

[0019] The L can be optionally replaced by one or more R. L Replace, each R LEach group is independently selected from: halogen, cyano, hydroxyl, amino, carboxyl, C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkoxy, hydroxyl-substituted C1-C6 alkyl, hydroxyl-substituted C1-C6 alkoxy, deuterated C1-C6 alkyl, deuterated C1-C6 alkoxy, or two R groups. L The groups and the atoms they are attached to together form 3-8 membered cycloalkyl groups containing 0-4 heteroatoms.

[0020] In some of these embodiments, X is selected from: 5-6 membered cycloalkyl groups, 5-12 membered heterocyclic groups having 1-2 nitrogen atoms, -NHC(O)-, -C(O)NH-, -C(O)O-, -OC(O)-.

[0021] In some embodiments, X is selected from: -NHC(O)-,

[0022] In some of these embodiments, Z is selected from: 5-6 membered cycloalkyl groups, 5-12 membered heterocyclic groups having 1-2 nitrogen atoms, -CH2-, -NH-, -O-, -NHC(O)-, -C(O)NH-, -C≡C-, -CH=CH-, -C(O)O-, -OC(O)-.

[0023] In some embodiments, Z is selected from: -O-, -NH-, -C≡C-, -CH=CH-,

[0024] In some embodiments, L1 is selected from: 5-6 membered cycloalkyl groups, 5-6 membered heterocyclic groups having 1-2 heteroatoms, and -(CH2). m -、-(CH2-L2-CH2) n -、-(CH2) m1 -L2-(CH2) m1 -、-(CH2-L2) p -CH2-, -CH2-(CH2-L2-CH2) q -CH2-, -(CH2) m1 -L2-C(O)-(CH2) a -;

[0025] L2 is selected from: 5-6 membered cycloalkyl groups, 5-6 membered heterocyclic groups with 1-2 heteroatoms, -O-, -NH-, -NHC(O)-, -C(O)NH-, -C≡C-, -CH=CH-, -C(O)O-, -OC(O)-;

[0026] m is a positive integer from 1 to 14; each m1 is independently selected from a positive integer from 1 to 5; n is a positive integer from 1 to 5; p is a positive integer from 1 to 5; q is a positive integer from 1 to 5; a is a positive integer from 1 to 5.

[0027] In some embodiments, L1 is selected from: -(CH2) m -、-(CH2) m1 -L2-(CH2) m1 -、-(CH2) m1 -L2-C(O)-(CH2) a -, -CH2-(CH2-O-CH2) q -CH2-; L2 is m is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; each m1 is independently selected from: 1, 2, 3; a is selected from: 1, 2, 3, 4, 5; q is selected from: 1, 2, 3, 4, 5.

[0028] In some embodiments, L is selected from:

[0029]

[0030] Each m is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14;

[0031] q is selected from: 1, 2, 3, 4, 5;

[0032] Each m1 is independently selected from: 1, 2, 3;

[0033] a is selected from: 1, 2, 3, 4, 5.

[0034] In some embodiments, L is selected from:

[0035] m is selected from: 10, 11 or 12; each m1 is independently selected from: 1 or 2; a is selected from: 2, 3 or 4.

[0036] In some embodiments, L is selected from:

[0037] In some embodiments, the protein degradation targeting chimera has the structure shown in formula (II), formula (III), formula (IV), or formula (V):

[0038]

[0039] The present invention also provides the application of the above-mentioned protein degradation targeting chimera, or its pharmaceutically acceptable salt, or its stereoisomer, or its prodrug molecule, including the following technical solutions.

[0040] The above-mentioned protein degradation-targeting chimeras, or their pharmaceutically acceptable salts, or their stereoisomers, or their prodrug molecules, are used in the preparation of RXRs degrading agents or RXRs inhibitors.

[0041] The use of the aforementioned protein degradation-targeting chimeras, or their pharmaceutically acceptable salts, or their stereoisomers, or their prodrug molecules in the preparation of drugs for the prevention and / or treatment of tumors.

[0042] In some embodiments, the tumor is a tumor mediated by abnormal expression of RXRs.

[0043] In some embodiments, the RXRs are RXRγ.

[0044] In some embodiments, the tumor is lung cancer, prostate cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, or thyroid cancer.

[0045] In some of these embodiments, the tumor is lung cancer.

[0046] In some embodiments, the lung cancer is small cell lung cancer.

[0047] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of tumors, comprising the following technical solutions.

[0048] A pharmaceutical composition for the prevention and / or treatment of tumors, prepared from an active ingredient and pharmaceutically acceptable excipients, said active ingredient comprising the aforementioned protein degradation-targeting chimera, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof.

[0049] The protein degradation targeting chimera provided by this invention can effectively degrade RXRs proteins, thereby effectively downregulating the expression of RXRs in tumor cells. It has a good inhibitory effect on a variety of tumor cells (especially a variety of small cell lung cancer cells) and can be used as a potential drug for anti-tumor (especially small cell lung cancer) treatment. Attached Figure Description

[0050] Figure 1 This diagram illustrates the effect of the compounds provided in this invention on the degradation of RXRγ protein in small cell lung cancer cells.

[0051] Figure 2 This diagram illustrates the effect of the compound of the present invention on the formation of small cell lung cancer cell colonies on a plate.

[0052] Figure 3This is a diagram showing the effect of the compound of the present invention on small cell lung cancer tumors in mice. Detailed Implementation

[0053] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0054] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0055] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0056] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0057] In the compounds described in this invention, when any variable appears more than once in any component, the definition of each occurrence is independent of the definitions of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. A line drawn from a substituent into the ring system indicates that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It will be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is itself substituted by more than one group, it should be understood that these groups can be on the same carbon atom or different carbon atoms, as long as structural stability is achieved.

[0058] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0059] The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group with a specific number of carbon atoms. For example, "5-6 membered cycloalkyl" includes cyclopentyl and cyclohexyl.

[0060] The term "alkoxy" refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0061] The term "heterocyclic alkyl" or "heterocyclic group" refers to a cyclic substituent, such as a saturated or partially unsaturated monocyclic, fused, spirocyclic, or bridged ring, wherein one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. Examples include: morpholinyl, piperidinyl, tetrahydropyrrolyl, oxadiazolyl, piperazine, pyrrolyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydropyrazine, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophene, dihydrotriazolyl, dihydroazacyclobutane, tetrahydrofuranyl, tetrahydrothiophene, etc., and their N-oxides. The connection of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

[0062] The term “substituted” as used in this article refers to the replacement of a hydrogen group in a specific structure with a group of a specified substituent.

[0063] As will be understood by those skilled in the art, the term “halo” or “halogen” as used herein refers to chlorine, fluorine, bromine, and iodine.

[0064] This invention includes the free forms of compounds of formulas (I)-(V), as well as their pharmaceutically acceptable salts and stereoisomers. The term "free form" refers to carboxylic acid compounds existing in a non-salt form. "Pharmaceutically acceptable salt" includes typical pharmaceutically acceptable salts of the free forms of all compounds of formulas (I)-(V). The pharmaceutically acceptable salts of this invention can be synthesized from the compounds of this invention containing the acidic moiety using conventional chemical methods. That is, "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali, including inorganic and organic bases, with the compounds of this invention. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic alkaloids include salts of primary, secondary, and tertiary amines. Substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, aminobutanetriol, etc.

[0065] Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci. '1977: 66: 1–19, describe in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0066] Since the deprotonated acidic portion of the compound, such as the carboxyl group, can be anionic under physiological conditions, and this charge can then be balanced by the protonated or alkylated basic portion, such as the tetravalent nitrogen atom, which carries a cation, it should be noted that the compounds of the present invention are potential internal salts or zwitterions.

[0067] In some embodiments, the present invention provides a method for treating hyperproliferative diseases or symptoms such as tumors in humans or other mammals using compounds having the structures shown in formulas (I)-(V) and their pharmaceutically acceptable salts.

[0068] In some of these embodiments, the compounds of the present invention and their pharmaceutically acceptable salts can be used to treat or control hyperproliferative diseases such as lung cancer (especially small cell lung cancer), prostate cancer, hepatocellular carcinoma, breast cancer, ovarian cancer, and thyroid cancer.

[0069] Metabolites of the compounds and pharmaceutically acceptable salts involved in this invention, as well as prodrugs that can be converted in vivo into structures of the compounds and pharmaceutically acceptable salts involved in this invention, are also included in the claims of this invention.

[0070] The terms "produce molecule" or "drug precursor" in this invention refer to compounds obtained by chemically modifying the chemical structure of the compounds of this invention. These compounds are inactive or have low activity in vitro, but release an active drug in vivo through enzymatic or non-enzymatic conversion to exert their pharmacological effects. Drug precursors can be prepared using techniques known to those skilled in the art. These techniques typically modify appropriate functional groups in a particular compound. However, these modified functional groups can be regenerated back to their original functional groups through routine manipulation or in vivo.

[0071] The compounds of this invention can be administered in bulk or as a pharmaceutical prodrug, for example, as in vivo hydrolyzable esters or in vivo hydrolyzable amides. In vivo hydrolyzable esters of the compounds of this invention containing a carboxyl group are pharmaceutically acceptable esters that hydrolyze in the human or animal body to form a parent acid or alcohol. Suitable pharmaceutically acceptable esters with a carboxyl group include C... 1-6 -Alkoxymethyl esters (e.g., methoxymethyl), C 1-6 -alkyl oxymethyl esters (e.g., trimethylacetyloxymethyl), phthaloyl esters, C 3-8 -cycloalkoxycarbonyloxy-C 1-6 -Alkyl esters (e.g., 1-cyclohexylcarbonyloxyethyl); 1,3-dioxolane-2-oxymethyl esters (e.g., 5-methyl-1,3-dioxolane-2-oxymethyl); and C 1-6 -Alkoxycarbonyloxyethyl esters (e.g., 1-methoxycarbonyloxyethyl), and can be formed on any carboxyl group in the compounds of the present invention.

[0072] Synthetic methods: In addition to standard methods known in the literature or illustrated in experimental procedures, the compounds of the present invention can be prepared using the methods in the following synthetic schemes (Schemes 1-2). A better understanding of the compounds and synthetic methods described in the present invention can be achieved by referring to the following synthetic schemes. The synthetic schemes described herein depict methods that can be used to prepare the compounds of the present invention. These methods are merely illustrative descriptions for illustrative purposes and do not constitute a limitation on the scope of the present invention.

[0073]

[0074] Option 1

[0075]

[0076] Option 2

[0077] The following are specific examples.

[0078] Example 1. Preparation of 4-(2-(4-((2-(2,6-dioxopyridine)-3-yl)-1,3-dioxoisoindol-5-yl)oxy)butamido)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 1)

[0079] Step 1.4 Preparation of tert-butyl benzoate (compound 1a) (5,7,7,10,10-pentamethyl-2-nitro-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)

[0080]

[0081] 320 mg (0.7 mmol) of 4-(5,7,7,10,10-pentamethyl-2-nitro-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (synthesis reference: Chem. Pharm. Bull. 47(12) 1778-1796(1999)) was dissolved in 50 mL of toluene. 0.23 mL (1.0 mmol) of di-tert-butyl dicarbonate, 16 mg (0.1 mmol) of 4-dimethylaminopyridine, and 0.16 mL (1.0 mmol) of tert-butanol were added sequentially. The mixture was stirred at 100 °C for 12 h. After the reaction was complete, the mixture was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. Rapid silica gel column chromatography yielded 238 mg of a yellow solid, compound 1a, in 67% yield. 1 HNMR (400MHz, CDCl3) δ8.17–1.14(m,1H),8.06(d,J=7.6Hz,2H),8.04–8.00(m,1H),7.87(d,J=7.5Hz,2H),7.02(d,J=8.9Hz,1H) ,6.95(s,1H),6.94(s,1H),3.34(s,3H),1.68–1.63(m,13H),1.34(s,3H),1.30(s,3H),1.17(s,3H),1.09(s,3H).LC-MS(ESI)m / z 539.8[M+H] + .

[0082] Step 2.4 Preparation of tert-butyl benzoate (compound 1b) (2-amino-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)

[0083]

[0084] 120 mg (0.2 mmol) of compound 1a was dissolved in 10 mL of ethanol and 4 mL of water. Then, 64 mg (1.1 mmol) of iron powder and 96 mg (1.8 mmol) of ammonium chloride were added sequentially. The mixture was stirred at 50 °C for 2 h. The reaction was stopped after the starting material disappeared as indicated by TLC. The reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness. Rapid silica gel column chromatography was used to obtain 105 mg of orange-red solid compound 1b, with a yield of 93%. 1 H NMR (400MHz, DMSO) δ7.97(d,J=8.5Hz,2H),7.74(d,J=8.5Hz,2H),6.99(s,1H),6.82(s,1H),6.77(d,J=8.6Hz,1H),6.48(d,J=2.6Hz,1H),6. 42(dd,J=8.6,2.7Hz,1H),4.85(s,2H),3.10(s,3H),1.63–1.56(m,13H),1.30(s,3H),1.26(s,3H),1.10(s,3H),1.02(s,3H).LC-MS(ESI)m / z 510.2[M+H] + .

[0085] Step 3.4 Preparation of tert-butyl butyrate (compound 1c) of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)butyrate

[0086]

[0087] 194 mg (0.87 mmol) of tert-butyl 4-bromobutyrate and 194 mg (0.73 mmol) of 4-hydroxythalidomide were dissolved in dry DMF, and 109 mg (1.09 mmol) of KHCO3 and 12 mg (0.07 mmol) of KI were added. The mixture was reacted overnight at 90 °C under argon protection. After the reaction was complete, the system was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. Rapid silica gel column chromatography was used to obtain 189 mg of white solid compound 1c, with a yield of 62%. 1H NMR (400MHz, DMSO) δ11.11(s,1H),7.83(d,J=8.3Hz,1H),7.42(d,J=2.3Hz,1H),7.35(dd,J=8.4,2.3Hz,1H),5.12(dd,J=12.9,5.3Hz,1 H),4.18(t,J=6.4Hz,2H),2.96–2.82(m,1H),2.65–2.53(m,2H),2.39(t,J=7.3Hz,2H),2.09–1.93(m,3H),1.40(s,9H).LC-MS(ESI)m / z 416.9[M+H] + .

[0088] Step 4.4 Preparation of ((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)butyric acid (compound 1d)

[0089]

[0090] 150 mg of compound 1c was dissolved in 10 mL of dichloromethane, 5 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. The system was then directly evaporated to dryness. Dichloromethane and methanol were added repeatedly to dissolve the compound, and the solvent was evaporated to dryness until the system became a solid, yielding 124 mg of white solid compound 1d, with a yield of 95%. 1 HNMR (400MHz, DMSO) δ12.17(s,1H),11.11(s,1H),7.84(d,J=8.2Hz,1H),7.43(s,1H),7.35(d,J=8.4Hz,1H),5.12(dd,J=13.0,5 .3Hz,1H),4.20(t,J=6.5Hz,2H),2.96–2.82(m,1H),2.65–2.52(m,2H),2.41(t,J=7.3Hz,2H),2.09–1.94(m,3H).LC-MS(ESI)m / z 359.1[MH] - .

[0091] Step 5.4 Preparation of tert-butyl benzoate (compound 1e) of 2-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxaindol-5-yl)oxy)butyramide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoate

[0092]

[0093] 17 mg (0.05 mmol) of compound 1d was dissolved in dry DMF, 22 mg (0.06 mmol) of HATU and 0.01 mL (0.08 mmol) of DIPEA were added, and the mixture was stirred at room temperature for 10 min. Then, 20 mg (0.04 mmol) of compound 1b was added, and the mixture was stirred at room temperature for another 30 min. After the reaction was complete, the mixture was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The solution was then purified by silica gel column chromatography using rapid chromatography to obtain 28 mg of yellow solid compound 1e, with a yield of 85%. 1 H NMR (400MHz, DMSO) δ11.11(s,1H),9.96(s,1H),8.02(d,J=8.1Hz,2H),7.83(d,J=8.2Hz,1H),7.75( d,J=7.9Hz,2H),7.56(s,1H),7.43(s,1H),7.34(t,J=10.3Hz,2H),7.08–6.97(m,2H),6.86(s,1H), 5.11(d,J=14.0Hz,1H),4.22(t,J=6.6Hz,2H),3.17(s,3H),2.95–2.83(m,1H),2.64–2.53(m,1H),2 .10–2.01(m,2H),1.66–1.49(m,13H),1.31–1.19(m,10H),1.10(s,3H),1.00(s,3H).LC-MS(ESI)m / z 851.8[M+H] + .

[0094] Step 6.4 Preparation of 2-(4-((2-(2,6-dioxopyridine)-3-yl)-1,3-dioxoisoindol-5-yl)oxy)butamido)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 1)

[0095]

[0096] Replace compound 1c in step 4 of Example 1 with compound 1e, and use the same reagents and preparation methods as in step 4 to prepare compound 1. 1HNMR (400MHz, DMSO) δ11.11(s,1H),9.95(s,1H),8.02(d,J=8.0Hz,2H),7.82(d,J=8.3Hz,1H),7.75 (d,J=8.0Hz,2H),7.55(s,1H),7.43(s,1H),7.33(t,J=10.3Hz,2H),7.09–6.96(m,2H),6.86(s,1H), 5.11(d,J=14.0Hz,1H),4.22(t,J=6.6Hz,2H),3.17(s,3H),2.94–2.82(m,1H),2.63–2.54(m,1H),2 .10–2.02(m,2H),1.66–1.52(m,4H),1.31–1.19(m,10H),1.10(s,3H),1.00(s,3H).HRMS(ESI)calcd for C 46 H 45 N5O8[M+H] + :796.3341,found 796.3311.

[0097] Example 2.4 Preparation of 4-(2-(6-((2-(2,6-dioxadiazine)-3-yl)-1,3-dioxoisoindoline-5-yl)oxy)hexaamino)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 2)

[0098]

[0099] Compound 2 was prepared according to Example 1. 1H NMR (400MHz, DMSO) δ11.11(s,1H),9.84(s,1H),8.01(d,J=8.0Hz,2H),7.81(d,J=8.2Hz,1H),7.75(d,J=8.1Hz,2H),7.54( s,1H),7.42(s,1H),7.36–7.26(m,2H),7.03(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.11(dd,J=13.0,5.4Hz,1H),4.18 (t,J=6.5Hz,2H),3.17(s,3H),2.95–2.81(m,1H),2.69–2.53(m,2H),2.32(t,J=7.2Hz,2H),2.07–1.99(m,1H),1.83–1.74( m,2H),1.66–1.60(m,2H),1.50–1.44(m,2H),1.30–1.23(d,J=18.1Hz,10H),1.11(s,3H),1.01(s,3H).HRMS(ESI)calcdfor C 48 H 49 N5O8[M+H] + :824.3654,found 824.3661.

[0100] Example 3.4 Preparation of 4-(2-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)oxy)octamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 3)

[0101]

[0102] Compound 3 was prepared according to Example 1. 1H NMR (400MHz, DMSO) δ11.11(s,1H),9.82(s,1H),8.01(d,J=8.0Hz,2H),7.81(d,J=8.3Hz,1H),7.74(d,J=8.0Hz,2H ),7.55(s,1H),7.41(s,1H),7.36–7.27(m,2H),7.03(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.11(dd,J=12.9, 5.4Hz,1H),4.16(t,J=6.6Hz,2H),3.17(s,3H),2.93–2.82(m,1H),2.64–2.52(m,2H),2.28(t,J=7.3Hz,2H),2.07– 2.00(m,1H),1.79–1.70(m,2H),1.64–1.56(m,4H),1.31–1.22(m,14H),1.11(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 50 H 53 N5O8[M+H] + :852.3967,found 852.3974.

[0103] Example 4. Preparation of 4-(2-(10-((2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindol-5-yl)oxy)decanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 4)

[0104]

[0105] Compound 4 was prepared according to Example 1. 1HNMR (400MHz, DMSO) δ13.15(s,1H),11.11(s,1H),9.80(s,1H),8.02(d,J=8.0Hz,2H),7.83–7.74(m,3H),7.55(d,J=2. 4Hz,1H),7.40(s,1H),7.34–7.29(m,2H),7.03(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.12(dd,J=13.0,5.4Hz,1H) ,4.14(t,J=6.5Hz,2H),3.17(s,3H),2.94–2.82(m,1H),2.63–2.53(m,2H),2.27(t,J=7.3Hz,2H),2.11–1.99(m,1H),1 .77–1.69(m,2H),1.65–1.53(m,6H),1.44–1.37(m,2H),1.31–1.22(m,14H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 52 H 57 N5O8[M+H] + :880.4280,found 880.4248.

[0106] Example 5. Preparation of 4-(2-(12-((2-(2,6-dioxopyridine)-3-yl)-1,3-dioxoisoindol-5-yl)oxy)dodecanoyl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 5)

[0107]

[0108] Compound 5 was prepared according to Example 1. 1HNMR(400MHz,DMSO)δ13.14(s,1H),11.11(s,1H),9.79(s,1H),8.02(d,J=8.0Hz,2H),7.8 3–7.74(m,3H),7.55(s,1H),7.40(s,1H),7.34–7.28(m,2H),7.03(s,1H),6.99(d,J=8.8H z,1H),6.86(s,1H),5.12(dd,J=12.9,5.4Hz,1H),4.14(t,J=6.5Hz,2H),3.17(s,3H),2.9 4–7.82(m,1H),2.64–2.53(m,2H),2.26(t,J=7.4Hz,2H),2.07–1.98(m,1H),1.77–1.68(m 2H),1.65–1.53(m,6H),1.43–1.36(m,2H),1.30–1.22(m,18H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 54 H 61 N5O8[M+H] + :908.4593,found908.4582.

[0109] Example 6.4 Preparation of 4-(2-(14-((2-(2,6-dioxopyridine)-3-yl)-1,3-dioxoisoindol-5-yl)oxy)tetradecanoyl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 6)

[0110]

[0111] Compound 6 was prepared according to Example 1. 1HNMR (400MHz, DMSO) δ13.12(s,1H),11.11(s,1H),9.79(s,1H),8.02(d,J=8.0Hz,2H),7.81(d,J=8.3Hz,1H),7.77(d,J=8.0Hz ,2H),7.54(d,J=2.4Hz,1H),7.40(s,1H),7.35–7.26(m,2H),7.03(s,1H),6.99(d,J=8.7Hz,1H),6.86(s,1H),5.11(dd,J=13. 0,5.4Hz,1H),4.14(t,J=6.4Hz,2H),3.17(s,3H),2.95–2.83(m,1H),2.66–2.53(m,2H),2.26(t,J=7.3Hz,2H),2.08–1.98(m, 1H),1.77–1.67(m,2H),1.64–1.53(m,5H),1.44–1.36(m,2H),1.32–1.20(m,23H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 56 H 65 N5O8[M+H] + :936.4906,found 936.5882.

[0112] Example 7.4 Preparation of 4-(2-(12-((2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindoline-4-yl)oxy)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 7)

[0113]

[0114] Compound 7 was prepared according to Example 1. 1HNMR (400MHz, DMSO) δ13.13(s,1H),11.10(s,1H),9.79(s,1H),8.02(d,J=8.3Hz,2H),7.82–7.74(m,3H),7.55(d,J=2.5Hz,1H),7 .50(d,J=8.6Hz,1H),7.43(d,J=7.2Hz,1H),7.31(dd,J=8.7,2.5Hz,1H),7.04(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.08(d d,J=12.9,5.4Hz,1H),4.18(t,J=6.4Hz,2H),3.17(s,3H),2.93–2.83(m,1H),2.65–2.54(m,2H),2.27(t,J=7.4Hz,2H),2.06–1.9 9(m,1H),1.77–1.71(m,2H),1.64–1.53(m,6H),1.48–1.39(m,2H),1.31–1.24(m,18H),1.11(s,3H),1.02(s,3H).HRMS(ESI)calcd for C 54 H 61 N5O8[M+H] + :908.4593,found 908.4583.

[0115] Example 8.4 Preparation of 4-(2-(12-((2-(2,6-dioxadiazin-3-yl)-1-oxoisoindoline-4-yl)oxy)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 8)

[0116] Step 1.3 Preparation of methyl 1,3-((12-(tert-butoxy)-12-oxododecyl)oxy)-2-methylbenzoate (compound 8a)

[0117]

[0118] 1.1 g (3.3 mmol) of tert-butyl 12-bromododecanoate and 500 mg (3.0 mmol) of methyl 3-hydroxy-2-methylbenzoate were dissolved in 100 mL of LMF. 624 mg (4.5 mmol) of potassium carbonate was added, and the mixture was stirred at 60 °C for 8 h. The reaction was stopped after the starting material disappeared as indicated by TLC. The mixture was poured into water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The solution was purified by rapid silica gel column chromatography to give 1.1 g of a colorless, transparent oily compound 8a, in 86% yield. 1 HNMR (400MHz, DMSO) δ7.29(d,J=7.7Hz,1H),7.21(t,J=8.0Hz,1H),7.11(d,J=8.1Hz,1H),3.95(t,J=6.3Hz,2H),3.80(s,3H),2.3 1(s,3H),2.13(t,J=7.3Hz,2H),1.71(p,J=6.6Hz,2H),1.48–1.42(m,2H),1.39–1.34(m,11H),1.26–1.18(m,12H).LC-MS(ESI)m / z 420.9[M+H] + .

[0119] Step 2. Preparation of methyl 2-(bromomethyl)-3-((12-(tert-butoxy)-12-oxododecyl)oxy)benzoate (compound 8b)

[0120]

[0121] 500 mg (1.2 mmol) of compound 8a was dissolved in 50 mL of carbon tetrachloride, and 254 mg (1.4 mmol) of N-bromosuccinimide (NBS) and 20 mg (0.12 mmol) of azobisisobutyronitrile (AIBN) were added. The reaction was carried out overnight under argon protection. After the starting material was observed to disappear on TLC, the solvent was evaporated to dryness, sodium thiosulfate solution was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to give 235 mg of a yellow oily compound 8b, which was used directly in the next step, with a yield of 40%. LC-MS (ESI) m / z 499.2 [M+H] + .

[0122] Step 3.1 Preparation of tert-butyl dodecanoate (compound 8c) of 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)oxy)

[0123]

[0124] 120 mg (0.25 mmol) of compound 8b and 61 mg (0.37 mmol) of 3-amino-2,6-piperidinidone hydrochloride were dissolved in 20 mL of acetonitrile, and 52 mg (0.37 mmol) of potassium carbonate were added. The mixture was reacted at 80 °C for 4 h. After the reaction was completed, the mixture was filtered through a diatomaceous earth filter, and the filtrate was evaporated to dryness. The filtrate was purified by rapid silica gel column chromatography to obtain 64 mg of a pale purple solid, compound 8c, in 50% yield. 1 HNMR (400MHz, DMSO) δ10.97(s,1H),7.47(t,J=7.8Hz,1H),7.30(d,J=7.4Hz,1H),7.23(d,J =8.1Hz,1H),5.11(dd,J=13.3,5.1Hz,1H),4.40–4.17(m,2H),4.10(t,J=6.4Hz,2H),2.96–2 .85(m,1H),2.63–2.54(m,1H),2.47–2.39(m,1H),2.16(t,J=7.3Hz,2H),2.04–1.91(m,1H) ,1.73(p,J=6.5Hz,2H),1.51–1.40(m,4H),1.38(s,9H),1.31–1.21(m,12H).LC-MS(ESI)m / z 515.2[M+H] + .

[0125] Step 4.1 Preparation of 2-((2-(2,6-dioxoperidin-3-yl)-1-oxoisoindoline-4-yl)oxy)dodecanoic acid (compound 8d)

[0126]

[0127] By replacing compound 1c in step 4 of Example 1 with compound 8c, and using the same reagents and preparation methods as in Example 1, compound 8d was obtained. 1HNMR (400MHz, DMSO) δ10.97(s,1H),7.47(t,J=7.8Hz,1H),7.30(d,J=7.5Hz,1H),7.23( d,J=8.2Hz,1H),5.10(dd,J=13.2,5.1Hz,1H),4.41–4.17(m,2H),4.11(t,J=6.4Hz,2H), 2.96–2.85(m,1H),2.63–2.54(m,1H),2.47–2.39(m,1H),2.18(t,J=7.4Hz,2H),2.03–1. 93(m,1H),1.73(p,J=6.6Hz,2H),1.52–1.39(s,4H),1.32–1.21(m,12H).LC-MS(ESI)m / z 456.8[MH] - .

[0128] Step 5.4 Preparation of 2-(12-((2-(2,6-dioxadiazin-3-yl)-1-oxoisoindoline-4-yl)oxy)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 8)

[0129]

[0130] Compound 8 was obtained by amide condensation and hydrolysis of tert-butyl ester following steps 5 and 6 of Example 1. 1H NMR (400MHz, DMSO) δ13.12(s,1H),10.97(s,1H),9.79(s,1H),8.02(d,J=8.1Hz,2H),7.77(d,J=8.1Hz,2H),7.55(d,J=2.5Hz,1H),7.46( t,J=7.8Hz,1H),7.35–7.26(m,2H),7.21(d,J=8.1Hz,1H),7.03(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.10(dd,J=13.2,5.1Hz,1H), 4.38–4.19(m,2H),4.08(t,J=6.4Hz,2H),3.17(s,3H),2.96–2.85(m,1H),2.69–2.57(m,1H),2.47–2.40(m,1H),2.26(t,J=7.3Hz,2H),2. 02–1.97(m,1H),1.75–1.68(m,2H),1.63–1.55(m,5H),1.44–1.38(m,2H),1.34–1.21(m,19H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 54 H 63 N5O7[M+H] + :894.4800,found 894.4798.

[0131] Example 9.4 Preparation of 2-(12-((2-(2,6-dioxadiazin-3-yl)-1-oxoisoindoline-5-yl)oxy)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 9)

[0132]

[0133] Compound 9 was prepared according to Example 8. 1H NMR (400MHz, DMSO) δ10.96(s,1H),9.81(s,1H),8.02(d,J=8.2Hz,2H),7.77(d,J=8.2Hz,2H),7.61(d,J=8.4Hz,1H),7.54(d,J=2.5H z,1H),7.31(dd,J=8.7,2.5Hz,1H),7.13(d,J=2.2Hz,1H),7.04–6.97(m,3H),6.85(s,1H),5.06(dd,J=13.3,5.2Hz,1H),4.40–4.21 (m,2H),4.03(t,J=6.5Hz,2H),3.16(s,3H),2.94–2.84(m,1H),2.67–2.58(m,1H),2.38–2.33(m,1H),2.26(t,J=7.3Hz,2H),2.00–1 .93(m,1H),1.74–1.66(m,2H),1.63–1.52(m,5H),1.42–1.36(m,2H),1.31–1.21(m,19H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 54 H 63 N5O7[M+H] + :894.4800,found894.4782.

[0134] Example 10. Preparation of 4-(2-(12-((2-(2,6-dioxadiazin-3-yl)-3-oxoisoindoline-5-yl)oxy)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 10)

[0135]

[0136] Compound 10 was prepared according to Example 8. 1H NMR (400MHz, DMSO) δ13.14(s,1H),10.98(s,1H),9.79(s,1H),8.02(d,J=8.0Hz,2H),7.77(d,J=8.1Hz,2H),7.55(s,1H),7.48(d,J=8.3 Hz,1H),7.31(d,J=8.8Hz,1H),7.24–7.12(m,2H),7.03(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.10(dd,J=13.2,5.0Hz,1H),4.41–4 .19(m,2H),4.02(t,J=6.7Hz,2H),3.17(s,3H),2.95–2.84(m,1H),2.63–2.55(m,1H),2.41–2.32(m,1H),2.27(t,J=7.3Hz,2H),2.03–1 .94(m,1H),1.71(t,J=7.0Hz,2H),1.65–1.51(m,6H),1.44–1.35(m,2H),1.33–1.19(m,18H),1.11(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 54 H 63 N5O7[M+H] + :894.4800,found 894.4775.

[0137] Example 11. Preparation of 11.4-(2-(12-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindoline-4-yl)oxy)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 11)

[0138]

[0139] Compound 11 was prepared according to Example 8. 1H NMR (400MHz, DMSO) δ13.14(s,1H),10.96(s,1H),9.79(s,1H),8.02(d,J=8.0Hz,2H),7.77(d,J=8.0Hz,2H),7.59–7.47(m,2H), 7.31(d,J=8.8Hz,1H),7.08(d,J=7.6Hz,1H),7.05–7.96(m,3H),6.86(s,1H),4.99(dd,J=13.4,5.1Hz,1H),4.42–4.18(m,2H),4 .07(t,J=6.5Hz,2H),3.17(s,3H),2.98–2.83(m,1H),2.65–2.54(m,1H),2.42–2.31(m,1H),2.25(d,J=7.5Hz,2H),2.01–1.89( m,1H),1.77–1.67(m,2H),1.66–1.52(m,6H),1.49–1.37(m,2H),1.35–1.18(m,18H),1.11(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 54 H 63 N5O7[M+H] + :894.4800,found 894.4775.

[0140] Example 12. Preparation of 4-(2-(12-((2-(2,6-dioxadiazin-3-yl)-1-oxoisoindoline-4-yl)amino)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 12)

[0141] Step 1.1 Preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)dodecanoic acid (compound 12a)

[0142]

[0143] 464 mg (1.4 mmol) of tert-butyl 12-bromododecanoate and 300 mg (1.2 mmol) of lenalidomide were dissolved in NMP, and 0.6 mL (3.5 mmol) of DIPEA was added. The mixture was reacted overnight at 100 °C under argon protection. After the reaction was complete, the system was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. Rapid silica gel column chromatography yielded 386 mg of a creamy white solid compound, which was then hydrolyzed with trifluoroacetic acid to give 370 mg of a white solid compound 12a, with a yield of 70%. 1 HNMR(400MHz,DMSO)δ11.95(s,1H),11.00(s,1H),7.27(t,J=7.7Hz,1H),6.92(d,J=7.4Hz,1H), 6.74(d,J=7.9Hz,1H),5.54(t,J=5.6Hz,1H),5.11(dd,J=13.3,5.1Hz,1H),4.28–4.05(m,2H),3. 11(q,J=6.6Hz,2H),2.97–2.86(m,1H),2.69–2.56(m,1H),2.36–2.26(m,1H),2.15(t,J=7.3Hz,2 H),2.08–1.98(m,1H),1.62–1.52(m,2H),1.50–1.42(m,2H),1.30–1.21(m,14H).LC-MS(ESI)m / z 455.9[MH] - .

[0144] Step 2.4 Preparation of 2-(12-((2-(2,6-dioxadiazin-3-yl)-1-oxoisoindoline-4-yl)amino)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 12)

[0145]

[0146] Compound 12 was obtained by amide condensation and hydrolysis of tert-butyl ester following steps 5 and 6 of Example 1. 1H NMR (400MHz, DMSO) δ13.12(s,1H),10.99(s,1H),9.78(s,1H),8.03(d,J=8.0Hz,2H),7.77(d,J=8.2Hz,2H),7.55(s,1H),7 .35–7.22(m,2H),7.03(s,1H),6.99(d,J=8.8Hz,1H),6.92(d,J=7.5Hz,1H),6.86(s,1H),6.72(d,J=8.1Hz,1H),5.52(s,1 H),5.11(dd,J=13.3,5.1Hz,1H),4.27–4.08(m,2H),3.17(s,3H),3.13–3.04(m,2H),2.99–2.84(m,1H),2.67–2.55(m,1H) ,2.35–2.21(m,3H),2.05–1.96(m,1H),1.65–1.51(m,7H),1.35–1.20(m,21H),1.11(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 54 H 64 N6O6[M+H] + :893.4960,found 893.4930.

[0147] Example 13. Preparation of 4-(2-(12-((2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)dodecanoamide)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 13)

[0148]

[0149] In Example 12, tert-butyl bromododecanoate and lenalidomide were replaced with tert-butyl 12-aminododecanoate and pomalidomide, while the other reagents and preparation methods were the same as in Example 12, to prepare compound 13. 1H NMR (400MHz, DMSO) δ13.13(s,1H),11.08(s,1H),9.78(s,1H),8.02(d,J=8.0Hz,2H),7.77(d,J=8.0Hz,2H),7.59– 7.53(m,2H),7.31(d,J=8.9Hz,1H),7.07(d,J=8.5Hz,1H),7.04–6.96(m,3H),6.86(s,1H),6.51(t,J=6.1Hz,1H), 5.05(dd,J=13.1,5.4Hz,1H),3.29–3.22(m,2H),3.17(s,3H),2.95–2.79(m,1H),2.64–2.53(m,2H),2.27(t,J=7. 4Hz,2H),2.06–1.96(m,1H),1.65–1.49(m,8H),1.32–1.20(m,20H),1.11(s,3H),1.01(s,3H).HRMS(ESI)calcdfor C 54 H 62 N6O7[M+H] + :907.4753,found 907.4726.

[0150] Example 14. Preparation of 4-(2-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 14)

[0151] Step 1.4 Preparation of ((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)piperazine-1-carboxylic acid benzyl ester (compound 14a)

[0152]

[0153] 10 g (47 mmol) of 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde and 13 g (62 mmol) of N-cbz-piperazine were dissolved in 200 mL of dry dichloromethane. 2 mL of glacial acetic acid was added, and the mixture was stirred at room temperature for 30 min. Then, 48 g (226 mmol) of sodium triacetoxyborohydride was added in portions, and the mixture was stirred for another 5 h at room temperature. After the reaction was complete, the system was poured into water, extracted three times with dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The solution was purified by rapid silica gel column chromatography to give 11.2 g of a white solid, compound 14a (57% yield). 1H NMR (400MHz, CDCl3) δ7.43–7.29(m,5H),5.15(s,2H),4.20–4.00(m,2H),3.51(t,J=5.0Hz,4H),2.70(t,J=12.5Hz,2H),2.4 4–2.30(m,4H),2.19(d,J=7.1Hz,2H),1.79–1.68(m,2H),1.69–1.59(m,1H),1.47(s,9H),1.15–1.01(m,2H).LC-MS(ESI)m / z 418.3[M+H] + .

[0154] Step 2.4 Preparation of 4-(piperidin-4-ylmethyl)piperazine-1-carboxylic acid benzyl ester (compound 14b)

[0155]

[0156] 6 g of compound 14a was dissolved in 50 mL of dichloromethane, and 20 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the solvent was evaporated to dryness, and dichloromethane was added repeatedly to dissolve the compound, followed by evaporation to dryness. Then, dichloromethane and methanol were added to dissolve the compound, and an appropriate amount of potassium carbonate aqueous solution was added. The mixture was stirred at room temperature for 20 min, and the system was evaporated to dryness. Dichloromethane was added again to dissolve the compound, and the solution was filtered through a diatomaceous earth filter. The filtrate was evaporated to dryness and used directly in the next step. LC-MS (ESI) m / z 316.3 [MH] - .

[0157] Step 34 - Preparation of tert-butyl benzoate (compound 14c) (2-bromo-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)

[0158]

[0159] In Example 1, step 1, 4-(5,7,7,10,10-pentamethyl-2-nitro-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid was replaced with 4-(2-bromo-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (synthesis reference: Chem. Pharm. Bull. 47(12) 1778-1796(1999)), and the other reagents and preparation methods were the same as in Example 1, to prepare compound 14c. 1H NMR (400MHz, DMSO) δ7.98(d,J=8.1Hz,2H),7.79(d,J=8.1Hz,2H),7.38–7.32(m,2H),7.06(s,1H),7.02(d,J=8.4Hz,1 H),6.85(s,1H),3.19(s,3H),1.65–1.53(s,13H),1.29(s,3H),1.25(s,3H),1.10(s,3H),0.99(s,3H).LC-MS(ESI)m / z 573.3[M+H] + .

[0160] Step 4.4 Preparation of ((1-(12-(4-(tert-butoxycarbonyl)phenyl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diazepine-2-yl)piperidin-4-yl)methyl)piperazine-1-carboxylic acid benzyl ester (compound 14d)

[0161]

[0162] 400 mg (0.69 mmol) of compound 14c and 332 mg (1.1 mmol) of compound 14b were dissolved in 40 mL of dry 1,4-dioxane. 64 mg (0.07 mmol) of Pd2(dba)3, 21 mg (0.04 mmol) of 2-(di-tert-butylphosphine)biphenyl and 134 mg (1.4 mmol) of sodium tert-butoxide were added. The mixture was reacted at 80 °C for 1 h under argon protection. After the TLC showed that the starting material 14c had disappeared, the system was cooled to room temperature and filtered. The filtrate was evaporated to dryness and passed through a column to give 435 mg of orange-red solid compound 14d, with a yield of 77%. 1 H NMR (400MHz, DMSO) δ7.97(d,J=8.1Hz,2H),7.78(d,J=8.1Hz,2H),7.41–7.29(m,5H),7.01(s,1H) ,6.93–6.88(m,1H),6.83(s,1H),6.80–6.75(m,2H),5.08(s,2H),3.64–3.54(m,2H),3.44–3.35(m ,4H),3.14(s,3H),2.64–2.54(m,2H),2.35–2.29(m,3H),2.19–2.13(m,2H),1.77(d,J=13.1Hz,2 H),1.65–1.54(m,13H),1.30(s,3H),1.27–1.22(m,7H),1.11(s,3H),1.01(s,3H).LC-MS(ESI)m / z 810.2[M+H] +.

[0163] Step 5.4 Preparation of tert-butyl benzoate (compound 14e) of (5,7,7,10,10-pentamethyl-2-(4-(piperazin-1-ylmethyl)piperidin-1-yl)-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)

[0164]

[0165] 400 mg of compound 14d was dissolved in 50 mL of methanol, and 50 mg of Pd / C (10%) was added. The mixture was stirred at room temperature under a hydrogen atmosphere for 12 h. When the starting material disappeared as shown by TLC, the mixture was filtered, and the filtrate was evaporated to dryness and passed through a column to obtain 280 mg of orange-red solid compound 14e, with a yield of 84%. 1 HNMR(400MHz,DMSO)δ7.97(d,J=8.1Hz,2H),7.78(d,J=8.1Hz,2H),7.01(s,1H),6.9 5–6.88(m,1H),6.83(s,1H),6.80–6.75(m,2H),3.63–2.55(m,2H),3.14(s,3H),3.0 8–3.00(m,3H),2.62–2.52(m,4H),2.24–2.16(m,2H),1.81–1.71(m,2H),1.64–1.52 (m,16H),1.29(s,3H),1.26–1.18(m,7H),1.10(s,3H),1.00(s,3H).LC-MS(ESI)m / z 675.9[M+H] + .

[0166] Step 6.4 Preparation of 2-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 14)

[0167]

[0168] 120 mg (0.18 mmol) of compound 14e and 74 mg (0.26 mmol) of 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione were dissolved in 10 mL of dry DMF. 0.12 mL (0.71 mmol) of DIPEA was added, and the mixture was reacted at 90 °C for 2 h under argon protection. After the reaction was complete, the mixture was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. Rapid silica gel column chromatography yielded 70 mg of a yellow solid compound, which was then hydrolyzed to give yellow solid compound 14, in 41% yield. 1 HNMR (400MHz, DMSO) δ11.08(s,1H),8.01(d,J=8.0Hz,2H),7.76(d,J=8.0Hz,2H),7.68(d,J=8.5Hz,1H),7.34(s,1H),7. 26(d,J=8.6Hz,1H),7.02(s,1H),6.94–6.89(m,1H),6.85(s,1H),6.81–6.75(m,2H),5.07(dd,J=12.9,5.4Hz,1H),3.61( d,J=11.7Hz,2H),3.53–3.38(m,4H),3.15(s,3H),2.93–2.81(m,1H),2.68–2.55(m,4H),2.22(d,J=7.1Hz,2H),2.06–1. 95(m,2H),1.81(d,J=11.8Hz,2H),1.61(d,J=9.2Hz,2H),1.31–1.20(m,14H),1.11(s,3H),1.02(s,3H).HRMS(ESI)calcd for C 52 H 57 N7O6[M+H] + :876.4443,found 876.4471.

[0169] Example 15. Preparation of 4-(2-(4-((4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 15)

[0170] Step 1.2 Preparation of (2,6-dioxadipinidin-3-yl)-5-(4-(hydroxymethyl)piperidin-1-yl)isodihydroindole-1,3-dione (compound 15a)

[0171]

[0172] By replacing compound 14e in step 6 of Example 14 with compound 4-hydroxymethylpiperidine, and using the same reagents and preparation methods as in Example 14, compound 15a was prepared. 1 H NMR (400MHz, DMSO) δ11.08(s,1H),7.64(d,J=8.5Hz,1H),7.30(d,J=2.3Hz,1H) ,7.22(dd,J=8.6,2.4Hz,1H),5.06(dd,J=12.9,5.4Hz,1H),4.50(t,J=5.3Hz,1H ),4.05(d,J=13.8Hz,2H),3.27(t,J=5.7Hz,2H),3.00–2.83(m,3H),2.65–2.52( m,2H),2.06–1.97(m,1H),1.74–1.60(m,3H),1.26–1.11(m,2H).LC-MS(ESI)m / z 370.1 [MH] - .

[0173] Step 2.1 Preparation of (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidine-4-carboxaldehyde (compound 15b)

[0174]

[0175] 500 mg (1.58 mmol) of compound 15a was dissolved in 20 mL of dry dichloromethane. 1.3 g (0.31 mmol) of Des Martin oxidant was slowly added in portions at room temperature, and the mixture was stirred for 1 h at room temperature. After the starting material disappeared as indicated by TLC, the mixture was poured into a saturated sodium thiosulfate solution and extracted three times with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. Rapid silica gel column chromatography yielded 490 mg of a yellow solid, compound 15b, in 84% yield. 1H NMR (400MHz, DMSO) δ11.08(s,1H),9.62(s,1H),7.66(d,J=8.5Hz,1H),7.34(d,J=2.3Hz,1H),7.25(dd,J=8.6,2.4Hz,1H),5.07(dd,J=12.9,5.4Hz,1H) ,3.98–3.89(m,2H),3.22–3.11(m,2H),2.95–2.82(m,1H),2.69–2.52(m,3H ),2.06–1.97(m,1H),1.97–1.88(m,2H),1.62–1.49(m,2H).LC-MS(ESI)m / z 368.2[MH] - .

[0176] Step 3.4 Preparation of 2-(4-((4-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)methyl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 15)

[0177]

[0178] Compound 15 was obtained by reductive amination and hydrolysis of tert-butyl ester, as described in Example 14. 1 HNMR (400MHz, DMSO) δ11.07(s,1H),8.01(d,J=7.9Hz,2H),7.76(d,J=8.0Hz,2H),7.64(d,J=8.5Hz,1H),7.30(s,1H),7.22(d ,J=8.9Hz,1H),7.01(s,1H),6.91(d,J=8.5Hz,1H),6.84(s,1H),6.80–7.72(m,2H),5.11–5.01(m,1H),4.07–3.95(m,2H),3.6 4–3.52(m,2H),3.14(s,3H),3.02–2.81(m,3H),2.65–2.54(m,4H),2.43–2.26(m,6H),2.18–2.07(m,3H),2.03–1.95(m,1H),1 .93–1.87(m,1H),1.84–1.69(m,4H),1.66–1.54(m,4H),1.36–1.20(m,12H),1.17–1.07(m,5H),1.01(s,3H).HRMS(ESI)calcd for C 58H 68 N8O6[M+H] + :973.5335,found 973.5320.

[0179] Example 16. Preparation of 4-(2-(4-((4-(((2-(2,6-dioxoperidinyl)-3-yl)-1-oxoisoindoline-4-yl)oxy)butyryl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 16)

[0180]

[0181] Compound 16 was obtained by amide condensation and hydrolysis of tert-butyl ester, referring to Examples 8 and 14. 1 H NMR (400MHz, DMSO) δ10.98(s,1H),8.01(d,J=8.0Hz,2H),7.78(d,J=8.1Hz,2H),7.48(t,J=7.9Hz,1H),7.31(d,J=7.5Hz,1H),7.24(d,J=8.1H z,1H),7.01(s,1H),6.91(d,J=9.1Hz,1H),6.84(s,1H),6.81–6.74(m,2H),5.11(dd,J=13.5,5.0Hz,1H),4.42–4.25(m,2H),4.14(t,J=6.5Hz, 2H),3.63–3.54(m,2H),3.53–3.43(m,2H),3.14(s,3H),2.97–2.85(m, 1H),2.65–2.55(m,3H),2.47–2.41(m,1H),2.36–2.23(m,4H),2.21–2.0 8(m,3H),2.05–1.93(m,4H),1.80–1.72(m,2H),1.66–1.56(m,4H),1.30(s,3H),1.27–1.21(m,8H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcd for C 56 H 65 N7O7[M+H] + :948.5018,found 948.5006.

[0182] Example 17. Preparation of 4-(2-(4-((4-(5-(2-(2,6-dioxoperidin-3-yl)-1-oxoisoindoline-4-yl)pent-4-ynyl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 17)

[0183] Step 1.5 Preparation of 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)pentanetraenoic acid (compound 17a)

[0184]

[0185] 200 mg (0.62 mmol) of 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione and 115 mg (0.74 mmol) of tert-butyl 4-pentyneate were dissolved in 10 mL of dry DMF, and 0.26 mL (1.87 mmol) of triethylamine were added. The mixture was reacted overnight at 80 °C under argon protection. After the reaction was complete, the system was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting product was prepared by rapid silica gel column chromatography and high-performance liquid chromatography to give 165 mg of a brown solid compound, which was then hydrolyzed to give 137 mg of a white solid compound 17a, with a yield of 65%. 1 HNMR (400MHz, DMSO) δ12.37(s,1H),11.01(s,1H),7.72(d,J=7.3Hz,1H),7.62(d,J=7.5Hz,1H),7.52(t,J=7.5Hz,1H),5.15(dd,J=13.6,4. 9Hz,1H),4.47–4.22(m,2H),2.99–2.85(m,1H),2.72–2.64(m,2H),2.64–2.54(m,3H),2.44–2.35(m,1H),2.07–1.97(m,1H).LC-MS(ESI)m / z 339.0[MH] - .

[0186] Step 2.4 Preparation of 2-(2-(4-((4-(5-(2-(2,6-dioxoperidin-3-yl)-1-oxoisoindoline-4-yl)pent-4-ynyl)piperazin-1-yl)methyl)piperidin-1-yl)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 17)

[0187]

[0188] Compound 17 was obtained by amide condensation and hydrolysis of tert-butyl ester, as described in Examples 1 and 14. 1 H NMR (400MHz, DMSO) δ11.02(s,1H),8.02(d,J=8.0Hz,2H),7.78(d,J=8.0Hz,2H),7.71(d,J=7.5Hz,1H),7.62(d,J=7.7Hz,1H),7.52(t,J=7.5H z,1H),7.02(s,1H),6.91(d,J=9.1Hz,1H),6.84(s,1H),6.81–7.74(m,2H),5.16(dd,J=13.6,5.1Hz,1H),4.47–4.30(m,2H),3.64–3.55(m,2H) ),3.51–3.42(m,4H),3.15(s,3H),2.98–2.88(m,1H),2.72–2.65(m,4H ),2.64–2.56(m,3H),2.45–2.38(m,1H),2.37–2.25(m,4H),2.19–2.10 (m,2H),2.05–1.97(m,1H),1.81–1.72(m,2H),1.65–1.56(m,4H),1.30(s,3H),1.28–1.22(m,6H),1.11(s,3H),1.02(s,3H).HRMS(ESI)calcd for C 57 H 63 N7O6[M+H] + :942.4913,found 942.4881.

[0189] Example 18. Preparation of 4-(2-(3-(2-(2-((2-(2,6-dioxopyridine)-3-yl)-1-oxoisoindoline-4-yl)oxy)ethoxy)ethoxy)ethoxy)propamido)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 18)

[0190] Step 1.3 Preparation of 2-(2-(2-(2-((2,6-dioxopiridine-3-yl)-1-oxoisoindoline-4-yl)oxy)ethoxy)ethoxy)ethoxy)propionic acid (compound 18a)

[0191]

[0192] 560 mg (2.2 mmol) of 3-(4-hydroxy-1-oxoisoindol-2-yl)piperidine-2,6-dione and 600 mg (2.2 mmol) of tert-butyl 12-hydroxy-4,7,10-trioxadodecanoate were dissolved in 10 mL of dry THF under argon protection and placed in an ice bath. 846 mg (3.2 mmol) of triphenylphosphine was added and stirred for 10 min, followed by the addition of 0.1 mL (3.2 mmol) of diisopropyl azodicarbonate. After stirring for 30 min, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the system was poured into water and extracted three times with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The solution was then prepared as a white solid (136 mg) by rapid silica gel column chromatography and high-performance liquid chromatography. Hydrolysis yielded 120 mg of a white solid (18a), with a yield of 12%. 1 H NMR (400MHz, DMSO) δ12.07(s,1H),10.97(s,1H),7.48(t,J=7.8Hz,1H),7.32( d,J=7.5Hz,1H),7.26(d,J=8.1Hz,1H),5.11(dd,J=13.5,5.1Hz,1H),4.42–4.1 9(m,4H),3.77(t,J=4.4Hz,2H),3.64–3.55(m,6H),3.17(s,4H),2.97–2.84(m ,1H),2.63–2.54(m,1H),2.49–2.38(m,3H),2.04–1.95(m,1H).LC-MS(ESI)m / z 463.1[MH] - .

[0193] Step 2.4 Preparation of 2-(3-(2-(2-(2-((2-(2,6-dioxopyridine)-3-yl)-1-oxoisoindoline-4-yl)oxy)ethoxy)ethoxy)ethoxy)propamido)-5,7,7,10,10-pentamethyl-7,8,9,10-tetrahydro-5H-benzo[b]naphtho[2,3-e][1,4]diaza-12-yl)benzoic acid (compound 18)

[0194]

[0195] Compound 18 was obtained by amide condensation and hydrolysis of tert-butyl ester, referring to Example 1. 1H NMR (400MHz, DMSO) δ13.14(s,1H),10.97(s,1H),9.86(s,1H),8.02(d,J=8.0Hz,2H),7.77(d,J=8.0Hz,2H),7.55(s,1H),7.45(t,J=7.8Hz ,1H),7.34–7.21(m,2H),7.21(d,J=8.1Hz,1H),7.04(s,1H),6.99(d,J=8.8Hz,1H),6.86(s,1H),5.10(dd,J=13.3,5.1Hz,1H),4.39–4.22( m,2H),4.20(4,J=4.5Hz,2H),3.73(t,J=4.6Hz,2H),3.68(t,J=6.3Hz,2H),3.59–3.46(m,8H),3.17(s,3H),2.96–2.85(m,1H),2.67–2.53 (m,3H),2.46–2.36(m,1H),2.01–1.94(m,1H),1.66–1.54(m,4H),1.30(s,3H),1.24(s,3H),1.10(s,3H),1.01(s,3H).HRMS(ESI)calcdfor C 51 H 57 N5O 10 [M+H] + :900.4178,found 900.4160.

[0196] Experiment Example 1: Cytotoxicity Inhibition Experiment of Small Cell Lung Cancer

[0197] Experimental Methods: Small cell lung cancer cells in logarithmic growth phase, including H446, H128, H1048, H69, and H128-sg (RXRα+RXRβ) cells (double knockout of RXRα and RXRβ), were digested and seeded into 6-well plates at 150,000 cells per well (2 mL). The plates were incubated at 37°C, 5% CO2, and saturated humidity. After 24 hours, medium containing different concentration gradients of the test compound was added to each well, resulting in final concentrations of 0, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. Four days after compound treatment, a live cell count was performed, with each group repeated three times. The IC50 was calculated. 50 value.

[0198] Table 1 shows the IC50 of the compound (RXRγ degrader) of this invention on small cell lung cancer cells. 50The experimental results show that the compounds (RXRγ degraders) provided by this invention have good anti-small cell lung cancer activity. Most of the compounds have a stronger inhibitory effect on tumor cells than the positive control drug HX531, indicating that the compounds of this invention have a good inhibitory effect on small cell lung cancer.

[0199] Table 1. IC50 of the compounds of the present invention against tumor cells 50 value

[0200]

[0201]

[0202] Experimental Example 2: Degradation of RXRγ protein by the compound

[0203] H128 and H69 cells in the logarithmic growth phase were used to prepare a cell concentration of 1.0 × 10⁻⁶ cells. 5 2 mL of cell suspension was seeded into each well of a 6-well plate, resulting in a cell count of 2.0 × 10⁶ cells per well. 5 Cells were cultured in a 5% CO2 environment at 37°C and saturated humidity to allow them to adhere to the culture vessel. Different concentrations of the compound (0, 2.5 μM, 5 μM, 10 μM and 0, 1.25 μM, 2.5 μM, 5 μM) were then added, and the cells were cultured for another 24 h. Adhering cells were scraped off the vessel and collected into EP tubes. The cells were centrifuged at 5000 rpm for 5 min at 4°C, the supernatant was discarded, and 100 μL of cell lysis buffer (prepared by mixing RIPA:PMSF:phosphatase inhibitor A:phosphatase inhibitor B = 100:1:1:1) was added to each well. The cells were then lysed on ice for 15 min. The cells were then centrifuged at 15000 rpm for 15 min at 4°C. The protein concentration of the collected protein lysate was determined using the Bradford method, and the loading volume of different protein concentrations for the same mass was calculated. The experimental results were obtained by Tris-glycine SDS-polyacrylamide gel electrophoresis.

[0204] like Figure 1 As shown, Figure 1 This diagram illustrates the effect of the compounds provided in this invention on the degradation of RXRγ protein in small cell lung cancer cells; from Figure 1 Western blot analysis showed that after 48 hours of treatment with different concentrations of the compound, the expression level of RXRγ protein decreased in human small cell lung cancer cells. Furthermore, the expression of RXRγ protein decreased significantly in a dose-dependent manner with increasing compound concentration, and the compound exhibited selectivity for RXRγ protein compared to RXRα and RXRβ. This indicates that the compound of the present invention can significantly degrade RXRγ protein, thereby inhibiting the proliferation of small cell lung cancer cells.

[0205] Experiment Example 3: Inhibition of Clonal Formation in Small Cell Lung Cancer by Compounds

[0206] Small cell lung cancer cells H446 and H1048 in logarithmic growth phase were prepared into a single-cell suspension of 500 cells / mL, and 2 mL of cell suspension was added to each well of a six-well plate. After culturing for 24 h, the medium was replaced with fresh medium containing different concentrations of the compound, with the fresh medium serving as an experimental control group. The compound concentrations were 0, 2.5 μM, 5 μM, 10 μM and 0, 1.25 μM, 2.5 μM, 5 μM, with three replicates for each group. The cells were incubated at 37°C in a 5% CO2 incubator for 10 days. The medium containing different concentrations of the compound was replaced every three days. After 10 days of culture, the six-well plates were removed, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS. 1 mL of 4% paraformaldehyde (PA) was added to each well for fixation for 15 min. After fixation, the fixative was removed, and each well was stained with 0.1% crystal violet for 30 min. After air drying, the cells were photographed.

[0207] See results Figure 2 , Figure 2 This is a graph showing the effect of the compound of the present invention on the formation of small cell lung cancer cell colonies on a plate; from Figure 2 As can be seen, the compounds of this invention exhibit a good inhibitory effect on the formation of small cell lung cancer cell clones. The number and size of small cell lung cancer cell clones decrease with increasing compound concentration, meaning that the compounds of this invention can significantly reduce the survival rate of small cell lung cancer cells.

[0208] Experiment Example 4: Experiment on the Inhibition of Tumor Growth in Mice by Compounds

[0209] Human small cell lung cancer cells (H128) were subcutaneously inoculated into nude mice. After approximately one week, mice were randomly divided into three groups of seven per group: a control group, a compound 8 degrader group (5 mg / kg), and a compound 8 degrader group (10 mg / kg). The drugs were administered daily via intraperitoneal injection. Tumor size and mouse weight were measured every three days using calipers. After 19 days of drug treatment, the mice were euthanized, and the tumors were removed, weighed, and photographed.

[0210] The results are as follows Figure 3 As shown, Figure 3 To investigate the effects of the compounds of this invention on small cell lung cancer tumors in mice, from... Figure 3 It can be seen that compound 8 can inhibit tumor growth in mice in vivo without affecting the weight of the mice. **P<0.05 compared with the control group.

[0211] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0212] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A protein degradation-targeting chimera having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof: ； in, Y is selected from: -CH2-, -C(O)-; L is -X-L1-Z-, where X is attached to a phenyl group in a diazapyridine ring and Z is attached to a phenyl group in a benzo[5]-membered ring; X is selected from: , , -NHC(O)-, -C(O)NH-, -C(O)O-, -OC(O)-; Z is selected from: , , -NH-, -O-, -C≡C-, -CH=CH-; L1 is selected from: -(CH2) m -、-(CH2) m1 -L2-(CH2) m1 -、-(CH2) m1 -L2-C(O)-(CH2) a -, -CH2-(CH2-O-CH2) q -CH2-; L2 is ; m is selected from: 10, 11, 12; each m1 is independently selected from: 1, 2, 3; a is selected from: 1, 2, 3, 4, 5; q is selected from: 2, 3, 4.

2. The protein degradation-targeting chimera or its pharmaceutically acceptable salt according to claim 1, characterized in that, X is selected from: -NHC(O)-, .

3. The protein degradation-targeting chimera or its pharmaceutically acceptable salt according to claim 1, characterized in that, Z is selected from: -O-, -NH-, -C≡C-, -CH=CH-, .

4. The protein degradation-targeting chimera or its pharmaceutically acceptable salt according to claim 1, characterized in that, L is selected from: , , , , , ; Each m is independently selected from: 10, 11, and 12; q is selected from: 2, 3, 4; Each m1 is independently selected from: 1, 2, 3; a is selected from: 2, 3, 4.

5. The protein degradation-targeting chimera according to claim 4, or its pharmaceutically acceptable salt, characterized in that, L is selected from: , , , , ; m is selected from: 10, 11 or 12; each m1 is independently selected from: 1 or 2; a is selected from: 2, 3 or 4.

6. The protein degradation-targeting chimera according to claim 5, or a pharmaceutically acceptable salt thereof, characterized in that, L is selected from: , , , , .

7. The protein degradation-targeting chimera or its pharmaceutically acceptable salt according to any one of claims 1-6, characterized in that, The protein degradation targeting chimera has the structure shown in formula (II), formula (III), formula (IV) or formula (V): .

8. The protein degradation-targeting chimera according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The protein degradation-targeting chimera is selected from the following compounds: 。 9. The use of the protein degradation targeting chimera according to any one of claims 1-8 or its pharmaceutically acceptable salt in the preparation of a medicament for treating tumors, wherein the tumor is lung cancer.

10. The application according to claim 9, characterized in that, The lung cancer in question is small cell lung cancer.

11. A pharmaceutical composition for treating tumors, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the protein degradation-targeting chimera as described in any one of claims 1-8 or a pharmaceutically acceptable salt thereof.

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