Degradation agent antibody coupling medicine for targeting GSPT1 protein as well as preparation method and application of degradation agent antibody coupling medicine

Abstract

The invention relates to a degradation agent antibody coupling drug targeting GSPT1 protein as well as a preparation method and application of the degradation agent antibody coupling drug. According to the invention, a GSPT1 molecular glue degradation agent with high degradation activity is coupled with a high-specificity targeting antibody to prepare a degradation agent antibody coupling drug (DAC). According to the degradation agent antibody coupling medicine, the GSPT1 molecular glue degradation agent can be accurately and efficiently delivered into tumor cells through the targeting effect of the antibody, so that the specific enrichment of the medicine in tumor tissues is realized, and the exposure of the medicine in non-targeted tissues is remarkably reduced. The targeted delivery strategy of the invention is expected to significantly reduce the toxic and side effects possibly generated by systemic administration of the GSPT1 molecular glue degradation agent, thereby effectively widening the treatment safety window of the GSPT1 molecular glue degradation agent. The tumor targeting property of the protein degradation agent is improved through the DAC technology, the patent medicine property of the protein degradation agent is improved, the systematic toxic and side effects of the protein degradation agent are reduced, a new solution is provided for clinical treatment of intractable or drug-resistant tumors, and the protein degradation agent has important clinical value and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to an antibody-drug conjugate targeting the GSPT1 protein, its preparation method, and its application. Specifically, this application relates to bioactive compounds with GSPT1 protein degradation function (or referred to as GSPT1 molecular glue degraders or GSPT1 molecular glue compounds), drug-linker conjugates, antibody-drug conjugates (DACs) based on protein degraders, their preparation methods, and their applications. Background Technology

[0002] GSPT1 is a translation termination factor encoded by G1toSphasetransition1. It mediates the termination of protein synthesis by binding to eukaryotic peptide chain releasing factor 1 (eRF1) and recognizing stop codons in mRNA. GSPT1 plays several key biological roles in normal development and growth, including: (1) regulating mRNA degradation; (2) regulating apoptosis; (3) terminating protein translation; and (4) participating in chromosome segregation and cytoskeleton formation regulation. Studies have shown that GSPT1 is closely related to the development and progression of various common malignant tumors such as lung cancer, liver cancer, breast cancer, and acute myeloid leukemia (AML), and is therefore considered an important anti-tumor target with broad potential. However, due to the lack of pockets suitable for small molecule binding on the surface of the GSPT1 protein, developing its small molecule inhibitors is extremely difficult, making it a traditionally "difficult-to-drug" target.

[0003] The emerging targeted protein degradation technology in recent years has provided new ideas for tackling such "difficult-to-drug" targets. Clinical studies have shown that while the GSPT1-targeting gel CC-885 possesses degradation activity, it exhibits certain systemic off-target toxicity, resulting in a narrow safety window for tumor treatment. Another gel, CC-90009, has shown poor efficacy, limiting its clinical application.

[0004] Antibody-drug conjugates (ADCs) are effective targeted therapies for tumors, consisting of a specific monoclonal antibody, a highly active cytotoxic drug, and a linker that modulates the drug's properties. ADCs leverage the high affinity and specificity of antibodies to precisely deliver highly active cytotoxic drugs to tumor tissues, thereby improving efficacy while significantly reducing systemic toxicity and off-target risks, and broadening the therapeutic window.

[0005] However, existing GSPT1 molecular gel degraders targeting the GSPT1 protein have problems such as high toxicity, narrow therapeutic window, and insufficient efficacy. Summary of the Invention

[0006] To address the problems of high toxicity, narrow therapeutic window, and insufficient efficacy of existing GSPT1 molecular gel degraders targeting the GSPT1 protein, this invention provides an antibody-drug conjugate targeting the GSPT1 protein, its preparation method, and its application.

[0007] Specifically, this invention prepares a degradation agent antibody-drug conjugate (DAC) by conjugating a highly degrading GSPT1 molecular glue degrader with a highly specific targeting antibody. This DAC can precisely and efficiently deliver the GSPT1 molecular glue degrader to tumor cells through the targeting action of the antibody, thereby achieving specific enrichment of the drug in tumor tissues and significantly reducing exposure in non-target tissues.

[0008] This targeted delivery strategy is expected to significantly reduce the potential toxic side effects of systemic administration of GSPT1 molecular gel degraders, thereby effectively broadening their therapeutic safety window. This invention enhances the tumor targeting of protein degraders, improves their drug-like properties, and reduces their systemic toxic side effects through DAC technology, providing a new solution for the clinical treatment of refractory or drug-resistant tumors, and possesses significant clinical value and application prospects.

[0009] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides an antibody-drug conjugate targeting the GSPT1 protein, which is a compound of Formula I, or a stereoisomer of a compound of Formula I, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. (Formula I) In Formula I, Ab refers to a macromolecular compound that can recognize and bind to cell-associated antigens or receptors, namely a highly specific targeting antibody or its antigen-binding fragment. The antibody may be selected from chimeric antibodies, humanized antibodies, fully human antibodies or murine antibodies. S represents the sulfur atom on Ab; L represents either a cuttable connector or a non-cuttable connector; R1 is selected from hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 Cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl, C(O)R6, C(O)OR6, C(O)NHR6, S(O)2R6 or C(O)C(O)R6; wherein each R6 is independently selected from: hydrogen, C1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; R2 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; X is selected from: -CH 2- -CD 2- -C(O)-, -CCH3-; Y is selected from: -CH2-, -NH-, -O-, -S-, -S(O)2-, -C(O)NH-, -C(O)O-, -NHC(O)-; R3, R4, and R5 are each independently selected from: H, D, and C. 1-3 alkyl; m1 and m2 are each independently 0, 1, 2, 3 or 4; q is an integer selected from 1 to 20.

[0010] In some embodiments of the present invention, the antibody may be selected from antibodies targeting the following targets or their antigen-binding fragments: HER2, Trop-2, Claudin-6, Claudin-9, Claudin-18.2, EGFR, c-Met, CD19, PSMA, Muc1, BCMA, PD-L1, CD33, CD30, CD22, CD79b, Nectin-4, CD19, tissue factor TF, FRα, B7-H3, B7-H4, CDH3, CDH6, CDH17, ALPP, CD56, CD37, HER3, ROR1. MSLN, TNF-α, CD25, ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM5, CEACAM6, CD138, GC-C, LIV-1, CA6, FUT3, IGF-1R, CTLA4, RNF43, D PEP3, 5T4, ITGB6, EFNA4, CD228, Notch3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, TIM1, sTn, ETB, Globo H, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD5 1. SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, LYFD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, Muc1, LRRC15, CD44, CD70, EphA2, CXCR4, DDR1, DKL1, FOLR, CD45, DSG2, ALK, TRAIL, EpCAM, VEGFR2, CD47, CD49, SSEA-4, DCLK1, OacGD2, CD73, ENO1, BSG, CD24, GLUT1 and GPRC5D, preferably HER2, HER3, EGFR, Trop-2, Claudin-6, Claudin-18.2, B7-H3, B7-H4, CDH3, CDH6, CDH17, FRα, ROR1, ALPP, CEACAM5, CEACAM6 or FOLR; In Formula I, the linker L is covalently bonded to the S atom on the antibody (the sulfur atom on the Ab) and also covalently bonded to the GSPT1 molecular glue degrader (or GSPT1 molecular glue compound).

[0011] In a more specific embodiment of the present invention, the antibody-drug conjugate targeting the GSPT1 protein is a compound of Formula II, or a stereoisomer of a compound of Formula II, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. (Formula II) In Formula II, L1 is a linker unit that covalently binds an antibody or its antigen-binding fragment (Ab) to L2; L2 is a connecting unit, which covalently connects the connector unit (L1) to L3; L3 is selected from amino acid residues or short peptides consisting of 2-5 amino acid residues; the amino acids are selected from natural amino acids and / or non-natural amino acids; L4 is a chemical bond or spacer unit that covalently binds the GSPT1 molecular glue degrader (or GSPT1 molecular glue compound) to L3; R1 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 Cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl, C(O)R6, C(O)OR6, C(O)NHR6, S(O)2R6, C(O)C(O)R6; wherein each R6 is independently selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; R2 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; X is selected from: -CH 2- -CD 2- -C(O)-, -CCH3-; Y is selected from: -CH2-, -NH-, -O-, -S-, -S(O)2-, -C(O)NH-, -C(O)O-, -NHC(O)-; R3, R4, and R5 are each independently selected from: H, D, and C. 1-3 alkyl; m1 and m2 are each independently 0, 1, 2, 3 or 4; q is an integer selected from 1 to 20.

[0012] In one embodiment of the present invention, L1 is selected from one of the following structures: , ; In one embodiment of the present invention, L2 is selected from one of the following structures: , , W is selected from -CH2- and O, and p is any integer between 0 and 20; In one embodiment of the present invention, L3 is selected from one of the following structures: , , , ; In one embodiment of the present invention, L4 is selected from one of the following structures: , .

[0013] In one embodiment of the present invention, the GSPT1 molecular glue degrader (or GSPT1 molecular glue compound, or simply drug payloads) is a compound represented by Formula III, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof; (Formula III) In Equation III, R1 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 Cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl, C(O)R6, C(O)OR6, C(O)NHR6, S(O)2R6, C(O)C(O)R6; wherein each R6 is independently selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; R2 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; X is selected from: -CH 2- -CD 2- -C(O)-, -CCH3-; Y is selected from: -CH2-, -NH-, -O-, -S-, -S(O)2-, -C(O)NH-, -C(O)O-, -NHC(O)-; R3, R4, and R5 are each independently selected from: H, D, and C. 1-3 alkyl; m1 and m2 are each independently 0, 1, 2, 3 or 4.

[0014] In one embodiment of the present invention, the GSPT1 molecular glue degrader (or GSPT1 molecular glue compound, or simply drug payloads) is further selected from one of the following structures: In one embodiment of the present invention, the drug-linker conjugate is selected from one of the following structures: This invention further provides a method for preparing the antibody-drug conjugate targeting the GSPT1 protein degrader, comprising the following steps: Synthesize drug-linker conjugates from drugs and linkers; The antibody is combined with a drug-linker conjugate to obtain the antibody-drug conjugate targeting the GSPT1 protein.

[0015] The present invention further provides the application of the antibody-drug conjugate targeting the GSPT1 protein degrader, and the application of the antibody-drug conjugate targeting the GSPT1 protein degrader in the preparation of medicaments for the prevention and / or treatment of diseases related to abnormal cell activity.

[0016] In one embodiment of the present invention, the disease associated with abnormal cell activity is a tumor.

[0017] In one embodiment of the present invention, the use of the antibody-drug conjugate targeting the GSPT1 protein in the preparation of a medicament for the prevention and / or treatment of tumor-related diseases.

[0018] In one embodiment of the present invention, the tumor is preferably a solid tumor, including pancreatic cancer, ovarian cancer, or lung cancer.

[0019] This invention fully utilizes the targeted delivery advantages of ADC drugs and the potential of protein degradation technology in overcoming "difficult-to-drug" targets. It conjugates a highly active GSPT1 molecular gel degrader (i.e., the drug described in this application) with a highly specific monoclonal antibody through a suitable linker to construct a novel DAC drug (degrader antibody-drug conjugate) targeting GSPT1.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: Based on the proposed method, the GSPT1 degrading agent (or GSPT1 molecular glue degrading agent or GSPT1 molecular glue compound) can be precisely delivered to tumor cells through the targeting action of antibodies, thereby achieving specific enrichment of the GSPT1 degrading agent in tumor tissues and significantly reducing exposure in non-target tissues. This targeted delivery strategy is expected to significantly reduce the potential toxic side effects of systemic administration of GSPT1 molecular glue degrading agents, thus effectively broadening its therapeutic safety window.

[0021] This invention improves the tumor targeting of protein degrading agents, enhances their drug properties, and reduces their systemic toxicity through DAC technology, providing a new solution for the clinical treatment of refractory or drug-resistant tumors and possessing significant clinical value and application prospects. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the action mode of the GSPT1 molecular gel antibody conjugate (DAC).

[0023] Figure 2 Figure 1 shows the DAR value determination results of the GSPT1 molecular gel antibody conjugate. Figure A shows the RP-HPLC detection results of FD40-ZYP-005-056; Figure B shows the LC-MS detection results of FD40-ZYP-005-056; Figure C shows the HIC-HPLC detection results of HuSC1-39-ZYP-005-056; Figure D shows the LC-MS detection results of HuSC1-39-ZYP-005-056.

[0024] Figure 3 For some TF-DACs, the inhibitory effect on tumor cell growth is IC50. 50 Figures A and B show the anti-proliferative activity of FD40-ZYP-031-179 against HPAF-II and MDA-MB-468 cells; Figures C and D show the anti-proliferative activity of HuSC1-39-ZYP-005-056 against HPAF-II and MDA-MB-468 cells; Figures E and F show the anti-proliferative activity of HuSC1-39-ZYP-031-179 against HPAF-II and MDA-MB-468 cells.

[0025] Figure 4 For the partial HER2-DAC inhibitory effect on tumor cell growth IC 50Figures A and B show the antiproliferative activity of pertocilizumab conjugate against BT-474 cells; Figure C shows the antiproliferative activity of trotocilizumab conjugate against BT-474 cells; Figures D and E show the antiproliferative activity of 1-G07-FC nanobody conjugate against BT-474 cells; Figure F shows the antiproliferative activity of pertocilizumab conjugate and Orum-5029 in BT-474 cells.

[0026] Figure 5 This represents the affinity of some DACs for tumor cell surface antigens. Figures A and B show the affinity of FD40-ZYP-005-056, FD40-ZYP-031-179 for MDA-MB-468 cell surface antigen; Figures C and D show the affinity of FD40-ZYP-005-056, FD40-ZYP-031-179, HuSC1-39-ZYP-005-056, HuSC1-39-ZYP-031-179 for HPAF-II cell surface antigen; Figure E shows the affinity of Pertuzumab-ZYP-005-056, Pertuzumab-ZYP-031-179, 1-G07-FC-ZYP-005-056, 1-G07-FC-ZYP-031-179 for BT-474 cell surface antigen. Detailed Implementation

[0027] In this application, the term "TF" generally refers to tissue factor. TF is a transmembrane glycoprotein that, in addition to participating in the coagulation process, is overexpressed in a variety of tumors, particularly solid tumors such as pancreatic cancer, ovarian cancer, and lung cancer.

[0028] In this application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (HER2). This term encompasses any naturally occurring HER2 derived from humans, including its full-length, unprocessed form and the mature protein produced through intracellular processing. Furthermore, HER2 also includes naturally occurring variants and isotypes, such as splice variants or allele variants. Sequence information for HER2 can be found in Uniprot accession number P04626.

[0029] In this application, the term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody constructed by grafting the complementarity-determining region (CDR) sequence of a murine antibody into the variable region framework of a human antibody. This type of antibody can significantly reduce the heterologous immune response that may be triggered by chimeric antibodies carrying a large amount of murine protein components. Human framework sequences can be obtained from public DNA databases or relevant references. For example, germline gene sequences of the human heavy and light chain variable regions can be found in the VBase human germline sequence database.

[0030] The abbreviations used in this application have the following meanings: The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] Example 1: Synthesis of Drugs (Payloads) Example 1-1 (ZYP-005-010): (2 S )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -Indole-3-yl)-1-oxopropyl-2-yl)benzyl carbamate Step 1: Preparation of methyl 2-(bromomethyl)-4-cyanobenzoate (1b) Under nitrogen protection, methyl 4-cyano-2-methylbenzoate (1a) (3.0 g, 17.1 mmol) was... N 1,4-bromosuccinimide (3.6 g, 20.6 mmol) and benzoyl peroxide (0.8 g, 3.4 mmol) were dissolved in carbon tetrachloride (40.0 mL) at 85°C. o The reaction mixture was stirred overnight. The reaction solution was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (PE / EA = 98.5 / 1.5 elution) to give intermediate 1b (3.3 g, yield 75.0%), a white solid. LCMS (ESI) m / z: 253.9, 255.9 [M + H] + .

[0033] Step 2: Preparation of 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-nitrile (1d) Under nitrogen protection, 1b (3.3 g, 12.9 mmol), 3-aminopiperidine-2,6-dione hydrochloride (1c) (2.1 g, 12.9 mmol), and anhydrous potassium carbonate (5.3 g, 38.7 mmol) were dissolved in DMF (20.0 mL) at 75 °C. o The reaction mixture was stirred at C for 3 hours. The reaction solution was concentrated under reduced pressure. The residue was mixed with water (50.0 mL) and stirred at room temperature for 0.5 h. After filtration and drying, 1d (3.0 g, yield 86.5%), a grayish-white solid, was obtained.

[0034] Step 3: Preparation of ((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)carbamate tert-butyl ester (1e) Under hydrogen atmosphere, 1d (2.5 g, 9.3 mmol), Raney nickel (4.0 mL), and di-tert-butyl dicarbonate (3.9 g, 18.6 mmol) were dissolved in THF (40.0 mL) and reacted with stirring at room temperature for 14 h. The mixture was filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (DCM / MeOH = 96.0 / 4.0) to give intermediate 1e (1.9 g, yield 54.8%) as a white solid. LCMS (ESI) m / z: 374.0 [M + H] + .

[0035] Step 4: Preparation of 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione (1f) 1e (1.8 g, 4.8 mmol) was dissolved in DCM (10.0 mL), and TFA (5 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (DCM / MeOH = 82.0 / 18.0 elution) to give intermediate 1f (1.3 g, 100% yield), a white solid. LCMS (ESI) m / z: 274.4 [M + H] + .

[0036] Step 5: (2) S )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H Preparation of (-indol-3-yl)-1-oxopropyl-2-yl)benzyl carbamate (ZYP-005-010) Intermediate 1f (100.0 mg, 0.36 mmol), (benzyloxycarbonyl)- LTryptophan (1 g) (123.0 mg, 0.36 mmol), DIPEA (139.0 mg, 1.08 mmol), PyBOP (225.0 mg, 0.43 mmol), and HOBT (58.0 mg, 0.43 mmol) were dissolved in DMF (5.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 95.0 / 5.0 elution) to give compound ZYP-005-010 (165.0 mg, yield 77.5%), a white solid. LCMS (m / z): 593.9 [M + H + ] + ; 1 H NMR (400MHz, DMSO- d 6 ) d 11.00 (s, 1H), 10.86 (s, 1H), 8.63 (t, J = 5.6 Hz, 1H), 7.64(t, J = 6.9 Hz, 2H), 7.51 (d, J = 8.1 Hz, 1H), 7.41 - 7.24 (m, 8H), 7.19 (s,1H), 7.09 (t, J = 7.4 Hz, 1H), 6.99 (t, J = 7.3 Hz, 1H), 5.12 (dd, J = 13.2,4.9 Hz, 1H), 5.05 - 4.95 (m, 2H), 4.41 (m, 4H), 4.24 (dd, J = 17.3, 4.9 Hz, 1H), 3.18 (dd, J = 14.4, 5.6 Hz, 1H), 3.06 - 2.87 (m, 2H), 2.63 (d, J = 16.9Hz, 1H), 2.41 (td, J = 13.2, 9.0 Hz, 1H), 2.06 - 1.97 (m, 1H).

[0037] Example 1-2 (ZYP-005-011): 3-((2S )-2-((tert-Butoxycarbonyl)amino)-3-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-oxopropyl)-1 H -tert-butyl indole-1-carboxylate Synthesis method: Refer to Example 1-1, step 5: From raw materials N -Boc-1-Boc- L - Tryptophan was condensed with intermediate 1f to give compound ZYP-005-011 (130.0 mg, yield 54.9%), a white solid. LCMS (ESI) m / z: 658.8 [M - H + ] - . 1 H NMR (400 MHz, DMSO - d 6) d 10.98 (s, 1H), 8.62 (t, J = 5.5 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.63 (d, J = 7.7 Hz, 1H),7.54 (s, 1H), 7.42 (s, 1H), 7.32 (t, J = 7.2 Hz, 2H), 7.25 (t, J = 7.4 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 5.12 (dd, J = 13.2, 4.9 Hz, 1H), 4.44 - 4.36(m, 4H), 4.30 (s, 1H), 3.10 (dd, J = 14.5, 4.3 Hz, 1H), 3.00 - 2.86 (m, 2H), 2.61 (d, J = 16.9 Hz, 1H), 2.39 (qd, J = 13.1, 4.1 Hz, 1H), 2.04 - 1.95 (m,1H), 1.62 (s, 9H), 1.32 (s, 9H).

[0038] Example 1-3 (ZYP-005-029): (2 R)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxo-3-phenylprop-2-yl)carbamate Synthesis method, refer to Example 1-1, step 5: from raw materials ( R 3-oxo-1-phenylbut-2-yl)carbamate benzyl ester was condensed with intermediate 1f to give compound ZYP-005-029 (166.0 mg, yield 73.1%), a white solid. LCMS (ESI) m / z: 554.8 [M + H] + ] + .

[0039] Example 1-4 (ZYP-005-076): (phenyl- d 5) Methyl ((1) S )-2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Step 1: (1) S Preparation of tert-butyl carbamate (4b) of 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Intermediate 1f (1.0 g, 3.60 mmol), S 2-((tert-Butoxycarbonyl)amino)-2-phenylacetic acid (4a) (0.9 g, 3.60 mmol), DIPEA (1.4 g, 10.80 mmol), HATU (1.6 g, 4.32 mmol), and HOBT (0.6 g, 4.32 mmol) were dissolved in DMF (5.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give intermediate 4b (747.0 mg, yield 41.0%), a white solid. LCMS (ESI) m / z: 406.9 [M + H + - 100] + .

[0040] Step 2: (2) SPreparation of 2-amino-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-2-phenylacetamide (4c) Intermediate 4b (747.0 mg, 1.48 mmol) was dissolved in DCM (4.0 mL), and TFA (2.0 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (DCM / MeOH = 81.0 / 19.0 elution) to give intermediate 4c (590.0 mg, yield 98.3%), a white solid. LCMS (ESI) m / z: 406.9 [M + H + ] + .

[0041] Step 3: 4-Nitrophenyl ((phenyl- d 5) Preparation of methyl carbonate (4f) Deuterated benzyl alcohol (4d) (280.0 mg, 2.46 mmol), p-nitrophenyl chloroformate (4e) (1.0 g, 5.05 mmol), and pyridine (1.0 g, 12.60 mmol) were dissolved in DCM (20.0 mL) and stirred at room temperature for 2 h. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE / EA = 94.0 / 6.0 elution) to give intermediate 4f (385.0 mg, yield 56.3%) as a white solid.

[0042] Step 4: (Phenyl- d 5) Methyl ((1) S Preparation of 2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (ZYP-005-076) Intermediate (4c) (81.0 mg, 0.20 mmol), intermediate (4f) (67.0 mg, 0.24 mmol), and DIPEA (129.0 mg, 1.0 mmol) were dissolved in DMSO (2.0 mL) and stirred at room temperature for 2 hours. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give compound ZYP-005-076 (100.0 mg, yield 91.7%), a white solid. LCMS (ESI) m / z: 545.9 [M + H + ] + .

[0043] Example 1-5 (ZYP-005-088): 3-((2 S )-2-((benzyloxy)carbonyl)amino)-3-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)3-oxopropyl)-1 H -tert-butyl indole-1-carboxylate Step 1: 3-((2) S )-2-(((9) H -fluorene-9-yl)methoxy)carbonyl)amino)-3-(((2-(2,6-dioxopiridine-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)3-oxopropyl)-1 H Preparation of tert-butyl indole-1-carboxylate (5b) Intermediate 1f (120.0 mg, 0.44 mmol) was administered. N a -(9) H -fluorene-9-yl)methoxy)carbonyl)-1-(tert-butoxycarbonyl)- LTryptophan (5a) (230.0 mg, 0.44 mmol), DIPEA (170.0 mg, 1.32 mmol), HATU (201.0 mg, 0.53 mmol), and HOBT (72.0 mg, 0.53 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give intermediate 5b (335.0 mg, 97.4% yield), a white solid. LCMS (ESI) m / z: 682.0 [M + H + - 100] + .

[0044] Step 2: 3-((2) S Preparation of tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-oxopropyl)-1H-indole-1-carboxylic acid (5c) Intermediate 5b (335.0 mg, 0.43 mmol) and piperidine (0.5 mL) were dissolved in DMF (2.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by silica gel column chromatography (DCM / MeOH = 93.0 / 7.0 elution) to give intermediate 5c (228.0 mg, yield 94.6%) as a white solid.

[0045] Step 3: 3-((2) S )-2-((benzyloxy)carbonyl)amino)-3-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)3-oxopropyl)-1 H Preparation of tert-butyl indole-1-carboxylate (ZYP-005-088) Intermediate 5c (228.0 mg, 0.41 mmol), benzyl chloroformate (69.2 mg, 0.41 mmol), and DIPEA (158.7 mg, 1.23 mmol) were dissolved in 1,4-dioxane (3.0 mL) and reacted at room temperature under nitrogen for 2 hours. After the reaction was complete, the reactants were purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give compound ZYP-005-088 (54.0 mg, yield 19.0%), a white solid. LCMS (ESI) m / z: 594.0 [M + H + - 100] + .

[0046] Example 1-6 (ZYP-005-115): (2 S )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1-methyl-1 H -Indole-3-yl)-3-oxopropyl-2-yl)tert-butyl carbamate Synthesis method: Refer to Example 1-1, step 5: From raw materials N a -(tert-Butoxycarbonyl)-1-methyl- L - Tryptophan condensed with intermediate 1f to give compound ZYP-005-115 (137.0 mg, yield 77.1%), a white solid. LCMS (ESI) m / z: 474.2 [M + H] + - 100] - .

[0047] Example 1-7 (ZYP-005-127): (2 S )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1-methyl-1 H (-indol-3-yl)-3-oxopropyl-2-yl)benzyl carbamate Step 1: (9) H -fluorene-9-yl)methyl((2) S )-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1-methyl-1 H Preparation of (7b) indole-3-yl)-1-oxopropyl-2-yl)carbamate Intermediate 1f (93.0 mg, 0.34 mmol).N a -(9) H -fluorene-9-yl)methoxy)carbonyl)-1-methyl- L Tryptophan (7a) (150.0 mg, 0.34 mmol), DIPEA (132.0 mg, 1.02 mmol), and HATU (155.0 mg, 0.41 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give intermediate 13b (224.0 mg, 94.7% yield), a white solid. LCMS (ESI) m / z: 696.3 [M + H + ] + .

[0048] Step 2: 3-((2) S )-2-amino-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-oxopropyl)-1 H Preparation of tert-butyl indole-1-carboxylate (7c) Intermediate 7b (224.0 mg, 0.32 mmol) and piperidine (0.5 mL) were dissolved in DMF (2.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by silica gel column chromatography (DCM / MeOH = 80.0 / 20.0 elution) to give intermediate 7c (144.0 mg, yield 94.7%), a white solid. LCMS (ESI) m / z: 474.1 [M + H + ] + .

[0049] Step 3: (2) S )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1-methyl-1 H Preparation of (-indol-3-yl)-3-oxopropyl-2-yl)benzyl carbamate (ZYP-005-127) Intermediate 7c (184.0 mg, 0.39 mmol), benzyl chloroformate (79.0 mg, 0.47 mmol), and DIPEA (151.0 mg, 1.17 mmol) were dissolved in 1,4-dioxane (5.0 mL) and reacted at room temperature under nitrogen for 2 hours. After the reaction was complete, the reactants were purified by silica gel column chromatography (DCM / MeOH = 97.3 / 2.7 elution) to give compound ZYP-005-127 (118.0 mg, yield 49.9%), a white solid. LCMS (ESI) m / z: 608.6 [M + H] + ] + .

[0050] Example 1-8 (ZYP-005-128): ((2 R )-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1 H -Indole-3-yl)-3-oxopropyl-2-yl)tert-butyl carbamate Synthesis method: Refer to Example 1-1, step 5: From the starting material (tert-butyloxycarbonyl) - D - Tryptophan condensed with intermediate 1f to give compound ZYP-005-128 (64.5 mg, yield 34.9%), a white solid. LCMS (ESI) m / z: 460.2 [M + H] + -100] + .

[0051] Example 1-9 (ZYP-005-140): ((2 R 3-(diphenylmethylthio)-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)tert-butyl carbamate For the synthesis method, refer to step 5 of Example 1-1: from S -diphenylmethyl- N -(tert-Butoxycarbonyl)- L -Cysteine ​​was condensed with intermediate 1f to give compound ZYP-005-140 (98.0 mg, yield 58.7%), a white solid. LCMS (m / z): 543.3 [M+ H] + - 100] + . 1 H NMR (400 MHz, DMSO - d 6) d11.01 (s, 1H), 8.68 (s, 1H), 7.62(d, J = 7.7 Hz, 1H), 7.50 - 7.39 (m, 6H), 7.33 (s, 4H), 7.25 (t, J = 6.9 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 5.40 (s, 1H), 5.12 (dd, J = 13.2, 4.8 Hz, 1H), 4.50 - 4.12 (m, 5H), 2.99 - 2.87 (m, 1H), 2.65 (dd, J = 12.6, 6.9 Hz, 2H),2.49 (s, 1H), 2.38 (td, J = 13.0, 9.2 Hz, 1H), 2.07 - 1.96 (m, 1H), 1.41 (s, 9H).

[0052] Example 1-10 (ZYP-005-167): ((2 R 3-(benzylthio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)tert-butyl carbamate For the synthesis method, refer to step 5 of Example 1-1: from S -Benzyl- N -(tert-Butoxycarbonyl)- L -Cysteine ​​was condensed with intermediate 1f to give compound ZYP-005-167 (75.0 mg, yield 41.4%), a white solid. LCMS (ESI) m / z: 467.1 [M + H] + - 100] + .

[0053] Example 1-11 (ZYP-005-168): ((2S)-3-(benzyloxy)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)tert-butyl carbamate For the synthesis method, refer to step 5 of Example 1-1: from O -Benzyl- N -(tert-Butoxycarbonyl)- L-The condensation of serine with intermediate 1f yielded compound ZYP-005-168 (98.4 mg, 52.6% yield), a white solid. LCMS (ESI) m / z: 451.1 [M+ H] + - 100] + .

[0054] Example 1-12 (ZYP-006-003): ((2 R )-1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxy-3-(triphenylthio)propyl-2-yl)carbamate tert-butyl For the synthesis method, refer to step 5 of Example 1-1: from N -(tert-Butoxycarbonyl)- S -triphenylmethyl- L -Cysteine ​​was condensed with intermediate 1f to give compound ZYP-006-003 (126.1 mg, yield 79.8%), a white solid. LCMS (m / z): 618.3 [M + H] + - 100] + Example 1-13 (ZYP-006-048): ((2 R )-3-(benzylthio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)benzyl carbamate For the synthesis method, refer to step 5 of Example 1-1: from S -Benzyl- N -(benzyloxy)carbonyl)- L -Cysteine ​​was condensed with intermediate 1f to give compound ZYP-006-048 (167.3 mg, 96.0% yield), a white solid. LCMS (ESI) m / z: 601.3 [M + H + ] + .

[0055] Example 1-14 (ZYP-031-029): (2 S )-3-((4-(aminomethyl)benzyl)thio)-1-(((2-(2,6-dioxopiridin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)benzyl carbamate Step 1: (2) RPreparation of 1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxy-3-(triphenylthio)propyl-2-yl)carbamate (14b) Intermediate 1f (274.0 mg, 1.0 mmol) was administered. N -(benzyloxycarbonyl)- S -triphenylmethyl- L Cysteine ​​(14a) (500.0 mg, 1.0 mmol), DIPEA (387.0 mg, 3.0 mmol), and HATU (570.0 mg, 1.5 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.5 / 2.5 elution) to give intermediate 14b (465.9 mg, yield 62.0%), a white solid. LCMS (ESI) m / z: 774.9 [M + Na] + ] + .

[0056] Step 2: (2) R Preparation of 1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-mercapto-1-oxopropyl-2-yl)carbamate (14c) Intermediate 14b (80.0 mg, 0.11 mmol) was dissolved in TFA / TIPS / H2O / DCM (0.5 / 0.1 / 0.1 / 0.5 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 14c (16.6 mg, yield 29.6%), a white solid. LCMS (ESI) m / z: 511.0 [M + H] + .

[0057] Step 3: Benzyl ((2) SPreparation of 14e-3-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate Intermediate 14c (100.0 mg, 0.20 mmol), tert-butyl (4-(bromomethyl)benzyl)carbamate (14d) (59.0 mg, 0.20 mmol), and anhydrous potassium carbonate (83.0 mg, 0.60 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature under nitrogen for 2 hours. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 96.5 / 3.5 elution) to give intermediate 14e (76.0 mg, yield 52.1%), a white solid. LCMS (ESI) m / z: 630.2 [M + H + - 100] + .

[0058] Step 4: (2) S Preparation of Benzyl Carbamate (ZYP-031-029) - 3-((4-(aminomethyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)carbamate Intermediate 14e (76.0 mg, 0.10 mmol) and TFA (0.5 mL) were dissolved in DCM (2.0 mL) and reacted at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give compound ZYP-031-029 (50.0 mg, yield 79.5%), a white solid. LCMS (ESI) m / z: 630.1 [M + H+] + ] + . 1 H-NMR (400 MHz, DMSO- d 6) d 10.99 (s, 1H), 8.79 (t, J = 5.9Hz, 1H), 8.16 (s, 3H), 7.67 (d, J= 7.7 Hz, 2H), 7.50 (s, 1H), 7.44 - 7.28 (m, 10H), 5.15 - 5.06 (m, 3H), 4.42 (d, J = 5.7 Hz, 2H), 4.36 - 4.24 (m, 3H), 4.01 (d, J = 5.6 Hz, 2H), 3.78 (s, 2H), 2.98 - 2.86 (m, 1H), 2.76 (dd, J =14.1, 6.1 Hz, 1H), 2.65 - 2.55 (m, 2H), 2.46 - 2.31 (m, 1H), 2.05 - 1.94 (m, 1H).

[0059] Example 1-15 (ZYP-031-046): (2 R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxy-3-(phenylthio)prop-2-yl)carbamate For the synthesis method, refer to step 5 of Example 1-1: from N -(benzyloxy)carbonyl)- S -phenyl- L -Cysteine ​​was condensed with intermediate 1f to give compound ZYP-031-046 (31.0 mg, yield 6.2%), a white solid. LCMS (ESI) m / z: 587.0 [M + H] + ] + .

[0060] Example 1-16 (ZYP-033-111): cyclopentyl ((2 R )-3-((4-(aminomethyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)carbamate Step 1: (9) H -fluorene-9-yl)methyl((2) R Preparation of 16b of 1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxo-3-(triphenylthio)propyl-2-yl)carbamate Intermediate 1f (274.0 mg, 1.0 mmol) was administered. N-(((9 H -fluorene-9-yl)methoxy)carbonyl)- S -triphenylmethyl- L Cysteine ​​(16a) (586.0 mg, 1.0 mmol), DIPEA (387.0 mg, 3.0 mmol), and HATU (456.0 mg, 1.2 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 98.0 / 2.0 elution) to give compound 16b (428.8 mg, yield 50.9%) as a white solid.

[0061] Step 2: (2) R Preparation of 2-amino-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-3-(triphenylthio)propionamide (16c) Intermediate 16b (428.0 mg, 0.51 mmol) and piperidine (0.5 mL) were dissolved in DMF (2.0 mL) and reacted at room temperature for 1 hour. After the reaction was complete, the mixture was purified by silica gel column chromatography (DCM / MeOH = 97.5 / 2.5 elution) to give compound 16c (194.5 mg, yield 61.7%) as a white solid. LCMS (ESI) m / z: 617.1 [M - H + ] - .

[0062] Step 3: Cyclopentyl ((2) R Preparation of 1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxy-3-(triphenylthio)propyl-2-yl)carbamate (16e) Intermediate 16c (100.0 mg, 0.16 mmol), cyclopentyl chloroformate (16d) (29.0 mg, 0.19 mmol), and DIPEA (62.0 mg, 0.48 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.5 / 2.5 elution) to give compound 16e (101.0 mg, yield 86.5%) as a white solid. LCMS (ESI) m / z: 753.2 [M + Na] + ] + .

[0063] Step 4: Cyclopentyl ((2) R Preparation of 16f of 1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-mercapto-1-oxopropyl-2-yl)carbamate Intermediate 16e (197.0 mg, 0.27 mmol) was dissolved in TFA / TIPS / H₂O / DCM (0.5 / 0.1 / 0.1 / 0.5 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 96.5 / 3.5 elution) to give intermediate 16f (124.0 mg, yield 94.1%) as a white solid. LCMS (ESI) m / z: 489.1 [M + H] + .

[0064] Step 5: Cyclopentyl ((2) R Preparation of 16g ​​of 3-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxoperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate Intermediate 16f (124.0 mg, 0.25 mmol), intermediate (16a) (76.0 mg, 0.25 mmol), and anhydrous potassium carbonate (104.0 mg, 0.75 mmol) were dissolved in DMF (2.0 mL) and reacted at room temperature under nitrogen for 2 hours. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.5 / 2.5 elution) to give intermediate 16 g (132.7 mg, yield 78.1%) as a white solid. LCMS (ESI) m / z: 608.2 [M + Na] + - 100] + .

[0065] Step 6: Cyclopentyl ((2) R Preparation of 3-((4-(aminomethyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)carbamate (ZYP-033-111) 16 g (132.7 mg, 0.19 mmol) of the intermediate and 0.5 mL of TFA were dissolved in 2.0 mL of DCM and reacted at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 85.0 / 15.0 elution) to give compound ZYP-033-111 (50.0 mg, yield 43.5%), a white solid. LCMS (ESI) m / z: 633.2 [M + H₂] + ] + .

[0066] Example 2: Synthetic route of drug-linker conjugate (Payload-Linker) Example 2-1 (ZYP-005-056): (1 S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( R )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Step 1: (1) S Preparation of tert-butyl carbamate (17b) of 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Intermediate 1f (946.0 mg, 3.5 mmol), S 2-((tert-Butoxycarbonyl)amino)-2-phenylacetic acid (17a) (867.0 mg, 3.5 mmol), DIPEA (1.4 g, 10.4 mmol), and HATU (1.6 g, 4.1 mmol) were dissolved in DMF (10.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 96.5 / 3.5 elution) to give intermediate 17b (1.2 g, yield 70.6%), a white solid. MS (m / z): 406.9 [M + H₂] + - 100] + ; 1 H NMR (400 MHz, DMSO- d 6 ) d 10.99 (s, 1H),8.76 (s, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 7.3 Hz, 2H), 7.38 - 7.25(m, 6H), 5.20 (d, J = 7.9 Hz, 1H), 5.10 (dd, J = 13.2, 4.9 Hz, 1H), 4.46 -4.30 (m, 3H), 4.22 (dd, J = 17.3, 2.8 Hz, 1H), 2.98 - 2.84 (m, 1H), 2.59 (d, J = 17.3 Hz, 1H), 2.39 (dd,J = 13.2, 4.1 Hz, 1H), 2.04 - 1.93 (m, 1H), 1.39(s, 9H). Step 2: (2) S Preparation of 2-amino-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)-2-phenylacetamide (17c) 17b (1.2 g, 2.4 mmol) was dissolved in DCM (4.0 mL), and TFA (2.0 mL) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (DCM / MeOH = 85.0 / 15.0 elution) to give intermediate 17c (0.7 g, yield 71.9%), a white solid. LCMS (ESI) m / z: 274.4 [M + H + ] + . Step 3: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S Preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (17e) Intermediate 17c (400.0 mg, 1.0 mmol), (1 S ,2 R 5 S 2-Isopropyl-5-methylcyclohexylcarbonyl chloride (17d) (220.0 mg, 1.0 mmol), DIPEA (400.0 mg, 3.0 mmol), and HATU (1.6 g, 4.1 mmol) were dissolved in DMF (10.0 mL) and reacted at room temperature for 2 hours. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give intermediate 17e (410.0 mg, yield 69.7%) as a white solid. LCMS (ESI) m / z: 589.0 [M + H + ] + . Step 4: Preparation of (allyloxycarbonyl)glycylglycine (17g) Glycylglycine (17f) (5.0 g, 25.7 mmol), NaHCO3 (4.5 g, 51.4 mmol), and allyl chloroformate (3.8 g, 30.8 mmol) were dissolved in H2O / THF (40.0 / 20.0 mL) and reacted at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was adjusted to pH ~5 with dilute HCl (aq. 1N) solution, extracted with EA (50.0 mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 17 g (3.5 g, yield 63.6%) of intermediate, a white solid. LCMS (ESI) m / z: 217.0.0 [M + H + ] + . Step 5: Preparation of (2-(((allyloxy)carbonyl)amino)acetamido)methyl acetate (17h) Intermediate 17 g (1.0 g, 4.6 mmol) and Cu(OAc)₂ (100 mg, 0.6 mmol) were dissolved in anhydrous THF (20.0 mL). The reaction was carried out in a nitrogen atmosphere at 60 °C in an oil bath for 1 hour. After cooling the reaction solution to room temperature, Pb(OAc)₄ (2.5 g, 5.5 mmol) was added to the reaction solution, and the reaction was continued at room temperature for 2 hours. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 99.0 / 9.0 elution) to give intermediate 17 h (900.0 mg, yield 85.0%) as a white solid. LCMS (ESI) m / z: 253.1 [M + Na] + ] + . Step 6: Preparation of allyl (2-(chloromethyl)amino)-2-oxoethyl)carbamate (17i) The intermediate (17h) (0.9 g, 3.9 mmol) and TMSCl (1.5 mL, 15.6 mmol) were dissolved in anhydrous DCM (5.0 mL). The reaction was carried out under nitrogen atmosphere at 0 °C in an ice bath for 1.5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain a white solid 17i, which could be used directly for subsequent reactions without purification. LCMS (ESI) m / z: 225.1 [M + Na] + (Methanol derivative).

[0067] Step 7: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 SPreparation of 2-((2-(1-(((allyloxycarbonyl)amino)acetamido)methyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (17j) Intermediate 17i (410.0 mg, 0.7 mmol), intermediate 17e, and K₂CO₃ (483.0 mg, 3.5 mmol) were dissolved in NMP (10.0 mL) and reacted in an oil bath at 60 °C for 2 hours under nitrogen atmosphere. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 94.0 / 6.0 elution) to give intermediate 17j (170.0 mg, yield 32.0%) as a white solid. LCMS (ESI) m / z: 759.0 [M + H₂] + ] + . Step 8: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S Preparation of 2-((2-(1-(((allyloxycarbonyl)amino)acetamido)methyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (17k) Intermediate 17j (120.0 mg, 0.16 mmol), Pd(PPh3)4 (20.0 mg, 0.02 mmol), and phenylsilane (35.0 mg, 0.32 mmol) were dissolved in THF (3.0 mL) and reacted at room temperature under nitrogen for 3 hours. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 80.0 / 20.0 elution) to give intermediate 17k (65.0 mg, yield 60.2%) as a white solid. LCMS (ESI) m / z: 675.2 [M + H + ] + . Step 9: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 SPreparation of 2-((2-(1-((S)-11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxy-2-oxa-4,7,10,13,16-pentazaheptadecane-17-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindole-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (17m) Intermediate 17k (65.0 mg, 0.1 mmol), ((9H-fluorene-9-yl)methoxy)carbonyl)glyceroglycero- L -Phenylalanine (17l) (48.4 mg, 0.1 mmol), HOBT (16.0 mg, 0.12 mmol), DIPEA (37.0 mg, 0.3 mmol), and HATU (44.0 mg, 0.12 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours. Water (10.0 mL) was added to the reaction system, and the mixture was extracted with EA (20.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 95.0 / 5.0 elution) to give intermediate 17m (50.0 mg, yield 45.0%) as a white solid.

[0068] Step 10: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S Preparation of 17n-13-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (17n) Intermediate 17m (50.0 mg, 0.04 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was completed, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 17n (27.0 mg, yield 73.0%), a white solid.

[0069] Step 11: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S Preparation of 7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (ZYP-005-056) Intermediate 17n (27.0 mg, 0.03 mmol), 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (27.7 mg, 0.09 mmol) and DIPEA (23.2 mg, 0.18 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-005-056 (22.0 mg, yield 65.1%), a white solid. LCMS (ESI) m / z: 1129.51 [M + H + ] + . 1 H-NMR (400 MHz, DMSO- d 6) d 8.75 (t, J = 5.7 Hz, 1H), 8.21(t, J = 5.4 Hz, 1H), 8.07 (dd, J = 15.3, 6.9 Hz, 2H), 7.98 (t, J = 5.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 2H), 7.44 (d, J= 7.1 Hz, 2H), 7.38 - 7.11 (m, 9H), 6.99 (s, 2H), 5.28 - 5.10 (m, 3H), 4.98 (dd, J = 12.3, 5.3 Hz, 1H), 4.59 -4.16 (m, 9H), 3.81 - 3.44 (m, 6H), 3.36 (t, J = 7.0 Hz, 2H), 3.01 (t, J =12.5 Hz, 2H), 2.78 (dd, J = 13.5, 9.5 Hz, 2H), 2.42 - 2.27 (m, 1H), 2.09 (dd, J = 13.1, 5.6 Hz, 2H), 2.04 - 1.97 (m, 1H), 1.90 (d, J = 4.4 Hz, 2H), 1.61(t, J = 11.9 Hz, 2H), 1.47 (dq, J = 14.4, 7.2 Hz, 5H), 1.21 (m, 3H), 1.06 -0.93 (m, 2H), 0.85 (dd, J = 16.4, 6.4 Hz, 6H), 0.68 (d, J = 6.6 Hz, 3H). Example 2-2 (ZYP-005-068): (1 S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S )-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15,18-hexaoxo-2,5,8,11,14,17-hexaazatrisaccharide)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Step 1: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S)-11-benzyl-1-(9 H Preparation of fluorene-9-yl)-3,6,9,12,15,18-hexaoxo-2-oxa-4,7,10,13,16,19-hexaazaeicosano-20-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (18b) Intermediate 17k (79.0 mg, 0.12 mmol), ((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycerol- L -Phenylalanylglycine (18a) (65.4 mg, 0.12 mmol), HOBT (18.9 mg, 0.14 mmol), DIPEA (45.3 mg, 0.35 mmol), and HATU (53.4 mg, 0.14 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours. Water (10.0 mL) was added to the reaction system, and the mixture was extracted with EA (20.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 92.0 / 8.0 elution) to give intermediate 18b (100.0 mg, yield 70.4%) as a white solid.

[0070] Step 2: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S Preparation of 18c of 16-amino-10-benzyl-3,6,9,12,15-pentoxy-2,5,8,11,14-pentazahexadecyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Intermediate 18b (100.0 mg, 0.08 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 18c (70.0 mg, yield 88.6%), a white solid.

[0071] Step 3: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S )-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1 H Preparation of (-pyrrolo-1-yl)-3,6,9,12,15,18-hexaoxo-2,5,8,11,14,17-hexaazatrisacchariyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (ZYP-005-068) Intermediate 18c (70.0 mg, 0.07 mmol), 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (93.0 mg, 0.30 mmol) and DIPEA (64.5 mg, 0.50 mmol) were dissolved in DMSO (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-005-068 (18.0 mg, yield 21.7%), a white solid. LCMS (ESI) m / z: 1186.34 [M + H + ] + . 1H-NMR (400 MHz, DMSO-d6) d 8.78 (t, J = 5.9 Hz, 1H), 8.31(d, J = 4.5 Hz, 1H), 8.20 (d, J = 3.8 Hz, 1H), 8.14 - 8.05 (m, 2H), 7.98 (d, J = 15.9 Hz, 2H), 7.64 (dd, J = 16.6, 7.7 Hz, 2H), 7.45 (d, J = 7.1 Hz, 2H),7.34 (dd, J = 12.6, 5.0 Hz, 3H), 7.28 (d, J = 12.1 Hz, 2H), 7.26 - 7.21 (m,4H), 7.18 (d, J = 6.0 Hz, 1H), 5.27 - 5.10 (m, 3H), 4.97 (d, J = 12.3 Hz,1H), 4.53 - 4.28 (m, 5H), 4.22 (dd, J = 17.1, 4.7 Hz, 1H), 3.78 - 3.65 (m,7H), 3.59 (dd, J = 16.7, 5.2 Hz, 1H), 3.35 (d, J = 7.1 Hz, 3H), 3.08 - 2.94(m, 2H), 2.79 (t, J = 11.9 Hz, 2H), 2.55 (s, 1H), 2.34 (d, J = 10.9 Hz, 1H),2.10 (t, J = 7.4 Hz, 2H), 2.02 (s, 1H), 1.90 (s, 2H), 1.60 (d, J = 11.7 Hz,2H), 1.47 (dq, J = 14.7, 7.3 Hz, 5H), 1.34 - 1.14 (m, 3H), 0.98 (dd, J =22.0, 12.2 Hz, 2H), 0.85 (dd, J= 16.7, 6.7 Hz, 7H), 0.68 (d, J = 6.8 Hz, 3H). Example 2-3 (ZYP-031-084): Benzyl ((2 R )-3-((4-( S )-2-(( S )-2-(6-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)hexamido)-3-methylbutamido)-5-ureopentamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2,6-dioxopiridin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate Step 1: Benzyl ((2) R )-3-((4-( S )-2-(( S )-2-(((9) H Preparation of -fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (19b) Intermediate ZYP-031-029 (30.0 mg, 0.05 mmol), intermediate (9 H -fluorene-9-yl)methyl( S )-3-methyl-1-( S ethyl carbamate (19a) (36.6 mg, 0.05 mmol) and dipebamate (31.0 mg, 0.25 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 1 hour under nitrogen protection. After the reaction was complete, the reaction solution was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give intermediate 19b (48.0 mg, yield 76.4%), a white solid. LCMS (ESI) m / z: 629.9 1 / 2 [M + 2H + ] + . Step 2: Benzyl ((2) R)-3-((4-( S )-2-(( S Preparation of 2-amino-3-methylbutamido)-5-ureidopentamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (19c) Intermediate 19b (48.0 mg, 0.04 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 19c (35.0 mg, yield 85.4%), a white solid. LCMS (ESI) m / z: 1035.2 [M + H + ] + . Step 3: Benzyl ((2) R )-3-((4-( S )-2-(( S )-2-(6-(2,5-dioxo-2,5-dihydro-1 H Preparation of -pyrrolo-1-yl)hexamido)-3-methylbutamido)-5-ureidopentamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (ZYP-031-084) Intermediate 19c (35.0 mg, 0.034 mmol), 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (16.4 mg, 0.034 mmol) and DIPEA (20.0 mg, 0.15 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-031-084 (13.9 mg, yield 22.8%), a white solid. LCMS (ESI) m / z: 615.0 1 / 2 [M + 2H+] + ] + . 1 H₂NMR (400 MHz, DMSO-) d 6) d 10.98 (s, 1H), 9.98 (s,1H), 8.76 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.73(d, J = 5.7 Hz, 1H), 7.68 - 7.63 (m, 1H), 7.62 - 7.55 (m, 3H), 7.49 (s, 1H),7.41 (d, J = 7.7 Hz, 1H), 7.39 - 7.31 (m, 5H), 7.28 (d, J = 8.3 Hz, 3H), 7.23(d, J = 7.8 Hz, 2H), 7.17 (d, J= 7.6 Hz, 2H), 6.99 (s, 2H), 5.16 - 5.05 (m,3H), 4.96 (s, 2H), 4.44 - 4.34 (m, 4H), 4.34 - 4.23 (m, 2H), 4.22 - 4.12 (m,3H), 3.73 (s, 2H), 3.09 - 2.85 (m, 3H), 2.80 - 2.71 (m, 1H), 2.63 - 2.56 (m,2H), 2.43 - 2.31 (m, 1H), 2.14 (m, J = 13.7, 6.7 Hz, 2H), 2.03 - 1.91 (m,2H), 1.75 - 1.55 (m, 2H), 1.53 - 1.32 (m, 7H), 1.31 - 1.12 (m, 5H), 0.85 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.7 Hz, 3H). Example 2-4 (ZYP-031-179): Benzyl ((2 R )-3-((4-(2) S 5 S )-15-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureopropyl)-10,13-dioxa-3,6-diazapentadecanoamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiridin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate) The synthetic method follows the steps of Example 2-1, step 11: Condensation of 2,5-dioxopyrrolidone-1-yl-3-(2-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionate (20a) with 19c yields compound ZYP-031-179 (39.1 mg, 59.1% yield), a white solid. LCMS (ESI) m / z: 637.8 1 / 2 [M + 2H] + . 1 H₂-NMR (400 MHz, DMSO- d 6) d10.99 (s, 1H), 9.99 (s, 1H), 8.77 (s, 1H), 8.12 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.75 (t, J = 6.0 Hz, 1H), 7.65 (t, J = 7.5 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.49 (s, 1H), 7.37 - 7.34 (m, 8H), 7.20 (dd, J =27.6, 7.7 Hz, 4H), 7.02 (s, 2H), 5.15 - 5.01 (m, 3H), 4.96 (s, 2H), 4.46 -4.34 (m, 4H), 4.33 - 4.19 (m, 3H), 4.19 - 4.12 (m, 2H), 3.62 - 3.52 (m, 8H), 3.51 - 3.39 (m, 7H), 3.09 - 2.84 (m, 3H), 2.80 - 2.71 (m, 1H), 2.65 - 2.54(m, 2H), 2.45 - 2.31 (m, 3H), 2.03 - 1.91 (m, 2H), 1.75 - 1.65 (m, 1H), 1.64- 1.51 (m, 1H), 1.50 - 1.30 (m, 2H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J =6.7 Hz, 3H). Example 2-5 (ZYP-031-183): Benzyl ((2 R )-3-((4-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate Step 1: Benzyl ((2) R )-3-((4-(( S)-11-benzyl-1-(9 H Preparation of -fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazaheptadecane-17-yl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (21b) Intermediate ZYP-031-029 (63.0 mg, 0.10 mmol), intermediate ((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanylglycine (21a) (56.0 mg, 0.10 mmol), DIPEA (31.0 mg, 0.25 mmol), and HATU (46.0 mg, 0.12 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 1 hour under nitrogen protection. After the reaction was complete, the reaction solution was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 5% ~ 100% elution) to give intermediate 21b (72.5 mg, yield 62.0%), a white solid. LCMS (ESI) m / z: 585.5 1 / 2 [M + 2H] + .

[0072] Step 2: Benzyl ((2) R )-3-((4-(( S Preparation of 1,3-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)benzyl)thio)-1-(((2-(2,6-dioxopiridin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (21c) Intermediate 21b (72.5 mg, 0.06 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min; flow rate: 30 mL / min) to obtain intermediate 21c (45.0 mg, yield 76.7%), a white solid. LCMS (ESI) m / z: 948.0 [M + H] + .

[0073] Step 3: Benzyl ((2) R )-3-((4-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H Preparation of pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (ZYP-031-183) Intermediate 21c (45.0 mg, 0.05 mmol), 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (19.0 mg, 0.05 mmol) and DIPEA (39.0 mg, 0.30 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-031-183 (28.8 mg, yield 50.5%), a white solid. LCMS (ESI) m / z: 571.3 1 / 2 [M + 2H+] + ] + . 1 H₂NMR (400 MHz, DMSO-) d 6) d 10.99 (s, 1H), 8.77 (s, 1H), 8.29 (d, J = 6.0 Hz, 1H), 8.18 (t, J = 5.9 Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.07 (t, J = 5.5 Hz, 1H), 8.00 (t, J = 5.5 Hz, 1H), 7.65 (t, J = 7.2 Hz,2H), 7.50 (s, 1H), 7.41 (d, J= 7.8 Hz, 1H), 7.36 (d, J = 5.6 Hz, 3H), 7.33(s, 1H), 7.23 (s, 7H), 7.18 (d, J = 7.5 Hz, 3H), 6.99 (s, 2H), 5.14 - 5.04(m, 3H), 4.42 (d, J = 5.4 Hz, 6H), 3.73 (d, J = 7.7 Hz, 5H), 3.66 (d, J = 5.5Hz, 2H), 3.62 - 3.49 (m, 1H), 3.36 (t, J = 7.0 Hz, 2H), 3.09 - 3.01 (m, 1H), 2.97 - 2.86 (m, 1H), 2.84 - 2.71 (m, 2H), 2.68 - 2.55 (m, 4H), 2.41 - 2.30(m, 1H), 2.10 (t, J = 7.4 Hz, 2H), 2.00 (d, J = 6.5 Hz, 1H), 1.52 - 1.41 (m,4H), 1.27 - 1.13 (m, 2H). Example 2-6 (ZYP-033-119): 4-((2 S 5 S )-15-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-5-isopropyl-2-methyl-4,7-dioxo-10,13-dioxo-3,6-diazapentadecanoamido)benzyl(4-(((2) R )-2-(((benzyloxy)carbonyl)amino)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-oxopropyl)thio)methyl)benzyl)carbamate) Step 1: (9) H -fluorene-9-base) S Preparation of 1-(1-(1-oxo)-1-oxopropyl-2-yl)carbamate-CTCResin(22c) 2-CTC Resin (22a) (3.0 g, 3.47 mmol) was swollen in DCM (40.0 mL) at room temperature for 0.5 hours. After swelling, intermediate (22b) (9... H -fluorene-9-yl)methoxy)carbonyl)- L -Alanine (1.1 g, 3.47 mmol) and DIPEA (1.4 g, 10.41 mmol) were added to the reaction solution. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered, dried, and intermediate 22c was obtained, which was then directly used for the next reaction.

[0074] Step 2: Preparation of (S)-2-amino-1-(oxido)prop-1-one-CTC Resin (22d) Intermediate 22c and piperidine (5.0 mL) were dissolved in DMF (20.0 mL) and reacted at room temperature for 0.5 hours. After the reaction was complete, the mixture was filtered and dried to obtain intermediate 22d, which was then directly used for the next reaction.

[0075] Step 3: (9) H -fluorene-9-base)(( S )-1-(( S Preparation of ethyl carbamate-CTC Resin (22f) - 1-(oxidoyl)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate Intermediate 22d, ((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (22e) (1.2 g, 3.47 mmol), PyBOP (2.7 g, 4.1 mmol), HOBT (0.7 g, 4.1 mmol), and DIPEA (1.4 g, 10.4 mmol) were dissolved in DMF (10.0 mL). The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered and dried to obtain intermediate 22f, which was then directly used for the next reaction.

[0076] Step 4: (((9)) H -fluorene-9-yl)methoxy)carbonyl)- L -valine- L Preparation of α-alanine (22g) Intermediate 22f and trifluoroethanol (10.0 mL) were dissolved in DCM (10.0 mL). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the mixture was filtered and concentrated under reduced pressure to give intermediate 22 g (450.0 mg, yield 31.6%) as a white solid. LCMS (ESI) m / z: 411.1 [M + H + ] +.

[0077] Step 5: (9) H -fluorene-9-yl)methyl( S )-1-((( S Preparation of 1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (22i) Intermediate 22 g (450.0 mg, 1.1 mmol), p-aminobenzyl alcohol (22 h) (150.0 mg, 1.1 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (500.0 g, 2.2 mmol) were dissolved in DCM / MeOH (10.0 / 5.0 mL). The reaction was carried out under nitrogen protection at room temperature in the dark for 16 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 98.0 / 2.0 elution) to give intermediate 22i (337.0 mg, yield 59.5%) as a white solid. LCMS (ESI) m / z: 516.2 [M + H + ] + .

[0078] Step 6: (9) H -fluorene-9-yl)methyl( S )-3-methyl-1-( S Preparation of ethyl carbamate (22k) of 1-(4-((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (22k) Intermediate 22i (100.0 mg, 0.19 mmol), di(p-nitrobenzene) carbonate (22j) (124.0 mg, 0.40 mmol), and DIPEA (74.0 mg, 0.57 mmol) were dissolved in DMF (2.0 mL). The reaction was carried out at room temperature for 16 hours under nitrogen protection. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 5% ~ 100% elution) to give intermediate 22k (79.5 mg, yield 61.5%), a white solid. LCMS (ESI) m / z: 703.1 [M + Na] + ] + .

[0079] Step 7: Benzyl ((2) R )-3-((4-( S )-2-(( S )-2-(((9)H Preparation of -fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (22l) Intermediate 22k (47.0 mg, 0.07 mmol), ZYP-031-029 (43.5 mg, 0.07 mmol), and DIPEA (44.5 mg, 0.35 mmol) were dissolved in DMF (1.0 mL). The reaction was carried out at room temperature for 2 hours under nitrogen protection. After the reaction was complete, water (10.0 mL) was added to the reaction system, and the mixture was extracted with EA (20.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 96.3 / 3.7 elution) to give intermediate 22l (68.2 mg, yield 83.2%) as a white solid.

[0080] Step 8: 4-( S )-2-(( S -2-amino-3-methylbutyramido)propionamido)benzyl(4-(((2) R Preparation of 2-((benzyloxy)carbonyl)amino)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3,3-oxopropyl)thio)methyl)benzyl)carbamate (22m) Intermediate 22l (68.2 mg, 0.06 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL). The reaction was carried out at room temperature for 0.5 h under nitrogen protection. After the reaction was complete, the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give intermediate 22m (45.0 mg, yield 79.1%), a white solid. LCMS (ESI) m / z: 949.2 [M + H] + .

[0081] Step 9: Benzyl ((2) R )-3-((4-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 HPreparation of pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (ZYP-033-119) Intermediate 22m (35.0 mg, 0.04 mmol), 2,5-dioxopyrrolidone-1-yl-3-(2-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionate (20a) (13.0 mg, 0.04 mmol) and DIPEA (15.0 mg, 0.12 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-033-119 (23.3 mg, yield 53.1%), a white solid. LCMS (ESI) m / z: 594.7 1 / 2 [M + 2H+] + ] + . 1 H-NMR (400 MHz, DMSO- d 6) d 11.01 (s, 1H), 9.95 (s, 1H), 8.79 (s, 1H), 8.20 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.77 (t, J = 6.0 Hz, 1H), 7.67 (t, J = 8.5 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 7.52 (s,1H), 7.38 (d, J = 6.1 Hz, 3H), 7.33 (dd, J = 13.5, 6.9 Hz, 3H), 7.26 (d, J =7.7 Hz, 2H), 7.19 (d, J= 7.8 Hz, 2H), 7.04 (s, 2H), 5.17 - 5.04 (m, 3H), 4.99 (s, 2H), 4.48 - 4.36 (m, 4H), 4.32 (d, J = 10.2 Hz, 1H), 4.27 - 4.14 (m,3H), 3.76 (s, 2H), 3.58 (dd, J = 10.7, 5.6 Hz, 4H), 3.49 - 3.45 (m, 6H), 2.99- 2.88 (m, 1H), 2.78 (dd, J = 13.4, 5.6 Hz, 1H), 2.67 - 2.55 (m, 3H), 2.44 -2.41 (m, 4H), 1.98 (dt, J = 13.2, 6.9 Hz, 2H), 1.32 (d, J = 7.0 Hz, 3H), 1.30- 1.24 (m, 1H), 0.89 (d, J = 6.7 Hz, 3H), 0.85 (d, J = 6.7 Hz, 3H). Example 2-7 (ZYP-033-126): cyclopentyl ((2 R )-3-((4-((2) S 5 S )-15-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureopropyl)-10,13-dioxa-3,6-diazapentadecanoamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiridin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate) Step 1: Cyclopentyl ((2) R )-3-((4-( S )-2-( S )-2-((9) HPreparation of -fluorene-9-ylmethoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (23a) Intermediate ZYP-033-111 (50.0 mg, 0.08 mmol), intermediate (9 H -fluorene-9-yl)methyl( S )-3-methyl-1-( S ethyl carbamate (19a) (63.0 mg, 0.08 mmol) and DIPEA (32.0 mg, 0.25 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 1 hour under nitrogen protection. After the reaction was completed, the reaction solution was purified by Pre-HPLC (Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 23a (80.0 mg, yield 79.2%), a white solid.

[0082] Step 2: Cyclopentyl ((2) R )-3-((4-(( S )-2-( S Preparation of 2-amino-3-methylbutamido)-5-ureidopentamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (23b) Intermediate 23a (64.0 mg, 0.05 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL). The reaction was carried out at room temperature for 0.5 h under nitrogen protection. After the reaction was complete, the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give intermediate 23b (45.1 mg, yield 89.1%), a white solid. LCMS (ESI) m / z: 507.2 1 / 2 [M + 2H+] +] + . Step 3: Cyclopentyl ((2) R )-3-((4-((2) S 5 S Preparation of 15-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureopropyl)-10,13-dioxa-3,6-diazapentadecanoamido)benzyl)oxy)carbonyl)amino)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxypropyl-2-yl)carbamate (ZYP-033-126) Intermediate 23b (45.0 mg, 0.04 mmol), 2,5-dioxopyrrolidone-1-yl-3-(2-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionate (20a) (21.0 mg, 0.06 mmol) and DIPEA (23.0 mg, 0.18 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-033-126 (40.0 mg, yield 80.0%), LCMS (ESI) m / z: 626.8 1 / 2 [M + 2H+] + ] + White solid. 1 H₂NMR (400 MHz, DMSO-) d 6) d 10.99 (s, 1H), 9.98 (s,1H), 8.69 (s, 1H), 8.12 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.75(t, J = 5.9 Hz, 1H), 7.67 - 7.55 (m, 3H), 7.49 (d, J = 4.7 Hz, 1H), 7.39 (t, J= 9.6 Hz, 1H), 7.27 (dd, J = 15.5, 8.0 Hz, 5H), 7.18 (d, J = 7.3 Hz, 3H),7.02 (s, 2H), 5.11 (dd, J = 13.2, 4.9 Hz, 1H), 4.97 (s, 3H), 4.73 (dd, J A =10.1, 4.5 Hz, 1H), 4.62 - 4.52 (m, 1H), 4.37 (d, J = 15.0 Hz, 4H), 4.30 -4.13 (m, 4H), 3.73 (s, 2H), 3.56 (dd, J = 11.4, 5.7 Hz, 4H), 3.50 (d, J = 5.2Hz, 3H), 3.46 (d, J = 9.4 Hz, 2H), 3.42 (dd, J = 7.8, 4.4 Hz, 2H), 3.06 -2.86 (m, 2H), 2.74 (d, J = 11.8 Hz, 1H), 2.57 (dd, J = 13.6, 8.9 Hz, 2H),2.44 (t, J = 7.0 Hz, 1H), 2.36 (dd, J = 13.6, 7.0 Hz, 1H), 2.22 (t, J = 16.7Hz, 1H), 2.09 - 1.91 (m, 2H), 1.81 (d, J = 5.9 Hz, 2H), 1.61 (dd, J = 33.8,24.5 Hz, 8H), 1.47 - 1.30 (m, 2H), 1.28 - 1.21 (m, 1H), 0.86 (d, J = 6.7 Hz,3H), 0.82 (d, J = 6.7 Hz, 3H). A

[0083] Example 2-8 (ZYP-043-199): (phenyl- d 5)methyl((1 S )-2-((2-(1-((S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate Step 1: Allyl (2-((2,6-dioxo-3-(1-oxo-5-((( S )-2-phenyl-2-(((phenyl- d 5) Preparation of methoxy)carbonyl)amino)acetamido)methyl)isoindoline-2-yl)piperidin-1-yl)methyl)amino)-2-oxoethyl)carbamate (24a) Intermediate ZYP-005-076 (183.0 mg, 0.34 mmol), intermediate 17i (206.0 mg, 1.0 mmol), and K2CO3 (139.0 mg, 1.0 mmol) were dissolved in NMP (10.0 mL) and reacted in an oil bath at 60 °C for 2 hours under nitrogen atmosphere. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 94.0 / 6.0 elution) to give intermediate 24a (61.0 mg, yield 25.1%), a white solid. LCMS (ESI) m / z: 716.2 [M + H + ] + .

[0084] Step 2: (Phenyl- d 5) Methyl ((1) S Preparation of 2-((2-(1-((2-aminoacetamido)methyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (24b) Intermediate 24a (100.0 mg, 0.14 mmol), Pd(PPh3)4 (16.0 mg, 0.01 mmol), and phenylsilane (30.0 mg, 0.28 mmol) were dissolved in anhydrous THF (3.0 mL) and reacted at room temperature under nitrogen for 3 hours. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 80.0 / 20.0 elution) to give intermediate 24b (28.8 mg, yield 32.6%), a white solid. LCMS (ESI) m / z: 632.2 [M + H + ] + .

[0085] Step 3: (9) H -fluorene-9-yl)methyl((7) S )-7-benzyl-1-(2,6-dioxo-3-(1-oxo-5-((( S )-2-phenyl-2-(((phenyl- d 5) Preparation of methoxy)carbonyl)amino)acetamido)methyl)isoindoline-2-yl)piperidin-1-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecane-13-yl)carbamate (24c) Intermediate 24b (28.8 mg, 0.05 mmol), ((9H-fluorene-9-yl)methoxy)carbonyl)glyceroglycero- L -Phenylalanine (17l) (22.9 mg, 0.05 mmol), HOBT (7.4 mg, 0.06 mmol), DIPEA (17.8 mg, 0.14 mmol), and HATU (21.0 mg, 0.06 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. Water (10.0 mL) was added to the reaction system, and the mixture was extracted with EA (20.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 95.0 / 5.0 elution) to give intermediate 24c (23.5 mg, yield 45.9%) as a white solid.

[0086] Step 4: (Phenyl- d 5) Methyl ((1) S )-2-((2-(1-(( SPreparation of 1,3-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (24d) Intermediate 24c (23.5 mg, 0.02 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 24d (15.4 mg, yield 86.5%), a white solid.

[0087] Step 5: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-(( S Preparation of 7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (ZYP-043-199) Intermediate 24d (15.4 mg, 0.02 mmol), 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (6.4 mg, 0.02 mmol) and DIPEA (11.0 mg, 0.09 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-043-199 (3.0 mg, yield 16.3%), a white solid. LCMS (ESI) m / z: 543.6 1 / 2 [M + 2H] + . Example 2-9 (ZYP-052-031): tert-butyl 3-((2 S )-3-((2-(1-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-(((tert-butoxycarbonyl)amino)-3-oxopropyl)-1 H -Indole-1-carboxylate Step 1: Preparation of allyl (2-(((3-(5-(((tert-butoxycarbonyl)amino)methyl)-1-oxoisoindoline-2-yl)-2,6-dioxopiperidin-1-yl)methyl)amino)-2-oxoethyl)carbamate (25a) Intermediate 1e (346.0 mg, 0.9 mmol), intermediate 17i (412.0 mg, 2.0 mmol), and K₂CO₃ (384.0 mg, 2.8 mmol) were dissolved in NMP (10.0 mL) and reacted in an oil bath at 60 °C for 2 hours under nitrogen atmosphere. After the reaction was complete, water (20.0 mL) was added to the reaction system, and the mixture was extracted with EA (30.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 94.0 / 6.0 elution) to give intermediate 25a (275.0 mg, yield 54.5%) as a white solid. LCMS (ESI) m / z: 566.0 [M + Na + ] + .

[0088] Step 2: Preparation of tert-butyl carbamate (25b) Intermediate 25a (275.0 mg, 0.5 mmol), Pd(PPh3)4 (58.0 mg, 0.05 mmol), and phenylsilane (108.0 mg, 1.0 mmol) were dissolved in anhydrous THF (3.0 mL) and reacted at room temperature under nitrogen for 3 hours. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 80.0 / 20.0 elution) to give intermediate 25b (70.0 mg, yield 30.4%), a white solid. LCMS (ESI) m / z: 460.1 [M + H + ] + .

[0089] Step 3: (2-(1-(( S )-11-benzyl-1-(9 H Preparation of 25c fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazaheptadecane-17-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)carbamate tert-butyl (25c) Intermediate 25b (70.0 mg, 0.15 mmol), ((9H-fluorene-9-yl)methoxy)carbonyl)glyceroglycero- L-Phenylalanine (17l) (76.0 mg, 0.15 mmol), HOBT (24.0 mg, 0.18 mmol), DIPEA (58.0 mg, 0.45 mmol), and HATU (68.0 mg, 0.18 mmol) were dissolved in DMF (2.0 mL) and reacted at room temperature for 2 hours. Water (10.0 mL) was added to the reaction system, and the mixture was extracted with EA (20.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 92.0 / 8.0 elution) to give intermediate 25c (96.0 mg, yield 68.1%) as a white solid.

[0090] Step 4: (9) H -fluorene-9-yl)methyl((7) S Preparation of 1-(3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)-2,6-dioxopiperidin-1-yl)-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecane-13-yl)carbamate (25d) Intermediate 25c (96.0 mg, 0.1 mmol) and TFA (1.0 mL) were dissolved in DCM (2.0 mL) and reacted at room temperature under nitrogen for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 80.0 / 20.0 elution) to give intermediate 25d (147.0 mg, yield 55.9%) as a white solid. LCMS (ESI) m / z: 843.2 [M+ H+] + ] + .

[0091] Step 5: tert-butyl 3-((2) S )-3-((2-(1-(( S )-11-benzyl-1-(9 H Preparation of -fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazaheptadecane-17-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-(((tert-butoxycarbonyl)amino)-3-oxopropyl)-1H-indole-1-carboxylate (25f) The intermediate was administered for 25 days (25.0 mg, 0.03 mmol). N -Boc-1-Boc- L-Tryptophan (25e) (12.0 mg, 0.03 mmol), HATU (14.0 mg, 0.04 mmol), HOBT (5.0 mg, 0.04 mmol), and DIPEA (11.0 mg, 0.09 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was completed, the reactants were purified by Pre-HPLC (instrument model: Agilent Prep 1200 series; UV lamp: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 25f (9.9 mg, yield 26.8%), a white solid.

[0092] Step 6: 3-((2) S )-3-((2-(1-(( S )-13-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-(((tert-butyloxycarbonyl)amino)-3-oxopropyl)-1 H Preparation of tert-butyl indole-1-carboxylate (25g) Intermediate 25f (9.9 mg, 0.008 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 25 g (7.1 mg, yield 88.8%) as a white solid.

[0093] Step 7: tert-butyl 3-((2) S )-3-((2-(1-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 HPreparation of (-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-(((tert-butoxycarbonyl)amino)-3-oxopropyl)-1H-indole-1-carboxylate (ZYP-052-031) 25 g (7.1 mg, 0.007 mmol) of intermediate, 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (2.6 mg, 0.009 mmol) and DIPEA (4.5 mg, 0.035 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-052-031 (5.0 mg, yield 59.5%), a white solid. LCMS (ESI) m / z: 600.8 1 / 2 [M + 2H] + .

[0094] Example 2-10 (ZYP-052-032): Benzyl ((2 R )-1-((2-(1-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindol-5-yl)methyl)amino)-3-(benzylthio)-1-onepropyl-2-yl)carbamate The synthesis method refers to steps five, six, and seven of Example 2-9: from raw materials S -Benzyl- N -(benzyloxy)carbonyl)- L -Cysteine ​​yielded compound ZYP-052-032 (2.1 mg, yield 36.8%), a white solid. LCMS (ESI) m / z: 571.2 1 / 2 [M + 2H+] + ] + .

[0095] Example 2-11 (ZYP-052-052): Benzyl ((2 R )-1-((2-(1-(( S 7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindol-5-yl)methyl)amino)-1-oxo-3-(phenylthio)propyl-2-yl)carbamate The synthesis method refers to steps five, six, and seven of Example 2-9: from raw materials N -(benzyloxy)carbonyl)- S -phenyl- L -Cysteine ​​yielded compound ZYP-052-052 (3.0 mg, yield 44.4%), a white solid. LCMS (ESI) m / z: 564.3 1 / 2 [M + 2H+] + ] + .

[0096] Example 2-12 (ZYP-052-053): Benzyl ((2 R )-1-((2-(1-(( S )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindol-5-yl)methyl)amino)-3-(benzylthio)-1-onepropyl-2-yl)carbamate The synthesis method refers to steps five, six, and seven of Example 2-9: from the starting material 1-[2-(1,1-dimethylethoxy)-2-oxoethyl]- N -[(9 H [-fluorene-9-ylmethoxy)carbonyl]- L -Tryptophan yielded compound ZYP-052-053 (5.3 mg, yield 61.6%), a white solid. LCMS (ESI) m / z: 568.2 1 / 2 [M + 2H+] + - 100] + .

[0097] Example 2-13 (ZYP-052-082): Benzyl ((2 S )-1-((2-(1-((R )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-(1-methyl-1 H -Indole-3-yl)-1-oxopropyl-2-yl)carbamate The synthesis method refers to steps five, six, and seven of Example 2-9: obtaining compound N from the starting materials. a -((benzyloxycarbonyl)-1-methyl- L -Tryptophan yielded ZYP-052-082 (3.3 mg, 41.3% yield), a white solid. LCMS (ESI) m / z: 574.71 / 2 [M + 2H⁺] + .

[0098] Example 2-14 (ZYP-052-089): Benzyl ((2 R )-3-((4-(aminomethyl)benzyl)thio)-1-((2-(1-(( R )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)carbamate first step: N -(benzyloxy)carbonyl)- S -triphenylmethyl- L Preparation of 26b-cysteine ​​methyl ester Will N -(benzyloxycarbonyl)- S -triphenylmethyl- LCysteine ​​(26a) (500.0 mg, 1.0 mmol), EDCI (288.0 mg, 1.5 mmol), MeOH (48.0 mg, 1.5 mmol), and DMAP (25.0 mg, 0.2 mmol) were dissolved in DMF (3.0 mL) and stirred overnight at room temperature. After the reaction was complete, the reaction solution was purified by silica gel column chromatography (PE / EA = 85.0 / 15.0 elution) to give intermediate 26b (532.8 mg, yield 104.3%), a white solid. LCMS (ESI) m / z: 534.2 [M+Na] + ] + .

[0099] Step 2: Methyl (benzyloxycarbonyl) - L Preparation of β-cysteine ​​(26c) Intermediate 26b (532.8 mg, 1.04 mmol), TFA (2.0 mL), TIPS (0.2 mL), and H₂O (0.2 mL) were dissolved in DCM (2.0 mL) and reacted at room temperature for 0.5 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE / EA = 90.0 / 10.0 elution) to give intermediate 26c (280.0 mg, 99.9% yield), a white solid. LCMS (ESI) m / z: 268.0 [M - H + ] + . Step 3: N -(benzyloxy)carbonyl)- S -(4-((tert-butoxycarbonyl)amino)methyl)benzyl)- L Preparation of β-cysteine ​​methyl ester (26d) Intermediate 26c (280.0 mg, 1.0 mmol), tert-butyl (4-(bromomethyl)benzyl)carbamate (16a) (300.0 mg, 1.0 mmol), and K₂CO₃ (414.0 mg, 3.0 mmol) were dissolved in DMF (3.0 mL) and reacted at room temperature for 2 hours under nitrogen protection. After the reaction was complete, water (10.0 mL) was added to the reaction system, and the mixture was extracted with EA (20.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE / EA = 70.0 / 30.0 elution) to give intermediate 26d (416.2 mg, yield 85.3%) as a white solid. LCMS (ESI) m / z: 389.2 [M + H + - 100]+ . Step 4: N- ((benzyloxy)carbonyl)- S -(4-((tert-butoxycarbonyl)amino)methyl)benzyl)- L Preparation of β-cysteine ​​(26e) Intermediate 26d (416.2 mg, 0.85 mmol) and lithium hydroxide monohydrate (179.0 mg, 4.26 mmol) were dissolved in MeOH / H₂O (3.0 mL / 2.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the pH of the reaction solution was adjusted to ~3.0 with 1N hydrochloric acid, extracted with EA (20.0 mL × 3), the organic phases were combined, washed with saturated sodium chloride solution (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (DCM / MeOH = 97.0 / 3.0 elution) to give intermediate 26e (315.0 mg, yield 78.2%), a white solid. LCMS (ESI) m / z: 375.2 [M + H₂] + - 100]+. Step 5: Benzyl ((2) R )-1-((2-(1-(( R )-11-benzyl-1-(9 H Preparation of -fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazaheptadecane-17-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)thio)-1-oxypropyl-2-yl)carbamate (26f) Intermediate 25d (23.0 mg, 0.027 mmol), intermediate (26e) (13.0 mg, 0.027 mmol), HATU (13.0 mg, 0.032 mmol), HOBT (5.0 mg, 0.032 mmol), and DIPEA (11.0 mg, 0.081 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 26f (13.3 mg, yield 38.0%), a white solid.

[0100] Step 6: Benzyl ((2) R )-1-((2-(1-(( R Preparation of 1,3-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)thio)-1-oxypropyl-2-yl)carbamate (26 g) Intermediate 26f (13.3 mg, 0.01 mmol) and piperidine (0.2 mL) were dissolved in DMF (1.0 mL) and reacted at room temperature under nitrogen for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 26 g (6.0 mg, yield 55.6%), a white solid. LCMS (ESI) m / z: 489.5 1 / 2 [M + 2H₂O]₂ + - 100] + .

[0101] Step 7: Benzyl ((2) R )-1-((2-(1-(( R)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H Preparation of (-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)thio)-1-oxopropyl-2-yl)carbamate (26h) 26 g (6.0 mg, 0.006 mmol) of intermediate, 2,5-dioxopyrrolidone-1-yl-6-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (17o) (2.6 mg, 0.008 mmol) and DIPEA (2.2 mg, 0.017 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was completed, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to give intermediate 26 h (3.4 mg, yield 47.9%) as a white solid. LCMS (ESI) m / z: 585.7 1 / 2[M+ 2H + - 100] + .

[0102] Step 8: Benzyl ((2) R )-3-((4-(aminomethyl)benzyl)thio)-1-((2-(1-(( R )-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1 H Preparation of (-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropyl-2-yl)carbamate (ZYP-052-089) The intermediate (3.4 mg, 0.002 mmol) was reacted with TFA (0.5 mL) and DCM (1.0 mL) at room temperature for 0.5 h. After the reaction was complete, the reactants were purified by Pre-HPLC (Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20%~100% B gradient in 30 min; flow rate: 30 mL / min) to obtain intermediate ZYP-052-089 (2.1 mg, yield 91.3%), a white solid. LCMS (ESI) m / z: 585.7 1 / 2 [M + 2H₂O]₂ + ] + .

[0103] Example 2-15 (WJ-039-98): Benzyl ((2 R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-((4-((7 S 10 S 7-(4-(dipropylamino)butyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)benzyl)thio)-1-oxopropane-2-yl)carbamate Step 1: Benzyl ((2) R )-3-((4-((2-((((9)) H Preparation of -fluorene-9-yl)methoxy)carbonyl)amino)acetamyl)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (27b) ((9H-fluorene-9-yl)methoxy)carbonyl)glycine (27a) (47.0 mg, 0.1 mmol), intermediate ZYP-031-029 (90.0 mg, 0.1 mmol), EDCI (29.0 mg, 0.2 mmol), HOAT (29.0 mg, 0.2 mmol), and DMAP (29.0 mg, 0.3 mmol) were dissolved in DMF (2.0 mL) and reacted at room temperature for 1 hour. Water (5.0 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (10.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (10.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (MeOH / DCM = 1 / 20 elution) to obtain intermediate 27b (92.0 mg, yield 71.0%) as a white solid.

[0104] Step 2: Benzyl ((2) R Preparation of 3-((4-((2-aminoacetamido)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (27c) Intermediate 27b (92.0 mg, 0.1 mmol) was dissolved in DMF (1 mL), and piperidine (0.2 mL) was added. The reaction mixture was reacted at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (MeOH / DCM = 1 / 20 elution) to obtain intermediate 27c (55.0 mg, yield 79.0%), a white solid. LCMS (ESI) m / z: 687.1 [M + H] + ] + , 1 H NMR (400MHz, DMSO d 6) d 10.97 (s, 1H), 8.75 (t, J = 8.0 Hz, 2H), 7.98 (s, 2H), 7.66(d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.41 (d, J = 8.0Hz, 1H), 7.37 - 7.32 (m, 5H), 7.26 (d, J = 7.6 Hz, 2H), 7.21 (d, J= 7.6 Hz,2H), 5.13 - 5.08 (m, 1H), 5.08 (s, 2H), 4.45 - 4.35 (m, 3H), 4.34 - 4.28 (m,4H), 3.74 (s, 2H), 2.97 - 2.85 (m, 1H), 2.82 - 2.72 (m, 2H), 2.69 - 2.55 (m,3H), 2.44 - 2.34 (m, 1H), 2.04 - 1.96 (m, 1H).

[0105] Step 3: N 2 -((((9 H -fluorene-9-yl)methoxy)carbonyl)- L -Valyl)- N 6 Preparation of -(tert-butoxycarbonyl)-L-lysine-CTC Resin(27e) 2-Chlorotriphenylmethyl chloride resin (22a) (1.5 g, 1.74 mmol) was swollen in DCM (20.0 mL) at room temperature for 0.5 hours. After swelling was complete, the intermediate (27d) was... N 2 -((((9 H -fluorene-9-yl)methoxy)carbonyl)- L -Valyl)- N 6 -(tert-butoxycarbonyl)-L-lysine (0.8 g, 1.74 mmol) and DIPEA (1.1 g, 8.70 mmol) were added to the reaction solution. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered, dried, and intermediate 27e was obtained, which was then directly used for the next reaction.

[0106] Step 4: ( S Preparation of tert-butyl carbamate (5-amino-6-(oxide)-6-oxohexyl)carbamate-CTC Resin (27f) Intermediate 27e and piperidine (5.0 mL) were dissolved in DMF (20.0 mL) and reacted at room temperature for 0.5 hours. After the reaction was complete, the mixture was filtered and dried to obtain intermediate 27f, which was then directly used for the next reaction.

[0107] Step 5: N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- L -valine)- N 6-(tert-butoxycarbonyl)- L Preparation of lysine-CTC Resin (27h) Intermediate 27f, ((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (27 g) (0.60 g, 1.47 mmol), PyBOP (1.5 g, 2.2 mmol), HOBT (0.38 g, 2.2 mmol), and DIPEA (0.95 g, 7.4 mmol) were dissolved in DMF (20.0 mL). The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered and dried to obtain intermediate 27f, which was then directly used for the next reaction.

[0108] Step 6: N 2 -(((9 H -fluorene-9-yl)methoxy)carbonyl)- L -valine)- N 6 -(tert-butoxycarbonyl)- L Preparation of lysine (27i) Intermediate 27i and trifluoroethanol (10.0 mL) were dissolved in DCM (10.0 mL). The reaction was carried out at room temperature for 3 hours. After the reaction was completed, the mixture was filtered and concentrated under reduced pressure to give intermediate 27i (560.0 mg, yield 44.1%) as a white solid.

[0109] Step 7: (((9)) H -fluorene-9-yl)methoxy)carbonyl)- L -Vialenyl- L Preparation of lysine (27j) Intermediate 27i (0.6 g, 1.1 mmol) was dissolved in DCM (3.0 mL), and TFA (1.0 mL) was added. The mixture was reacted at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give intermediate 27j (411.0 mg, yield 83.0%), a white solid. LCMS (ESI) m / z: 467.5 [M + H + ] + .

[0110] Step 8: N 2 -((((9 H -fluorene-9-yl)methoxy)carbonyl)- L -Valyl)- N 6 , N6 -Dipropyl- L Preparation of lysine (27kJ) Intermediate 27j (411.0 mg, 0.9 mmol) and propionaldehyde (280.0 mg, 4.8 mmol) were dissolved in DCE (5.0 mL), and 2 drops of acetic acid were added. The mixture was stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (933.0 mg, 4.4 mmol) was then added in portions to the reaction mixture, and the mixture was reacted overnight at room temperature. Water (5.0 mL) was added to the reaction mixture, and the mixture was extracted with DCM (15.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give intermediate 27k (240.0 mg, yield 49.0%), a white solid. LCMS (ESI) m / z: 551.6 [M + H + ] + .

[0111] Step 9: Benzyl ((2) R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-((4-((5 S 8 S )-8-(4-(dipropylamino)butyl)-1-(9 H Preparation of fluorene-9-yl)-5-isopropyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradecane-14-yl)benzyl)thio)-1-oxopropane-2-yl)carbamate (27l) Intermediate 27k (30.0 mg, 0.05 mmol), intermediate 27c (41.0 mg, 0.06 mmol), EDCI (16.0 mg, 0.08 mmol), HOAT (11.0 mg, 0.08 mmol), and N-methylmorpholine (11.0 mg, 0.11 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. Water (5.0 mL) was added to the reaction mixture, and the mixture was extracted with EA (10.0 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (10.0 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (MeOH / DCM = 1 / 15 elution) to obtain intermediate 27l (27.0 mg, yield 41.0%) as a white solid. LCMS (ESI) m / z: 610.5 1 / 2 [M + 2H+] + ] + .

[0112] Step 10: Benzyl ((2) R )-3-((4-((2-(( S )-2-(( S Preparation of 2-amino-3-methylbutamido)-6-(dipropylamino)hexamido)acetamido)methyl)benzyl)thio)-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (27m) Intermediate 27l (27.0 mg, 0.02 mmol) was dissolved in DMF (1.0 mL), and piperidine (0.2 mL) was added. The reaction mixture was reacted at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by C18 reversed-phase silica gel column chromatography (0.1% TFA in H2O / ACN = 20% ~ 100% elution) to give intermediate 27m (9.0 mg, yield 41.0%), a white solid. LCMS (ESI) m / z: 499.5 1 / 2 [M + 2H+] + ] + .

[0113] Step 11: Benzyl ((2) R )-1-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-3-((4-((7 S 10 S Preparation of 7-(4-(dipropylamino)butyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)benzyl)thio)-1-oxopropane-2-yl)carbamate (WJ-039-98) Intermediate 27m (9.0 mg, 0.01 mmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (27n) (3.0 mg, 0.01 mmol), EDCI (3.0 mg, 0.01 mmol), HOAT (2.0 mg, 0.01 mmol), and N-methylmorpholine (2.0 mg, 0.02 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. The reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to give compound WJ-039-98 (2.0 mg, yield 18.0%). LCMS (ESI) m / z: 625.0 1 / 2[M + 2H + ] + .

[0114] Example 2-16 (WJ-039-106): Benzyl ((2) R )-3-((4-((7) S 10 S )-20-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)-7-(4-(dipropylamino)butyl)-10-isopropyl-3,6,9,12-tetraoxo-15,18-dioxa-2,5,8,11-tetraazaeicosyl)benzyl)thio)-1-(((2-(2,6-dioxopiridin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-1-oxopropane-2-yl)carbamate) Synthetic method as described in Example 2-1, step 11: Compound WJ-039-106 (3.0 mg, yield 20.0%) was obtained by condensation of intermediate 27m and 20a. LCMS (ESI) m / z: 619.5 1 / 2 [M + 2H + ] + .

[0115] Example 2-17 (ZYP-052-142): (1 S ,2 R 5 S )-2-Isopropyl-5-methylcyclohexyl((1 S )-2-(((2-(1-((41 R50 R )-50-benzyl-41-(3-(2,5-dioxo-2,5-dihydro-1 H -pyrrolo-1-yl)propamido)-38,42,45,48,51,54-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxo-39,43,46,49,52,55-hexaazapentadecane-56-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate first step:( R )-3-((((9 H Preparation of -fluorene-9-yl)methoxycarbonyl)amino)-2-((tert-butoxycarbonyl)amino)propionate-CTC Resin (28b) 2-CTC Resin (24a) (1.0 g, 1.0 mmol) was swollen in DCM (20.0 mL) at room temperature for 0.5 hours. After swelling, the intermediate (28a) was... R 3-(((9H-fluorene-9-yl)methoxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)amino)propionic acid (0.43 g, 1.0 mmol) and DIPEA (0.6 g, 5.0 mmol) were added to the reaction solution. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered and dried to obtain intermediate 28b, which was then directly used for the next reaction.

[0116] Step Two: ( R Preparation of 3-amino-1-(-oxo)-1-oxopropyl-2-yl)carbamate tert-butyl ester-CTCResin (28c) Intermediate 28b and piperidine (1.0 mL) were dissolved in DMF (4.0 mL) and reacted at room temperature for 0.5 hours. After the reaction was complete, the mixture was filtered and dried to obtain intermediate 28c, which was then directly used for the next reaction.

[0117] Step 3: ( R Preparation of tert-butyl carbamate (38,42-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39-azatricarbamate-41-yl)-CTC Resin(28e) Intermediate 28c, 2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxytridecane-38-acid (28d) (0.25 g, 0.42 mmol), PyBOP (1.0 g, 1.5 mmol), HOBT (0.2 g, 1.5 mmol), and DIPEA (0.4 g, 3.0 mmol) were dissolved in DMF (10.0 mL). The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was filtered and dried to obtain intermediate 28e, which was then directly used for the next reaction.

[0118] Step 4: ( R Preparation of 41-((tert-butoxycarbonyl)amino)-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39-azatetradecane-42-acid (28f) Intermediate 28e and trifluoroethanol (10.0 mL) were dissolved in DCM (10.0 mL). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the mixture was filtered and concentrated under reduced pressure to give intermediate 28f (353.8 mg, yield 45.7%), a white solid. LCMS (ESI) m / z: 675.5 [M + H] + - 100] + .

[0119] Step 5: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-((41) R 50 R Preparation of 50-benzyl-41-(tert-butoxycarbonyl)amino)-38,42,45,48,51,51,54-hexyloxy-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39,43,46,49,52,55-hexaazahexapentane-56-y)-2,6-dioxopiperidin-3-yl)-1-oxoisoindole-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (28g) Intermediate 28f (25.0 mg, 0.03 mmol), intermediate (17n) (30.0 mg, 0.03 mmol), PyBOP (19.0 mg, 0.04 mmol), HOBT (5.0 mg, 0.04 mmol), and DIPEA (20.0 mg, 0.15 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 28 g (37.0 mg, yield 72.8%) as a white solid.

[0120] Step 6: (1) S ,2 R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-((2-(1-((41) R 50 R Preparation of 41-amino-50-benzyl-38,42,45,48,51,51,54-hexyloxy-2,5,8,11,14,17,20,23,26,29,32,35-dodecyloxy-39,43,46,49,52,52,55-hexaazahexapentane-56-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (28h) 28 g (37.0 mg, 0.02 mmol) of intermediate and 0.2 mL of TFA were dissolved in 1.0 mL of DCM and reacted at room temperature under nitrogen for 0.5 h. After the reaction was completed, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 80% B gradient in 30 min); flow rate: 30 mL / min) to obtain intermediate 28 h (25.4 mg, yield 80.0%) as a white solid.

[0121] Step 7: (1) S ,2R 5 S )-2-isopropyl-5-methylcyclohexyl ((1 S )-2-(((2-(1-((41) R 50 R ))-50-benzyl-41-(3-(2,5-dioxo-2,5-dihydro-1 H Preparation of pyrrolo-1-yl)propamido)-38,42,45,48,51,54-hexaoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxo-39,43,46,49,52,55-hexaazapentadecane-56-yl)-2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)methyl)amino)-2-oxo-1-phenethyl)carbamate (ZYP-052-142) Intermediate 28h (45.4 mg, 0.028 mmol), 2,5-dioxopyrrolidone-1-yl-3-(2,5-diketone-2,5-dihydro-1H-pyrrolo-1-yl)propionate (28i) (24.0 mg, 0.086 mmol) and DIPEA (18.0 mg, 0.140 mmol) were dissolved in DMF (1.0 mL) and reacted at room temperature for 2 hours. After the reaction was complete, the reactants were purified by Pre-HPLC (instrument: Agilent Prep 1200 series; UV lamps: 214 and 254 nm; mobile phase: solvent A (0.1% of TFA in water) and solvent B (acetonitrile), 20% ~ 100% B gradient in 30 min); flow rate: 30 mL / min) to obtain compound ZYP-052-142 (23.0 mg, yield 56.4%), a white solid. LCMS (ESI) m / z: 872.3 1 / 2 [M + 2H+] + ] + .

[0122] Example 3: Method for fabricating a DAC The antibody-drug conjugate targeting the GSPT1 protein provided in this embodiment consists of an antibody and a drug-linker conjugate.

[0123] In this invention, a preferred type of antibody for DAC is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof. Preferably, the antibody is selected from the following targets: TF, HER2, HER3, CD33, CD30, CD22, Trop2, EGFR, CD79b, Nectin4. The antibody is selected from the following group: FD40, HuSC1-39, 1-G07-FC, Pertuzumab, Trastuzumab, Disitamab, Gemtuzumab, Sacituzumab, Patritumab, Cetuximab, Polatuzumab, Brentuximab, Inotuzumab, Enfortumab. The corresponding antibody-drug conjugates targeting the GSPT1 protein degrader are: FD40-ZYP-005-056, FD40-ZYP-005-068, FD40-ZYP-031-084, FD40-ZYP-031-179, FD40-ZYP-031-183, FD40-ZYP-033-119, FD40-ZYP-033-126, FD40-ZYP-043-199, FD40-ZYP-052-031, FD40-Z YP-052-032, FD40-ZYP-052-052, FD40-ZYP-052-053, FD40-ZYP-052-082, FD40-ZYP-052-089, FD40-ZYP-052 -142, FD40-WJ-039-098, FD40-WJ-039-106, HuSC1-39-ZYP-005-056, HuSC1-39-ZYP-031-084, HuSC1-39-ZYP- 031-179, HuSC1-39-ZYP-031-183, HuSC1-39-ZYP-033-119, HuSC1-39-ZYP-033-126, HuSC1-39-ZYP-043-199 , HuSC1-39-ZYP-052-031, HuSC1-39-ZYP-052-032, HuSC1-39-ZYP-052-052, HuSC1-39-ZYP-052-053, HuSC1-3 9-ZYP-052-082, HuSC1-39-ZYP-052-089, HuSC1-39-ZYP-052-142, HuSC1-39-WJ-039-098, HuSC1-39-WJ-039- 106. 1-G07-FC-ZYP-005-056, 1-G07-FC-ZYP-031-179, Pertuzumab-ZYP-005-056, Pertuzumab-ZYP-031-179,Trastuzumab-ZYP-005-056, Trastuzumab-ZYP-031-179, Disitamab-ZYP-005-056, Disitamab-ZYP-031-179, Gemtuzumab-ZYP-005-056, G emtuzumab-ZYP-031-179, Sacituzumab-ZYP-005-056, Sacituzumab-ZYP-031-179, Patritumab-ZYP-005-056, Patritumab-ZYP-031-179, Cetuximab-ZYP-005-056, Cetuximab-ZYP-031-179, Polatuzumab-ZYP-005-056, Polatuzumab-ZYP-031-179, Brentuximab-ZYP-005-056, Brentuximab-ZYP-031-179, Inotuzumab-ZYP-005-056, Inotuzumab-ZYP-031-179, Enfortumab-ZYP-005-056, Enfortumab-ZYP-031-179. ,

[0124] A typical method for preparing DAC is as follows: The antibody is replaced with a reaction buffer solution of 50 mM phosphate-buffered saline (PBS), 2 mM EDTA, and pH 7.4. Tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) is added in excess at a molar ratio of 2-20, and the mixture is incubated at 37°C for 2 hours to completely reduce interchain disulfide bonds. The reduced antibody reaction solution is cooled to room temperature, and a drug linker conjugate pre-dissolved in DMA is added in excess at a molar ratio of 5-12, ensuring that the volume percentage of DMA does not exceed 10% of the reaction system. The resulting reaction mixture is incubated at room temperature for 2 hours. Excess small molecules are removed from the conjugate using a desalting column or ultrafiltration tube, and the conjugate is then replaced with a PBS / 8% sucrose buffer solution at pH 7.4. Analysis is performed using UV / Vis spectrophotometer, LC-MS, HIC-HPLC, RP-HPLC, and SE-HPLC (e.g., Figure 2 The average drug / antibody ratio of the purified DAC was 2-8. The average drug-antibody ratio (DAR) of the GSPT1 molecular gel degradation agent antibody conjugate obtained from the obtained nanobody fraction is shown in Table 1. Then, the DAC was sterilized through a 0.22-micron pore size needle filter, the concentration of DAC was determined by Nano-Drop, and it was stored at -80℃.

[0125] Table 1: Average Drug-Antibody Conjugate Ratio of Selected GSPT1 Molecular Gel Antibody Conjugates Example 4: In vitro antitumor activity of GSPT1 molecular gel and antibody-drug conjugate (DAC) The cell lines used in this embodiment were purchased from the American Type Culture Collection (ATCC) or Nanjing Kebai Biotechnology Co., Ltd., and cultured according to the corresponding instructions. These included HPAF-II, MDA-MB-468, and BT-474.

[0126] GSPT1 molecular gel degradation activity assay (Western blotting): Logarithmically growing MDA-MB-468 cells were trypsinized and counted, then seeded at an appropriate density in 24-well plates. After cell adhesion, drug was administered at concentrations of 0 nM, 10 nM, 100 nM, and 1000 nM. Cells were washed once with PBS 12 h after drug administration. RIPA lysis buffer was mixed with a protease inhibitor and added to the cells, then lysed on ice. The lysis buffer was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. After adding loading buffer, the sample was boiled at 100 °C for 10 min. Protein bands were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, followed by incubation with antibody. Finally, scanning and exposure analysis were performed using a Tanon 5200 imaging system. Table 2 shows the degradation levels of the target protein by the GSPT1 molecular gel compound in MDA-MB-468. "+++" indicates that 1 nM ≤ DC 50 ≤10 nM; "+++" means 1nM≤DC 50 ≤10 nM; "++" means 10 nM ≤ DC 50 ≤100 nM; "+" indicates DC 50 ≥100 nM.

[0127] Table 2: Degradation activity data of GSPT1 molecular adhesive compounds Cell proliferation assay: Cells in the logarithmic growth phase were seeded into 96-well cell culture plates at appropriate cell numbers according to their growth rate, with 150 μL of culture medium per well, and incubated at 37°C. oC. After culturing in 5% CO2 for 5 hours, different concentrations of DACs were added, and corresponding replicates and solvents were set up, along with control and blank control wells. After 6-9 days of incubation, the culture medium was removed, and CCK8 reagent (purchased from Meilunbio) was added to each well (100 μL). The mixture was incubated at 37℃ until the desired color depth was reached. Cell viability (OD 450 nm) of each group was then measured using a multi-functional microplate reader, and cell viability was calculated using the following formula: Viability = (OD drug - OD blank) / (OD control - OD blank) × 100%. The above data were analyzed using GraphPad Prism8 software, and calculations were performed accordingly.

[0128] IC50 of different drugs on cell lines with high expression of corresponding antibody antigens 50 Value. The results showed (Figures 3 and 4) that the preferred GSPT1 molecular gel antibody conjugates of this invention, HuSC1-39-ZYP-005-056, HuSC1-39-ZYP-031-179, Pertuzumab-ZYP-005-056, Trastuzumab-ZYP-005-056, 1-G07-FC-ZYP-005-056, and 1-G07-FC-ZYP-031-179, exhibited strong cell proliferation inhibitory activity against antigen-positive pancreatic cancer HPAF-II, breast cancer MDA-MB-468, and breast cancer BT-474 cells, with IC50 values. 50 Values ​​below 0.1 nM were observed, with Pertuzumab-ZYP-005-056 showing superior inhibitory activity against BT-474 breast cancer cells compared to Orum-5029. Table 3 summarizes the IC50 values ​​for some cell proliferation inhibition. 50 value.

[0129] Table 3: In vitro antitumor activity of GSPT1 molecular gel antibody conjugates Example 5: Determination of the binding affinity of GSPT1 molecular gel antibody conjugate (DAC) to tumor cell surface antigens Using MDA-MB-468 breast cancer cells with high TF expression as target cells, 100 μL of the test antibody, serially diluted 5-fold, was used as the primary antibody and reacted with 1×10⁶ cells of RPMI-1640 serum-free medium suspended in 100 μL of the medium. 5 Mix the tumor cells thoroughly, then at 4 o Incubate at C for 1 h, wash cells twice with PBS to remove unbound primary antibody, then incubate target cells with 200 μL of 2 μg / mL PE-labeled secondary antibody. oCells were incubated at C for 30 min, washed twice with PBS to remove unbound secondary antibody, and finally resuspended in 200 μL PBS. The binding affinity of the test antibody to the corresponding cell surface TF was determined by flow cytometry. Further serially diluted FACS analysis yielded the cell binding affinity. The results are shown below. Figure 5 As shown, FD40, FD40-ZYP-031-179, HuSC1-39, HuSC1-39-ZYP-005-056, and HuSC1-39-ZYP-031-179 exhibit EC50 binding affinity to MDA-MB-468 cells. 50 0.131 μg / mL, 0.136 μg / mL, 0.201 μg / mL, 0.223 μg / mL, and 0.203 μg / mL respectively.

[0130] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0131] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An antibody-drug conjugate targeting a degrader of GSPT1 protein, characterized in that, It is a compound with the structure shown in Formula I, or a stereoisomer of a compound with the structure shown in Formula I, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. (Equation I) In Formula I, Ab is a highly specific targeting antibody or its antigen-binding fragment; S represents the sulfur atom on Ab; L represents either a cuttable connector or a non-cuttable connector; R1 is selected from hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 Cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl, C(O)R6, C(O)OR6, C(O)NHR6, S(O)2R6 or C(O)C(O)R6; wherein each R6 is independently selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; R2 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; X is selected from: -CH 2- -CD 2- -C(O)-, -CCH3-; Y is selected from: -CH2-, -NH-, -O-, -S-, -S(O)2-, -C(O)NH-, -C(O)O-, -NHC(O)-; R3, R4, and R5 are each independently selected from: H, D, and C. 1-3 alkyl; m1 and m2 are each independently 0, 1, 2, 3 or 4; q is an integer selected from 1 to 20.

2. The antibody-drug conjugate targeting the GSPT1 protein degrader according to claim 1, characterized in that, The antibodies are selected from antibodies or antigen-binding fragments targeting the following targets: HER2, Trop-2, Claudin-6, Claudin-9, Claudin-18.2, EGFR, c-Met, CD19, PSMA, Muc1, BCMA, PD-L1, CD33, CD30, CD22, CD79b, Nectin-4, CD19, tissue factor TF, FRα, B7-H3, B7-H4, CDH3, CDH6, CDH17, ALPP, CD56, CD37, HER3, ROR1, MSLN, TNF. -α, CD25, ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM5, CEACAM6, CD138, GC-C, LIV-1, CA6, FUT3, IGF-1R, CTLA4, RNF43, DPEP3 , 5T4, ITGB6, EFNA4, CD228, Notch3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, TIM1, sTn, ETB, Globo H, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD5 1. SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, LYFD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, Muc1, LRRC15, CD44, CD70, EphA2, CXCR4, DDR1, DKL1, FOLR, CD45, DSG2, ALK, TRAIL, EpCAM, VEGFR2, CD47, CD49, SSEA-4, DCLK1, OacGD2, CD73, ENO1, BSG, CD24, GLUT1 and GPRC5D, preferably HER2, HER3, EGFR, Trop-2, Claudin-6, Claudin-18.2, B7-H3, B7-H4, CDH3, CDH6, CDH17, FRα, ROR1, ALPP, CEACAM5, CEACAM6 or FOLR.

3. The antibody-drug conjugate targeting the GSPT1 protein degrader according to claim 1, characterized in that, Antibody-drug conjugate targeting GSPT1 protein, which is a compound with the structure shown in Formula II, or a stereoisomer of a compound with the structure shown in Formula II, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. (Formula II) In Formula II, L1 is a linker unit that covalently binds an antibody or its antigen-binding fragment (Ab) to L2; L2 is a connecting unit, which covalently connects to connector units L1 and L3; L3 is selected from amino acid residues or short peptides consisting of 2-5 amino acid residues; the amino acids are selected from natural amino acids and / or non-natural amino acids; L4 is a chemical bond or spacer unit that covalently binds the GSPT1 molecular adhesive degrader to L3; R1 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 Cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl, C(O)R6, C(O)OR6, C(O)NHR6, S(O)2R6, C(O)C(O)R6; wherein each R6 is independently selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; R2 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; X is selected from: -CH 2- -CD 2- -C(O)-, -CCH3-; Y is selected from: -CH2-, -NH-, -O-, -S-, -S(O)2-, -C(O)NH-, -C(O)O-, -NHC(O)-; R3, R4, and R5 are each independently selected from: H, D, and C. 1-3 alkyl; m1 and m2 are each independently 0, 1, 2, 3 or 4; q is an integer selected from 1 to 20.

4. The antibody-drug conjugate targeting the GSPT1 protein degrader according to claim 3, characterized in that, L1 is selected from one of the following structures: 、 ； L2 is selected from one of the following structures: 、 ， W is selected from -CH2- and O, and p is any integer between 0 and 20; L3 is selected from one of the following structures: 、 、 、 ； L4 is selected from one of the following structures: 、 。 5. The antibody-drug conjugate targeting the GSPT1 protein degrader according to claim 3, characterized in that, The GSPT1 molecular gel degrader is a compound represented by Formula III, or a pharmaceutically acceptable salt, solvate, or stereoisomer of the compound represented by Formula III; (Formula III) In Equation III, R1 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 Cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl, C(O)R6, C(O)OR6, C(O)NHR6, S(O)2R6, C(O)C(O)R6; wherein each R6 is independently selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-8 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; R2 is selected from: hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 3-10 cycloalkyl, 3- to 8-membered heterocyclic, aryl, heteroaryl; X is selected from: -CH 2- -CD 2- -C(O)-, -CCH3-; Y is selected from: -CH2-, -NH-, -O-, -S-, -S(O)2-, -C(O)NH-, -C(O)O-, -NHC(O)-; R3, R4, and R5 are each independently selected from: H, D, and C. 1-3 alkyl; m1 and m2 are each independently 0, 1, 2, 3 or 4.

6. The antibody-drug conjugate targeting the GSPT1 protein degrader according to claim 3, characterized in that, GSPT1 molecular adhesive degrader is selected from one of the following structures: 。 7. The antibody-drug conjugate targeting the GSPT1 protein degrader according to claim 6, characterized in that, Drug-linker conjugates are selected from one of the following structures: 。 8. The use of the antibody-drug conjugate targeting the GSPT1 protein as described in any one of claims 1-7, characterized in that, The use of the antibody-drug conjugate targeting the GSPT1 protein in the preparation of drugs for the prevention and / or treatment of diseases related to abnormal cell activity.

9. The application according to claim 8, characterized in that, The use of the antibody-drug conjugate targeting the GSPT1 protein in the preparation of drugs for the prevention and / or treatment of tumor diseases.

10. The application according to claim 9, characterized in that, The tumor is selected from pancreatic cancer, ovarian cancer, or lung cancer.

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