CN114249798A - Crystal form of irinotecan analogue and preparation method thereof - Google Patents

Abstract

The present disclosure relates to crystalline forms of an irinotecan analog and processes for their preparation. In particular, the present disclosure relates to crystalline forms of the compounds of formula I and methods for their preparation. The novel crystal form disclosed by the invention has good physicochemical properties, and is more favorable for storage and utilization of raw materials.

Description

Crystal form of irinotecan analogue and preparation method thereof

Technical Field

The present disclosure relates to crystalline forms of an irinotecan analog and processes for their preparation, and in particular to crystalline forms of the compound of formula I and processes for their preparation.

Background

Chemotherapy remains one of the most important anticancer modalities, including surgery, radiation therapy, and targeted therapies. Although the variety of high-efficiency cytotoxins is large, the difference between tumor cells and normal cells is small, and the wide clinical application of the anti-tumor compounds is limited due to toxic and side effects. The specificity of the anti-tumor monoclonal antibody to the surface antigen of tumor cells, the antibody drug has become the front-line drug of anti-tumor therapy, but the curative effect is often not satisfactory when the antibody is used alone as the anti-tumor drug.

Antibody Drug Conjugate (ADC) connects monoclonal antibody or antibody fragment with biologically active cytotoxin through stable chemical linker compound, fully utilizes the specificity of antibody for combining normal cell and tumor cell surface antigen and high efficiency of cytotoxin, and avoids the defects of low curative effect of the former and overlarge toxic and side effect of the latter. This means that the antibody Drug conjugate binds tumor cells precisely and has a reduced effect on normal cells compared to conventional chemotherapeutic drugs (Mullard A, (2013) Nature Reviews Drug Discovery,12: 329-; DiJoseph JF, Armellono DC, (2004) Blood,103: 1807-; 1814).

The first antibody drug conjugate in 2000, Mylotarg (gemtuzumab ozogamicin, Hewlett packard, Inc.) was approved by the U.S. FDA for marketing for the treatment of acute myeloid leukemia (Drugs of the Future (2000)25(7): 686; US 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).

In 8 months of 2011, Adcetris (Brentuximab vedotin, Seattle Gene genetics) was used through the U.S. FDA quick review channel for the treatment of Hodgkin lymphoma and recurrent anaplastic large cell lymphoma (nat. Biotechnol (2003)21(7): 778-784; WO 2004010957; WO 2005001038; US 7090843A; US 7659241; WO 2008025020).

Is a novel targeting ADC drug, and can lead the drug to generate endocytosis after directly acting on a target spot CD30 on a lymphoma cell so as to induce the apoptosis of the tumor cell.

Both Mylotarg and Adcetris are targeted therapies against hematological tumors, which are relatively simple in tissue structure compared to solid tumors. In month 2 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) obtained U.S. FDA approval for the treatment of advanced or metastatic breast cancer patients who were positive for HER2 while being resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel (WO 2005037992; US 8088387). Kadcula is the first ADC drug approved by the FDA in the united states for the treatment of solid tumors.

There are several classes of cytotoxic small molecules for antibody drug conjugates, one of which is camptothecin derivatives, which have anti-tumor effects by inhibiting topoisomerase I. The application of camptothecin derivative, irinotecan (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) to antibody-conjugated drugs (ADC) is reported in WO 2014057687; clinical Cancer Research (2016)22 (20): 5097-5108; cancer Sci (2016)107: 1039-1046.

WO2020063676 relates to a series of novel ligand-drug conjugates, wherein a class of ligand-drug conjugates represented by the general formula (Pc-L-Y-Dr) have good anti-tumor activity, and the structure thereof is shown as follows:

wherein Pc represents a ligand, and n is 1 to 10, and may be an integer or a decimal.

In addition, the compound shown in the formula I can be used for preparing the ligand-drug conjugate, and the structure of the compound shown in the formula I is shown as follows:

the crystal form structures of the active ingredients and the intermediates thereof for medical use often influence the chemical stability of the active ingredients and the intermediates, and the difference of crystallization conditions and storage conditions can cause the change of the crystal form structures of the compounds and sometimes bring about other crystal forms. Generally, amorphous products have no regular crystal structure and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, poor flowability and the like. Therefore, it is necessary to improve various properties of the above products, and intensive research is needed to find new crystal forms with high purity and good chemical stability.

Disclosure of Invention

The purpose of the present disclosure is to provide a novel crystal form of the compound represented by formula I, which has good stability.

The disclosure provides a crystal form A of the compound shown in the formula I, and an X-ray powder diffraction pattern of the crystal form A has characteristic peaks at 6.626 and 7.572 angles of 2 theta.

In certain embodiments, the present disclosure provides a crystalline form a of the compound of formula I having an X-ray powder diffraction pattern as shown in figure 2, figure 4, or figure 5.

The present disclosure further provides a method of preparing form a of the compound of formula I, the method comprising: mixing the compound shown in the formula I with a proper amount of solvent, and pulping at room temperature, wherein the solvent can be dichloromethane.

The disclosure provides a B crystal form of a compound shown in a formula I, and an X-ray powder diffraction pattern of the B crystal form has characteristic peaks at 2 theta angles of 9.607, 11.064, 13.240, 14.446, 16.255 and 20.379.

In certain embodiments, the present disclosure provides a form B of the compound of formula I having an X-ray powder diffraction pattern as shown in figure 3.

The present disclosure further provides a method of preparing form B of the compound of formula I, the method comprising: mixing the compound shown in the formula I with a proper amount of solvent, and pulping at room temperature, wherein the solvent can be acetonitrile.

The crystal form obtained by the present disclosure is subjected to structure determination and crystal form research through X-ray powder diffraction pattern (XRPD) and Differential Scanning Calorimetry (DSC).

The crystallization method of the crystalline form in the present disclosure is conventional, such as volatile crystallization, temperature-reduced crystallization or room temperature crystallization.

The starting materials used in the preparation methods of the crystalline forms of the present disclosure may be any form of the compound of formula I, including but not limited to: amorphous, random crystalline, hydrate, solvate, and the like.

The present disclosure also provides a method of preparing a ligand-drug conjugate shown in formula II, or a pharmaceutically acceptable salt or solvate thereof, comprising: a step of coupling reaction with the crystal form of the compound shown in the formula I after the ligand is reduced,

wherein Pc represents a ligand, and n is 1 to 10, and may be an integer or a decimal.

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

In certain embodiments, wherein the antibody or antigen binding fragment thereof is selected from an anti-HER 2(ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3(ErbB3) antibody, an anti-HER 4(ErbB4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-cl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-nmb antibody, an anti-gpmutrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-mescalin 3644 a4 antibody or an anti-mescalin antibody or an antigen binding fragment thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, enobilituzumab, Emibetuzumab, Inotuzumab, pintuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and glemtuzumab, or an antigen-binding fragment thereof.

In the description and claims of this application, unless otherwise indicated, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. However, for a better understanding of the present disclosure, definitions and explanations of some of the relevant terms are provided below. In addition, where the definitions and explanations of terms provided herein are inconsistent with the meanings that would normally be understood by those skilled in the art, the definitions and explanations of terms provided herein shall control.

The term "pulping" as used in the present disclosure refers to a method of purification by utilizing the characteristic that a substance has poor solubility in a solvent, but impurities have good solubility in a solvent, and pulping purification can remove color, change crystal form or remove a small amount of impurities.

The term "X-ray powder diffraction pattern or XRPD" as used herein means that when X-rays are incident on an atomic plane having a d-lattice interplanar spacing of a crystal or a portion of a crystal sample at a grazing angle θ (complementary angle of incidence, also referred to as bragg angle), the bragg equation is satisfied according to bragg formula 2d sin θ ═ n λ (where λ is the wavelength of the X-rays, and n is any positive integer of the diffraction orders, and is generally a first-order diffraction peak, and n is 1), so that the set of X-ray powder diffraction patterns is obtained.

The "X-ray powder diffraction pattern or XRPD" described in this disclosure is a pattern obtained by using Cu-ka radiation in an X-ray powder diffractometer.

The differential scanning calorimetry or DSC in the present disclosure refers to measuring the temperature difference and heat flow difference between a sample and a reference substance during the temperature rise or constant temperature process of the sample to characterize all the physical changes and chemical changes related to the thermal effect and obtain the phase change information of the sample.

The "2 theta or 2 theta angle" referred to in the present disclosure means the diffraction angle, theta is the bragg angle in degrees or degrees, and the error range of 2 theta is ± 0.3 or ± 0.2 or ± 0.1.

The term "interplanar spacing or interplanar spacing (d value)" as used in this disclosure means that the spatial lattice selects 3 non-parallel unit vectors a, b, c connecting two adjacent lattice points, which divide the lattice into juxtaposed parallelepiped units, called interplanar spacing. The space lattice is divided according to the determined connecting lines of the parallelepiped units to obtain a set of linear grids called space grids or lattices. The lattice and the crystal lattice respectively reflect the periodicity of the crystal structure by using geometrical points and lines, and the surface spacing (namely the distance between two adjacent parallel crystal surfaces) of different crystal surfaces is different; has a unit of

Or angstroms.

The term "ligand-drug conjugate," means that the ligand is linked to the biologically active drug through a stable linking unit. In the present disclosure, "ligand-drug conjugate" is preferably Antibody Drug Conjugate (ADC), which means that a monoclonal antibody or antibody fragment is linked to a toxic drug having biological activity through a stable linking unit.

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

The term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The antibodies described in the present disclosure are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples being the following antibodies: one or more of an anti-HER 2(ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3(ErbB3) antibody, an anti-HER 4(ErbB4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44a4 antibody, or an anti-sothelin antibody; preferred are Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name taoxin), enobiluzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and glemtuzumab.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.

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

The term "murine antibody" is used in this disclosure to prepare antibodies from mice according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with a particular antigen and then isolating hybridomas that express antibodies having the desired sequence or functional properties.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into expression vector, and finally expressing chimeric antibody molecule in eukaryotic system or prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of genes such as the human heavy and light chain variable regions can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/VBase), as well as in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. Humanized antibodies of the present disclosure also include humanized antibodies after further affinity maturation of the CDRs by phage display. Further literature describing methods involving humanization of usable mouse antibodies includes, for example, Queen et al, Proc., Natl.Acad.Sci.USA, 88, 2869, 1991 and Winter and co-workers' methods [ Jones et al, Nature, 321, 522(1986), Riechmann, et al, Nature, 332, 323-.

The terms "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", have both the variable and constant regions of the antibody being of human origin, eliminating immunogenicity and toxic side effects. Monoclonal antibodies have progressed through four stages, respectively: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The present disclosure is fully human monoclonal antibodies. The related technologies for preparing 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), single B cell antibody preparation technology, and the like.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al, (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, enabling their production as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science242: 423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA85: 5879-. 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 skilled in the art, and the fragments are screened for utility 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. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

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

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

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

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

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), Proc. Natl. Acad. Sci. USA90: 6444-. Can be usedOther linkers in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. One of the most common definitions of the 6 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 the CDRs 1,2, and 3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), and the CDRs 2 and 3 of the heavy chain variable domain (CDR H2, CDR H3 or H2, H3).

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

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epipope Mapping Protocols in Methods in Molecular B biology, volume 66, g.e. morris, Ed. (1996).

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is administered at a rate of about less than 10^-7M, e.g. less than about 10^-8M、10^-9M or 10^-10M or less affinity (KD) binding.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is 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 it 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 may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold spring harbor antibody protocols, chapters 5-8 and 15. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen binding fragment of the invention is genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequences can be obtained from the website http:// IMGT. cities.fr of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN012441351, by aligning the IMGT human antibody variable region germline gene database with the MOE software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

Engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting antibody fragments by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

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

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

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the ligand-drug conjugates of the present disclosure, or salts of the compounds described in the present disclosure, which salts are safe and effective for use in the mammalian body and possess the requisite biological activity, and the antibody-antibody drug conjugate compounds of the present disclosure contain at least one amino group and thus can form salts with acids, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulphate, hydrogen sulphate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate.

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

The term "drug loading" refers to the average number of cytotoxic drugs loaded per ligand in the molecule of formula (I) and can also be expressed as the ratio of drug amount to antibody amount, and the drug loading can range from 0 to 12, preferably from 1 to 10 cytotoxic drugs (D) attached per ligand (Pc). In an embodiment of the present invention, the drug loading is represented as n, which may be an exemplary mean value of 1,2, 3, 4, 5, 6,7, 8, 9, 10. The average amount of drug per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays and HPLC characterization.

In one embodiment of the present disclosure, the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the epsilon-amino group of the lysine residue via a linker unit, and generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum.

The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

(1) controlling the molar ratio of the connecting reagent to the monoclonal antibody,

(2) the reaction time and the temperature are controlled,

(3) different reagents were selected.

The preparation of the conventional pharmaceutical composition is shown in Chinese pharmacopoeia.

The term "carrier" as used with respect to a drug of the present disclosure refers to a system that alters the manner and distribution of the drug into the body, controls the rate of release of the drug, and delivers the drug to the targeted organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects and improve bioavailability. For example, the polymeric surfactant used as a carrier can self-assemble due to its unique amphiphilic structure to form aggregates in various forms, such as micelles, microemulsions, gels, liquid crystals, vesicles, and the like. The aggregates have the capacity of encapsulating drug molecules, have good permeability to membranes and can be used as excellent drug carriers.

Advantageous effects of the invention

The compound shown in the formula I prepared by the method has the advantages of high purity of crystal forms A and B, good crystal form stability under the conditions of illumination, high temperature and high humidity, small HPLC purity change and high physical and chemical stability, and is more favorable for storage and use of raw materials.

Drawings

FIG. 1is an amorphous XRPD pattern for a compound of formula I;

figure 2 is the XRPD pattern of form a of the compound of formula I obtained in example 2;

FIG. 3 is an XRPD pattern for form B of the compound of formula I;

figure 4 is the XRPD pattern of form a of the compound of formula I obtained in example 4;

figure 5 is the XRPD pattern of form a of the compound of formula I obtained in example 5;

FIG. 6 is a comparison of XRPD patterns of form A of the compound of formula I before and after DVS detection;

figure 7 is a comparison of XRPD patterns of form B of the compound of formula I before and after DVS detection.

Detailed Description

The present disclosure will be explained in more detail with reference to examples, which are provided only for illustrating the technical solutions of the present disclosure and do not limit the spirit and scope of the present disclosure.

Test conditions of the apparatus used for the test:

1. differential Scanning Calorimeter (DSC)

The instrument model is as follows: mettler Toledo DSC 3+

And (3) purging gas: nitrogen gas

The heating rate is as follows: 10.0 ℃/min

Temperature range: 25-350 deg.C

2. X-ray Diffraction Spectroscopy (XRPD)

The instrument model is as follows: BRUKER D8 DISCOVERY X-RAY POWDER DIFFRACTOMETER

Ray: monochromatic Cu-Kalpha radiation (Cu-Kalpha 1 wavelength of

Cu-Kalpha 2 wavelength of

The Cu-Ka wavelength is taken as the weighted average value of Ka 1 and Ka 2

)

The scanning mode is as follows: θ/2 θ, scan range: 3-48 degrees and 5-48 degrees

Voltage: 40KV, current: 40 mA;

example 1

The compound of formula I was prepared according to example 9 of WO2020063676 and the resulting product was amorphous as determined by X-ray powder diffraction.

Example 2

The compound of formula I (12.2mg, 11. mu. mol) was added to 0.4mL of dichloromethane, slurried at room temperature with stirring, centrifuged, the supernatant was aspirated off, and the residual solid was dried under vacuum to give 10mg of form A of the compound of formula I.

Example 3

Adding the compound shown in the formula I (13.5mg, 13 mu mol) into 0.4mL acetonitrile, pulping and stirring at room temperature, centrifuging, sucking away a supernatant, and drying a residual solid in vacuum to obtain 12mg of a B crystal form of the compound shown in the formula I, wherein the characteristic peak positions are shown in the following table:

table 1: XRD characteristic peak position of B crystal form

Example 4

The compound of formula I (11.5mg, 11 μmol) was added to 0.4mL dichloromethane, 2mg seed crystals (example 2) were added, slurried at room temperature, stirred, centrifuged, the supernatant was aspirated off, and the residual solid was dried under vacuum to give 12mg of compound of formula I as form a. The characteristic peak positions are shown in the following table:

table 2: XRD characteristic peak position of crystal form A

Example 5

The compound of formula I (103.3mg, 96 μmol) was added to 40mL dichloromethane, 5mg seed crystals (example 4) were added, slurried at room temperature, centrifuged, the supernatant was aspirated off, and the residual solid was dried under vacuum to give 102mg of the compound of formula I as form a. The characteristic peak positions are shown in the following table:

table 3: XRD characteristic peak position of crystal form A

Example 6

The compound of formula I (11.1mg, 10 μmol) was added to 0.4mL acetonitrile, 2mg seed crystals (example 3) were added, slurried at room temperature, stirred, centrifuged, the supernatant was aspirated off, and the residual solid was dried under vacuum to give 11mg of form B of the compound of formula I.

Example 7

The compound of formula I (90mg, 84. mu. mol) was added to 4mL acetonitrile, 5mg seed crystals (example 6) were added, slurried and stirred at room temperature, centrifuged, the supernatant was aspirated off, and the residual solid was dried under vacuum to give 88mg of form B of the compound of formula I.

EXAMPLE 8 hygroscopicity study of the crystalline form

And detecting the hygroscopicity of each crystal form by adopting a DVS dynamic moisture adsorption analyzer, detecting XRPD (X-ray diffraction pattern) of a sample after DVS detection of each crystal form, and comparing XRPD results of crystal forms before and after DVS detection.

After DVS detection, XRD of the crystal form A is retested, and the crystal form is not transformed, as shown in figure 6.

After DVS detection, B crystal form XRD is retested, and crystal form transformation is found, as shown in figure 7.

Example 9

The A, B crystal form sample is placed open and spread, the stability of the sample under the conditions of high temperature (40 ℃, 60 ℃) and high humidity (RH 75 percent and RH 92.5 percent) is respectively inspected, and the sampling inspection period is 30 days.

Table 4: stability study of influencing factors

Claims (9)

1. A crystal form A of a compound shown as a formula I has characteristic peaks at 6.626 and 7.572 angles of 2 theta in an X-ray powder diffraction pattern,

2. the crystalline form a of the compound of formula I according to claim 1 having an X-ray powder diffraction pattern as shown in figure 2, figure 4 or figure 5.

3. A crystal form B of the compound shown as a formula I has characteristic peaks at 2 theta angles of 9.607, 11.064, 13.240, 14.446, 16.255 and 20.379 in an X-ray powder diffraction pattern.

4. The crystal form B of the compound shown in the formula I according to claim 3, and the X-ray powder diffraction pattern of the crystal form B is shown in figure 3.

5. A crystalline form of the compound of formula I according to any one of claims 1 to 4, wherein the 2 θ angle has a tolerance of ± 0.2.

6. A process for preparing form a of the compound of formula I as claimed in any one of claims 1-2, which process comprises: mixing the compound shown in the formula I with a proper amount of solvent, and pulping at room temperature, wherein the solvent is dichloromethane.

7. A process for preparing form B of a compound of formula I according to any one of claims 3 to 4, which process comprises: mixing the compound shown in the formula I with a proper amount of solvent, and pulping at room temperature, wherein the solvent is acetonitrile.

8. A preparation method of a ligand-drug conjugate shown in a formula II or pharmaceutically acceptable salt or solvate thereof comprises the step of coupling reaction with a crystal form of a compound shown in a formula I after reducing a ligand,

wherein Pc represents a ligand, and n is 1 to 10, and may be an integer or a decimal.

9. The method of claim 8, wherein the Pc is an antibody or an antigen-binding fragment thereof selected from the group consisting of a chimeric, humanized or fully human antibody, preferably a monoclonal antibody, more preferably a HER2(ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3(ErbB3) antibody, an anti-HER 4(ErbB4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti PSMA antibody, an anti-C antibody, an anti-SLC antibody, or an anti-Mesollin 36 binding antibody or an antigen-Mestrin 4 antibody thereof.

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