Patent Application
20250205349
The present invention relates to the field of biopharmaceuticals, and in particular, to an antibody-drug conjugate formed by an anti-human Trop2 antibody and a camptothecin drug, as well as a preparation method and use of the antibody-drug conjugate. The present invention also relates to a linker-drug compound that can be conjugated to Ab to form an antibody-drug conjugate.
Human trophoblast cell surface antigen 2 (hTROP2) is a single-transmembrane type I cell membrane protein encoded by the TACSTD2 gene. It was first discovered by M. Lipinski et al. during the study on normal human and cancerous trophoblast cells. During subsequent research, the hTROP2 molecule was also known as the tumor antigen GA733-1 that was recognized by the mouse monoclonal antibody GA733 obtained by immunizing a gastric cancer cell line, as well as the epidermal glycoprotein that was recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunizing a non-small cell lung cancer cell line. After the gene was cloned in 1995, it was confirmed that they were of the same molecule.
The hTROP2 protein has a full length of 323 amino acid residues, including an intracellular domain composed of 26 amino acid residues at the N terminus, an extracellular domain composed of 248 amino acid residues at the C terminus, and a transmembrane domain composed of 23 amino acid residues. The hTROP2 has four N-glycosylation sites at amino acid residues 33, 120, 168 and 208 of the extracellular domain. The TROP2 gene belongs to the TACSTD gene family and has about 50% homology with human trophoblast surface antigen 1 (hTROP1), another member of this gene family.
At present, the physiological ligands and molecular functions of the hTROP2 protein are still unclear, but it can regulate calcium signaling in tumor cells. The intracellular domain of hTROP2 has a PIP2 (phosphatidylinositol 4,5-bisphosphate) binding sequence. Protein kinase C of the Ca2+-dependent kinase can phosphorylate the serine residue 303. Phosphorylation of serine residue 303 causes PIP2 to be hydrolyzed by phospholipase C (PLC) into IP3 (inositol trisphosphate) and DAG (diacylglycerol), thereby regulating the process of intracellular calcium signaling.
Immunohistochemical analysis of clinical specimens shows that hTROP2 is only limitedly expressed in certain epithelial cells in normal tissues, but is overexpressed in a variety of epithelial cell cancers such as cervical cancer, breast cancer, gastric cancer, lung cancer, prostate cancer, and pancreatic cancer. Reports have shown that the expression level of hTROP2 is closely related to the invasive ability of tumors, the degree of malignancy and the poor prognosis of patients. In the meanwhile, hTROP2 can be used as a marker of prostate cancer stem cells during the initiation and development of tumors. Currently, using Trop-2 as a target for tumor therapy, anti-Trop-2 antibody-drug conjugates (ADC) are being used in the treatment of a variety of metastatic tumors, including triple-negative breast cancer (TNBC), non-small cell lung cancer, and small cell lung cancer. The American Society of Clinical Oncology (ASCO) announced relevant research on IMMU-132 in the treatment of triple-negative breast cancer in 2016. IMMU-132 is an antibody-drug conjugate that conjugates the monoclonal antibody RS7 to the drug SN-38. RS7 can specifically target hTROP2. hTROP2 has been found to be abundantly expressed in more than 80% of triple-negative breast cancer tumor cells.
Camptothecin (CPT) is a DNA topoisomerase I (TOPI) inhibitor. Camptothecin derivatives can inhibit TOPI and achieve anti-tumor effects. TOPI can catalyze the breakage and joining of single-stranded DNA during DNA translation and transcription to relax the DNA supercoil, thus promoting replication and transcription. The TOPI inhibitor SN-38 can cause DNA damage by blocking DNA chain reconnection, thereby inhibiting DNA replication and transcription and inducing cell apoptosis. In the clinical study of ADC targeting Trop-2 with humanized RS7 antibody, IMMU-132 uses a moderately stable linker. The half-life of IMMU-132 releasing SN-38 in human serum is about 24 h, and the elimination half-life in humans and mice is about 11 h. IMMU-132 has fast elimination rate and its mean residence time is about 15.4 h, which indicates that IMMU-132 will be administered more frequently in clinical treatment. In the meanwhile, during the use of topoisomerase inhibitors such as SN-38, IMMU-132 is often accompanied by serious side effects, such as diarrhea. The use of more stable linkers is expected to increase mean residence time and reduce drug use. Therefore, further research on safer and more effective Trop-2 targeting technology is urgent.
The ADC consists of three parts: an antibody, a cytotoxic small molecule drug, and a linker that connects the antibody and the drug. The small molecule drug is bound to the antibody protein through chemical coupling. The ADC guides the small molecule drug to a cancer cell target by antibody specific recognition, enters the cancer cell through endocytosis, and releases the cytotoxic drug to specifically kill the cancer cell. Clinical studies have proven that the ADC is relatively stable and highly effective in the blood, and can effectively reduce the toxicity of the cytotoxic small molecule drug to healthy tissues. ADCs are currently a hot topic in anti-cancer drug research. Trop-2, as a highly expressed protein specifically in a variety of epithelial cell cancers, is an excellent candidate target for the ADC.
Based on the comprehensive understanding of ADCs, the inventors disclose an anti-human Trop2 antibody-drug conjugate, a preparation method of the conjugate, a pharmaceutical composition comprising the conjugate, and use of the conjugate or the pharmaceutical composition. The present invention further relates to a linker-drug compound that can be coupled to an anti-human Trop2 antibody to form an antibody-drug conjugate.
A first aspect of the present invention discloses a ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof;
o is selected from integers between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably between 2 and 8;
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ab is an antibody targeting TROP2 or an antigen-binding fragment thereof.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises: two IgG1 heavy chains; and two κ light chains.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein in the antibody of the Ab, the κ light chain comprises: CDRs as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, and the IgG1 heavy chain comprises CDRs as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the light chain comprises CDRL1, CDRL2 and CDRL3 having nucleic acid coding sequences as shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and the heavy chain comprises CDRH1, CDRH2 and CDRH3 having nucleic acid coding sequences as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprises a VH having a sequence as shown in SEQ ID NO: 13 and the light chain comprises a VL having a sequence as shown in SEQ ID NO: 14.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprises a VH having a nucleic acid coding sequence as shown in SEQ ID NO: 33, and the light chain comprises a VL having a nucleic acid coding sequence as shown in SEQ ID NO: 34.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein in the antibody of the Ab, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 17, and the light chain has an amino acid sequence as shown in SEQ ID NO: 18.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein in the antibody of the Ab, the heavy chain has a nucleic acid coding sequence as shown in SEQ ID NO: 37, and the light chain has a nucleic acid coding sequence as shown in SEQ ID NO: 38.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises: two IgG1 heavy chains; and two κ light chains.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein in the antibody of the Ab, the κ light chain comprises CDRs as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and the IgG1 heavy chain comprises CDRs as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the light chain comprises CDRL1, CDRL2 and CDRL3 having nucleic acid coding sequences as shown in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, and the heavy chain comprises CDRH1, CDRH2 and CDRH3 having nucleic acid coding sequences as shown in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprises a VH having a sequence as shown in SEQ ID NO: 15 and the light chain comprises a VL having a sequence as shown in SEQ ID NO: 16.
In some embodiments of the first aspect of the present invention, a ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the antibody of the Ab comprises a heavy chain and a light chain, the heavy chain comprises a VH having a nucleic acid coding sequence as shown in SEQ ID NO: 35, and the light chain comprises a VL having a nucleic acid coding sequence as shown in SEQ ID NO: 36.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein in the antibody of the Ab, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 19, and the light chain has an amino acid sequence as shown in SEQ ID NO: 20.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein in the antibody of the Ab, the heavy chain has a nucleic acid coding sequence as shown in SEQ ID NO: 39, and the light chain has a nucleic acid coding sequence as shown in SEQ ID NO: 40.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the X is selected, without limitation, from the following structures or isomers thereof:
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein L4 is selected, without limitation, from peptide residues comprising an amino acid,
Particularly preferably, the peptide residue is -GGFG-.
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein
L5 is selected, without limitation, from —NR5(CR6R7)q— and a chemical bond, q is selected from integers between 0 and 6 (e.g., 0, 1, 2, 3, 4, 5 or 6).
R5, R6 and R7 are the same or different and each independently selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3-to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;
In some embodiments, L1 is selected, without limitation, from the group consisting of:
In some embodiments of the first aspect of the present invention, the ligand-camptothecin derivative conjugate shown in general formula I or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the linking unit -L1-L2-L3-L4-L5- is selected, without limitation, from the following structures:
A second aspect of the present invention discloses a ligand-camptothecin derivative conjugate shown in general formula II or a pharmaceutically acceptable salt or solvate thereof;
and o is selected from integers between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably between 2 and 8;
In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ac has a structure shown in formula B,
In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ac is selected, without limitation, from the group consisting of glycine, (D/L) alanine, (D/L) leucine, (D/L) isoleucine, (D/L) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) cystine, (D/L) arginine, (D/L) histidine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or unnatural amino acid derivatives or the following structures or isomers thereof,
In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ac is selected, without limitation, from the group consisting of glycine, phosphoric acid, (D/L) glutamic acid and polyethylene glycol hydrophilic structure.
In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the
R2 has a structure shown in formula d;
In some embodiments of the first and second aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the structural formula d is selected, without limitation, from the following compounds:
In certain embodiments of the present invention, L1 may comprise a succinimide group. In these embodiments, the ligand-drug conjugate can be hydrolyzed under easily hydrolyzable conditions, and hydrolysis occurs at the succinimide group of the linking unit. When the ligand comprises multiple linker-drug units, the following cases may occur depending on the degree of hydrolysis:
and other succinimide groups are in an open-ring form
Therefore, when there are multiple L1's containing succinimide groups in the ADC (i.e., the Ab is connected to multiple drug-linker units containing succinimide groups), these succinimide groups may be all in closed-ring forms, partially in open-ring forms or all in open-ring forms.
It should be understood that although the succinimide group appearing in the chemical structural formula of ADC in the present application is in a closed ring form, it actually covers three cases: fully closed-ring, partially open ring and fully open ring.
A third aspect of the present invention discloses a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof, having a structure shown in formula III,
and o is selected from integers between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);
In some embodiments of the third aspect of the present invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ac has a structure shown in formula B,
In some embodiments of the third aspect of the present invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ac is selected, without limitation, from glycine, (D/L) alanine, (D/L) leucine, (D/L) isoleucine, (D/L) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) cystine, (D/L) arginine, (D/L) histidine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or unnatural amino acid derivatives or the following structures,
In some embodiments of the third aspect of the present invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the Ac is selected, without limitation, from the group consisting of glycine, phosphoric acid, (D/L) glutamic acid and polyethylene glycol hydrophilic structure.
In some embodiments of the third aspect of the present invention, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the linker-drug compound is selected, without limitation, from the following structures or isomers thereof,
The linker-drug compound disclosed in the third aspect of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used as an intermediate for coupling with the ligand Ab to form the ligand-camptothecin derivative conjugate shown in the formula I as described in the first aspect or in the formula II as described in the second aspect.
A fourth aspect of the present invention discloses a method for preparing the ligand-camptothecin derivative conjugate shown in the general formula I as described in the first aspect or in the general formula II as shown in the second aspect, or a pharmaceutically acceptable salt or solvate, comprising the following steps,
The present application further relates to use of the linker-drug compound as disclosed in the third aspect or a pharmaceutically acceptable salt or solvate thereof as an intermediate in the preparation of a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is the ligand-camptothecin derivative conjugate as described in the first, second and fourth aspects of the present invention or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the preparation is carried out according to the preparation method as disclosed in the fourth aspect.
In some embodiments of the first, second and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is selected, without limitation, from the following structures, their derived structures in which rings in succinimide groups are in open form, and their isomers,
In some embodiments of the first, second and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is selected, without limitation, from the following structures, their derived structures in which rings in succinimide groups are in open form, and their isomers,
In some embodiments of the first, second and fourth aspects of the present invention, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof or the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is disclosed, wherein the pharmaceutically acceptable salt includes sodium salts, potassium salts, calcium salts or magnesium salts formed with acidic functional groups in the structural formula, and acetates, trifluoroacetates, citrates, oxalates, tartrates, malates, nitrates, chlorides, bromides, iodides, sulfates, bisulfates, phosphates, lactates, oleates, ascorbates, salicylates, formates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates or p-toluenesulfonates formed with basic functional groups in the structure.
A fifth aspect of the present invention discloses a pharmaceutical composition comprising the ligand-camptothecin derivative conjugate as described in the first aspect and the second aspect or a pharmaceutically acceptable salt or solvate thereof or the linker-drug compound as described in the third aspect or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable carrier.
A sixth aspect of the present invention discloses a pharmaceutical formulation comprising the ligand-camptothecin derivative conjugate as described in the first aspect and the second aspect or a pharmaceutically acceptable salt or solvate thereof or the linker-drug compound as described in the third aspect or a pharmaceutically acceptable salt or solvate thereof.
A seventh aspect of the present invention discloses use of the ligand-camptothecin derivative conjugate as described in the first aspect and the second aspect or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound as described in the third aspect or a pharmaceutically acceptable salt or solvate thereof, the pharmaceutical composition as described in the fifth aspect and/or the pharmaceutical formulation as described in the sixth aspect in preparation of a medicament for treating or preventing a cancer or tumor;
An eighth aspect of the present invention discloses a method for treating or preventing a cancer or tumor, including: administrating to a subject in need thereof a prophylactically or therapeutically effective amount of the ligand-camptothecin derivative conjugate as described in the first aspect and the second aspect or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound as described in the third aspect or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition as described in the fifth aspect and/or the pharmaceutical formulation as described in the sixth aspect;
In the above aspects of the present invention and their embodiments,
The anti-human Trop2 antibody-drug conjugate provided by the present invention is an antibody-drug conjugate obtained by coupling a humanized anti-human Trop2 antibody and a camptothecin drug. Compared with existing drugs of the same type, the anti-human Trop2 antibody-drug conjugate has good molecular stability and good preclinical efficacy, and is expected to have excellent clinical therapeutic effects.
Unless otherwise stated, the following terms and phrases, as used herein, are intended to have the following meanings. When a trade name is used herein, the trade name includes the product formulation, generics, and active ingredients of the product with the trade name, unless the context indicates otherwise.
Unless stated to the contrary, the terms used in the claims and description herein have the following meanings.
The term “ligand” generally refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor related to a target cell. The role of ligands is to deliver drugs to the target cell populationbound to the ligands. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules capable of binding to cells. In the embodiments of the present invention, the ligand is represented as Ab. The ligand can form a linking bond with a linking unit through a heteroatom on the ligand, and is preferably an antibody or an antigen-binding fragment thereof. The antibody is selected from chimeric antibodies, humanized antibodies, fully human antibodies or murine antibodies. Preferably, the antibody is a monoclonal antibody.
A ligand unit is a targeting agent that specifically binds to a target moiety. The ligand is capable of specifically binding to a cellular component or to a cellular component or to other target molecules of interest. The targeting moiety or target is usually on the cell surface. In some aspects, the ligand unit functions to deliver the drug unit to a specific target cell population with which the ligand unit interacts. The ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a non-antibody targeting agent, it may be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecules or substances. In some embodiments, the linker is covalently attached to the sulfur atom of the ligand. In some aspects, the sulfur atom is that of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom that has been introduced into a cysteine residue of the ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom that has been introduced into a cysteine residue of the ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the sulfur atom bound by the linker is selected from cysteine residues that form interchain disulfide bonds of an antibody or cysteine residues that have been introduced into a ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the EU index numbering system is in accordance with Kabat {[Kabat E. A et al., (1991)] Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242”}.
As used herein, “antibody” or “antibody unit” includes, within its scope, any part of the antibody structure. This unit can bind, reactively associate, or complex a receptor, antigen, or other receptor unit present in the target cell population. The antibody may be any protein or proteinaceous molecule that binds, complexes, or reacts with a portion of the cell population to be treated or biomodified. The antibody constituting the antibody-drug conjugate of the present invention retains its original antigen-binding ability in the wild state. Therefore, the antibody of the present invention can specifically bind to an antigen. Antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (such as known or predicted functional molecules), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known or predicted functional molecules). Tumor-associated factors may be cluster differentiation factors (such as CD proteins).
Antibodies for use in antibody-drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art, and can be prepared by well-known antibody production methods and information in the art. In order to develop effective cellular-level targets for cancer diagnosis and treatment, researchers strive to find transmembrane peptides or other tumor-associated peptides. These targets can be specifically expressed on the surface of one or more cancer cells, with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides may be more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Identifying such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatment. For convenience, antigen-related information well known in the industry is marked as follows, including name, other names, and Genbank accession numbers. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases, such as Genbank. The tumor-associated antigens targeted by the antibody include all amino acid sequence variants and homologs, and have at least 70%, 80%, 85%, 90% or 95% homology with the sequence confirmed in the reference, or have completely consistent biological properties and characteristics with the tumor-associated antigen sequences in the reference cited.
The term “inhibit” or “inhibition of” means reduction in a detectable amount or complete prevention.
The term “cancer” refers to a physiological condition or disease characterized by unregulated cell growth. The “tumor” includes cancer cells.
The term “autoimmune disease” is a disease or disorder arising from and directed against an individual's own tissues or proteins.
The term “drug” refers to cytotoxic drug, represented by d, which is a chemical molecule that has strong ability to disrupt the normal growth of tumor cells. In principle, the cytotoxic drug can kill tumor cells at high enough concentrations. However, due to lack of specificity, while killing tumor cells, the cytotoxic drug can also cause apoptosis of normal cells, leading to serious side effects. The term includes toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and Lu176), toxic drugs, chemotherapeutic drugs, antibiotics and ribozymes, preferably toxic drugs.
The term “linker” or “linking fragment” or “linking unit” refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end. It can also be connected to other linkers and then connected to the drug.
The linker, including extenders, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a “cleavable linker” that facilitates release of the drug in cells. For example, acid-labile linkers (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al. Cancer Research 52:127-131, 1992); U.S. Pat. No. 5,208,020.
According to the mechanism of intracellular drug release, “linker” or “linker of the antibody drug conjugate” as used herein can be divided into two categories: non-cleavable linkers and cleavable linkers. For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed by cells, the antibody is enzymatically hydrolyzed in lysosomes, releasing active molecules composed of small molecule drugs, linkers, and antibody amino acid residues. The resulting change in the structure of the drug molecule does not weaken its cytotoxicity, but because the active molecule is charged (amino acid residue), it cannot penetrate into neighboring cells. Therefore, such active drugs cannot kill adjacent tumor cells (bystander effect) that do not express the target antigen (antigen-negative cells) (Ducry et al., 2010, Bioconjugate Chem. 21: 5-13). For antibody-drug conjugates containing cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed by cells, it is broken in the target cell and releases the active ingredient (the small molecule drug itself). The cleavable linkers are mainly divided into: chemosensitive linkers and enzyme-sensitive linkers. The chemosensitive linkers can be selectively cleaved due to differences in the properties of plasma and cytoplasm or tumor microenvironments. Such properties include pH, glutathione concentration, etc. Linkers that are sensitive to pH are relatively stable in the neutral or weakly alkaline environment of blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleaved linkers, antibody-drug conjugates based on this type of linker usually have a short half-life (2-3 days). This short half-life limits the application of pH-sensitive linkers in new generation antibody-drug conjugates to a certain extent. Glutathione-sensitive linkers are also called disulfide linkers. Drug release is caused by the difference between high intracellular glutathione concentration (millimolar range) and relatively low glutathione concentration in the blood (micromolar range). This is especially true for tumor cells, where low oxygen content leads to increased activity of reductase enzymes and thus higher glutathione concentration. Disulfide bonds are thermodynamically stable and therefore have good stability in plasma. Enzyme-labile linkers, such as peptide linkers, enable better control of drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B. Such as peptide linkers are believed to be very stable in the plasma circulation because proteases are usually inactive outside cells due to inappropriate extracellular pH and serum protease inhibitors. Enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates due to their high plasma stability and good intracellular cleavage selectivity and effectiveness.
The term “antibody-drug conjugate” refers to connecting an antibody to a biologically active drug by a stable linking unit. In the present invention, “ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which refers to connecting a monoclonal antibody or antibody fragment to a biologically active toxic drug by a stable linking unit.
The three-letter and single-letter codes for amino acids used in the present disclosure are as described in J. boil. Chem. 1968, 243, 3558.
The term “alkyl” refers to a saturated aliphatic hydrocarbon group, which is a linear or branched chain group containing 1 to 20 carbon atoms (i.e., “C1-C20 alkyl”), preferably an alkyl containing 1 to 12 carbon atoms (i.e., “C1-C12 alkyl”), more preferably an alkyl containing 1 to 10 carbon atoms (i.e., “C1-C10 alkyl”), most preferably an alkyl containing 1 to 6 carbon atoms (i.e., “C1-C6 alkyl”). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferably, the alkyl group is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. The alkyl can be substituted or unsubstituted. When substituted, the substituent group(s) can be substituted at any available connection point. The substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.
The term “substituted alkyl” refers to that hydrogen in an alkyl group is replaced by a substituent group. Unless otherwise stated in the context, the substituent of the alkyl group may be a variety of groups selected from the group consisting of: -halogen, —OR′, —NR′R″, —SR′, —SiR′R″R″′, —OC(O)R′, —C(O)R′, —CO2R′, ˜CONR′R″, —OC(O)NR′R″, —NR″C (O) R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NR′S(O)2R″, —CN and —NO2. The number of substituents ranges from 0 to (2m′+1), where m′ is the total number of carbon atoms in the group. R′, R″ and R″′ each independently refer to hydrogen, unsubstituted C1-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted by one to three halogens, unsubstituted C1-8 alkyl, C1-8 alkoxy or C1-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-C1-4 alkyl. When R′ and R″ are connected to the same nitrogen atom, they can form a 3-, 4-, 5-, 6- or 7-membered ring together with the nitrogen atom. For example, —NR′R″ includes 1-pyrrolidinyl and 4-morpholinyl.
The term “alkylene” refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. The alkylene is a linear or branched group having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (—CH2—, 1,1-ethylene (—CH(CH3)—), 1,2-ethylene (—CH2CH2)—, 1,1-propylene (—CH(CH2CH3)—), 1,2-propylene (—CH2CH(CH3)—), 1,3-propylene (—CH2CH2CH2—), 1,4-butylene (—CH2CH2CH2CH2—), 1,5-pentylene (—CH2CH2CH2CH2CH2—), and the like. The alkylene can be substituted or unsubstituted. When substituted, the substituent group(s) can be substituted at any available connection point. The substituent group(s) is preferably one or more groups independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
The term “alkoxy” refers to an —O-(alkyl) and an —O-(cycloalkyl) group, wherein the alkyl and cycloalkyl are as defined above. Non-limiting examples of C1-C6 alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. The alkoxy can be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more group(s) independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.
The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent group having 3 to 20 carbon atoms (i.e., “C3-C20 cycloalkyl”), preferably 3 to 12 carbon atoms (i.e. “C3-C12 cycloalkyl”), more preferably 3 to 10 carbon atoms (i.e. “C3-C10 cycloalkyl”), and most preferably 3 to 8 carbon atoms (i.e. “C3-C8 cycloalkyl”). Non-limiting examples of monocyclic cycloalkyl (e.g., “C3-C8 cycloalkyl”) include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexenyl Dialkenyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc. Polycyclic cycloalkyl includes a cycloalkyl having a spiro ring, fused ring or bridged ring.
The term “heterocyclyl” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group containing 3 to 20 ring atoms (i.e., “3- to 20-membered heterocyclyl”), in which one or more ring atoms are heteroatoms selected from the group consisting of N, O and S(O)m (where m is an integer of 0 to 2), but excluding —O—O—, —O—S— or —S—S— in the ring, with the remaining ring atoms being carbon atoms. Preferably, the heterocyclyl contains 3 to 12 ring atoms (i.e. “3- to 12-membered heterocyclyl”), of which 1 to 4 atoms are heteroatoms; and more preferably the cycloalkyl ring contains 3 to 10 ring atoms (i.e. “3- to 10-membered heterocyclyl”). Non-limiting examples of monocyclic heterocyclyl (e.g., 3- to 7-membered heterocyclyl) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyl includes a heterocyclyl having a spiro ring, fused ring or bridged ring.
The term “cycloalkylalkyl” refers to an alkyl group substituted by one cycloalkyl or more, preferably one cycloalkyl, wherein the alkyl and the cycloalkyl are as defined above. Examples of cycloalkylalkyl include C3-C8 cycloalkyl C1-C6 alkyl.
The term “haloalkyl” refers to an alkyl group substituted by one or more halogen(s), where the alkyl is as defined above. Examples of haloalkyl include halogenated C1-C6 alkyl.
The term “deuterated alkyl” refers to an alkyl group substituted by one or more deuterium atom(s), where the alkyl is as defined above. Examples of deuterated alkyl include deuterated C1-C6 alkyl.
The term “C6-C12 aryl” refers to a group of a carbocyclic aromatic system having 6 to 12 carbon atoms.
The term “C6-C10 aryl” refers to a group of a carbocyclic aromatic system having 6 to 10 carbon atoms. Examples of C6-C10 aryl are phenyl, naphthyl, etc.
The term “5- to 10-membered heteroaryl” refers to an aromatic heterocyclic ring, usually a 5-, 6-, 7-, 8-, 9-, or 10-membered heterocyclic ring with 1 to 3 heteroatoms selected from N, O or S. A heteroaryl ring may optionally be further fused or attached to aromatic and nonaromatic carbocyclic and heterocyclic rings. Non-limiting examples of the 5- to 10-membered heteroaryl are, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyl, phenyl-pyrrolyl, furyl, phenyl-furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzofuranyl, benzothienyl, benzo-1,3-dioxolane (benzodioxin), isoindolyl, benzimidazolyl, indazolyl, quinolyl, isoquinolyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-indolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, benzopyrin pyryl, 2,3-dihydrobenzoxazinyl, 2,3-dihydroquinoxalinyl, etc.
The term “substituted C6-C10 aryl” or “substituted 5- to 10-membered heteroaryl” or “substituted 3- to 7-membered heterocyclyl” refers to that hydrogen in aryl or heteroaryl or heterocyclyl is substituted by a substituent group, unless otherwise stated in the context, the substituent of aryl or heteroaryl or heterocyclyl may be a variety of groups selected from the group consisting of: -halogen, —OR′, —NR′R″, —SR′, —SiR′R″R′, —OC(O)R′, —C(O)R′, —CO2R′, ˜CONR′R″, —OC(O)NR′R″, —NR″C (O) R′, —NR′—C(O)NR″R″′, —NR″C(O)2R′, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH— C(NH2)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NR′S(O)2R″, —CN and —NO2. The number of substituents ranges from 0 to (2m′+1), where m′ is the total number of carbon atoms in the group. R′, R″ and R″′ each independently refer to hydrogen, unsubstituted C1-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted by one to three halogens, unsubstituted C1-8 alkyl, C1-8 alkoxy or C1-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-C1-4 alkyl. when R′ and R″ are connected to the same nitrogen atom, they can form a 3-, 4-, 5-, 6- or 7-membered ring together with the nitrogen atom. For example, —NR′R″ includes 1-pyrrolidinyl and 4-morpholinyl.
The term “hydroxy” refers to an —OH group.
The term “halogen” refers to fluorine, chlorine, bromine or iodine.
The term “amino” refers to a —NH2 group. The term “nitro” refers to a —NO2 group.
The term “amide” refers to a —C(O)N(alkyl) or —C(O)N(cycloalkyl) group, where the alkyl and cycloalkyl are as defined above.
The term “carboxylate group” refers to a —C(O)O(alkyl) or —C(O)O(cycloalkyl) group, where the alkyl and cycloalkyl are as defined above.
The present invention also comprises formula I in various deuterated forms. Each of the available hydrogen atoms attached to the carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize formula I in a deuterated form with reference to the relevant literatures. The formula I in deuterated form can be prepared by employing commercially available deuterated raw materials, or they can be synthesized by conventional techniques with deuterated reagents. Non-limiting examples of deuterated reagents include: deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane and the like.
The term “antibody” refers to immunoglobulin, a four-peptide chain structure connected together by interchain disulfide bond between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and sequences, hence presenting different antigenicity. Accordingly, immunoglobulins can be divided into five types, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, with corresponding heavy chains μ, δ, γ, α and ε, respectively. According to the amino acid composition of hinge region and the number and location of heavy chain disulfide bonds, the same type of Ig can further be divided into different sub-types, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. Light chain can be divided into κ or λ chain based on different constant region. Each of the five types of Ig can have a κ or λ chain. The antibodies described in the present invention are preferably specific antibodies against the cell surface antigens on the target cells, and non-limiting examples are one or more of the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2(ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER3(ErbB3) antibody, anti-MUC1/CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3(CD276) antibody, anti-PTK7/CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRa antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, and anti-CD123 antibody.
The term “solvate” or “solvent compound” refers to a pharmaceutically acceptable solvate formed by a ligand-drug conjugate of the present invention with one or more solvent molecule(s). Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.
The term “drug loading” refers to the average number of cytotoxic drugs loaded on each ligand in formula I, and can also be expressed as a drug to antibody ratio (DAR). The drug loading can range from 0 to 12, preferably from 1 to 10 cytotoxic drugs (d) per antibody (Ab). In an embodiment of the present invention, the drug loading is expressed as n, and exemplary values can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs per ADC molecule after coupling reaction can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization.
In an embodiment of the present invention, the cytotoxic drug is conjugated to an opened cysteine thiol-SH between antibody chains and/or the thiol-SH of site-directed mutated cysteine residue by a linking unit. Typically, the number of drug molecules capable of being conjugated to the antibody in a coupling reaction will be less than the theoretical maximum.
The following non-limiting methods can be used to control the loading of the ligand-cytotoxic drug conjugates:
The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.
The term “pharmaceutically acceptable salt” or “medicinal salt” generally refers to the salts of the ligand-drug conjugate of the present invention or salts of the compound of the present invention. Such salts may have safety and/or effectiveness when used in mammals, and may have corresponding biological activity. The ligand-drug conjugate compound of the present invention comprises at least one carboxyl, so it can form a salt with a base. Non-limiting examples of the pharmaceutically acceptable salt include: sodium salt, potassium salt, calcium salt, and magnesium salt.
The term “pharmaceutically acceptable salt” or “medicinal salt” generally refers to the salts of the antibody-drug conjugate of the present invention or salts of the compound of the present invention. Such salts may have safety and/or effectiveness when used in mammals, and may have corresponding biological activity. The ligand-drug conjugate compound of the present invention comprises at least one amino, so it can form a salt with an acid. Non-limiting examples of the pharmaceutically acceptable salt include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.
“Acidic amino acid” refers to an amino acid with an isoelectric point less than 7. An acidic amino acid molecule often has one or more acidic groups such as carboxyl, which can be effectively ionized into negative ions in the structure to increase hydrophilicity. Acidic amino acids can be natural or unnatural amino acids.
“Natural amino acids” refer to amino acids synthesized by organisms. Natural amino acids are generally in the L-form, but there are a few exceptions, such as glycine. The natural amino acids include natural and biologically synthesized amino acids.
“Unnatural amino acids” refer to amino acids obtained by synthetic means.
The present invention will be further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods without specifying specific conditions in the following examples are generally conducted under conventional conditions or conditions recommended by the manufacturers. Unless otherwise stated, all percentages, proportions, ratios, or parts are calculated by weight.
In a 5000 mL single-neck flask, N-fluorenylmethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 395 mmol, 1.4 eq), 2000 mL of dry tetrahydrofuran and 670 mL of toluene were mixed and stirred adequately, and then heated to 85° C. in the presence of N2 to react for 2.5 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was cooled to room temperature and filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain Compound M1 (87 g); LC-MS: [M+NH4]+=386.0.
In a 1000 mL single-neck flask, Compound SM-2 (prepared according to the synthesis route disclosed by patent application CN108452321A) (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were mixed and then heated to 120° C. to reflux for 2 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was cooled to 50° C. and rotary evaporation under reduced pressure was performed to remove the solvent. The reaction product was then dissolved in ethyl acetate (150 mL) and water (40 mL), and 1M of HCl was then added to adjust the pH to 2-3 with stirring in an ice bath. The resulting solution was allowed for liquid separation, the aqueous layer was extracted once more with ethyl acetate, the organic layers were combined, and dried with anhydrous Na2SO4. After filtration, the filtrate was concentrated to obtain a light yellow oily rude product. The rude product was purified by column chromatography (DCM:MeOH=40:1) to obtain Compound M2 (26.6 g); LC-MS: [M+H]+=399.3.
In a 1000 mL single-neck flask, Compound M2 (26.5 g, 60.5 mmol, 1.0 eq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq) and THE (300 mL) were mixed and the reaction was allowed at room temperature for 30 min and monitored by TLC. The insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound M3 (31.5 g) with a yield of 64%; LC-MS: [M+H]+=565.1.
Synthesis of Compound ent-M3:
Compound ent-M3 (27.8 g) was obtained by referring to the synthesis route of example 2; LC-MS: [M+H]+=565.2.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., benzyl glycolate (5.4 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought naturally to room temperature and stirred for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was quenched with a saturated NaHCO3 solution, and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-1:1) to obtain 1a (4 g), with a yield of 52%; LC-MS: [M+H]+=475.18.
In a 25 mL single-neck flask, Compound 1a (2 g, 4.2 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (766 mg, 5.04 mmol) was then added to have a reaction for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use.
In another 25 mL single-neck flask, M4 (prepared according to the synthesis route disclosed in CN111051330 A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol) and 10 mL of DMF were mixed, DIPEA (830 uL, 5.04 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 30 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through preparative liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain a solid product 1b (1.7 g), with a yield of 63%; LCMS: [M+H]+=648.26.
In a 25 mL single-neck flask, Compound 1b (900 mg, 1.39 mmol) was completely dissolved in 15 mL of DMF and 900 mg of 5% Pd/C was then added to have a hydrogenation reaction for 2 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 1c was placed in a flask in an ice-water bath, followed by addition of DIPEA (235 uL, 1.39 mmol), and then Compound M3 (784 mg, 1.39 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 1d (504 mg); LC-MS: [M+H]+=804.4.
In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 1e. The preparation solution was lyophilized to obtain 1e (210 mg); LC-MS: [M+H]+=1221.6.
In a 25 mL single-neck flask, 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 1 (60 mg); LC-MS: [M+H]+=1065.3.
Compound 2 (51 mg) was obtained by referring to the synthesis route of example 4; LC-MS: [M+H]+=1065.3.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 2-hydroxy-2-methylpropionic acid benzyl ester (6.3 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought naturally to room temperature to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was quenched with a saturated NaHCO3 solution, and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 3a (4.2 g), with a yield of 52%; LC-MS: [M+H]+=503.3.
In a 25 mL single-neck flask, Compound 3a (2 g, 4.0 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (760 mg, 5.0 mmol) was then added to have a reaction for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use.
In another 25 mL single-neck flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol) and 10 mL of DMF were mixed, DIPEA (823 uL, 5.04 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 30 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain a solid product 3b (1.4 g), with a yield of 53%; LC-MS: [M+H]+=676.2.
In a 25 mL single-neck flask, Compound 3b (700 mg, 1.04 mmol) was completely dissolved in 10 mL of DMF and 700 mg of 5% Pd/C was then added to have a hydrogenation reaction for 1.5 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 3c was placed in a flask in an ice-water bath, followed by addition of DIPEA (210 uL, 1.25 mmol), and then Compound M3 (704 mg, 1.25 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 3d (486 mg); LC-MS: [M+H]+=830.5.
In a 50 mL single-neck flask, 3d (300 mg, 0.36 mmol), M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol) and 10 mL of DMF were mixed and then DIPEA (219.5 uL, 1.33 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 3e. The preparation solution was lyophilized to obtain 3e (157 mg); LC-MS: [M+H]+=1249.6.
In a 25 mL single-neck flask, 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 3 (64 mg); LC-MS: [M+H]+=1093.1.
Compound 4 (60 mg) was obtained by referring to the synthesis route of example 6; LC-MS: [M+H]+=1093.2.
In a 25 mL single-neck flask, M1 (500 mg, 1.4 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 eq) and 10 mL of TIF were mixed and stirred and then cooled to 0° C., L-lactic acid benzyl ester (1.2 g, 7.0 mmol, 5 eq) was then added slowly. The resulting solution was stirred and brought to room temperature. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was quenched with a saturated NaHCO3 solution, and then extracted with ethyl acetate, the product was dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a reversed phase column to obtain 5a (400 mg);
LC-MS: [M+NH4]=506.2.
1H NMR (400 Mz, CDCl3/CD3OD): 1.39 (3H, d, J=6.8 Hz), 3.78 (2H, t, J=4.0 Hz), 4.17-4.27 (2H, m), 4.42 (2H, d, J=4.0 Hz), 4.72-4.85 (2H, m), 5.11-5.58 (2H, m), 5.43 (1H, s), 7.06 (1H, t, J=8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t, J=8.0 Hz), 7.57 (2H, d, J=8.0 Hz), 7.75 (2H, d, J=8.0 Hz).
In a 25 mL single-neck flask, Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were mixed and stirred and then cooled to 0° C., DBU (137 mg, 0.9 mmol, 1.1 eq) was then added slowly. The resulting solution was then brought to room temperature to have a reaction. Marking as reaction solution 1 after the reaction was found to be completed from the monitoring of the TLC;
In another 25 mL single-neck flask, M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq) and 3 mL of DMF were mixed and stirred at room temperature for 5 min, reaction solution 1 was then added, the reaction system was allowed at room temperature. After the reaction was found to be completed from the monitoring of the TLC, the reaction solution was purified by high-performance liquid chromatography to obtain Compound 5b (326 mg); LC-MS: [M+NH4]+=679.2.
In a 100 mL single-neck flask, 5b (4.0 g, 6.05 mmol, 1.0 eq) was dissolved in DMF (60 mL) and 5% Pd/C (4 g) was then added to have a hydrogenation reaction for 4 h (reaction progress was monitored by HPLC). Pd/C was filtered, and the filtrate without concentration was directly put in an ice water bath (about 0° C.) for further use.
The crude product 5c was placed in a flask in an ice-water bath, followed by addition of DIPEA (1.1 mL, 1.1 eq), and then Compound M3 (3.4 g, 6.05 mmol). The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 5d (3.15 g); LC-MS: [M−H]−=816.3.
In a 100 mL single-neck flask, 5d (2.07 g, 2.53 mmol, 1.0 eq), M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol, 1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq) and DMF (40 mL) were added, and DIPEA (1.05 mL, 1.5 eq) was added in condition of an ice-water bath, the resulting solution was brought to room temperature to have a reaction for 2 h (monitored by HPLC). the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound 5e (1.92 g) with a yield of 61%; LC-MS: [M+H]+=1235.4.
In a 100 mL single-neck flask, Compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were mixed and dissolved, followed by addition of zinc bromide (3.64 g, 16 mmol, 20.0 eq). Reaction was allowed in an oil bath at 40° C. (the reaction system was preheated and stabilized in advance) for 30 min. The reaction solution was concentrated in a reduced-pressure (water pump) water bath at 45° C. to remove nitromethane to obtain a yellow solid residue (under the monitoring of HPLC). The preparation solution of Compound 5A was obtained by acid preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound 5A (786 mg) with a yield of 90%.
LC-MS: [M+H]+=1079.4;
1H NMR (400 MHz, DMSO-d6) δ 9.39-9.02 (m, 1H), 8.70 (t, J=6.5 Hz, 1H), 8.64 (t, J=5.7 Hz, 1H), 8.56 (d, J=8.8 Hz, 1H), 8.34 (t, J=5.7 Hz, 1H), 8.16 (d, J=8.2 Hz, 1H), 8.01 (t, J=5.5 Hz, 1H), 7.71 (d, J=10.9 Hz, 1H), 7.30 (s, 1H), 7.28-7.15 (m, 4H), 7.14 (s, 2H), 5.53 (dd, J=14.5, 6.4 Hz, 1H), 5.49-5.34 (m, 2H), 5.22 (d, J=18.8 Hz, 1H), 5.09 (d, J=18.7 Hz, 1H), 5.03 (dd, J=9.6, 3.9 Hz, 1H), 4.73 (dd, J=9.9, 6.9 Hz, 1H), 4.59 (dd, J=10.1, 6.5 Hz, 1H), 4.49 (ddd, J=13.2, 8.6, 4.4 Hz, 1H), 4.14 (dd, J=13.3, 6.6 Hz, 2H), 3.93 (s, 2H), 3.84 (dd, J=16.5, 6.3 Hz, 1H), 3.76 (dd, J=16.9, 5.7 Hz, 2H), 3.70 (d, J=5.2 Hz, 2H), 3.60 (dd, J=16.7, 5.4 Hz, 1H), 3.52 (dd, J=16.4, 5.1 Hz, 1H), 3.45 (dd, J=12.8, 10.1 Hz, 1H), 3.25-3.15 (m, 1H), 3.14-3.05 (m, 1H), 3.01 (dd, J=13.7, 4.1 Hz, 1H), 2.73 (dd, J=13.5, 9.8 Hz, 1H), 2.54-2.47 (m, 1H), 2.33 (s, 2H), 2.17 (d, J=5.5 Hz, 2H), 1.91-1.79 (m, 2H), 1.33 (d, J=6.6 Hz, 2H), 0.87 (t, J=7.3 Hz, 2H).
In a 25 mL single-neck flask, Compound 5b (300 mg, 0.45 mmol, 1.0 eq) was stirred and completely dissolved in DMF (3 mL), 5% Pd/C (300 mg) was then added, hydrogen was replaced three times, and hydrogenation reaction was carried out for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was filtered to remove Pd/C, the filtrate was cooled to 0-5° C., DIPEA (65 mg, 0.5 mmol, 1.1 eq) was then added, followed by addition of ent-M3 (255 mg, 0.45 mmol), and the resulting solution was brought to 20±5° C. to have a reaction for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by HPLC, and the preparation solution of the product was collected and lyophilized to obtain Compound 5d-1 (200 mg) with a yield of 54%;
LC-MS: [M−H]−=816.3.
In a 25 mL single-neck flask, Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), M5 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were mixed and then cooled to 0-5° C. in an ice water bath, followed by addition of DIPEA (62 mg, 0.48 mmol, 2.0 eq). The reaction was allowed at 20±5° C. for 2 h. After the reaction was found to be completed from the monitoring of the HPLC. The reaction solution was directly purified by HPLC preparation. The product preparation solution was collected and then lyophilized to obtain Compound 5e-1 (162.8 mg); LC-MS: [M+H]+=1235.4.
In a 25 mL single-neck flask, Compound 5e-1 (110 mg, 0.089 mmol, 1.0 eq), ZnBr2 (400 mg, 1.78 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in sequence. The reaction was allowed at 40° C. for 0.5 h and then terminated. Reduced-pressure rotary evaporation was performed at 45° C. to obtain a yellow solid, then sampled, and monitored by HPLC. The solid subjected to rotary evaporation was directly purified by HPLC preparation. The product preparation solution was collected and then lyophilized to obtain Compound 5B (73.4 mg), with a yield of 76.5%; LC-MS: [M+H]+=1079.4.
Compound 6A (71 mg) was obtained by referring to the synthesis route of example 8; LC-MS: [M+H]+=1079.4.
Compound 6B (59 mg) was obtained by referring to the synthesis route of example 9; LC-MS: [M+H]+=1079.4.
In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 3, 3, 3-trifluorolactic acid benzyl ester (prepared according to the synthesis route disclosed in patent application WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were mixed and heated to 100° C. to react for 4 h. After the reaction was found to be completed, the reaction solution was cooled to room temperature, then filtered to remove the insoluble matter, the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=10:1-5:1-2:1) to obtain 5.15 g of the target, with a yield of 35.1%; LC-MS: [M+H]+=543.17.
In a 50 mL single-neck flask, Compound 7a (5 g, 9.2 mmol) was completely dissolved in 15 mL of DMF and DBU (1.68 g, 11 mmol) was then added in an ice water bath to react for 1 h, marking as reaction solution 1; In another 50 mL single-neck flask, M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL DMF were mixed and dissolved completely. DIPEA (1.82 mL, 11 mmol) was then added in an ice water bath to further react for 30 min, reaction solution 1 was added, and the reaction was allowed at room temperature for 2 h and monitored by HPLC. After the reaction was found to be completed, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated to obtain 4.1 g of a solid product with a yield of 62.3%; LC-MS: [M+H]+=716.25.
In a 25 mL single-neck flask, 7b (900 mg, 1.26 mmol) was completely dissolved in 15 mL of DMF and 900 mg of 5% Pd/C was then added to have a hydrogenation reaction for 2 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was put into a flask in an ice-water bath, followed by addition of DIPEA (228 uL, 1.38 mmol), and then M3 (712 mg, 1.26 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 525 mg of the product, with a yield of 47.9%; LC-MS: [M−H]−=870.33.
In a 50 mL single-neck flask, 7d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 7e-1 and Compound 7e-2. The preparation solutions were lyophilized separately to obtain 150 mg of Compound 7e-1, LC-MS: [M+H]+=1289.46; 220 mg of Compound 7e-2, LC-MS: [M+H]+=1289.46.
In a 25 mL single-neck flask, 7e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 52 mg of a solid; TOF result: 1133.3613.
In a 25 mL single-neck flask, 7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 63 mg of a solid; TOF result: 1133.3668.
In a 25 mL single-neck flask, 7c (900 mg, 1.83 mmol) was completely dissolved in 20 mL of DMF, followed by addition of DIPEA (303 uL, 1.83 mmol), and then ent-M3 (1034 mg, 1.83 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 613 mg of the product, with a yield of 38.5%; LC-MS: [M−H]−=870.32.
In a 50 mL single-neck flask, 8d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 8e-1 and Compound 8e-2. The preparation solutions were lyophilized separately to obtain 140 mg of Compound 8e-1 and 210 mg of Compound 8e-2. LC-MS of Compound 8e-1: [M+H]+=1289.47; LC-MS of Compound 8e-2: [M+H]+=1289.47.
In a 25 mL single-neck flask, 8e-1 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 50 mg of a solid; TOF result: 1133.3623.
In a 25 mL single-neck flask, Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 58 mg of a solid; TOF result: 1133.3653.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 2-hydroxy-2-cyclopropylacetic acid benzyl ester (prepared according to the synthesis route disclosed in patent application US20050020645 A1) (6.3 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought naturally to room temperature to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was quenched with a saturated NaHCO3 solution, and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 9a (3.7 g), with a yield of 45%; LC-MS: [M+H]+=501.5.
In a 25 mL single-neck flask, Compound 9a (2 g, 4.0 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (760 mg, 5.0 mmol) was then added to have a reaction for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use.
In another 25 mL single-neck flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol) and 10 mL of DMF were mixed, DIPEA (823 uL, 5.04 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 30 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain 1.5 g of a solid product, with a yield of 56%; LC-MS: [M+H]+=674.7.
In a 25 mL single-neck flask, Compound 9b (900 mg, 1.3 mmol) was completely dissolved in 10 mL of DMF and 900 mg of 5% Pd/C was then added to have a hydrogenation reaction for 1.5 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 9c was placed in a flask in an ice-water bath, followed by addition of DIPEA (223 uL, 1.3 mmol), and then Compound M3 (750 mg, 1.3 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 9d (529 mg); LC-MS: [M+H]+=828.4.
In a 50 mL single-neck flask, 9d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol) and 15 mL of DMF were mixed and then DIPEA (351 uL, 2.13 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 9e. The preparation solution was lyophilized to obtain 9e (257 mg); LC-MS: [M+H]+=1247.5.
In a 25 mL single-neck flask, 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 9A (55 mg); LC-MS: [M+H]+=1091.3.
Compound 9B (44 mg) was obtained by referring to the synthesis route of example 14; LC-MS: [M+H]+=1091.3.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 3-hydroxy-2-cyclopropylpropionic acid benzyl ester (prepared according to the synthesis route disclosed in patent application WO2013187496A1) (6.7 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought to room temperature naturally to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, saturated NaHCO3 solution was added to the reaction system, and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue was purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 10a (4.9 g), with a yield of 58%; LC-MS: [M+H]+=515.4.
In a 25 mL single-neck flask, Compound 10a (4 g, 7.8 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (1.2 g, 8.0 mmol) was then added. The reaction was carried out for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use. In another 25 mL single-neck flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol) and 10 mL of DMF were mixed, DIPEA (1.65 mL, 10.1 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 50 min and then placed in a reaction flask to have a reaction at room temperature. After the completion of the reaction monitored by HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, the organic layers was dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain 2.3 g of a solid product, with a yield of 42%; LCMS: [M+H]+=688.8.
In a 25 mL single-neck flask, Compound 10b (1.0 g, 1.45 mmol) was completely dissolved in 15 mL of DMF and 1.0 g of 5% Pd/C was then added to have a hydrogenation reaction for 1.5 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 10c was placed in a flask in an ice-water bath, followed by addition of DIPEA (258 uL, 1.5 mmol) and Compound M3 (837 mg, 1.45 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 10d (499 mg); LC-MS: [M−H]−=842.4.
In a 50 mL single-neck flask, 10d (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol) and 15 mL of DMF were mixed and then DIPEA (330 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 10e. The preparation solution was lyophilized to obtain 10e (188 mg); LC-MS: [M+H]+=1261.5.
In a 25 mL single-neck flask, 10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 10A (61 mg); LC-MS: [M+H]+=1105.4.
Compound 10B (75 mg) was obtained by referring to the synthesis route of example 16; LC-MS: [M+H]+=1105.4.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 2-hydroxy-2-cyclobutyl acetic acid benzyl ester (prepared according to the synthesis route disclosed by the literature: Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552.) (6.7 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought to room temperature to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, a saturated NaHCO3 solution was added, and organic layers was extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 11a (5.1 g), with a yield of 62%; LC-MS: [M+H]+=515.7.
In a 25 mL single-neck flask, Compound 11a (4 g, 7.8 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (1.2 g, 8.0 mmol) was then added. And the reaction was carried out for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use. In another 25 mL single-neck flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol) and 10 mL of DMF were mixed, DIPEA (1.63 mL, 10.0 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 40 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain 2.3 g of a solid product, with a yield of 42%; LCMS: [M+H]+=688.3.
In a 25 mL single-neck flask, Compound 11b (2.0 g, 2.9 mmol) was completely dissolved in 25 mL of DMF and 2.0 g of 5% Pd/C was then added to have a hydrogenation reaction for 3 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 11c was placed in a flask in an ice-water bath, followed by addition of DIPEA (516 uL, 3.0 mmol), and then Compound M3 (1.7 g, 2.9 mmol). The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 11d (934 mg); LC-MS: [M−H]−=842.4.
In a 50 mL single-neck flask, 11d (800 mg, 0.96 mmol), M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were mixed and then DIPEA (660 uL, 4.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 4 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 11e. The preparation solution was lyophilized to obtain 11e (401 mg); LC-MS: [M+H]+=1261.4.
In a 25 mL single-neck flask, 11e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 11A (86 mg); LC-MS: [M+H]+=1105.4.
Compound 11B (50 mg) was obtained by referring to the synthesis route of example 18. LC-MS: [M+H]+ 1105.4.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 3-hydroxy-2-cyclobutylpropionic acid benzyl ester (prepared according to the synthesis route disclosed in patent application WO2009011285A1) (7.2 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought to room temperature to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, a saturated NaHCO3 solution was added, then organic layers were washed with saturated NaCl solution, and extracted with ethyl acetate, dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 12a (4.5 g), with a yield of 52%; LC-MS: [M+H]+=529.4.
In a 25 mL single-neck flask, Compound 12a (4 g, 7.6 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (1.2 g, 8.0 mmol) was then added to have a reaction for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use.
In another 25 mL single-neck flask, M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol) and 10 mL of DMF were mixed, DIPEA (1.49 mL, 0.9 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 30 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain 2.0 g of a solid product, with a yield of 37%; LC-MS: [M+H]+=702.8.
In a 25 mL single-neck flask, Compound 12b (1.0 g, 1.43 mmol) was completely dissolved in 15 mL of DMF and 1.0 g of 5% Pd/C was then added to have a hydrogenation reaction for 1.5 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 12c was placed in a flask in an ice-water bath, followed by addition of DIPEA (258 uL, 1.5 mmol), and then Compound M3 (825 mg, 1.43 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 12d (522 mg); LC-MS: [M−H]−=856.4.
In a 50 mL single-neck flask, 12d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol) and 15 mL of DMF were mixed and then DIPEA (330 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 12e. The preparation solution was lyophilized to obtain 12e (198 mg); LC-MS: [M+H]+=1275.4.
In a 25 mL single-neck flask, 12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 12A (55 mg); LC-MS: [M+H]+=1119.4.
Compound 12B (50 mg) was obtained by referring to the synthesis route of example 20; LC-MS: [M+H]+=1119.4.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 2-hydroxy-2-cyclopentylacetic acid benzyl ester (prepared according to the synthesis route disclosed by the literature: Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552.) (7.2 g, 32.6 mmol) was then added dropwise. The resulting solution was brought naturally to room temperature to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was quenched with a saturated NaHCO3 solution, and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 13a (4.6 g), with a yield of 53%; LC-MS: [M+H]+=529.5.
In a 25 mL single-neck flask, Compound 13a (4 g, 7.6 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (1.17 g, 7.8 mmol) was then added to have a reaction for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use. In another 25 mL single-neck flask, M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol) and 10 mL of DMF were mixed, DIPEA (1.39 mL, 0.85 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 30 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain 2.1 g of a solid product, with a yield of 39%; LC-MS: [M+H]+=702.8.
In a 25 mL single-neck flask, Compound 13b (1.5 g, 1.87 mmol) was completely dissolved in 25 mL of DMF and 1.5 g of 5% Pd/C was then added to have a hydrogenation reaction for 3 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 13c was placed in a flask in an ice-water bath, followed by addition of DIPEA (333 uL, 1.93 mmol), and then Compound M3 (1.1 g, 1.87 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 13d (519 mg); LC-MS: [M−H]−=856.6.
In a 50 mL single-neck flask, 13d (400 mg, 0.47 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol) and 15 mL of DMF were mixed and then DIPEA (330 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 4 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 13e. The preparation solution was lyophilized to obtain 13e (187 mg); LC-MS: [M+H]+=1275.5.
In a 25 mL single-neck flask, 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 13A (60 mg); LC-MS: [M+H]+=1119.6.
Compound 13B (51 mg) was obtained by referring to the synthesis route of example 22; LC-MS: [M+H]+=1119.6.
In a 250 mL single-neck flask, M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were mixed and stirred and then cooled to 0° C., 3-hydroxy-2-cyclopentylpropanoic acid benzyl ester (prepared according to the synthesis route disclosed by patent application WO2009011285A1) (7.6 g, 32.6 mmol) was then added dropwise. The resulting solution was then brought to room temperature to have a reaction for about 2-4 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction system was quenched with a saturated NaHCO3 solution, and then extracted with ethyl acetate, the product was washed with saturated NaCl solution, then dried with anhydrous Na2SO4, filtered and concentrated, and the residue is purified on a silica gel column (PE:EA=10:1-5:1-2:1) to obtain 14a (4.4 g), with a yield of 49%; LC-MS: [M+H]+=543.6.
In a 25 mL single-neck flask, Compound 14a (4 g, 7.4 mmol) and 10 mL of DMF were mixed and stirred at 0° C., and DBU (1.2 g, 8.0 mmol) was then added to have a reaction for 1 h. After Fmoc deprotection was found to be completed from the monitoring of the TLC, the resulting product was ready for later use. In another 25 mL single-neck flask, M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol) and 10 mL of DMF were mixed, DIPEA (1.49 mL, 9.0 mmol) was then added in an ice water bath, the resulting reaction solution was stirred for 30 min and then placed in a reaction flask to have a reaction at room temperature. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified through liquid chromatography to obtain a product preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to obtain 2.6 g of a solid product, with a yield of 49%; LC-MS: [M+H]+=716.4.
In a 25 mL single-neck flask, Compound 14b (1.0 g, 1.4 mmol) was completely dissolved in 15 mL of DMF and 1.0 of 5% Pd/C was then added to have a hydrogenation reaction for 1.5 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was directly put into the next reaction without purification.
The crude product 14c was placed in a flask in an ice-water bath, followed by addition of DIPEA (248 uL, 1.5 mmol), and then Compound M3 (808 mg, 1.4 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 14d (500 mg); LC-MS: [M−H]−=870.5.
In a 50 mL single-neck flask, 14d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (99 mg, 0.46 mmol) and 15 mL of DMF were mixed and then DIPEA (331 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 14e. The preparation solution was lyophilized to obtain 14e (146 mg); LC-MS: [M+H]+=1289.5.
In a 25 mL single-neck flask, 14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 14A (52 mg); LC-MS: [M+H]+=1133.4.
Compound 14B (48 mg) was obtained by referring to the synthesis route of example 24; LC-MS: [M+H]+=1133.4.
In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxy-butyric acid benzyl ester (prepared according to the synthesis route disclosed in the literature Chemical Communications, 2019, 55 (53), 7699-7702.) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were mixed and heated to 100° C. to react for 4 h. After the reaction was found to be completed, the reaction solution was cooled to room temperature, then filtered to remove the insoluble matter, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=10:1-5:1-2:1) to obtain 5.67 g of the target, with a yield of 42%; LC-MS: [M+H]+=503.5.
In a 50 mL single-neck flask, Compound 15a (5 g, 9.95 mmol) was completely dissolved in 15 mL of DMF and DBU (1.68 g, 11 mmol) was then added in an ice water bath to react for 1 h, marking as reaction solution 1; In another 50 mL single-neck flask, M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL DMF were mixed and dissolved completely. DIPEA (1.82 mL, 11 mmol) was then added in an ice water bath to further react for 40 min, reaction solution 1 was added, and the reaction was allowed at room temperature for 2 h and monitored by HPLC. After the reaction was found to be completed, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated to obtain 4.6 g of a solid product with a yield of 68%; LC-MS: [M+H]+=676.7.
In a 25 mL single-neck flask, 15b (2.0 g, 2.96 mmol) was completely dissolved in 15 mL of DMF and 2.0 g of 5% Pd/C was then added to have a hydrogenation reaction for 2 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was put into a flask in an ice-water bath, followed by addition of DIPEA (496 uL, 3.0 mmol), and then M3 (1.7 g, 2.96 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 1120.0 mg of the product, with a yield of 45%; LC-MS: [M−H]−=830.3.
In a 50 mL single-neck flask, 15d (500 mg, 0.60 mmol), M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90 mmol) and 15 mL of DMF were mixed and then DIPEA (446 uL, 2.7 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 15e-1 and Compound 15e-2. The preparation solutions were lyophilized separately to obtain 138 mg of Compound 15e-1, LC-MS: [M+H]+=1249.5; 140 mg of Compound 15e-2, LC-MS: [M+H]+=1249.5.
In a 25 mL single-neck flask, 15e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 59 mg of a solid; LC-MS: [M+H]+=1093.4.
In a 25 mL single-neck flask, 15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 60 mg of a solid; LC-MS: [M+H]+=1093.4.
Compound 16A (55 mg) was obtained by referring to the synthesis route of example 26; LC-MS: [M+H]+=1093.4.
Compound 16B (54 mg) was obtained by referring to the synthesis route of example 26; LC-MS: [M+H]+=1093.4.
In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxy-benzenepropanoic acid benzyl ester (prepared according to the synthesis route disclosed in literature Nature Communications, 2020. 11 (1), 56.) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were mixed and heated to 100° C. to react for 4 h. After the reaction was found to be completed, the reaction solution was cooled to room temperature, then filtered to remove the insoluble matter, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=10:1-5:1-2:1) to obtain 6.13 g of the target, with a yield of 40%; LC-MS: [M+H]+=565.6.
In a 50 mL single-neck flask, Compound 17a (5 g, 8.86 mmol) was completely dissolved in 15 mL of DMF and DBU (1.53 g, 10 mmol) was then added in an ice water bath to react for 1 h, marking as reaction solution 1; In another 50 mL single-neck flask, M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol) and 10 mL of DMF were mixed and dissolved completely. DIPEA (1.65 mL, 10 mmol) was then added in an ice water bath to further react for 30 min, reaction solution 1 was added, and the reaction was allowed at room temperature for 2 h and monitored by HPLC. After the reaction was found to be completed, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated to obtain 5.0 g of a solid product with a yield of 77%; LC-MS: [M+H]+=738.3.
In a 25 mL single-neck flask, 17b (3.0 g, 4.07 mmol) was completely dissolved in 15 mL of DMF, and 3.0 g of 5% Pd/C was then added to have a hydrogenation reaction for 2 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was put into a flask in an ice-water bath, followed by addition of DIPEA (744 uL, 4.5 mmol), and then M3 (2.34 g, 4.07 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 1.2 g of the product, with a yield of 33%; LC-MS: [M−H]−=892.4.
In a 50 mL single-neck flask, 17d (500 mg, 0.56 mmol), M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol) and 15 mL of DMF were mixed and then DIPEA (330 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 17e-1 and Compound 17e-2. The preparation solutions were lyophilized separately to obtain 156 mg of Compound 17e-1, LC-MS: [M+H]+=1311.4; 150 mg of Compound 17e-2, LC-MS: [M+H]+=1311.7.
In a 25 mL single-neck flask, 17e-1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 43 mg of a solid; LC-MS: [M+H]+=1155.4.
In a 25 mL single-neck flask, 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 40 mg of a solid; LC-MS: [M+H]+=1155.4.
Compound 18A (54 mg) was obtained by referring to the synthesis route of example 28; LC-MS: [M+H]+=1155.4.
Compound 18B (55 mg) was obtained by referring to the synthesis route of example 28; LC-MS: [M+H]+=1155.4.
In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxyacetic acid benzyl ester (prepared according to the synthesis route disclosed in patent application WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were mixed and heated to 100° C. to react for 4 h. After the reaction was found to be completed, the reaction solution was cooled to room temperature, then filtered to remove the insoluble matter, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=10:1-5:1-2:1) to obtain 4.97 g of the target, with a yield of 36%; LC-MS: [M+H]+=515.2.
In a 50 mL single-neck flask, Compound 19a (4 g, 7.8 mmol) was completely dissolved in 10 mL of DMF and DBU (1.42 g, 9.3 mmol) was then added in an ice water bath to react for 1 h, marking as reaction solution 1; In another 50 mL single-neck flask, M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were mixed and dissolved completely. DIPEA (1.65 mL, 10 mmol) was then added in an ice water bath to further react for 30 min, reaction solution 1 was added, and the reaction was allowed at room temperature for 2 h and monitored by HPLC. After the reaction was found to be completed, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated to obtain 4.2 g of a solid product with a yield of 78%; LC-MS: [M+H]+=688.3.
In a 25 mL single-neck flask, 19b (1000 mg, 1.45 mmol) was completely dissolved in 15 mL of DMF and 1000 mg of 5% Pd/C was then added to have a hydrogenation reaction for 2 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was put into a flask in an ice-water bath, followed by addition of DIPEA (248 uL, 1.5 mmol), and then M3 (720 mg, 1.45 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 503 mg of the product, with a yield of 41%; LC-MS: [M−H]−=842.3.
In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were mixed and then DIPEA (292 uL, 1.77 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 19e-1 and Compound 19e-2. The preparation solutions were lyophilized separately to obtain 112 mg of Compound 19e-1, LC-MS: [M+H]+=1261.5; 131 mg of Compound 19e-2, LC-MS: [M+H]+=1261.5.
In a 25 mL single-neck flask, 19e-1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 55 mg of a solid; LC-MS: [M+H]+=1105.4.
In a 25 mL single-neck flask, 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 58 mg of a solid; LC-MS: [M+H]+=1105.4.
In a 250 mL single-neck flask, M1 (10 g, 27.1 mmol), 2-hydroxy-cyclopropylpropionic acid benzyl ester (prepared according to the synthesis route disclosed by the patent application WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were mixed and heated to 100° C. to react for 4 h. After the reaction was found to be completed, the reaction solution was cooled to room temperature, then filtered to remove the insoluble matter, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=10:1-5:1-2:1) to obtain 5.09 g of the target; LC-MS: [M+H]+=529.2.
In a 50 mL single-neck flask, Compound 20a (4 g, 7.6 mmol) was completely dissolved in 10 mL of DMF and DBU (1.39 g, 9.1 mmol) was then added in an ice water bath to react for 1 h, marking as reaction solution 1; In another 50 mL single-neck flask, M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were mixed and dissolved completely. DIPEA (1.65 mL, 10 mmol) was then added in an ice water bath to further react for 30 min, reaction solution 1 was added, and the reaction was allowed at room temperature for 2 h and monitored by HPLC. After the reaction was found to be completed, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated to obtain 4.5 g of a solid product with a yield of 84%; LC-MS: [M+H]+=702.3.
In a 25 mL single-neck flask, 20b (1000 mg, 1.42 mmol) was completely dissolved in 15 mL of DMF and 1000 mg of 5% Pd/C was then added to have a hydrogenation reaction for 2 h. After the reaction was found to be completed, the reaction solution was filtered and the filtrate was put into a flask in an ice-water bath, followed by addition of DIPEA (248 uL, 1.5 mmol), and then M5 (708 mg, 1.42 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 443 mg of the product, with a yield of 36%; LC-MS: [M−H]−=856.4.
In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), ixotecan mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were mixed and then DIPEA (248 uL, 1.5 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 20e-1 and Compound 20e-2. The preparation solutions were lyophilized separately to obtain 103 mg of Compound 20e-1, LC-MS: [M+H]+=1275.5; 103 mg of Compound 20e-2, LC-MS: [M+H]+=1275.5.
In a 25 mL single-neck flask, 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 51 mg of a solid; LC-MS: [M+H]+=1119.4.
In a 25 mL single-neck flask, 20e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 47 mg of a solid; LC-MS: [M+H]+=1119.4.
In a 2000 mL single-neck flask, 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol) and 1000 mL of toluene were mixed and heated to 100° C. to react for 5 h. After the reaction was found to be completed, the reaction solution was cooled to room temperature, then filtered to remove the insoluble matter, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=100:1-50:1-20:1) to obtain 75.6 g of the target; LC-MS: [M+H]+=299.1.
Second Step: Compound (R)-2-hydroxy-1, 5-glutaric acid tert-butyl ester
In a 2000 mL single-neck flask, 172793-31-6 (100 g, 338 mmol) and 1000 mL water were added, followed by addition of sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol), and the resulting solution was brought to room temperature to react for 24 h. After the reaction was found to be completed, the reaction solution was extracted three times with 500 mL of ethyl acetate. The organic phase was dried with anhydrous Na2SO4 and filtered, concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=50:1-30:1-2:1) to obtain 91.2 g of the target; LC-MS: [M+H]+=261.4.
In a 2000 mL single-neck flask, (R)-2-hydroxy-1, 5-glutaric acid tert-butyl ester (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran were mixed and cooled in an ice water bath to 0° C., then followed by addition of PPh3 (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol) and SM3-1 (57.3, 192 mmol), and the resulting solution was brought to room temperature to react for 13 h. After the reaction was found to be completed, the reaction solution was filtered to remove the insoluble matter. The filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA=50:1-30:1-1:1) to obtain 68.6 g of the product;
The above product was dissolved in 500 mL of methanol, then cooled in an ice water bath to 0° C., NaOH (64 mL, 190 mmol, 3 M/L) was added dropwise at this temperature to react for 12 h, HCl (6M/L) was added to adjust pH to 3. The reaction solution was extracted five times with 500 mL of dichloromethane, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (DCM/MeOH=50/1-20/1-2/1) to obtain 50.4 g of SM3; LC-MS: [M−H]−=525.5.
In a 2000 mL single-neck flask, SM3 (50 g, 95 mmol, 1.0 eq), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 eq), DCC (21.5 g, 104.5 mmol, 1.1 eq) and THE (600 mL) were mixed and the reaction was allowed at room temperature for 1 h and monitored by TLC. The insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound M6 (51.9 g) with a yield of 79%; LC-MS: [M+H]+=693.3.
In a 25 mL single-neck flask, 1c (1 g, 2.36 mmol) was completely dissolved in 25 mL of DMF, followed by addition of DIPEA (430 uL, 2.6 mmol) and M6 (1177 mg, 2.36 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 555 mg of the product; LC-MS: [M−H]−=931.0.
In a 100 mL single-neck flask, 21a (500 mg, 0.54 mmol), ixotecan mesylate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol) and 10 mL of DMF were mixed and then DIPEA (248 uL, 1.5 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 21b. The preparation solution was lyophilized to obtain 231 mg of the compound; LC-MS: [M+H]+=1349.5.
In a 25 mL single-neck flask, Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 103 mg of a solid; LC-MS: [M+H]+=1137.5.
Taking Compounds M6 and 3c as starting raw materials, Compound 22 (91 mg) was obtained by referring to the synthesis route of example 32; LC-MS: [M+H]+=1165.5.
Taking Compounds M6 and 5c as starting raw materials, 102 mg of Compound 23 was obtained by referring to the synthesis route of example 32, LC-MS: [M+H]+=1151.4; 99 mg of Compound 24 was obtained, LC-MS: [M+H]+=1151.4.
Taking Compounds M6 and 7c as starting raw materials, 83 mg of Compound 25 was obtained by referring to the synthesis route of example 32, LC-MS: [M+H]+=1205.7; 80 mg of Compound 26 was obtained, LC-MS: [M+H]+=1205.7.
Taking Compounds M6 and 19c as starting raw materials, 100 mg of Compound 27 was obtained by referring to the synthesis route of example 32, LC-MS: [M+H]+=1177.5; 101 mg of Compound 28 was obtained, LC-MS: [M+H]+=1177.5.
In a 5000 mL single-neck flask, maleic acid (50 g, 431 mmol, 1.0 eq), 114559-25-0 (110 g, 431 mmol, 1 eq), TEA (263 g, 2.16 mol, 5 eq) and toluene (2000 mL) were mixed and then heated to reflux for 5 h and monitored by TLC. The insoluble matter was filtered out. Rotatory evaporation under reduced pressure was directly performed on the reaction solution to remove the solvent, and the residue was subjected to silica gel column chromatography (PE/EA=50/1-20/1-1/1) to obtain SM4-1 (64.7 g), with a yield of 50%; LC-MS: [M+H]+=299.2.
In a 2000 mL single-neck flask, SM4-1 (64 g, 215 mmol) was completely dissolved in 1000 mL of DMF, then DIPEA (71 mL, 430 mmol) was added, followed by addition of nonane ethylene glycol monomethyl ether mesylate (111.5 g, 220 mmol), and the resulting solution was subjected to room temperature to react for 2 h. After the reaction was found to be completed from the monitoring of HPLC, the reaction solution was purified by silica gel column chromatography (PE/EA=50/1-20/1-1/1), to obtain 59.9 g of the product; LC-MS: [M+H]+=709.4.
In a 2000 mL single-neck flask, SM4-2 (59 g, 83 mmol) was completely dissolved in 1000 mL of MeOH, K2CO3 (11.75 g, 85 mmol) was added and the reaction was allowed at room temperature for 4 h and monitored by HPLC. After the reaction was found to be completed, the insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound SM4 (27 g); LC-MS: [M−H]−=693.5.
In a 500 mL single-neck flask, SM4 (25 g, 36 mmol, 1.0 eq), pentafluorophenol (7.3 g, 40 mmol, 1.1 eq), DCC (8.2 g, 40 mmol, 1.1 eq) and THE (200 mL) were mixed and the reaction was allowed at room temperature for 1 h and monitored by TLC. The insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound M7 (23.3 g) with a yield of 93%; LC-MS: [M+H]+=695.8.
In a 25 mL single-neck flask, 1c (1 g, 2.36 mmol) was completely dissolved in 25 mL of DMF, followed by addition of DIPEA (430 uL, 2.6 mmol) and then M7 (1640 mg, 2.36 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 609 mg of the product. LC-MS: [M−H]−=1098.5.
In a 100 mL single-neck flask, 29a (500 mg, 0.45 mmol), ixotecan mesylate M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol) and 10 mL of DMF were mixed and then DIPEA (248 uL, 1.5 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 29b. The preparation solution was lyophilized to obtain 187 mg of the compound; LC-MS: [M+H]+=1517.6.
In a 25 mL single-neck flask, Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 114 mg of a solid; LC-MS: [M+H]+=1517.9.
Taking Compounds M7 and 3c as starting raw materials, Compound 30 (125 mg) was obtained by referring to the synthesis route of example 37; LC-MS: [M+H]+=1445.6.
Taking Compounds M7 and 5c as starting raw materials, 61 mg of Compound 31 was obtained by referring to the synthesis route of example 37, LC-MS: [M+H]+=1431.7; 63 mg of Compound 32 was obtained, LC-MS: [M+H]+=1431.7.
Taking Compounds M7 and 7c as starting raw materials, 60 mg of Compound 33 was obtained by referring to the synthesis route of example 37, LC-MS: [M+H]+=1485.6; 58 mg of Compound 34 was obtained, LC-MS: [M+H]+=1485.6.
Taking Compounds M7 and 19c as starting raw materials, 102 mg of Compound 35 was obtained by referring to the synthesis route of example 37, LC-MS: [M+H]+=1457.8; 102 mg of Compound 36 was obtained, LC-MS: [M+H]+=1457.8.
In a 2000 mL single-neck flask, Compound 16947-84-5 (100 g, 295 mmol, 1.0 eq), DIPEA (50 mL, 300 mmol), Benzyl bromide (51.3 g, 300 mmol) and THE (1000 mL) were mixed, and the reaction was allowed at room temperature for 12 h and monitored by TLC. The insoluble matter was filled out. Rotatory evaporation under reduced pressure was directly performed on the reaction solution to remove the solvent, and the residue was subjected to silica gel column chromatography (PE/EA=50/1-20/1-2/1) to obtain SM5-1 (110.1 g), with a yield of 87%; LC-MS: [M+H]+=429.2.
In a 2000 mL single-neck flask, Compound SM5-1 (100 g, 233.4 mmol, 1.0 eq) and THE (1000 mL) were mixed and cooled in an ice water bath to 0° C., NaH (37.4 g, 933.5 mmol) and Mel (132.5 g, 933.5 mmol) were then added step by step, and the reaction was allowed at 0° C. for 24 h and monitored by TLC. 500 mL of saturated NH4C1 in water was added to quench the reaction. The reaction solution was extracted three times with 500 mL of ethyl acetate. The organic phase was dried with anhydrous Na2SO4 and filtered. Rotatory evaporation under reduced pressure was directly performed on the filtrate to remove the solvent and the residue was subjected to silica gel column chromatography (PE/EA=100/1-50/1-10/1) to obtain SM5-2 (37.1 g); LC-MS: [M+H]+=443.3.
Third Step: Compound SM5 (see Org. Lett., 2006, 8, 3387-3390.)
In a 1000 mL single-neck flask, Compound SM5-2 (35 g, 79 mmol, 1.0 eq) and DCE (500 mL) were mixed and then added in sequence with palladium diacetate, (180 mg, 0.8 mmol), 12 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol), and the reaction was allowed at 60° C. for 40 h and monitored by TLC. 500 mL of saturated aqueous sodium thiosulfate solution in water was added to quench the reaction. The reaction solution was extracted three times with 500 mL of dichloromethane. The organic phase was dried with anhydrous Na2SO4 and filtered. Rotatory evaporation under reduced pressure was directly performed on the filtrate to remove the solvent and the residue was subjected to silica gel column chromatography (PE/EA=100/1-50/1-10/1) to obtain SM5 (28 g); LC-MS: [M+H]+=501.3.
In a 500 mL single-neck flask, Compound SM5 (25 g, 50 mmol, 1.0 eq), di-tert-butyl phosphate potassium salt (13.66 g, 55 mmol, 1.1 eq), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq) and THE (200 mL) were mixed and the reaction was allowed at room temperature for 1 h and monitored by TLC. The insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound SM6 (15.1 g), with a yield of 46%; LC-MS: [M+H]+=651.4.
In a 250 mL single-neck flask, SM6 (15 g, 23 mmol) was completely dissolved in 100 mL of DMF, and then added with 15 g of 5% Pd/C in an ice water bath. The atmosphere in the system was replaced with hydrogen three times. The reaction was allowed at room temperature for 12 h. Pd/C was removed, and reduced-pressure (oil pump) rotary evaporation was performed to remove the solvent. The resulting crude product was ready for later use.
In another 250 mL single-neck flask, the crude product and 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol) were mixed and dissolved completely. The reaction was allowed at 100° C. for 2 h and monitored by HPLC. After the reaction was found to be completed, the reaction solution was purified through liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane, then washed with saturated NaCl solution, dried with anhydrous Na2SO4 and filtered. The filtrate was concentrated to obtain 4.2 g of a solid product with a yield of 36%; LCMS: [M+H]+=507.3.
In a 100 mL single-neck flask, Compound SM7 (4 g, 7.9 mmol, 1.0 eq), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 eq), DCC (1.8 g, 8.7 mmol, 1.1 eq) and THF (60 mL) were mixed and the reaction was allowed at room temperature for 1 h and monitored by TLC. The insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound M8 (3.7 g) with a yield of 70%; LC-MS: [M+H]+=673.2.
In a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol) was completely dissolved in 25 mL of DMF, followed by addition of DIPEA (430 uL, 2.6 mmol), and then Compound M8 (1.2 g, 2.36 mmol). The reaction was allowed at room temperature for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 488 mg of the product; LC-MS: [M−H]−=911.0.
In a 100 mL single-neck flask, Compound 37a (400 mg, 0.44 mmol), ixotecan mesylate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol) and 10 mL of DMF were mixed and then DIPEA (218 uL, 1.32 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 37b. The preparation solution was lyophilized to obtain 201 mg of the Compound; LC-MS: [M+H]+=1329.6.
In a 25 mL single-neck flask, Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 96 mg of a solid; LC-MS: [M+H]+=1117.4.
Taking Compounds M8 and 3c as starting raw materials, Compound 38 (51 mg) was obtained by referring to the synthesis route of example 42; LC-MS: [M+H]+=1145.6.
Taking Compounds M8 and 5c as starting raw materials, 57 mg of Compound 39 was obtained by referring to the synthesis route of example 42, LC-MS: [M+H]+=1131.4; 60 mg of Compound 40 was obtained, LC-MS: [M+H]+=1131.4.
Taking Compounds M7 and 7c as starting raw materials, 44 mg of Compound 41 was obtained by referring to the synthesis route of example 42, LC-MS: [M+H]+=1185.3; 44 mg of Compound 42 was obtained, LC-MS: [M+H]+=1185.3.
Taking Compounds M8 and 19c as starting raw materials, 62 mg of Compound 43 was obtained by referring to the synthesis route of example 42, LC-MS: [M+H]+=1157.4; 59 mg of Compound 44 was obtained, LC-MS: [M+H]+=1157.4.
Compound 45 was obtained by referring to the synthesis route of Example 58 in patent application CN104755494A.
In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), M9 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 46a (210 mg); LC-MS: [M+H]+=1221.6.
In a 25 mL single-neck flask, 46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 46 (120 mg); LC-MS: [M+H]+=1065.3.
Compound 47 (81 mg) was obtained by referring to the synthesis route of example 48; LC-MS: [M+H]+=1065.3.
In a 100 mL single-neck flask, 5d (1.66 g, 2.02 mmol, 1.0 eq), M9 (1.08 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq) and DMF (40 mL) were mixed and then DIPEA (0.84 mL, 1.5 eq) was added in an ice water bath, and the reaction was allowed at room temperature for 2 h and monitored by HPLC. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound 48a (1.54 g) with a yield of 61%; LC-MS: [M+H]+=1235.4.
In a 100 mL single-neck flask, Compound 48a (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were mixed and dissolved, followed by addition of zinc bromide (3.64 g, 16 mmol, 20.0 eq). Reaction was allowed in an oil bath at 40° C. (the reaction system was preheated and stabilized in advance) for 30 min. The reaction solution was concentrated in a reduced-pressure (water pump) water bath at 45° C. to remove nitromethane to obtain a yellow solid residue (under the monitoring of HPLC). The preparation solution was obtained by acid preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound 48A (786 mg) with a yield of 90%.
In a 25 mL single-neck flask, Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), M9 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were mixed and then cooled to 0-5° C. in an ice water bath, followed by addition of DIPEA (62 mg, 0.48 mmol, 2.0 eq). The reaction was allowed at 20±5° C. for 2 h. After the reaction was found to be completed from the monitoring of the HPLC. The reaction solution was directly purified by HPLC preparation. The product preparation solution was collected and then lyophilized to obtain Compound 48b (150.2 mg); LC-MS: [M+H]+=1235.4.
In a 25 mL single-neck flask, Compound 48b (100 mg, 0.081 mmol, 1.0 eq), ZnBr2 (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in sequence. The reaction was allowed at 40° C. for 0.5 h and then terminated. Reduced-pressure rotary evaporation was performed at 45° C. to obtain a yellow solid, then sampled, and monitored by HPLC. The solid subjected to rotary evaporation was directly purified by HPLC preparation. The product preparation solution was collected and then lyophilized to obtain Compound 48B (70.0 mg); LC-MS: [M+H]+=1079.4.
Compound 49A (71 mg) was obtained by referring to the route of example 50; LC-MS: [M+H]+=1079.4.
Compound 49B (65 mg) was obtained by referring to the synthesis route of example 51; LC-MS: [M+H]+=1079.4.
In a 50 mL single-neck flask, 7d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 50a and Compound 50b. The preparation solutions were lyophilized separately to obtain 170 mg of Compound 50a, LC-MS: [M+H]+=1289.46; 202 mg of Compound 50b, LC-MS: [M+H]+=1289.46.
In a 25 mL single-neck flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 44 mg of a solid.
In a 25 mL single-neck flask, 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 45 mg of a solid.
In a 50 mL single-neck flask, 8d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 51a and Compound 51b. The preparation solutions were separately lyophilized to obtain 190 mg of Compound 51a and 186 mg of Compound 51b. LC-MS of Compound 51a: [M+H]+=1289.47; LC-MS of Compound 51b: [M+H]+=1289.47.
In a 25 mL single-neck flask, Compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 39 mg of a solid; Third step: Compound 51B
In a 25 mL single-neck flask, Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 60 mg of a solid.
In a 50 mL single-neck flask, 11d (800 mg, 0.96 mmol), M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were mixed and then DIPEA (660 uL, 4.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 4 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 52a. The preparation solution was lyophilized to obtain 52a (388 mg); LC-MS: [M+H]+=1261.4.
In a 25 mL single-neck flask, Compound 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 52A (79 mg); LC-MS: [M+H]+=1105.4.
Compound 52B (50 mg) was obtained by referring to the synthesis route of example 56. LC-MS: [M+H]+ 1105.4.
In a 50 mL single-neck flask, 12d (400 mg, 0.47 mmol), M9 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol) and 15 mL of DMF were mixed and then DIPEA (330 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 53a. The preparation solution was lyophilized to obtain 53a (200 mg); LC-MS: [M+H]+=1275.4.
In a 25 mL single-neck flask, 53a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 53A (51 mg); LC-MS: [M+H]+=1119.4.
Compound 53B (50 mg) was obtained by referring to the synthesis route of example 58; LC-MS: [M+H]+=1119.4.
In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were mixed and then DIPEA (292 uL, 1.77 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compounds 54a and 54b. The preparation solutions were separately lyophilized to obtain 103 mg of Compound 54a; LC-MS: [M+H]+=1261.5; 111 mg of Compound 54b, LC-MS: [M+H]+=1261.5.
In a 25 mL single-neck flask, 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 61 mg of a solid; LC-MS: [M+H]+=1105.4.
In a 25 mL single-neck flask, 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 57 mg of a solid; LC-MS: [M+H]+=1105.4.
In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), M9 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were mixed and then DIPEA (248 uL, 1.5 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compounds 55a and 55b. The preparation solutions were separately lyophilized to obtain 100 mg of Compound 55a, LC-MS: [M+H]+=1275.5; 101 mg Compound 55b, LC-MS: [M+H]+=1275.5.
In a 25 mL single-neck flask, 55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 42 mg of a solid; LC-MS: [M+H]+=1119.4.
In a 25 mL single-neck flask, 55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 45 mg of a solid; LC-MS: [M+H]+=1119.4.
Compound 56 (50 mg) was obtained by referring to the synthesis route of example 60; LC-MS: [M+H]+=1119.3.
Compound 57 (50 mg) was obtained by referring to the synthesis route of example 61; LC-MS: [M+H]+=1119.4.
400 mL of DMF was added to ixotecan mesylate M5 (15 g, 28 mol, obtained by referring to the synthesis route disclosed by patent application EP0737683A1). The resulting solution was then cooled to 0° C. in an ice water bath, triethylamine was added dropwise to adjust the pH to 7-8, benzyl bromide (9.6 g, 56 mmol) was then added dropwise in an ice water bath, and the reaction was allowed at room temperature (25° C.) for 1 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction solution was concentrated under reduced pressure. The crude product obtained was purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system) and the target peak was collected. Acetonitrile was removed under reduced pressure and then lyophilization was performed to about 11 g of yellow solid Compound 58a, with a yield of 74%, MS m/z: [M+H]+ 526.3.
In a 250 mL single-neck flask, Compound 58a (11 g, 21 mol) and 120 mL of formic acid were added in sequence and dissolved at room temperature. 30 mL of formaldehyde (40% in water) was added to the resulting bright yellow solution. Reaction was allowed at 50° C. for 1 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction solution was cooled to room temperature and then purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system) and the target peak was collected. Acetonitrile was removed under reduced pressure and then lyophilization was performed to about 4.5 g of Compound 58b as yellow solid powder, with a yield of 40%, MS m/z: [M+H]+ 540.6.
In a 250 mL single-neck flask, Compound 58b (2.3 g, 4.3 mol) and 100 mL of DMF were added and dissolved at room temperature. 2.3 g of 5% Pd/C was added to the resulting bright yellow solution and the atmosphere in the system was replaced by a hydrogen balloon. Reaction was allowed at room temperature for 1.5 h. After the reaction was found to be completed from the monitoring of the HPLC, Pd/C was filtered out, the resulting reaction solution was concentrated and then purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system) and the target peak was collected. Acetonitrile was removed under reduced pressure and then lyophilization was performed to about 1.0 g of Compound 58 as yellow solid powder, with a yield of 52%, MS m/z: [M+H]+ 450.5.
In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 59a (166 mg); LC-MS: [M+H]+=1235.6.
In a 25 mL single-neck flask, 59a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 59 (43 mg); LC-MS: [M+H]+=1079.3.
Compound 60 (40 mg) was obtained by referring to the synthesis route of example 65; LC-MS: [M+H]+=1079.3.
In a 100 mL single-neck flask, 5d (1.66 g, 2.02 mmol, 1.0 eq), 58 (0.91 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq) and DMF (40 mL) were mixed and then DIPEA (0.84 mL, 1.5 eq) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h (monitored by HPLC).
The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound 61a (1.21 g); LC-MS: [M+H]+=1249.4.
In a 100 mL single-neck flask, Compound 61a (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were mixed and dissolved, followed by addition of zinc bromide (3.64 g, 16 mmol, 20.0 eq). Reaction was allowed in an oil bath at 40° C. (the reaction system was preheated and stabilized in advance) for 30 min. The reaction solution was concentrated in a reduced-pressure (water pump) water bath at 45° C. to remove nitromethane to obtain a yellow solid residue (under the monitoring of HPLC). The preparation solution was obtained by acid preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound 61 (786 mg), LC-MS: [M+H]+=1093.6.
In a 25 mL single-neck flask, Compound 5d-1 (200 mg, 0.24 mmol, 1.0 eq), 58 (110.3 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were mixed and then cooled to 0-5° C. in an ice water bath, followed by addition of DIPEA (62 mg, 0.48 mmol, 2.0 eq). The reaction was allowed at 20±5° C. for 2 h. After the reaction was found to be completed from the monitoring of the HPLC. The reaction solution was directly purified by HPLC preparation. The product preparation solution was collected and then lyophilized to obtain Compound 62a (120.9 mg); LC-MS: [M+H]+=1249.4.
In a 25 mL single-neck flask, Compound 62a (100 mg, 0.081 mmol, 1.0 eq), ZnBr2 (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in sequence. The reaction was allowed at 40° C. for 0.5 h and then terminated. Reduced-pressure rotary evaporation was performed at 45° C. to obtain a yellow solid, then sampled, and monitored by HPLC. The solid subjected to rotary evaporation was directly purified by HPLC preparation. The product preparation solution was collected and then lyophilized to obtain Compound 62 (61 mg); LC-MS: [M+H]+=1093.4.
Compound 63 (60 mg) was obtained by referring to the route of example 67; LC-MS: [M+H]+=1093.4.
Compound 64 (65 mg) was obtained by referring to the synthesis route of example 68; LC-MS: [M+H]+=1093.4.
In a 50 mL single-neck flask, 7d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 65a and Compound 65b. The preparation solutions were lyophilized separately to obtain 155 mg of Compound 65a, LC-MS: [M+H]+=1303.4; 158 mg of Compound 65b, LC-MS: [M+H]+=1303.6.
In a 25 mL single-neck flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the preparation solution. The preparation solution was lyophilized to obtain 49 mg of a solid.
In a 25 mL single-neck flask, 65b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the preparation solution. The preparation solution was lyophilized to obtain 47 mg of a solid.
In a 50 mL single-neck flask, 8d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were mixed and then DIPEA (378 uL, 2.29 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 66a and Compound 66b. The preparation solutions were lyophilized separately to obtain 160 mg of Compound 66a and 160 mg Compound 66b. LC-MS of Compound 66a: [M+H]+=1303.7; LC-MS of Compound 66b: [M+H]+=1303.6.
In a 25 mL single-neck flask, Compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 57 mg of a solid, LC-MS: [M+H]+=1147.5.
In a 25 mL single-neck flask, Compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 57 mg of a solid, LC-MS: [M+H]+=1147.5.
In a 50 mL single-neck flask, 11d (800 mg, 0.96 mmol), 58 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were mixed and then DIPEA (660 uL, 4.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 4 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 67a. The preparation solution was lyophilized to obtain 67a (402 mg); LC-MS: [M+H]+=1275.4.
In a 25 mL single-neck flask, 67a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 67A (47 mg); LC-MS: [M+H]+=1119.5.
Compound 67B (50 mg) was obtained by referring to the synthesis route of example 73. LC-MS: [M+H]+ 1119.4.
In a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol) and 15 mL of DMF were mixed and then DIPEA (330 uL, 2.0 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 3 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 68a. The preparation solution was lyophilized to obtain 68a (177 mg); LC-MS: [M+H]+=1289.4.
In a 25 mL single-neck flask, 68a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 68A (45 mg); LC-MS: [M+H]+=1133.4.
Compound 68B (50 mg) was obtained by referring to the synthesis route of example 75; LC-MS: [M+H]+=1133.4.
In a 50 mL single-neck flask, 19d (500 mg, 0.59 mmol), 58 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were mixed and then DIPEA (292 uL, 1.77 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 69a and Compound 69b. The preparation solutions were lyophilized separately to obtain 109 mg of Compound 69a, LC-MS: [M+H]+=1275.5; 111 mg of Compound 69b, LC-MS: [M+H]+=1275.7.
In a 25 mL single-neck flask, 69a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 53 mg of a solid; LC-MS: [M+H]+=1119.4.
In a 25 mL single-neck flask, 69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 54 mg of a solid; LC-MS: [M+H]+=1119.4.
In a 50 mL single-neck flask, 20d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were mixed and then DIPEA (248 uL, 1.5 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of Compound 70a and 70b, and the preparation solutions were lyophilized to obtain 106 mg of Compound 70a, LC-MS: [M+H]+=1289.5, and 101 mg of Compound 70b, LC-MS: [M+H]+=1289.4.
In a 25 mL single-neck flask, 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 39 mg of a solid; LC-MS: [M+H]+=1133.4.
In a 25 mL single-neck flask, 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain 35 mg of a solid; LC-MS: [M+H]+=1133.4.
Compound 71 (30 mg) was obtained by referring to the synthesis route of example 78; LC-MS: [M+H]+=1133.3.
Compound 72 (33 mg) was obtained by referring to the synthesis route of example 78; LC-MS: [M+H]+=1133.4.
In a 100 mL single-neck flask, Compound M3 (11.0 g, 19.5 mmol, 1.0 eq), DIPEA (2.8 g, 21.4 mmol, 1.1 eq), 27-amino-4, 7, 10, 13, 16, 19, 22, 25-octaoxaheptacosanoic acid (9.7 g, 20.5 mmol, 1.05 eq) and DMF (60 mL) were mixed and the reaction was allowed at room temperature for 20 min and monitored by TLC. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound M10 (13.2 g) with a yield of 78%; LC-MS: [M+H]+=866.5.
In a 100 mL single-neck flask, M10 (13.0 g, 15 mmol, 1.0 eq), pentafluorophenol (3 g, 16.5 mmol, 1.1 eq), DCC (3.4 g, 16.5 mmol, 1.1 eq) and THF (30 mL) were mixed and the reaction was allowed at room temperature for 30 min and monitored by TLC. The insoluble matter was filtered out. The reaction solution was directly purified by preparation. The preparation solution was concentrated in a reduced-pressure (water pump) water bath at 35° C. to remove acetonitrile, and then lyophilized to obtain Compound M11 (14.2 g) with a yield of 92%; LC-MS: [M+H]+=1032.5.
10 mL of DMF was added to M11 (1 g, 0.79 mol). The resulting solution was then cooled to 0° C. in an ice water bath, followed by addition of Compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol). Reaction was allowed under the same condition for 1 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system) and the target peak was collected. Acetonitrile was removed under reduced pressure and then lyophilization was performed to about 1.2 g of Compound 73a, MS m/z: [M+H]+=1271.9.
In a 25 mL single-neck flask, 73a (1.2 g, 0.94 mmol), M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), and 15 mL of DMF were mixed and then DIPEA (310 mg, 2.4 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 73b (709 mg); LC-MS: [M+H]+=1720.8.
In a 25 mL single-neck flask, 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 73 (88 mg); LC-MS: [M+H]+=1532.6.
Compound 74 (90 mg) was obtained by referring to the synthesis route of example 82; LC-MS: [M+H]+=1532.6.
10 mL of DMF was added to M11 (1 g, 0.79 mol). The resulting solution was then cooled to 0° C. in an ice water bath, followed by addition of Compound 5c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol). Reaction was allowed under the same condition for 1 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system) and the target peak was collected. Acetonitrile was removed under reduced pressure and then lyophilization was performed to obtain 0.9 g of Compound 75a, MS m/z: [M+H]+=1285.6.
In a 25 mL single-neck flask, 75a (700 mg, 0.54 mmol), M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and 10 mL of DMF were mixed and then DIPEA (155 mg, 1.2 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution of Compound 75b (304 mg); LC-MS: [M+H]+=1734.8.
In a 25 mL single-neck flask, 75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 75 (96 mg); LC-MS: [M+H]+=1546.6.
Compound 76 (92 mg) was obtained by referring to the synthesis route of example 84; LC-MS: [M+H]+=1546.5.
Compound 77 (87 mg) was obtained by referring to the synthesis route of example 84; LC-MS: [M+H]+=1546.5.
Compound 78 (94 mg) was obtained by referring to the synthesis route of example 84; LC-MS: [M+H]+=1546.7.
10 mL of DMF was added to M11 (1 g, 0.79 mol). The resulting solution was then cooled to 0° C. in an ice water bath, followed by addition of Compound 20c (377 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol). Reaction was allowed under the same condition for 1 h. After the reaction was found to be completed from the monitoring of the TLC, the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system) and the target peak was collected. Acetonitrile was removed under reduced pressure and then lyophilization was performed to obtain 783 mg of Compound 79a, MS m/z: [M+H]+=1325.8.
In a 25 mL single-neck flask, 79a (600 mg, 0.45 mmol), M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol) and 10 mL of DMF were mixed and then DIPEA (130 mg, 1 mmol) was added in condition of an ice-water bath. The reaction was allowed at room temperature for 2 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain preparation solutions of 79b-1 and 79b-2. The preparation solutions were lyophilized separately to obtain 79b-1 (124 mg); LC-MS: [M+H]+=1743.0; 79b-1 (122 mg); LC-MS: [M+H]+=1743.0.
In a 25 mL single-neck flask, 79b-1 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 79 (30 mg); LC-MS: [M+H]+=1586.9.
In a 25 mL single-neck flask, 79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were mixed, and reaction was allowed at 40° C. for 1 h. After the reaction was found to be completed from the monitoring of the HPLC, the reaction solution was concentrated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain the product preparation solution. The preparation solution was lyophilized to obtain a solid Compound 80 (33 mg); LC-MS: [M+H]+=1587.0.
Compound 81 (24 mg) was obtained by referring to the synthesis route of example 88; LC-MS: [M+H]+=1586.9.
Compound 82 (29 mg) was obtained by referring to the synthesis route of example 88; LC-MS: [M+H]+=1586.9.
1) Expression and Purification of Antibody hu4D3:
Cells were suspended with Expi293 (Shanghai OPM Biosciences Co., Ltd.) to express the antibody hu4D3. One day before transfection, the cells were inoculated into a 1 L shake flask containing 300 mL of OPM-293 CD05 Medium (81075-001, Shanghai OPM Biosciences Co., Ltd.) at a density of 0.9×106 cells/mL and cultured overnight on a cell culture shaker at 37° C., 5% CO2, and 120 rpm. The next day, PEI-MAX was used to transfect the antibody expression plasmid, in which the mass ratio of plasmid:PEI-MAX was 1:3. OPM-293 ProFeed was added at 5% (v/v) on day 1 after transfection, OPM-293 ProFeed was added again at 5% (v/v) on day 3 after transfection, and the supernatant was collected by centrifugation on day 6 after transfection.
The collected cell expression supernatant was loaded on a Protein A affinity chromatography column (UniMab 50, Suzhou NanoMicro Technology Co., Ltd), and eluted with 0.05 M sodium acetate (pH3.6). The captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH8.8) at a ratio of 0.7/10 (v/v), and then by using a gel filtration chromatography column SEC (Superdex 200, GE Company), impurities such as polymers were removed, and at the same time, the antibody Buffer was replaced with 20 mM PB (pH6.5).
Antibody hu4D3:
STSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQYDDLFTFG
The nucleic acid coding sequence of the variable region of the light chain described above is SEQ ID NO: 34
In the variable region of the light chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 8), and CDR3 (SEQ ID NO: 9) are as follows:
GEALYYFDYWGQGTLVTVSS
The nucleic acid coding sequence of the variable region of the heavy chain described above is SEQ ID NO: 33
In the variable region of the heavy chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3) are as follows:
2) Expression and Purification of Antibody hu7F11: The Antibody hu7F11 was Expressed and Purified in a Similar Way.
Antibody hu7F11:
YASNRYTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYCQQDYSSPWTF
The nucleic acid coding sequence of the variable region of the light chain described above is SEQ ID NO: 36
In the variable region of the light chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12) are as follows:
EGYGYGKNGVGYAMDYWGQGTLVTVSS
The nucleic acid coding sequence of the variable region of the heavy chain described above is SEQ ID NO: 35
In the variable region of the heavy chain described above, the nucleotide coding sequences of CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6) are as follows:
3) Expression and purification of antibody hTINA1: The antibody hTINA1 was expressed and purified in a similar way. For details, please see item “hTINA1-H1L1” disclosed in Table 2, patent application CN105849126A, description, pages 67/91.
Antibody hTINA1:
1) Synthesis of an Antibody hu4D3-Drug Conjugate Sample by Coupling Antibody hu4D3 and Payload:
After being expressed in cells and purified by Protein A affinity chromatography and molecular sieve chromatography, the anti-human Trop2 antibody was replaced in 20 mM NaAc—HAc, pH 6.0 buffer and then concentrated or diluted until the anti-human Trop2 antibody reached a protein concentration of 5 mg/mL. The linker-drug was a white powder and dissolved in DMA to the concentration of 10 mg/mL for later use. In order to open the inter-chain disulfide bond of the anti-human Trop2 antibody, 15-fold TCEP was first added according to the molecular ratio and reaction was allowed at room temperature for 2 h; then, 16-fold linker-drug solution was added according to the molecular ratio and reaction was allowed at room temperature for 2 h. After the reaction was found to be completed, the reaction solution was ultrafiltrated using a 30 KDa ultrafiltration centrifuge tube to remove the linker-drug not coupled to the anti-human Trop2 antibody, and then the anti-human Trop2 antibody-drug conjugate sample was obtained.
The resulting human Trop2 antibody-drug conjugate sample will be tested for the percentage of monomer by SEC-HPLC and for the drug loading by RP-HPLC or HIC-HPLC.
2) Synthesis of an Antibody hu7F11-Drug Conjugate Sample by Coupling Antibody hu7F11 and Payload in a Similar Way.
After being expressed in cells and purified by Protein A affinity chromatography and molecular sieve chromatography, the anti-human Trop2 antibody was replaced in 20 mM NaAc—HAc, pH 6.0 buffer and then concentrated or diluted until the anti-human Trop2 antibody reached a protein concentration of 5 mg/mL. The linker-drug was a white powder and dissolved in DMA to the concentration of 10 mg/mL for later use. In order to open the inter-chain disulfide bond of the anti-human Trop2 antibody, 8-12-fold TCEP was first added according to the molecular ratio and reaction was allowed at room temperature for 1-2 h; then, 5-8-fold linker-drug solution was added according to the molecular ratio and reaction was allowed at room temperature for 1-2 h. After the reaction was found to be completed, the reaction solution was ultrafiltrated using a 30 KDa ultrafiltration centrifuge tube to remove the linker-drug not coupled to the anti-human Trop2 antibody, and then the anti-human Trop2 antibody-drug conjugate sample was obtained.
The resulting human Trop2 antibody-drug conjugate sample will be tested for the percentage of monomer by SEC-HPLC and for the drug loading by RP-HPLC or HIC-HPLC.
Conclusion: The ADCs disclosed herein have the characteristics of low degradation and low aggregation and have the excellent property of high percentage of monomer.
Conclusion: The ADCs disclosed herein have the excellent property of high DAR, and can significantly increase the drug concentration at the target location under the same dosage of ADC.
The mixture of an ADC and IgG-depleted plasma were formulated with the final concentration of ADC being 0.6 mg/mL. The plasma mixture was incubated in a water bath box in a 37° C. incubator. The incubation time was set to 0 days, 3 days, and 7 days. At the same time, the unincubated plasma control was set. The incubated sample was purified and extracted and then tested for the DAR to reflect the stability of the ADC in plasma.
Conclusion: The ADCs disclosed herein have good plasma stability, and no major change in DAR occurs during incubation in plasma. However, the control DS-1062a has a significant decrease in DAR during incubation in plasma, indicating poor plasma stability.
The ADCs Maintain the Original Affinity of the Corresponding Anti-Trop2 Antibodies hu4D3 and hu7F11 to TROP2:
Comparison of relative affinity to TROP2 between hu4D3 and ADC-6/ADC10/ADC12 and between TINA1 and ADC-1/DS1062a was performed using double-antigen sandwich ELISA.
Specific procedures are as follows.
The recombinant TROP2-His*6 antigen was coated on the plate and then blocked with 1% bovine serum albumin; hu4D3 and the corresponding ADC and TINA1 and the corresponding ADC were diluted respectively, and then serially diluted 3 folds at a starting concentration of 2000 ng/mL respectively, for a total of 11 concentrations. After the sample was incubated on the coated elisa plate for a period of time, the secondary antibody labeled by goat anti-human Fc-HRP was added for incubation. Finally, TMB color development was performed and terminated by sulfuric acid solution, and the absorption value at 450 nm was detected on an elisa plate. The results are plotted on concentration at OD450 nm.
Conclusion: As shown in
In the present invention, a human tumor cell line (BxPC-3) was used as an experimental model to evaluate the in vitro efficacy of ADCs. A certain number of tumor cells were inoculated into a 96-well plate, and gradient dilutions of the test antibody and the corresponding ADC were added to the cells, the cells were treated for 5 days, cell viability was detected by using MTS, and IC50 was calculated to evaluate the in vitro inhibitory effect of the test antibody and ADCs on the tumor cell line. The cells were treated for 5 days with the antibody drugs having initial concentration of 500 nM, diluted 7 folds, with a total of 8 concentration points. Finally, the cell survival was calculated as: survival rate=(experimental group-blank group)/(control group-blank group)×100%. Then, by using Graph Pad Prism, the curve was fitted and the half inhibitory concentration (IC50) was calculated.
Conclusion: As shown in Table 7 and
Various human tumor cell lines (BXPC3, A431+SW620) were inoculated subcutaneously into BALB/c nude mice as experimental models to evaluate the in vivo efficacy of ADCs. In BALB/c nude mice inoculated with a certain number of tumor cell lines, when the tumor volume grew to 150-300 mm3, the antibodies or the corresponding ADCs were injected into the tail vein once a week, four times in total, and continuously observed. The tumors were measured twice a week to evaluate the inhibitory effects of test antibodies and ADCs on tumor cell lines.
In the efficacy experiment on tumor-bearing mice inoculated with the cell line BXPC3 having medium-high expression of TROP2, the in vivo efficacy experimental results of ADC-6, ADC-10 and ADC-12 were superior to those of DS1062a.
In the A431+SW620 heterogeneous tumor, the in vivo efficacy experimental results of ADC-6 and ADC-12 were superior to those of DS1062a, and ADC-6 and ADC-12 have a good “bystander effect”.
The UV light of the biosafety cabinet was turned on 30 min in advance, and then the ventilation system was enabled for 3 min. The growth medium, the detection medium, D-PBS and trypsin were preheated in a 37° C. constant-temperature water bath, and then had surface disinfected with alcohol and then placed in the biosafety cabinet. The cells with a confluence rate of about 80% were placed in the biosafety cabinet, the old culture medium was removed, the cells were rinsed with D-PBS, and the D-PBS was then discarded. The cells were digested with trypsin for 2-3 min, then neutralized with the growth medium and centrifuged at 1200 rpm for 3 min, and the supernatant was removed. The cell solution was mixed with 4 mL of test medium, and 100 μL of cell solution was taken for counting (where 50 μL of cell solution was well mixed with 50 μL of Trypan Blue Stain and then counted). According to a pre-optimized cell plating density, the cells were plated in a 96-well plate at 80 ul/well. Only 80 μL of test medium was added to wells E11 and F11, and 150 μL of D-PBS was added to wells on the edges. After 24 hours of plating, diluted antibodies were added with 20 uL per well and control was set. Only 20 μL of test medium was added to cells in column 11. Two duplicate wells were set at each concentration. The cells were then well mixed on the cell vortex shaker at 550 rpm for 3 min.
Dilution of the solution: The test medium was used to formulate 300 μL of a test solution with a starting concentration of 5 uM in the first column of the V-shaped 96-well plate. 240 μL of test medium was added to the subsequent columns 2 to 10. 60 μL of the well mixed test solution was taken from the first column and added to the second column, and the cell solution was mixed up and down with a pipette for 10 times, and then the pipette tip was discarded. The same operation was performed for other seven concentrations one by one.
After 4 days, the MTS reagent was taken out, thawed at room temperature in the dark, and vortex mixed thoroughly. In the biosafety cabinet, 20 μL of CellTiter One Solution Reagen MTS reagent was added per 100 μL cell culture volume along the side wall of each well, and the plate surface was gently tapped to well mix the MTS solution. The cells were incubated for 2 h in the dark in an incubator. After the reaction, the 96-well plate was taken out for testing the OD490 nm absorption on an elisa plate, and the test data were recorded, organized, analyzed and stored.
From Table 8, it shows that Compound 5A (Linker-drug) has good inhibitory effects on the above solid tumor cells and hematological tumor cells.
In vitro efficacy experiments of naked antibodies and Compound d3, d38, d39 and d44:
In a 96-well plate, cells were plated at an appropriate cell density. After 24 h, the drugs were added. After 24 h, the drugs were diluted with the test medium (1 uM starting concentration, 5-fold dilution, 9 concentrations, test medium was added in the tenth column as blank control). The diluted drugs were added to the corresponding cell wells and then shaken for 3 min with a microplate shaker (model: MX100-4A) at a shaking speed of 550 rpm/min. After shaking, the cells were incubated in a carbon dioxide incubator for 3 days.
After 4 days, the MTS reagent was taken out, thawed at room temperature in the dark, and vortex mixed thoroughly. In the biosafety cabinet, 20 μL of CellTiter One Solution Reagen MTS reagent per 100 L cell culture volume was added along the side wall of each well, and the plate surface was gently tapped to well mix the MTS solution. The cells were incubated for 2 h in the dark in an incubator. After the reaction, the 96-well plate was taken out for testing the OD490 nm absorption on an elisa plate, and the test data were recorded, organized, analyzed and stored.
Conclusion: As shown in Table 9 and
The in vitro efficacy of ADCs was evaluated by using experimental models of various human tumor cell lines (N87, SW620, Fadu, and HCC827) and SW620+A431 heterogeneous tumor.
A certain number of tumor cells were inoculated into a 96-well plate, and gradient dilutions of the test antibody and the corresponding ADC were added to the cells, the cells were treated for 5 days, cell viability was detected by using Alamar Blue or MTS, and IC50 was calculated to evaluate the inhibitory effect of the test ADCs on the tumor cell line. The cells were treated for 5 days with the antibody drugs having initial concentration of 500 nM, diluted at 7 times, with a total of 8 concentration points. Finally, the cell survival was calculated as: survival rate=(experimental group-blank group)/(control group-blank group)×100%. Then, by using Graph Pad Prism, the curve was fitted and the half inhibitory concentration (IC50) was calculated. The results are shown in Table 10 and
Conclusion The ADCs prepared by the present invention show excellent tumor inhibitory activity in the experimental models of various human tumor cell lines (N87, SW620, Fadu, and HCC827) and SW620+A431 heterogeneous tumor. Experimental data on SW620+A431 heterogeneous tumor show that the ADCs prepared by the present invention can well exert the bystander effect.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111469786.8 | Dec 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/136278 | 12/2/2022 | WO |
