CN117157304A

CN117157304A - Crystal form of irinotecan analogue and preparation method thereof

Info

Publication number: CN117157304A
Application number: CN202280028252.8A
Authority: CN
Inventors: 吴琪; 杨俊然; 杜振兴
Original assignee: Jiangsu Hengrui Medicine Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd
Priority date: 2021-04-15
Filing date: 2022-04-15
Publication date: 2023-12-01
Also published as: TW202302595A; WO2022218415A1

Abstract

A crystal form of an irinotecan analogue and a preparation method thereof, in particular to a crystal form of a compound shown in a formula I and a preparation method thereof. The crystal form has good physicochemical properties, and is more beneficial to the storage and utilization of raw materials.

Description

Crystal form of irinotecan analogue and preparation method thereof

The present application claims priority from chinese patent application 2021104069487, whose application date is 2021, 4, 15. The present application incorporates the entirety of the above-mentioned chinese patent application.

Technical Field

The present disclosure relates to crystalline forms of an irinotecan analog and methods of making the same, and in particular to crystalline forms of a compound of formula I and methods of making the same.

Background

Chemotherapy remains one of the most important anticancer means, including surgery, radiation therapy, and targeted therapies. Although the variety of highly potent cytotoxins is very large, the small difference between tumor cells and normal cells limits the wide clinical use of these antitumor compounds due to toxic side effects. The specificity of the anti-tumor monoclonal antibody to the surface antigen of the tumor cells is the front line medicine of anti-tumor treatment, but the curative effect is often unsatisfactory when the antibody is singly used as the anti-tumor medicine.

The antibody drug conjugate (antibody drug conjugate, ADC) connects monoclonal antibody or antibody fragment with cytotoxin with bioactivity through stable chemical joint compound, makes full use of the specificity of antibody on the surface antigen of normal cells and tumor cells and the high efficiency of cytotoxin, and simultaneously avoids the defects of low curative effect and overlarge toxic and side effect of the former. This means that the antibody drug conjugate binds tumor cells precisely and has reduced effect on normal cells compared to conventional chemotherapeutics (Mullard A, (2013) Nature Reviews Drug Discovery,12:329-332;DiJoseph JF,Armellino DC, (2004) Blood, 103:1807-1814).

The first antibody drug conjugate, mylotarg (gemtuzumab ozogamicin (gemtuzumab ozogamicin), of the wheatstone pharmaceutical company limited) was approved by the FDA in the united states for use in 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).

8 2011, adcetris (brentuximab vedotin, seattle Gene genetics Co.) was delivered by the United states FDA fastA rapid 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; WO 20080525020).Is a novel targeted ADC drug, and can lead the drug to directly act on target CD30 on lymphoma cells and then generate endocytosis so as to induce apoptosis of tumor cells.

Mylotarg and Adcetris are targeted therapies against hematological tumors, which are relatively simple in tissue structure compared to solid tumors. In 2013, kadcyla (ado-trastuzumab emtansine, T-DM 1) was approved by the United states FDA for the treatment of HER2 positive patients with advanced or metastatic breast cancer who were both Tratuzumab (trade name: herceptin) and paclitaxel resistant (WO 2005037992; US 8088387). Kadcyla is the first ADC drug approved by the FDA in the united states to treat solid tumors.

There are several classes of small molecules with cytotoxicity for antibody drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. The report that the 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) has been disclosed in WO2014057687 as being applied to antibody coupled drugs (ADC); 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 one class of ligand-drug conjugates of the general formula (Pc-L-Y-Dr) has good antitumor activity, and the structure is 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 as follows:

the crystal structure of pharmaceutically active ingredients and intermediates thereof often affects the chemical stability thereof, and differences in crystallization conditions and storage conditions may lead to changes in the crystal structure of the compounds, sometimes accompanied by other forms of the crystal. Generally, amorphous products have no regular crystal structure, and often have other defects such as poor product stability, finer crystallization, difficult filtration, easy caking, poor flowability, and the like. Therefore, there is a need to improve the properties of the above products in all aspects, and we need to find new crystal forms with higher purity and good chemical stability.

Disclosure of Invention

The present disclosure provides a novel crystalline form of a compound of formula I and methods for preparing the same.

The present disclosure provides a crystalline form C of a compound of formula I having an X-ray powder diffraction pattern with 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 crystalline form C of a compound of formula I having an X-ray powder diffraction pattern with 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 certain embodiments, the present disclosure provides a crystalline form C of a compound of formula I having an X-ray powder diffraction pattern as shown in fig. 4.

The present disclosure provides a D-form of a compound of formula I having an X-ray powder diffraction pattern with characteristic peaks at 2Θ angles of 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, 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 D crystalline form of a compound of formula I having an X-ray powder diffraction pattern as shown in figure 5.

The present disclosure provides a crystalline form E of a compound of formula I having an X-ray powder diffraction pattern with characteristic peaks at 2Θ angles of 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, 17.9, 19.3, 20.4, 21.3, and 22.3.

In certain embodiments, the present disclosure provides a crystalline form E of a compound of formula I having an X-ray powder diffraction pattern as shown in fig. 6.

The present disclosure provides a form F of a compound of formula I having an X-ray powder diffraction pattern with 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 form F of a compound of formula I having an X-ray powder diffraction pattern as shown in figure 7.

The present disclosure further provides a process for preparing form C of a compound of formula I, comprising: and mixing the compound shown in the formula I with a proper amount of solvent, and crystallizing out, wherein the solvent is tetrahydrofuran.

The present disclosure further provides a process for preparing form D of a compound of formula I, comprising: mixing the compound shown in the formula I with a proper amount of solvent, pulping overnight for crystallization, wherein the solvent is acetonitrile.

The present disclosure further provides a process for preparing form E of a compound of formula I, comprising: and placing the A crystal form of the compound shown in the formula I under a humidity condition, and carrying out crystal transformation.

The present disclosure further provides a process for preparing form F of a compound of formula I, comprising: mixing a compound shown in a formula I with a proper amount of solvent, pulping for 12 hours for crystallization, wherein the solvent is acetonitrile.

The crystalline forms obtained by the present disclosure were subjected to structural determination, crystalline form study by X-ray powder diffraction pattern (XRPD), differential Scanning Calorimetry (DSC).

The crystallization methods of the crystalline forms in this disclosure are conventional, such as volatile crystallization, temperature-reduced crystallization, or crystallization at room temperature.

The starting materials used in the methods of preparing the crystalline forms of the present disclosure may be any form of the compounds of formula I, including, but not limited to: amorphous, any crystalline form, hydrate, solvate, etc.

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

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

In certain embodiments, the Pc is an antibody or antigen-binding fragment thereof, selected from the group consisting of a chimeric, humanized or fully human antibody; monoclonal antibodies are preferred.

In certain embodiments, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an anti-HER 2 (ErbB 2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3 (ErbB 3) antibody, an anti-HER 4 (ErbB 4) 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-gpgpb antibody, an anti-intel antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, or an anti-Mesothelin antibody, or antigen binding fragment thereof.

In certain embodiments, wherein the antibody or antigen binding fragment thereof is selected from Trastuzumab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR and glematumaab, or antigen binding fragments thereof.

In the description and claims of the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. However, for a better understanding of the present disclosure, definitions and explanations of some related terms are provided below. In addition, when the definition and interpretation of terms provided by the present application are inconsistent with the meanings commonly understood by those skilled in the art, the definition and interpretation of terms provided by the present application is in control.

The term "beating" as used in this disclosure refers to a method of purifying by utilizing the characteristic that a substance is poorly soluble in a solvent, but impurities are well soluble in a solvent, and the beating purification can remove color, change a crystal form or remove a small amount of impurities.

The "X-ray powder diffraction pattern or XRPD" described in this disclosure refers to a bragg equation that is satisfied when X-rays are incident on an atomic plane of a crystal or a portion of a crystal sample having a d-lattice plane spacing at a glancing angle θ (the complementary angle of incidence, also called bragg angle) according to bragg formula 2dsin θ=nλ (where λ is the wavelength of the X-rays, and the number of diffraction orders n is any positive integer, typically taking the first order diffraction peak, n=1).

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 calorimetric analysis or DSC disclosed by the disclosure means that the temperature difference and the heat flow difference between a sample and a reference object are measured in the process of heating or constant temperature of the sample so as to represent all physical changes and chemical changes related to thermal effects and obtain phase change information of the sample.

The term "2θ or 2θ angle" as used in this disclosure refers to the diffraction angle, θ is the bragg angle, the units are degrees or degrees, and the error range of 2θ is ±0.3 or ±0.2 or ±0.1.

The term "interplanar spacing or interplanar spacing (d value)" as used in this disclosure refers to the fact that the spatial lattice selects 3 non-parallel unit vectors a, b, c that connect two adjacent lattice points, which divide the lattice into juxtaposed parallelepiped units, referred to as interplanar spacing. The space lattice is divided according to the determined parallelepipedal unit lines to obtain a set of rectilinear grids, called space lattices or lattices. The lattice and the lattice respectively reflect the periodicity of the crystal structure by using geometric points and lines, and the surface pitches (i.e. the distance between two adjacent parallel crystal surfaces) of different crystal surfaces are different; the unit is thatOr angstroms.

The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug through a stable linking unit. In the present disclosure "ligand-drug conjugate" is preferably an antibody-drug conjugate (antibody drug conjugate, ADC), meaning that a monoclonal antibody or antibody fragment is linked to a biologically active toxic drug via a stable linking unit.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in J.biol. Chem,243, p3558 (1968).

The term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed by joining two identical heavy chains and two identical light chains via interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α, and epsilon chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. 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 to cell surface antigens on target cells, non-limiting examples being the following antibodies: one or more of an anti-HER 2 (ErbB 2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3 (ErbB 3) antibody, an anti-HER 4 (ErbB 4) 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-intel antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, or an anti-Mesothelin antibody; trastuzumab (trade name Herceptin), pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), nimuzumab (Nimotuzumab, trade name tenascin), enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR and glematumaab are preferred.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. 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) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: 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 are referred to as HCDR1, HCDR2 and HCDR3.

Antibodies of the present disclosure include murine, chimeric, humanized and fully human antibodies, with humanized and fully human antibodies being preferred.

The term "murine antibody" is used in this disclosure to refer to antibodies prepared by murine methods according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a specific antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available in Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured for CDRs by phage display. Further references describing methods of using mouse antibodies for the humanization involved include, for example, queen et al, proc., natl. Acad. Sci. USA,88, 2869, 1991 and methods of Winter and co-workers [ Jones et al, nature,321, 522 (1986), riechmann et al, nature,332, 323-327 (1988), verhoeyen et al, science,239, 1534 (1988) ].

The terms "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", are used to eliminate immunogenicity and toxic side effects by making both the variable and constant regions of the antibody human. Monoclonal antibody development has undergone 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 related 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), 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 an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments contained in the "antigen-binding fragment" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of the single arm of the antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature 341:544-546) consisting of VH domains; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked 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, using recombinant methods, by a synthetic linker, so that they can produce a single protein chain (known as a single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and are prepared byFragments were screened for utility in the same manner as for intact antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

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

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 linked at hinge positions, 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 antigen binding activity obtained by cleavage of disulfide bonds in the hinge region of the above F (ab') 2.

In addition, the Fab ' may be produced by inserting DNA encoding a Fab ' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryotic organism 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) connected by a linker. Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -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. USA 90:6444-6448). 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, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancerImmunol.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily 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 interface. As used herein, the Kabat definition of a CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3, or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3, or H2, H3) of the heavy chain variable domain.

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 the variable domain. Essentially, it is a variable domain that does not have CDRs.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes generally 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., epitope Mapping Protocols in Methods in Molecular B iology, volume 66, g.e.Morris, ed. (1996).

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

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 may 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 and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate 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 guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the application are engineered to incorporate one or more human FR regions into the non-human CDR regions. Human FR germline sequences can be obtained from the website http:// IMGT. Cines. FR of ImMunoGeneTics (IMGT), or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with 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 that are susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); the family of bacillus (bacillus) such as bacillus subtilis (Bacillus subtilis); pneumococci (pneumococci); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

The 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 can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. 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 group" means that an alkyl group may be, but is not necessarily, present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

The term "pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients 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, which is safe and effective when used in a mammal, and which has the desired biological activity, an antibody-antibody drug conjugate compound of the present disclosure containing at least one amino group and thus being capable of forming a salt with an acid, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

The term "solvate" or "solvent 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 amount of cytotoxic drug loaded per ligand in the molecule of formula (I), which may also be expressed as a ratio of the amount of drug to the amount of antibody, and the drug loading may range from 0 to 12, preferably 1 to 10, cytotoxic drugs (D) per ligand (Pc). In embodiments of the present disclosure, the drug loading is expressed as n, and may be, for example, an average of 1,2,3,4,5,6,7,8,9, 10. The number of drug molecules per ADC molecule after the coupling 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 linking unit, and typically 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) The molar ratio of the connecting reagent to the monoclonal antibody is controlled,

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

(3) Different reagents are selected.

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

The term "carrier" as used in this disclosure refers to a system that alters the manner and distribution of a drug into the body, controls the release rate of the drug, and delivers the drug to a targeted organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects and improve bioavailability. For example, a polymer surfactant which can be used as a carrier can be self-assembled due to the unique amphiphilic structure of the polymer surfactant to form various forms of aggregates, and preferable examples are micelles, microemulsions, gels, liquid crystals, vesicles and the like. These aggregates have the ability to entrap drug molecules while having good permeability to membranes and can be used as good drug carriers.

Drawings

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

Figure 2 is an XRPD pattern of form a of the compound of formula I.

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

Fig. 4 is an XRPD pattern of form C of the compound of formula I.

Figure 5 is an XRPD pattern of form D of the compound of formula I.

FIG. 6 is an XRPD pattern for form E of the compound of formula I.

Figure 7 is an XRPD pattern of form F of the compound of formula I.

Detailed Description

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

Test conditions of the instrument used for the test:

1. differential scanning calorimeter (Differential Scanning Calorimeter DSC)

Instrument model: mettler Toledo DSC 3+

Sweep gas: nitrogen gas

Rate of temperature rise: 10.0 ℃/min

Temperature range: 25-300 ℃ (25-200 ℃)

2. X-ray diffraction spectrum (X-ray Powder Diffraction, XRPD)

Instrument model: BRUKER D8 ADVANCE X-ray powder diffractometer

Rays: monochromatic Cu-K alpha rays (Cu-K alpha 1 wavelength isCu-K alpha 2 wavelength isThe Cu-K alpha wavelength takes the weighted average of K alpha 1 and K alpha 2)

Scanning mode: θ/2θ, scan range: 3-45 degrees,

voltage: 40KV, current: 40mA;

example 1

The compound of formula I was prepared according to example 9 of WO2020063676 and the resulting product was amorphous as measured by X-ray powder diffraction and the XRPD pattern is shown in figure 1.

Example 2

10mg of the compound of formula I was added to 1mL of methylene chloride, stirred at room temperature, centrifuged, and dried in vacuo to give the product. The product was defined as form a as measured by X-ray powder diffraction, the XRPD pattern shown in figure 2 and the characteristic peak positions shown in table 1. DSC results show that the endothermic peak is 141.78 ℃ and 162.11 DEG C

TABLE 1

Example 3

10mg of the compound of formula I was added to 0.4mL of acetonitrile, stirred at room temperature, centrifuged, and dried in vacuo to give the product. The product was defined as form B as measured by X-ray powder diffraction, the XRPD pattern shown in figure 3 and the characteristic peak positions shown in table 2. DSC results showed an endothermic peak of 142.10 ℃.

TABLE 2

Example 4

10mg of the compound represented by formula I was added to 1mL of tetrahydrofuran, stirred at room temperature, centrifuged, and dried in vacuo to give the product. The product was defined as form C as measured by X-ray powder diffraction, the XRPD pattern shown in figure 4 and the characteristic peak positions shown in table 3. DSC results showed an endothermic peak of 153.85 ℃.

TABLE 3 Table 3

Example 5

10mg of the compound shown in the formula I is added into 1mL of tetrahydrofuran, heated to 50 ℃ for dissolving, cooled to room temperature for stirring, centrifuged and dried in vacuum to obtain the product. The product is C crystal form through X-ray powder diffraction detection.

Example 6

150mg of the compound of formula I was added to 4mL of tetrahydrofuran, 1mg of seed crystal (example 4) was added, stirred at room temperature, centrifuged, and dried in vacuo to give the product. The product is C crystal form through X-ray powder diffraction detection.

Example 7

150mg of the compound of formula I was added to 3mL of acetonitrile, stirred overnight at room temperature, and centrifuged to give the product. The product was defined as form D as measured by X-ray powder diffraction, the XRPD pattern shown in figure 5 and the characteristic peak positions shown in table 4.

TABLE 4 Table 4

Example 8

10mg of form A was placed under 75% RH and 93% RH humidity for 7 days and more to give the target product. The product was defined as form E as measured by X-ray powder diffraction, the XRPD pattern shown in figure 6 and the characteristic peak positions shown in table 5.

TABLE 5

Example 9

15mg of the crystal form A is placed in a dynamic moisture adsorption instrument, and a target product is obtained through a moisture absorption experiment (humidity gradient: 50% -95% -0% -95% -0% -50% RH). The product is E crystal form through X-ray powder diffraction detection.

Example 10

150mg of the compound of formula I was added to 4mL of acetonitrile, stirred at room temperature for 12 hours, centrifuged, and dried in vacuo to give the product. The product was defined as form F as measured by X-ray powder diffraction, the XRPD pattern shown in figure 7 and the characteristic peak positions shown in table 6.

TABLE 6

Example 11

The A, B, C crystal form sample is spread open and placed, the stability of the sample under the conditions of high temperature (40 ℃ C., 60 ℃) and high humidity (RH 75% and RH 92.5%) is inspected respectively, the sampling inspection period is 30 days, and the results are shown in Table 7.

TABLE 7

Example 11

The stability of the C-form samples was examined at-20deg.C, 4deg.C, 25deg.C/60% RH and 40deg.C/75% RH, respectively, and the results are shown in Table 8.

TABLE 8

Claims

A C crystal form of a compound shown in a formula I has characteristic peaks at 2 theta angles of 6.0, 8.8, 10.3, 12.2 and 15.5 in an X-ray powder diffraction pattern,
form C of the compound of formula I according to claim 1, having an X-ray powder diffraction pattern with 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.
Form C of the compound of formula I according to claim 1, having an X-ray powder diffraction pattern as shown in figure 4.
A crystalline form D of a compound of formula I having an X-ray powder diffraction pattern with characteristic peaks at 2Θ angles of 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, 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 D-form of the compound of formula I according to claim 4, having an X-ray powder diffraction pattern as shown in figure 5.
A crystalline form E of a compound of formula I having an X-ray powder diffraction pattern with characteristic peaks at 2Θ angles of 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, 17.9, 19.3, 20.4, 21.3 and 22.3.
The form E of the compound of formula I according to claim 6, having an X-ray powder diffraction pattern as shown in figure 6.
A form F of a compound of formula I having an X-ray powder diffraction pattern with 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.
Form F of the compound of formula I according to claim 8, having an X-ray powder diffraction pattern as shown in figure 7.
The crystalline form of the compound of formula I according to any one of claims 1-9, wherein the error range of the 2Θ angle is ± 0.2.
A process for preparing form C of a compound of formula I according to any one of claims 1 to 3, which process comprises: and mixing the compound shown in the formula I with a proper amount of solvent, and crystallizing out, wherein the solvent is tetrahydrofuran.
A method for preparing a ligand-drug conjugate shown in formula II or pharmaceutically acceptable salt or solvate thereof comprises the steps of reducing the ligand and then carrying out coupling reaction with the crystal form of the compound shown in formula I,

wherein Pc represents a ligand, and n is 1 to 10, and may be an integer or a decimal.
The method of preparation according to claim 12, wherein the Pc is an antibody or an antigen binding fragment thereof, said antibody being selected from the group consisting of chimeric, humanized or fully human antibodies, preferably monoclonal antibodies, more preferably HER2 (ErbB 2) antibodies, anti-EGFR antibodies, anti-B7-H3 antibodies, anti-C-Met antibodies, anti-HER 3 (ErbB 3) antibodies, anti-HER 4 (ErbB 4) antibodies, anti-CD 20 antibodies, anti-CD 22 antibodies, anti-CD 30 antibodies, anti-CD 33 antibodies, anti-CD 44 antibodies, anti-CD 56 antibodies, anti-CD 70 antibodies, anti-CD 73 antibodies, anti-CD 105 antibodies, anti-CEA antibodies, anti-a 33 antibodies, anti-Cripto antibodies, anti-EphA 2 antibodies, anti-G250 antibodies, anti-MUCl antibodies, anti-Lewis Y antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-gpgrin antibodies, PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC 44A4 antibodies or anti-Mesothelin antibodies or antigen binding fragments thereof.