TW202302595A - Crystalline form of exatecan analog and preparation method of the same - Google Patents

Abstract

The present disclosure relates to a crystalline form of an Exatecan analog and a preparation method thereof. In particular, the present disclosure relates to crystalline forms of compounds represented by formula I and methods for their preparation. The new crystal form of the present disclosure has good physical and chemical properties, and is more conducive to the storage and utilization of raw materials.

Description

Crystalline forms of exitecan analogues and methods for their preparation

The present disclosure relates to a crystalline form of exitecan analogues and a preparation method thereof, in particular to a crystalline form of the compound represented by formula I and a preparation method thereof.

This application claims the priority of Chinese patent application 2021104069487 with a filing date of April 15, 2021. This application cites the full text of the above-mentioned Chinese patent application.

Chemotherapy remains one of the most important anti-cancer methods including surgery, radiotherapy, and targeted therapy. Although there are many types of high-efficiency cytotoxins, the difference between tumor cells and normal cells is very small, which limits the wide application of these anti-tumor compounds in clinical practice due to their toxic side effects. Anti-tumor monoclonal antibodies are specific to tumor cell surface antigens, and antibody drugs have become the front-line drugs for anti-tumor treatment. However, when antibodies are used alone as anti-tumor drugs, the curative effect is often unsatisfactory.

Antibody drug conjugates (antibody drug conjugates, ADCs) link monoclonal antibodies or antibody fragments with biologically active cytotoxins through stable chemical linker compounds, making full use of the specificity of antibodies for binding to surface antigens on normal cells and tumor cells and the high efficiency of cytotoxin, while avoiding the defects of low curative effect of the former and excessive toxic side effects of the latter. This means that, compared with traditional chemotherapy drugs in the past, antibody drug conjugates can precisely bind tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332 ; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

In 2000, the first antibody-drug conjugate Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceutical Co., Ltd.) was approved by the US FDA for the treatment of acute myeloid leukemia (Drugs of the Future ( 2000) 25(7):686; US4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).

In August 2011, Adcetris (brentuximab vedotin, Seattle Genetics Co., Ltd.) passed the US FDA's fast-track review channel for the treatment of Hodgkin's lymphoma and recurrent anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7 ):778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). Adcetris® is a new type of targeted ADC drug, which can cause the drug to directly act on the target CD30 on lymphoma cells and then undergo endocytosis to induce tumor cell apoptosis.

Both Mylotarg and Adcetris are targeted therapies for hematological tumors, which have a relatively simple tissue structure compared with solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for the treatment of HER2-positive patients with advanced or metastatic disease resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel breast cancer patients (WO2005037992; US8088387). Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.

There are several types of cytotoxic small molecules used in antibody-drug conjugates, one of which is camptothecin derivatives, which have anti-tumor effects by inhibiting topoisomerase I. It is reported that the camptothecin derivative exitecan (chemical name: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl- 1H,12H-Benzo[de]pyrano[3,,4,:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) applied to antibody The literature on conjugated drug (ADC) includes WO2014057687; Clinical Cancer Research (2016) 22 (20): 5097-5108; Cancer Sci (2016) 107: 1039-1046.

WO2020063676 relates to a series of new ligand-drug conjugates, among which a ligand-drug conjugate with the general formula (Pc-LY-Dr) has good anti-tumor activity, and its structure is as follows:

Wherein, Pc represents a ligand, and n is 1 to 10, which can be an integer or a decimal.

。 In addition, the compound shown in formula I can be used to prepare the above-mentioned ligand-drug conjugate, and the structure of the compound shown in formula I is as follows:

.

The crystal structure of pharmaceutical active ingredients and their intermediates often affects their chemical stability. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by other forms crystal form. Generally speaking, amorphous products have no regular crystal structure and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, and poor fluidity. Therefore, it is necessary to improve the various properties of the above-mentioned products, and we need to conduct in-depth research to find new crystal forms with high purity and good chemical stability.

The present disclosure provides a new crystal form of the compound represented by formula I and a preparation method thereof.

The present disclosure provides a crystal form C of the compound represented by formula I, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.0, 8.8, 10.3, 12.2 and 15.5.

In certain embodiments, the present disclosure provides a crystal form C of the compound represented by formula I, whose X-ray powder diffraction pattern is 6.0, 7.3, 8.8, 9.2, 10.3, 12.2, 15.5, 18.5 and 24.6 at 2θ angle There are characteristic peaks.

In certain embodiments, the present disclosure provides a crystal form C of the compound represented by formula I, whose X-ray powder diffraction pattern is shown in FIG. 4 .

The present disclosure provides a D crystal form of the compound represented by formula I, and its X-ray powder diffraction pattern is 6.1, 7.3, 9.0, 10.6, 11.3, 11.5, 11.9, 12.5, 12.9, 14.6, 14.8, 15.9, There are characteristic peaks at 16.1, 16.6, 17.4, 18.7, 19.4, 20.9, 21.6, 22.3, 23.1, 23.8, 24.9, 26.7, 28.3 and 29.8.

In certain embodiments, the present disclosure provides a crystal form D of the compound represented by formula I, and its X-ray powder diffraction pattern is shown in FIG. 5 .

The present disclosure provides a crystal form E of the compound represented by formula I, and its X-ray powder diffraction pattern is 6.7, 7.1, 7.3, 7.6, 8.4, 10.2, 10.8, 11.9, 12.6, 13.4, 14.3, 15.2, There are characteristic peaks at 15.6, 17.0, 17.9, 19.3, 20.4, 21.3 and 22.3.

In certain embodiments, the present disclosure provides a crystal form E of the compound represented by formula I, and its X-ray powder diffraction pattern is shown in FIG. 6 .

The present disclosure provides a crystal form F of the compound represented by formula I, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 9.5, 9.7, 11.2, 14.6, 14.8, 16.5, 17.2, 20.5, 22.7 and 28.6.

In certain embodiments, the present disclosure provides a crystal form F of the compound represented by formula I, and its X-ray powder diffraction pattern is shown in FIG. 7 .

The present disclosure further provides a method for preparing crystal form C of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, and crystallizing out, and the solvent is tetrahydrofuran.

The present disclosure further provides a method for preparing crystal form D of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, beating for overnight crystallization, and the solvent is acetonitrile.

The present disclosure further provides a method for preparing crystal form E of the compound represented by formula I, the method comprising: placing crystal form A of the compound represented by formula I under humidity conditions, and performing crystallization.

The present disclosure further provides a method for preparing the F crystal form of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, beating for 12 hours to crystallize, and the solvent is acetonitrile.

Through X-ray powder diffraction pattern (XRPD) and differential scanning calorimetry (DSC), the structure determination and crystal form research of the crystal form obtained in the present disclosure are carried out.

The crystallization method of the crystal form in the present disclosure is conventional, such as volatilization crystallization, cooling crystallization or room temperature crystallization.

The starting material used in the preparation method of the disclosed crystal form can be any form of the compound represented by formula I, and the specific form includes but not limited to: amorphous, any crystal form, hydrate, solvate, etc.

The crystal form D in the present disclosure is prone to crystal transformation after drying, and the reproducibility of the crystal forms E and F is poor.

II The present disclosure also provides a method for preparing the ligand-drug conjugate represented by formula II or a pharmaceutically acceptable salt or solvate thereof, comprising: after reducing the ligand, combined with the compound represented by formula I described in the present disclosure Show the steps of the crystal form coupling reaction of the compound,

II

Wherein, Pc represents a ligand, and n is 1 to 10, which can be an integer or a decimal.

In some embodiments, the Pc is an antibody or an antigen-binding fragment thereof, and the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody.

In some embodiments, wherein said antibody or antigen-binding fragment thereof is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti- HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, Anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody or an antigen-binding fragment thereof.

In certain embodiments, wherein said antibody or antigen-binding fragment thereof is selected from the group consisting of Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumab, or an antigen-binding fragment thereof .

In the description of the present application and the scope of the patent application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the technical field to which the present invention belongs. However, for a better understanding of the present disclosure, definitions and explanations of some related terms are provided below. In addition, when the definitions and explanations of the terms provided in this application are inconsistent with the meanings commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs, the definitions and explanations of the terms provided in this application shall prevail.

The "beating" mentioned in the present disclosure refers to a method of purifying by using the property that substances have poor solubility in solvents but impurities have good solubility in solvents. The beating purification can remove color, change crystal form or remove a small amount of impurities.

The "X-ray powder diffraction pattern or XRPD" described in the present disclosure means that according to the Bragg formula 2d sin θ = nλ (wherein, λ is the wavelength of X-ray, and the order n of diffraction is any positive integer, generally one order Diffraction peak, n=1), when the X-ray is incident on an atomic surface with a d-lattice plane spacing of a crystal or a part of the crystal sample at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle) , can satisfy the Bragg equation, thus measuring this group of X-ray powder diffraction patterns.

An "X-ray powder diffraction pattern or XRPD" as used in this disclosure is a pattern obtained by using Cu-Kα radiation in an X-ray powder diffractometer.

"Differential scanning calorimetry or DSC" in this disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference during the heating or constant temperature of the sample to characterize all the physical changes and chemical changes related to thermal effects. change to obtain the phase change information of the sample.

The “2θ or 2θ angle” in this disclosure refers to the diffraction angle, θ is the Bragg angle, the unit is ° or degree, and the error range of 2θ is ±0.3 or ±0.2 or ±0.1.

The "interplanar spacing or interplanar spacing (d value)" in the present disclosure refers to that the spatial lattice selects 3 non-parallel unit vectors a, b, and c that connect two adjacent lattice points. The matrix is divided into juxtaposed parallelepiped units called interplanar spacing. The spatial lattice is divided according to the determined parallelepiped unit connection lines to obtain a set of linear grids, which are called spatial lattices or lattices. Lattice and lattice reflect the periodicity of the crystal structure with geometric points and lines respectively. Different crystal planes have different interplanar spacing (that is, the distance between two adjacent parallel crystal planes); the unit is Å or Angstrom.

The term "ligand-drug conjugate" means that a ligand is linked to a biologically active drug through a stable linker unit. In this disclosure, the "ligand-drug conjugate" is preferably an antibody-drug conjugate (antibody drug conjugate, ADC), which refers to linking a monoclonal antibody or antibody fragment with a toxic drug with biological activity through a stable linker unit .

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are µ chain, δ chain, γ chain, α chain, and ε chain. The same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa chains or lambda chains by difference in the constant region. Each of the five Ig classes can have either a kappa chain or a lambda chain. Antibodies of the present disclosure are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody, Anti-PSMA Antibody, Anti-Tenascin- One or more of C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody; preferably Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumab.

The sequence of about 110 amino acids near the N-terminal of the heavy and light chains of the antibody varies greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminal are relatively stable and are the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. The three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.

The term "murine antibody" in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.

The term "chimeric antibody" is an antibody that fuses the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, E.A. et al. Al, 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid decreased immunogenicity and decreased activity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity. The humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

The term "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody are all human, and the immunogen sex and side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed by "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments comprising A bivalent fragment of two Fab fragments connected by a disulfide bridge of (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of the antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A synthetic linker is a combination of two or more separate CDRs joined. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to link them via a synthetic linker, making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art to which the invention pertains, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of different isotypes, eg, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM antibodies.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (which cleaves the amino acid residue at position 224 of the H chain), wherein the H chain N-terminal About half of the sides and the entire L chain are held together by disulfide bonds.

F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin fragment.

Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

In addition, the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

The term "single-chain antibody", "single-chain Fv" or "scFv" is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448 ). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDRs applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the variable domain of the light chain, and to CDR1, CDR2, and L3 of the variable domain of the heavy chain. CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3).

The term "antibody framework" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about 10-7M, eg, less than about 10-8M, 10-9M, or 10-10M or less.

The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or can integrate into the genome of the host cell after introduction into the host cell, thereby following The host genome is replicated together (eg, non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor's Antibody Laboratory Technique Guide, Chapters 5-8 and Chapter 15. Antigen-binding fragments can also be prepared by conventional methods. In the antibody or antigen-binding fragment described in the invention, one or more human FR regions are added to the non-human CDR region by genetic engineering methods. The human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NSO cells.

Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production. The culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. The bound antibody was then eluted by the pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, a "heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes cases where the heterocycle group is substituted with an alkyl group and cases where the heterocycle group is not substituted with an alkyl group .

The term "pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and Excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of a ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, when such salt is used in a mammal With safety and efficacy, and proper biological activity, the antibody-antibody drug conjugate compound of the present disclosure contains at least one amine group, so it can form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: Hydrochloride, Hydrobromide, Hydroiodide, Sulfate, Bisulfate, Citrate, Acetate, Succinate, Ascorbate, Oxalate, Nitrate, Limate, Hydrogen Phosphate, Dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonic acid salt, p-toluenesulfonate.

The term "solvate" or "solvate" refers to the formation of a pharmaceutically acceptable solvate of the ligand-drug conjugate compound of the present disclosure with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, iso Propanol, DMSO, ethyl acetate.

The term "drug loading" refers to the average amount of cytotoxic drugs loaded on each ligand in the molecule of formula (I), and can also be expressed as the ratio of the amount of drug to the amount of antibody, and the range of drug loading can be (Pc) link 0-12, preferably 1-10 cytotoxic drugs (D). In the embodiments of the present disclosure, the drug loading amount is expressed as n, which may be an exemplary average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average amount of drug product per ADC molecule after conjugation can be characterized by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay and HPLC.

In one embodiment of the present disclosure, the cytotoxic drug is coupled to the N-terminal amine group of the ligand and/or the ε-amine group of the lysine residue through the linking unit. Generally, the coupling reaction can be coupled with the antibody The number of drug molecules linked will be less than the theoretical maximum.

The loading of ligand cytotoxic drug conjugates can be controlled by the following non-limiting methods, including: (1) Control the molar ratio of the linking reagent and the monoclonal antibody, (2) Control reaction time and temperature, (3) Choose different reagents.

The preparation of conventional pharmaceutical compositions can be found in Chinese Pharmacopoeia.

The term "carrier" used in the drug of the present disclosure refers to a system that can change the way the drug enters the human body and distributes the drug in the body, controls the release rate of the drug, and delivers the drug to the target organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble and form various forms of aggregates due to their unique amphiphilic structure. Preferred examples are micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to entrap drug molecules, and at the same time have good permeability to the membrane, which can be used as excellent drug carriers.

The following will explain the present disclosure in more detail in conjunction with the embodiments, and the embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the essence and scope of the present disclosure.

The test conditions of the instruments used in the test: 1. Differential Scanning Calorimeter (DSC) Instrument model: Mettler Toledo DSC 3+ Purge gas: Nitrogen Heating rate: 10.0 ℃/min Temperature range: 25-300°C (25-200°C) 2. X-ray Powder Diffraction (XRPD) Instrument model: BRUKER D8 ADVANCE X-ray powder diffractometer Ray: monochromatic Cu-Kα ray (the wavelength of Cu-Kα1 is 1.5406Å, the wavelength of Cu-Kα2 is 1.54439Å, and the wavelength of Cu-Kα is the weighted average of Kα1 and Kα2 λ=1.5418Å) Scanning mode: θ/2θ, scanning range: 3-45°, Voltage: 40KV, current: 40mA;

Example 1 The compound shown in formula I was prepared according to Example 9 of WO2020063676, and the obtained product was detected as amorphous by X-ray powder diffraction, and the XRPD spectrum is shown in FIG. 1 .

Example 2 Add 10 mg of the compound represented by formula I to 1 mL of dichloromethane, stir at room temperature, centrifuge, and vacuum dry to obtain the product. The product was defined as crystal form A by X-ray powder diffraction detection. The XRPD spectrum is shown in Figure 2, and the positions of its characteristic peaks are shown in Table 1. DSC results show that the endothermic peaks are 141.78°C and 162.11°C Table 1 Peak number 2θ[°] d[Å] Relative Strength% 1 6.797 12.99474 97.4 2 7.055 12.51885 71.1 3 7.958 11.10121 100.0 4 8.609 10.26307 11.4 5 10.364 8.52834 5.1 6 11.994 7.37298 20.3 7 12.785 6.91846 6.1 8 13.363 6.62063 7.5 9 13.697 6.45982 15.3 10 15.371 5.75989 11.0

Example 3 Add 10 mg of the compound represented by formula I into 0.4 mL of acetonitrile, stir at room temperature, centrifuge, and dry in vacuo to obtain the product. The product was defined as crystal form B by X-ray powder diffraction detection. The XRPD spectrum is shown in Figure 3, and the positions of its characteristic peaks are shown in Table 2. DSC results showed that the endothermic peak was 142.10°C. Table 2 Peak number 2θ[°] d[Å] Relative Strength% 1 9.763 9.05193 100.0 2 11.170 7.91524 21.7 3 11.879 7.44380 14.3 4 13.416 6.59449 19.4 5 14.685 6.02738 36.7 6 16.472 5.37728 40.9 7 17.173 5.15931 15.8 8 19.109 4.64075 13.3 9 20.475 4.33414 51.8 10 22.761 3.90381 21.1 11 24.617 3.61345 21.0 12 25.756 3.45613 14.3 13 27.013 3.29815 12.0 14 29.655 3.01008 9.9 15 32.729 2.73399 6.1

Example 4 10 mg of the compound represented by formula I was added to 1 mL of tetrahydrofuran, stirred at room temperature, centrifuged, and vacuum-dried to obtain the product. The product was defined as crystal form C by X-ray powder diffraction detection. The XRPD spectrum is shown in Fig. DSC results showed that the endothermic peak was 153.85°C. table 3 Peak number 2θ[°] d[Å] Relative Strength% 1 6.035 14.63223 100.00 2 7.294 12.11026 11.00 3 8.772 10.07193 30.70 4 9.235 9.56848 12.80 5 10.299 8.58218 27.50 6 12.156 7.27514 62.90 7 15.453 5.72935 24.70 8 18.47 4.79994 12.60 9 24.614 3.61388 12.60

Example 5 10 mg of the compound represented by formula I was added to 1 mL of tetrahydrofuran, heated to 50° C. to dissolve, cooled to room temperature, stirred, centrifuged, and vacuum-dried to obtain the product. X-ray powder diffraction detection showed that the product was C crystal form.

Example 6 Add 150 mg of the compound represented by formula I to 4 mL of tetrahydrofuran, add 1 mg of seed crystal (Example 4), stir at room temperature, centrifuge, and vacuum dry to obtain the product. X-ray powder diffraction detection showed that the product was C crystal form.

Example 7 Add 150 mg of the compound represented by formula I to 3 mL of acetonitrile, stir overnight at room temperature, and centrifuge to obtain the product. The product was defined as crystal form D by X-ray powder diffraction detection. The XRPD spectrum is shown in Figure 5, and the positions of the characteristic peaks are shown in Table 4. Table 4 Peak number 2θ[°] d[Å] Relative Strength% 1 6.127 14.41304 15.60 2 7.276 12.13909 14.50 3 8.977 9.84347 100.00 4 10.599 8.34019 55.10 5 11.253 7.85676 36.30 6 11.542 7.66081 22.60 7 11.907 7.42644 12.20 8 12.539 7.05353 33.70 9 12.895 6.85961 4.10 10 14.589 6.0668 30.00 11 14.793 5.9835 12.60 12 15.944 5.55414 30.20 13 16.084 5.50602 12.20 14 16.619 5.32995 5.50 15 17.416 5.08789 8.60 16 18.709 4.7391 26.10 17 19.449 4.5605 13.40 18 20.878 4.25143 24.50 19 21.579 4.11481 17.00 20 22.321 3.97971 18.20 twenty one 23.066 3.85285 18.80 twenty two 23.774 3.73965 6.80 twenty three 24.908 3.57182 21.90 twenty four 26.65 3.34225 12.00 25 28.3 3.15097 6.50 26 29.774 2.99827 4.60

Example 8 The target product was obtained by placing 10 mg of Form A under the humidity conditions of 75% RH and 93% RH for 7 days or more. Through X-ray powder diffraction detection, the product was defined as crystal form E. The XRPD spectrum is shown in Figure 6, and the positions of the characteristic peaks are shown in Table 5. table 5 Peak number 2θ[°] d[Å] Relative Strength% 1 6.704 13.17496 100.00 2 7.076 12.48215 26.20 3 7.311 12.08196 32.50 4 7.602 11.62067 94.50 5 8.411 10.50369 20.80 6 10.172 8.68927 16.10 7 10.805 8.18181 15.80 8 11.877 7.44507 34.80 9 12.593 7.02376 25.30 10 13.44 6.58258 33.00 11 14.32 6.18037 23.10 12 15.185 5.8299 50.30 13 15.623 5.66758 47.00 14 17.046 5.1976 37.70 15 17.929 4.94333 24.80 16 19.25 4.60711 20.60 17 20.379 4.35436 35.00 18 21.299 4.16821 34.00 19 22.331 3.97796 27.10

Example 9 Put 15 mg of crystal form A in a dynamic moisture adsorption apparatus, and after a hygroscopic experiment (humidity gradient: 50%-95%-0%-95%-0%-50%RH), the target product was obtained. X-ray powder diffraction detection showed that the product was E crystal form.

Example 10 Add 150 mg of the compound represented by formula I to 4 mL of acetonitrile, stir at room temperature for 12 hours, centrifuge, and dry in vacuo to obtain the product. Through X-ray powder diffraction detection, the product was defined as crystal form F, and the XRPD spectrum is shown in Figure 7, and the positions of the characteristic peaks are shown in Table 6. Table 6 Peak number 2θ[°] d[Å] Relative Strength% 1 9.53 9.27322 68.90 2 9.731 9.08196 100.00 3 11.167 7.91713 44.50 4 14.601 6.06193 41.00 5 14.848 5.96141 33.30 6 16.499 5.36862 38.70 7 17.183 5.15636 33.50 8 20.481 4.33286 81.30 9 22.716 3.91136 43.00 10 28.622 3.11631 43.50

Example 11 The samples of crystal forms A, B, and C were placed open and flat, and the stability of the samples under high temperature (40°C, 60°C) and high humidity (RH 75%, RH 92.5%) conditions were investigated respectively, and samples were taken The investigation period is 30 days, and the results are shown in Table 7. Table 7 condition time (days) Form A Form B Form C start 0 99.38% 99.61% 99.62% 40°C 7 98.78% 99.39% 99.41% 14 98.32% 99.02% 98.83% 30 96.57% 97.67% 98.30% 60°C 7 98.74% 99.21% 99.42% 14 97.89% 98.46% 98.86% 30 96.53% 97.36% 98.67% 75% RH 7 99.54% 99.62% 99.59% 14 99.51% 99.64% 99.62% 30 99.36% 99.57% 99.62% 92.5% RH 7 99.26% 99.60% 99.50% 14 99.17% 99.57% 99.59% 30 98.80% 99.42% 99.53%

Example 11 The crystal form C samples were placed at -20°C, 4°C, 25°C/60%RH and 40°C/75%RH to investigate their stability. The results are shown in Table 8. Table 8 Form C placement conditions purity% purity% purity% purity% crystal form start 7 days 14 days 1 month -20°C 99.62 99.62 99.59 99.57 C 4°C 99.62 99.59 99.60 99.54 C 25°C, 60%RH 99.62 99.60 99.62 99.59 C 40°C, 75%RH 99.62 99.58 99.59 99.54 C

none

FIG. 1 is an amorphous XRPD pattern of the compound represented by formula I. Fig. 2 is the XRPD spectrum of the crystal form A of the compound represented by formula I. Fig. 3 is the XRPD spectrum of the B crystal form of the compound represented by formula I. Fig. 4 is the XRPD spectrum of the C crystal form of the compound represented by formula I. Fig. 5 is the XRPD spectrum of the D crystal form of the compound represented by formula I. Fig. 6 is the XRPD pattern of the crystal form E of the compound represented by formula I. Fig. 7 is the XRPD spectrum of the F crystal form of the compound represented by formula I.

Claims (13)

A crystal form C of the compound shown in formula I, its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.0, 8.8, 10.3, 12.2 and 15.5,

. The crystal form C of the compound represented by formula I as claimed in Claim 1 has characteristic peaks at 2θ angles of 6.0, 7.3, 8.8, 9.2, 10.3, 12.2, 15.5, 18.5 and 24.6 in its X-ray powder diffraction pattern. The X-ray powder diffraction pattern of the crystal form C of the compound represented by formula I as described in Claim 1 is shown in FIG. 4 . A crystal form D of a compound represented by formula I, whose X-ray powder diffraction pattern is 6.1, 7.3, 9.0, 10.6, 11.3, 11.5, 11.9, 12.5, 12.9, 14.6, 14.8, 15.9, 16.1, 16.6, There are characteristic peaks at 17.4, 18.7, 19.4, 20.9, 21.6, 22.3, 23.1, 23.8, 24.9, 26.7, 28.3 and 29.8. The X-ray powder diffraction pattern of the crystal form D of the compound represented by formula I as described in Claim 4 is shown in FIG. 5 . A crystal form E of a compound represented by formula I, whose X-ray powder diffraction pattern is 6.7, 7.1, 7.3, 7.6, 8.4, 10.2, 10.8, 11.9, 12.6, 13.4, 14.3, 15.2, 15.6, 17.0, There are characteristic peaks at 17.9, 19.3, 20.4, 21.3 and 22.3. The X-ray powder diffraction pattern of the crystal form E of the compound represented by formula I as described in Claim 6 is shown in FIG. 6 . A crystal form F of the compound represented by formula I has characteristic peaks at 2θ angles of 9.5, 9.7, 11.2, 14.6, 14.8, 16.5, 17.2, 20.5, 22.7 and 28.6 in its X-ray powder diffraction pattern. The X-ray powder diffraction pattern of the crystal form F of the compound represented by formula I as described in Claim 8 is shown in FIG. 7 . The crystal form of the compound represented by formula I as described in any one of claim items 1-9, wherein the error range of the 2θ angle is ±0.2. A method for preparing the C crystal form of the compound shown in formula I as described in any one of claim items 1-3, the method comprising: mixing the compound shown in formula I with an appropriate amount of solvent, and crystallizing out, the solvent is Tetrahydrofuran. A method for preparing a ligand-drug conjugate represented by formula II or a pharmaceutically acceptable salt or solvate thereof, comprising reducing the ligand and reacting with the crystal form of the compound represented by formula I A step of,

II wherein, Pc represents a ligand, and n is 1 to 10, which can be an integer or a decimal. The preparation method according to claim 12, the Pc is an antibody or an antigen-binding fragment thereof, the antibody is selected from chimeric antibodies, humanized antibodies or fully human antibodies, preferably a monoclonal antibody, more preferably HER2 ( ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, Anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti- VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody or an antigen-binding fragment thereof.

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