Pharmaceutical composition containing an antibody-drug conjugate and its use

Abstract

The present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate in a buffer solution, the antibody-drug conjugate having a structure represented by the general formula (Pc-LYD), further comprising a sugar and a surfactant. [Formula 1] JPEG2023518583000121.jpg5564

Description

Technical Field

[0001] This application claims the priority of a Chinese patent application (Application No. CN 202010219601.7) filed on March 25, 2020 and a Chinese patent application (Application No. CN 202110287012.7) filed on March 17, 2021. The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing an antibody-drug conjugate, and its use as an anticancer agent.

Background Art

[0002] The descriptions herein do not necessarily constitute prior art, but only provide background information related to the present disclosure.

[0003] An antibody-drug conjugate (ADC) links a monoclonal antibody or antibody fragment to a cytotoxic agent having biological activity by a stable chemical linker compound, and utilizes the specificity of the binding of the antibody to the surface antigens of normal cells and tumor cells and the high efficacy of the cytotoxic agent, while avoiding the defects that the therapeutic effect of the former is relatively low and the toxicity and side effects of the latter are too large. This means that, compared with conventional chemotherapeutic drugs, the antibody-drug conjugate can accurately bind to 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).

[0004] In 2000, Mylotarg (R) (gemtuzumab ozogamicin, Wyeth Pharmaceuticals Inc.) was approved for sale by the US FDA 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).

[0005] In August 2011, Adcetris (R) (brentuximab vedotin, Seagen) was approved by the US FDA under accelerated review for use in the treatment of Hodgkin lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). Adcetris (R) is a novel ADC-targeted drug that can induce apoptosis of tumor cells by directly acting on the target CD30 in lymphoma cells and then generating endocytosis.

[0006] Mylotarg (R) and Adcetris (R) both perform targeted therapy for hematological malignancies, which have a simpler tissue structure compared to solid tumors. In February 2013, Kadcyla (R) (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for use in the treatment of patients with progressive or metastatic breast cancer that is HER2-positive and drug-resistant to trastuzumab (trade name: Herceptin) and taxol (WO2005037992, US8088387). Kadcyla (R) is the first ADC drug approved by the US FDA for the treatment of solid tumors.

[0007] There are several types of small molecules with cytotoxicity used in antibody-drug conjugates. One of them is a camptothecin derivative that has antitumor effects by inhibiting topoisomerase I. Documents reporting the application of exatecan, a camptothecin derivative (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-drug conjugates (ADCs) include WO2014057687, Clinical Cancer Research (2016) 22(20):5097-5108, and Cancer Sci (2016) 107:1039-1046. However, further development of ADC drugs with better therapeutic effects is still necessary.

[0008] However, since ADCs have a more complex heterostructure than antibodies, they pose an even greater challenge to therapeutic ADC formulations.

Summary of the Invention

[0009] The present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has the general formula (Pc-L-Y-D), that is,

Chemical formula

[0010] In alternative embodiments, the pH value of the buffer in the pharmaceutical composition is from about 4.5 to about 6.0, non-limiting examples include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, preferably from about 4.8 to about 5.3, more preferably from about 5.0 to about 5.1. In some embodiments, the pharmaceutical composition has a pH of 4.5 to 5.2, preferably a pH of 4.8 to 5.2, more preferably a pH of 5.0 to 5.1. In some embodiments, the pharmaceutical composition has a pH of 5.0.

[0011] In alternative embodiments, the pharmaceutical composition further comprises a surfactant selected from polysorbate, polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfonate, sodium dodecyl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoate sodium, methyl oleoyl taurine sodium, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0012] In alternative embodiments, the concentration of the surfactant in the pharmaceutical composition is from about 0.01 mg / mL to about 1.0 mg / mL. In alternative embodiments, the concentration of the surfactant in the pharmaceutical composition is from about 0.05 mg / mL to about 0.5 mg / mL, preferably from about 0.1 mg / mL to about 0.3 mg / mL, from about 0.2 mg / mL to about 0.6 mg / mL, from about 0.2 mg / mL to about 0.5 mg / mL or from about 0.2 mg / mL to about 0.3 mg / mL, more preferably about 0.2 mg / mL, and non-limiting examples include 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL.

[0013] In alternative embodiments, the pharmaceutical composition further comprises a sugar. The "sugar" of the present disclosure includes ordinary compositions (CH 2 O) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugar may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltose alcohol, lactose alcohol, iso-maltulose, etc. Preferred sugars are non-reducing disaccharides, more preferably trehalose or sucrose, and most preferably sucrose.

[0014] In alternative embodiments, the concentration of the sugar in the pharmaceutical composition is from about 60 mg / mL to about 90 mg / mL, and non-limiting examples include 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, preferably 80 mg / mL. In some embodiments, the concentration of the sugar is from 70 mg / mL to 90 mg / mL.

[0015] In alternative embodiments, the concentration of the antibody-drug conjugate in the pharmaceutical composition is from about 1 mg / mL to about 100 mg / mL, non-limiting examples include 1 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, preferably from about 10 mg / mL to about 30 mg / mL, more preferably from about 20 mg / mL to about 22 mg / mL. Specifically, non-limiting examples include 20.1 mg / mL, 20.2 mg / mL, 20.3 mg / mL, 20.4 mg / mL, 20.5 mg / mL, 20.6 mg / mL, 20.7 mg / mL, 20.8 mg / mL, 20.81 mg / mL, 20.82 mg / mL, 20.83 mg / mL, 20.84 mg / mL, 20.85 mg / mL, 20.86 mg / mL, 20.87 mg / mL, 20.88 mg / mL, 20.89 mg / mL, 20.9 mg / mL, 20.9 mg / mL, 20.91 mg / mL, 20.92 mg / mL, 20.93 mg / mL, 20.94 mg / mL, 20.95 mg / mL, 20.96 mg / mL, 20.97 mg / mL, 20.98 mg / mL, 20.99 mg / mL, 21 mg / mL. In alternative embodiments, the concentration of the antibody-drug conjugate in the pharmaceutical composition is, for the naked antibody (i.e., the antibody portion in the ADC), from about 10 mg / mL to about 30 mg / mL, preferably about 20 mg / mL.

[0016] In alternative embodiments, the buffer in the pharmaceutical composition is selected from histidine salt buffers, succinate buffers, and citrate buffers, preferably a succinate buffer, more preferably a succinic acid-sodium succinate buffer.

[0017] In alternative embodiments, the concentration of the buffer in the pharmaceutical composition is from about 5 mM to about 50 mM, and non-limiting examples include 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM, preferably from about 5 mM to about 20 mM, and most preferably about 10 mM.

[0018] In alternative embodiments, as the range of the drug loading amount (n), each antibody or its antigen-binding fragment (Pc) may bind to 3 to 8, 4 to 8, 5 to 7, more preferably 5.3 to 6.1, 5.7 cytotoxic agents. n is a decimal or an integer.

[0019] In alternative embodiments, the pharmaceutical composition (a) the antibody-drug conjugate of about 10 mg / mL to about 30 mg / mL, (b) polysorbate of about 0.05 mg / mL to about 0.5 mg / mL, (c) sugar of about 60 mg / mL to about 90 mg / mL, and (d) buffer of about 5 mM to about 20 mM, and the pH of the pharmaceutical composition is about 4.8 to about 5.3, In alternative embodiments, the pharmaceutical composition (a) the antibody-drug conjugate of about 10 mg / mL to about 30 mg / mL, (b) polysorbate of about 0.05 mg / mL to about 0.5 mg / mL, (c) sugar of about 60 mg / mL to about 90 mg / mL, and (d) buffer of about 5 mM to about 20 mM, and the pH of the pharmaceutical composition is 4.8 to 5.2, In alternative embodiments, the pharmaceutical composition (a) The above antibody-drug conjugate at about 20 mg / mL to about 22 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at 80 mg / mL, and (d) 10 mM succinate buffer, and the pH of the above pharmaceutical composition is about 5.0 to about 5.1. In some embodiments, the above pharmaceutical composition has a pH of 5.0 to 5.1.

[0020] In alternative embodiments, the antibody-drug conjugate has -Y- is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen, and an alkyl group, R 1 is a C 3-7 cycloalkylalkyl group or a C 3-7 cycloalkyl group, R 2 is selected from a hydrogen atom, a haloalkyl group, and a C 3-7 cycloalkyl group, preferably a hydrogen atom, or, R 1 and R 2 together with the carbon atom to which they are attached form a C 3-7 cycloalkyl group, m is 0 or 1.

[0021] In alternative embodiments, the antibody-drug conjugate has -Y- is -O-(CH 2 ) m -CR 1 R 2 -C(O)-, R 1 is a C 3-7 cycloalkylalkyl group or a C 3-7 cycloalkyl group, R 2 is selected from a hydrogen atom, a haloalkyl group, and a C 3-7selected from cycloalkyl groups, or R 1 and R 2 together with the carbon atoms to which they are attached form a C 3-7 cycloalkyl group, m is 0 or 1.

[0022] In an alternative embodiment, the antibody-drug conjugate comprises -Y- is -O-(CH 2 ) m -CR 1 R 2 -C(O)-, R 1 is a C 3-7 cycloalkylalkyl group or a C 3-7 cycloalkyl group, R 2 is a hydrogen atom, or R 1 and R 2 together with the carbon atoms to which they are attached form a C 3-7 cycloalkyl group, m is 0 or 1.

[0023] In an alternative embodiment, the antibody-drug conjugate comprises -Y- is -O-(CH 2 ) m -CR 1 R 2 -C(O)-, R 1 is a C 3-7 cycloalkylalkyl group or a C 3-7 cycloalkyl group, R 2 is a hydrogen atom, or R 1 and R 2 together with the carbon atoms to which they are attached form a C 3-7 cycloalkyl group, m is 0.

[0024] In an alternative embodiment, the antibody-drug conjugate comprises -Y- is [Chemical formula] selected from the following.

[0025] In an alternative embodiment, the antibody-drug conjugate comprises - The O-terminus of -Y is linked to the linker unit L.

[0026] In an alternative embodiment, the antibody-drug conjugate comprises -Y- is [Chemical formula] selected from the following.

[0027] In an alternative embodiment, the antibody-drug conjugate has a structure represented by the general formula (Pc-L-D 1 ), that is, [Chemical formula] Among them, R 1 is a cycloalkylalkyl group or a cycloalkyl group, preferably a C 3-7 cycloalkylalkyl group or a C 3-7 cycloalkyl group, R 2 is selected from a hydrogen atom, a haloalkyl group and a C 3-7 cycloalkyl group, preferably a hydrogen atom, Alternatively, R 1 and R 2 together with the carbon atom to which they are attached form a C 3-7 cycloalkyl group, m is 0 or 1, n is from 1 to 10, which may be an integer or a decimal, Pc is an antibody or an antigen-binding fragment thereof, and L is a linker unit.

[0028] In a selective embodiment, in the above antibody-drug conjugate, n is from 2 to 8, which may be an integer or a decimal, preferably from 3 to 8, which may be an integer or a decimal.

[0029] In a selective embodiment, in the above antibody-drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, and among them, L 1 is selected from -(succinimid-3-yl-N)-W-C(O)-, -CH 2 -C(O)-NR 3 -W-C(O)- and -C(O)-W-C(O)-, and among them, W is C 1-8 alkyl group, C 1-8 alkyl group-cycloalkyl group and linear heteroalkyl group with 1 to 8 atoms, and the above heteroalkyl group contains 1 to 3 heteroatoms selected from N, O and S. Among them, the above C 1-8 alkyl group, cycloalkyl group and linear heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group, L 2 is -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 )p 1 C(O)- and chemical bond, and among them, p 1 is an integer from 1 to 20, and L 2 is preferably a chemical bond, L 3is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group, L 4 is -NR 5 (CR 6 R 7 )t-, -C(O)NR 5 , -C(O)NR 5 (CH 2 )t- and a chemical bond, wherein t is an integer from 1 to 6, and L 4 is preferably -NR 5 (CR 6 R 7 )t-, R 3 , R 4 and R 5 are the same or different and each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group, R 6 and R 7 are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0030] In an alternative embodiment, the antibody-drug conjugate has a linker unit L 1 which is -(succinimid-3-yl-N)-(CH 2 )s 1 -C(O)-, -(succinimid-3-yl-N)-CH 2 -cyclohexyl-C(O)-, -(succinimid-3-yl-N)-(CH 2 CH 2 O)s 2 -CH 2 CH 2 -C(O)-, -CH 2 -C(O)-NR 3 -(CH 2 )s 3 -C(O)- and -C(O)-(CH 2 )s4 Selected from C(O)-, among which, s 1 is an integer from 2 to 8, s 2 is an integer from 1 to 3, s 3 is an integer from 1 to 8, s 4 is an integer from 1 to 8, and s 1 5 is preferred.

[0031] In an alternative embodiment, the antibody-drug conjugate comprises a linker unit L 2 is -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)- or a chemical bond, and p 1 is an integer from 6 to 12.

[0032] In an alternative embodiment, the antibody-drug conjugate comprises L 4 is -NR 5 (CR 6 R 7 )t-, R 5 is a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently is a hydrogen atom or an alkyl group, t is 1 or 2, preferably 2, and L 4 is -NR 5 CR 6 R 7 - is preferred, and -NHCH 2 - is more preferred.

[0033] In an alternative embodiment, the linker unit -L- of the antibody-drug conjugate is -L 1 -L 2 -L 3 -L 4 -, and L 1 is

Chemical formula

[0034] In an alternative embodiment, in the above antibody-drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -. L 1 is -(succinimid-3-yl-N)-CH 2 -cyclohexyl-C(O)-. L 2 is -NR 4 (CH 2 CH 2 O) 9 CH 2 C(O)-. L 3 is a tetrapeptide residue, L 4 is -NR 5 (CR 6 R 7 )t-, where R 5 is a hydrogen atom or an alkyl group, and R 6 and R 7 are the same or different and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2.

[0035] In an alternative embodiment, in the above antibody-drug conjugate, the above L 3The peptide residue consists of one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), aspartic acid (N), preferably an amino acid residue consisting of one, two or more amino acids selected from phenylalanine and glycine, more preferably a tetrapeptide residue, and most preferably a tetrapeptide residue of GGFG (glycine - glycine - phenylalanine - glycine).

[0036] In an alternative embodiment, in the above antibody - drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, and its L 1 terminus is linked to the antibody or its antigen - binding fragment, and the L 4 terminus is linked to Y.

[0037] In an alternative embodiment, in the above antibody - drug conjugate, the -L - Y- is

Chemical formula

[0038] In an alternative embodiment, in the above antibody-drug conjugate, the above -L-Y- is

Chemical formula

Chemical formula

[0039] In an alternative embodiment, in the above antibody-drug conjugate, the above -L-Y- is

Chemical formula

[0040] In a selected embodiment, in the above antibody-drug conjugate, the above -L-Y- is

Chemical formula

[0041] In a selective embodiment, in the above antibody-drug conjugate, the above -L-Y- is

Chemical formula

[0042] In a selective embodiment, the above antibody-drug conjugate has the general formula (Pc-L a -Y-D), that is,

Chemical formula

[0043] In an alternative embodiment, the antibody-drug conjugate has the structure represented by the general formula (Pc-L b -Y-D), i.e.,

Chemical formula

[0044] In alternative embodiments, the antibody-drug conjugate, the -L-Y- includes, but is not limited to, the following.

[0045]

Table 1-1

Table 1-2

[0046] In alternative embodiments, the antibody-drug conjugate has the following structure, namely,

Table 2-1

Table 2-2

Table 2-3

[0047] In alternative embodiments, the antibody-drug conjugate has the following formula, namely,

Chemical formula

[0048] In alternative embodiments, the Pc is an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody, preferably a monoclonal antibody.

[0049] In alternative embodiments, the Pc is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 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-SLC44A4 antibody, and an anti-Mesothelin antibody, or an antigen-binding fragment thereof.

[0050] In alternative embodiments, in the above antibody-drug conjugate, the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab, or an antigen-binding fragment thereof.

[0051] In alternative embodiments, the above antibody-drug conjugate has a structure represented by the following formula. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0052] Among them, n is a non-zero integer or decimal from 0 to 10, preferably an integer or decimal from 1 to 10, more preferably from 2 to 8, and can be either an integer or a decimal, most preferably from 3 to 8, and can be either an integer or a decimal.

[0053] In an alternative embodiment, the antibody-drug conjugate in the pharmaceutical composition has the following formula, namely,

Chemical formula

[0054] The present disclosure provides a pharmaceutical composition comprising (a) the antibody-drug conjugate at about 10 mg / mL to about 30 mg / mL, (b) polysorbate at about 0.05 mg / mL to about 0.5 mg / mL, (c) sugar at about 60 mg / mL to about 90 mg / mL, and (d) buffer at about 5 mM to about 20 mM, and the composition has a pH of 4.8 to 5.2, Among them, the antibody-drug conjugate has the following formula, namely,

Chemical formula

[0055] The present disclosure provides a pharmaceutical composition comprising (a) the antibody-drug conjugate at about 20 mg / mL to about 22 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 80 mg / mL, and (d) succinate buffer at about 10 mM, and the pharmaceutical composition has a pH of 5.0 to 5.1, Among them, the antibody-drug conjugate has the following formula, namely,

Chemical formula

[0056] The present disclosure further provides a lyophilized preparation comprising an antibody-drug conjugate, characterized in that the pharmaceutical composition can be formed after redissolving the above preparation.

[0057] The present disclosure further provides a method for preparing a lyophilized preparation comprising an antibody-drug conjugate, the method comprising the step of lyophilizing the above pharmaceutical composition.

[0058] In an alternative embodiment, the lyophilization in the method for preparing a lyophilized preparation comprising an antibody-drug conjugate comprises the steps of pre-freezing, primary drying and secondary drying in sequence. Lyophilization is carried out by freezing the preparation and subsequent sublimation of water at a temperature suitable for primary drying. Under these conditions, the temperature of the product is lower than the eutectic point or collapse temperature of the preparation. Usually, the temperature range for primary drying is about -30 to 25 °C (assuming the product remains frozen during the primary drying process). The time required for drying is determined by the preparation, the size and type of the container (e.g., glass vial) containing the sample and the volume of the liquid, and the range of the above time may be from several hours to several days (e.g., 40 to 60 hours). The secondary drying stage may be carried out at about 0 to 40 °C, which is mainly determined by the type and size of the container and the type of protein used. The time for secondary drying is determined by the desired remaining moisture level in the product and usually at least about 5 hours is required. Usually, the water content of the lyophilized preparation is lower than about 5%, preferably lower than about 3%. The pressure may be the same as the pressure applied in the primary drying step, and preferably, the pressure for secondary drying is lower than that for primary drying. The conditions for lyophilization may be varied depending on the preparation and the size of the vial.

[0059] In an alternative example of the present disclosure, 5 mL of the stock solution of the pharmaceutical composition is lyophilized, and the lyophilization process has a pre-freezing temperature of -5 °C or -45 °C, a primary drying temperature of -20 °C, a vacuum degree of 10 Pa, a secondary drying temperature of 25 °C, and a vacuum degree of 1 Pa.

[0060] In some embodiments, the lyophilized formulation is stable at 2 - 8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

[0061] The present disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, which is obtained by lyophilizing a pharmaceutical composition comprising the above anti-HER2 antibody-drug conjugate.

[0062] The present disclosure further provides a reconstituted solution comprising an antibody-drug conjugate, which is characterized by being obtained by reconstituting the above lyophilized formulation.

[0063] The present disclosure further provides a method for preparing the above reconstituted solution, which comprises a step of reconstituting the above lyophilized formulation, and the solution used for reconstitution is selected from, but not limited to, water for injection, physiological saline, or glucose solution.

[0064] In an alternative embodiment, the reconstituted solution comprises the following components, namely, (a) about 10 mg / mL - 30 mg / mL of the above antibody-drug conjugate, (b) about 0.05 mg / mL - 0.5 mg / mL of polysorbate, (c) about 60 mg / mL - 90 mg / mL of sugar, and (d) about 5 mM - 20 mM of a buffer, and the pH of the reconstituted solution is 4.8 - 5.2.

[0065] In an alternative embodiment, the reconstituted solution comprises the following components, namely, (a) about 20 mg / mL - 22 mg / mL of the antibody-drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, and (d) about 10 mM of a succinate buffer, and the pH of the reconstituted solution is 5.0 - 5.1.

[0066] The present disclosure further provides a product comprising a container containing the above pharmaceutical composition, freeze-dried formulation or reconstituted solution. In some embodiments, the container is an injection bottle made of neutral borosilicate glass tubing.

[0067] The present disclosure further provides the application of the above pharmaceutical composition, freeze-dried formulation, reconstituted solution or product in the preparation of a drug for treating or preventing tumors.

[0068] The present disclosure further provides a method for treating a disease, the method comprising providing the above pharmaceutical composition, freeze-dried formulation, reconstituted solution or product.

[0069] The present disclosure further provides the above pharmaceutical composition, or freeze-dried formulation, or reconstituted solution, or product as a drug, preferably, the drug is used for the treatment or prevention of tumor diseases.

[0070] In alternative embodiments, the disease or tumor is a cancer associated with HER2, HER3, B7H3 or EGFR expression.

[0071] In alternative embodiments, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

[0072] As is well known to those skilled in the art, one, some or all of the characteristics of each embodiment described in the present disclosure can be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by combination will be further described in the following detailed description.

[0073] The present disclosure provides a pharmaceutical composition that is advantageous in manufacturing and administration and has stable performance. Specifically, the pharmaceutical composition described in the present disclosure comprises an antibody-drug conjugate and a buffer. The term

[0074] To make the present disclosure more easily understood, several technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art.

[0075] This disclosure incorporates all the contents of Application PCT / CN2019 / 107873 (WO2020 / 063676) into this application.

[0076] "Antibody-drug conjugate (ADC)" refers to conjugating an antibody or antibody fragment to a cytotoxic agent with biological activity or a small molecule drug with cell killing activity by a stable chemical linker compound, utilizing the specificity of the antibody to tumor cells or the binding specificity to highly expressed antigens and the high efficacy of the cytotoxic agent to avoid toxicity and side effects on normal cells. Compared with conventional chemotherapeutic drugs, antibody-drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells.

[0077] "Buffer" refers to a buffer that withstands changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine salt, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0078] "Histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include buffers such as histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., preferably histidine-hydrochloride buffer, and histidine-acetate buffer is prepared from histidine and acetic acid, and histidine hydrochloride buffer is prepared from histidine and hydrochloric acid.

[0079] "Citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid - sodium citrate, citric acid - potassium citrate, citric acid - calcium citrate, citric acid - magnesium citrate, etc. A preferred citrate buffer is citric acid - sodium citrate.

[0080] "Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid - sodium succinate, succinic acid - potassium succinate, succinic acid - calcium succinate salt, etc. A preferred succinate buffer is succinic acid - sodium succinate. Exemplarily, the above - mentioned succinic acid - sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0081] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate - sodium dihydrogen phosphate, disodium hydrogen phosphate - potassium dihydrogen phosphate, disodium hydrogen phosphate - citric acid, etc. A preferred phosphate buffer is disodium hydrogen phosphate - sodium dihydrogen phosphate.

[0082] "Acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid - sodium acetate, acetic acid - histidine salt, acetic acid - potassium acetate, acetic acid - calcium acetate, acetic acid - magnesium acetate, etc. A preferred acetate buffer is acetic acid - sodium acetate.

[0083] "Pharmaceutical composition" refers to a mixture containing one or more antibody - drug conjugates or their physiologically / pharmaceutically acceptable salts or prodrugs described herein, and other chemical components such as physiologically / pharmaceutically acceptable vectors and excipients. The pharmaceutical composition is for maintaining the stability of the active ingredient of the antibody, facilitating administration to the living body, contributing to the absorption of the active ingredient, and further exerting biological activity.

[0084] In the present disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0085] The pharmaceutical compositions described in the present disclosure are in solution form, and unless otherwise specified, the solvent therein is water.

[0086] "Lyophilized preparation" refers to a preparation or pharmaceutical composition obtained after subjecting a pharmaceutical composition or liquid or solution preparation in liquid or solution form to a vacuum freeze-drying process.

[0087] As used herein, the terms "about" and "substantially" mean that a numerical value is within an acceptable error range of a specific value measured by a person skilled in the art, depending on how the above numerical part is measured or determined (i.e., the limitations of the measurement system). For example, in each execution in this field, "about" may mean within 1 or a standard deviation exceeding 1. Alternatively, "about" or "substantially includes" may mean a range of at most 20%. Further, particularly for biological systems or processes, the term may mean at most one order of magnitude or at most 5 times the numerical value. Unless otherwise specified, when a specific value appears in the present application and the claims, the meaning of "about" or "substantially includes" should be assumed to be within the acceptable error range of the specific value.

[0088] The pharmaceutical compositions described in the present disclosure can achieve the following stable effects, that is, after storage, the antibody-drug conjugate in the pharmaceutical composition basically retains its physical stability and / or chemical stability and / or biological activity. Preferably, after storage, the pharmaceutical composition basically retains its physical and chemical stability and its biological activity. The storage period is generally selected according to the predetermined shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring the stability of proteins, and the stability after storage for a selected period at a selected temperature can be measured.

[0089] A stable formulation is a formulation that shows no significant changes when stored at refrigerated temperature (2 - 8 °C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. Also, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after being stored for a period including 1 month, 3 months, and 6 months at temperatures including 25 °C. As a typical example of stability, it is measured by SEC-HPLC that the monomeric antibody that has aggregated or decomposed usually does not exceed about 10%, preferably does not exceed about 5%. By visual analysis, the formulation is a pale yellow, almost colorless and transparent liquid, or colorless, or clear to slightly milky white. The concentration, pH, and osmolality of the above formulation do not change by more than ±10%. Usually, a decrease not exceeding about 10%, preferably not exceeding about 5% is seen. Usually, aggregation not exceeding about 10%, preferably not exceeding about 5% is formed.

[0090] When the color and / or clarity is detected visually, or measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), and the antibody-drug conjugate shows no significant increase in aggregation, precipitation, and / or denaturation, the antibody-drug conjugate is considered to "maintain its physical stability" in the pharmaceutical formulation. The conformational change of the protein may be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0091] If the antibody-drug conjugate does not show significant chemical changes, the antibody is considered to "maintain its chemical stability" in the pharmaceutical formulation. The chemical stability can be evaluated by detecting and quantifying chemically modified proteins. The degradation process that constantly changes the chemical structure of the protein includes hydrolysis or cleavage (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as mass spectrometry or peptide mapping combined with MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing electrophoresis, peptide mapping, and measurement of isoaspartic acid), and isomerization (evaluated by measurement of the content of isoaspartic acid, peptide mapping, etc.).

[0092] If the biological activity of the antibody-drug conjugate at a given time is within a predetermined range of the biological activity shown during the preparation of the pharmaceutical formulation, the antibody-drug conjugate is considered to "maintain its biological activity" in the pharmaceutical formulation.

[0093] The three-letter and one-letter codes of amino acids used in the present disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0094] The "antibody" described in the present disclosure is an immunoglobulin. A complete antibody has a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. Immunoglobulins have different antigenicity because of the different amino acid compositions and sequence orders of their heavy chain constant regions. Thus, immunoglobulins can be divided into five types, or are called immunoglobulin isotypes of IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains according to the constant region. Each of the five types of Ig may have a κ chain or a λ chain. The antibody described in the present disclosure is preferably a specific antibody against a cell surface antigen in a target cell. As non-limiting examples, it includes one or more of 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-MUC1 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, and preferably Trastuzumab (Herceptin, trade name), Pertuzumab (also called 2C4, trade name: Perjeta), Nimotuzumab (trade name: Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab.

[0095] The heavy and light chains of an antibody have a large sequence variation in approximately 110 amino acids near the N-terminus, forming the variable region (Fv region), and the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also referred to as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and in the order from the amino group terminus to the carboxyl group terminus, they are FR1, CDR1, FR2, CDR2, FR 3 , CDR 3 , FR 4 . They are arranged in this order. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR 3 , and the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR 3 . The CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment described in the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in terms of quantity and position.

[0096] In the present disclosure, the antibody light chain described in the present disclosure may further include a light chain constant region, and the above light chain constant region includes human or mouse κ, λ chains or variants thereof.

[0097] In the present disclosure, the antibody heavy chain described in the present disclosure may further include a heavy chain constant region, and the above heavy chain constant region includes human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0098] The antibodies of the present disclosure include mouse antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0099] The term "mouse antibody" in the present disclosure refers to an antibody prepared using a mouse by the knowledge and technology in this field. After injecting a specific antigen as a test subject during preparation, hybridomas expressing antibodies with desired sequence or functional characteristics are isolated.

[0100] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, and can reduce the immune response induced by the mouse antibody. To create a chimeric antibody, first, a hybridoma that secretes a mouse-specific monoclonal antibody is created, and the variable region gene is cloned from the mouse hybridoma cells. Furthermore, if necessary, the constant region gene of the human antibody is cloned. After ligating the mouse variable region gene and the human constant region gene to form a chimeric gene, it is inserted into a human vector, and finally, the chimeric antibody molecule is expressed in a eukaryotic cell line or a prokaryotic cell line.

[0101] The term "humanized antibody", also called a CDR-grafted antibody, refers to an antibody generated by transplanting the CDR sequences of a mouse into the framework of a human antibody variable region, i.e., the antibody framework sequences of different types of human germline. It can overcome the strong heterologous reactivity induced by the fact that chimeric antibodies have a large amount of mouse protein components. Such framework sequences can be obtained from a common DNA database containing germline antibody gene sequences or from disclosed references. For example, the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database and discovered from Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing the least number of reverse mutations or revertant mutations on the above human antibody variable region framework sequences. The humanized antibodies of the present disclosure further include humanized antibodies with affinity maturation for CDRs presented by phage.

[0102] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous module (e.g., a cytotoxic module) or a radioactive marker.

[0103] The "antigen-binding fragment of an antibody" described in the present disclosure may refer to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment, a scFv fragment having antigen-binding activity. The Fv fragment contains the variable region of the antibody heavy chain and the variable region of the light chain, but has no constant region and has the smallest antibody fragment of all antigen-binding sites. Generally, the Fv antibody further contains a polypeptide linker between the VH and VL domains and can form a structure necessary for antigen binding. Two antibody variable regions may be linked by different linkers to form a single polypeptide chain called a single chain antibody or single chain Fv (sFv).

[0104] The term "antigen-binding site" of the present disclosure refers to a three-dimensional spatial site on the antigen that is continuous or discontinuous and is recognized by the antibody or antigen-binding fragment of the present disclosure.

[0105] "ADCC" described in the present disclosure, that is, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors directly kill target cells coated with an antibody by recognizing the Fc portion of the antibody. Modification of the Fc portion in IgG can reduce or eliminate the ADCC effector function of the antibody. The above modification refers to performing a mutation selected from the mutations N297A, L234A, L235A of IgG1, IgG2 / 4 chimeras, and F234A / L235A mutations of IgG4 in the heavy chain constant region of the antibody.

[0106] The "mutation" in the mutant sequence described in the present disclosure includes, but is not limited to, "reverse mutation", "conservative modification" or "conservative substitution or replacement". The "conservative modification" or "conservative substitution or replacement" described in the present disclosure means that by substituting an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), it can be frequently changed without changing the biological activity of the protein. Those skilled in the art generally know that a single amino acid substitution in a non-essential region of a polypeptide basically does not change its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p224, (4th edition)). Also, substitution of amino acids with similar structures or functions is less likely to destroy biological activity.

[0107] The "mutant sequence" described in the present disclosure refers to a nucleotide sequence and / or amino acid sequence having a certain percentage of sequence identity different from the nucleotide sequence and / or amino acid sequence of the present disclosure obtained by performing mutation modifications such as appropriate substitutions, insertions, and deletions on the nucleotide sequence and / or amino acid sequence of the present disclosure. The sequence identity described in the present disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The sequence comparison between two sequences and the measurement of the percentage of identity can be performed according to the default settings of the BLASTN / BLASTP algorithm available from the homepage of the National Center For Biotechnology Institute.

[0108] The term "linker unit" or "linker fragment" or "linking unit" refers to a chemical structural fragment or bond having one end linked to an antibody or an antigen-binding fragment thereof and the other end linked to a drug, which may be linked to the antibody or the drug after being linked to another linker. Preferred forms of the present disclosure are L and L 1 ~L 4 as shown, wherein the L 1 end is linked to the antibody and the L 4 end is linked to a compound or toxin after being linked to the structural unit Y.

[0109] The linker includes an extender, a spacer and an amino acid unit and can be synthesized by known methods in the art, such as the method described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photo-labile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0110] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As one of the more preferred prior arts, the mammalian expression system will cause glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. The positive clones are cultured in a serum-free medium in a bioreactor to expand the culture and produce the antibody. The culture supernatant secreting the antibody can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing a conditioned buffer. The non-specifically bound components are washed away. Further, the bound antibody is eluted by a pH gradient method, and the antibody fragment is detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers may be removed by conventional methods such as molecular sieving and ion exchange. The resulting product must be immediately frozen, for example, at -70°C or lyophilized.

[0111] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples thereof include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched-chain isomers thereof, etc.More preferably, it is a lower alkyl group containing 1 to 6 carbon atoms, and non-limiting examples thereof include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, etc. The alkyl group may or may not be substituted. When substituted, the substituent may be substituted at any available linking site. The above substituents are preferably one or more groups independently selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxy group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, oxo group.

[0112] The term "heteroalkyl group" is an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.

[0113] The term "alkylene group" is a saturated straight-chain or branched-chain aliphatic hydrocarbon group having a residue derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of the alkylene group include methylene (-CH 2 -), 1,1-ethylidene (-CH(CH 3 ))-), 1,2-ethylidene (-CH2 CH 2 )-, 1,1-propylidene (-CH(CH 2 CH 3 ))-, 1,2-propylidene (-CH 2 CH(CH 3 ))-, 1,3-propylidene (-CH 2 CH 2 CH 2 -)-, 1,4-butylidene (-CH 2 CH 2 CH 2 CH 2 -)- and 1,5-butylidene (-CH 2 CH 2 CH 2 CH 2 CH 2 -)-, etc., including but not limited to these. The alkylene group may or may not be substituted. When substituted, the substituent may be substituted at any available linking site. The above substituents are independently preferably substituted with one or more groups optionally selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, and an oxo group.

[0114] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where the definitions of the alkyl group or cycloalkyl group are as described above. Non-limiting examples of alkoxy groups include methoxy group, ethoxy group, propoxy group, butoxy group, cyclopropoxy group, cyclobutoxy group, cyclopentyloxy group, cyclohexyloxy group. The alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxy group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group.

[0115] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclopentenyl group, cyclohexyl group, cyclohexenyl group, cyclohexadienyl group, cycloheptyl group, cycloheptatrienyl group, cyclooctyl group, etc. Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

[0116] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, where one or more of the ring atoms are nitrogen, oxygen or S(O) m(Among them, m is an integer from 0 to 2), which is a heteroatom selected therefrom, does not contain a ring portion of -O-O-, -O-S- or -S-S-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, among which 1 to 4 are heteroatoms, and a more preferred cycloalkyl group ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterosilyl groups include pyrrolidinyl group, piperidinyl group, piperazinyl group, morpholinyl group, thiomorpholinyl group, homopiperazinyl group, etc. Polycyclic heterosilyl groups include spiro ring, fused ring and bridged ring heterosilyl groups.

[0117] The term "spiroheterosilyl group" refers to a polycyclic heterosilyl group in which two 5- to 20-membered monocyclic rings share one atom (referred to as a spiro atom), among which one or more ring atoms are nitrogen, oxygen or S(O) m (Among them, m is an integer from 0 to 2), which is a heteroatom selected therefrom, and the remaining ring atoms are carbon. It may contain one or more double bonds, but there is no ring having a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Spiroheterosilyl groups are divided into monospiroheterosilyl groups, bisspiroheterosilyl groups or polyspiroheterosilyl groups according to the number of spiro atoms shared by the rings, and preferably monospiroheterosilyl groups and bisspiroheterosilyl groups. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered or 5-membered / 6-membered monospiroheterosilyl group. Non-limiting examples of spiroheterosilyl groups are

Chemical formula

[0118] The term "fused heterosilyl group" refers to a polycyclic heterosilyl group having 5 to 20 members, in which each ring in the system shares a pair of adjacent atoms with another ring in the system, and one or more rings may contain one or more double bonds, but there is no ring having a completely conjugated π electron system, among which one or more ring atoms are nitrogen, oxygen, or S(O) mAmong them, m is an integer from 0 to 2, and the remaining ring atoms are carbon. Preferably, it is a 6- to 14-membered ring, more preferably a 7- to 10-membered ring. Depending on the number of rings formed, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl groups, preferably bicyclic or tricyclic, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl group. Non-limiting examples of the fused heterocyclyl group are

Chemical formula

[0119] The term "bridged heterocyclyl group" refers to a polycyclic heterocyclyl group having 5 to 14 members, in which any two rings share atoms that are not directly linked, and may contain one or more double bonds, but there is no ring having a completely conjugated π-electron system. Among them, one or more ring atoms are nitrogen, oxygen or S(O) m Among them, m is an integer from 0 to 2, and the remaining ring atoms are carbon. Preferably, it is a 6- to 14-membered ring, more preferably a 7- to 10-membered ring. Depending on the number of rings formed, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of the bridged heterocyclyl group are

Chemical formula

[0120] The above heterocyclyl ring may be condensed with an aryl group, a heteroaryl group or a cycloalkyl group ring. Among them, the ring linked to the parent structure is a heterocyclyl group, and non-limiting examples thereof are

Chemical formula

[0121] The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, and an oxo group.

[0122] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as a phenyl group and a naphthyl group, preferably a phenyl group. The aryl ring may be fused to a heteroaryl group, a heterocyclic group, or a cycloalkyl group ring, among which the ring linked to the parent structure is an aryl ring. Non-limiting examples thereof are

Chemical formula

[0123] The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a sulfhydryl group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocyclic alkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocyclic alkoxy group, a cycloalkylthio group, and a heterocyclic alkylthio group.

[0124] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, among which the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group preferably has 5 to 10 members, more preferably 5 or 6 members, and examples thereof include a furanyl group, a thienyl group, a pyridyl group, a pyrrolyl group, an N-alkylpyrrolyl group, a pyrimidinyl group, a pyrazinyl group, an imidazolyl group, a tetrazolyl group, etc. The above heteroaryl ring may be condensed with an aryl group, a heterocyclyl group or a cycloalkyl group ring, among which the ring linked to the parent structure is a heteroaryl group ring, and non-limiting examples thereof are [Chemical formula] including.

[0125] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a sulfhydryl group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocyclylalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocyclylalkoxy group, a cycloalkylthio group, a heterocyclylalkylthio group.

[0126] The term "amino protecting group" is a group that protects an amino group with a group that is easily removed so that the amino group does not change when other parts of the molecule react. Non-limiting examples include a 9-fluorenylmethyloxycarbonyl group, a tert-butoxycarbonyl group, an acetyl group, a benzyl group, an allyl group, a p-methoxybenzyl group, etc. These groups can be optionally substituted with 1 to 3 substituents selected from a halogen, an alkoxy group or a nitro group. The above amino protecting group is preferably a 9-fluorenylmethyloxycarbonyl group.

[0127] The term "cycloalkylalkyl group" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably substituted with one cycloalkyl group, wherein the alkyl group is as defined above, and wherein the cycloalkyl group is as defined above.

[0128] The term "haloalkyl group" refers to an alkyl group substituted with one or more halogen atoms, wherein the alkyl group is as defined above.

[0129] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0130] The term "hydroxy group" refers to the -OH group.

[0131] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0132] The term "amino group" refers to -NH 2 and means.

[0133] The term "nitro group" refers to -NO 2 and means.

[0134] "Optional" or "optionally" means that the event or circumstance described thereafter may occur, but is not necessarily required, and this description includes the case where the event or circumstance occurs or does not occur. For example, "optionally containing 1 to 3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may be present, but are not necessarily required.

[0135] "Substituted" means that one or more hydrogen atoms in the base, preferably no more than 5, more preferably 1 to 3 hydrogen atoms, are independently substituted with the corresponding number of substituents. Of course, the substituents are located only at chemically possible sites, and those skilled in the art can determine possible or impossible substitutions without much effort (either experimentally or theoretically). For example, an amino group or a hydroxy group having a free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0136] The term "drug loading amount" or "drug payload" refers to the average quantity of cytotoxic agent loaded on each antibody or its antigen-binding fragment in an antibody-drug conjugate molecule, and may be expressed as the ratio of the amount of drug to the amount of antibody. As the range of drug loading amount, each antibody or its antigen-binding fragment (Pc) may be linked to 0 to 12, preferably 1 to 10, more preferably 3 to 8, and most preferably 5.3 to 6.1 cytotoxic agents. In embodiments of the present disclosure, the drug loading amount is represented by n and is also referred to as the DAR (Drug-antibody Ratio) value, and may exemplarily be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average quantity of drug of each ADC molecule after the conjugation reaction can be characterized by ordinary methods such as ultraviolet-visible spectroscopy (UV-Vis), hydrophobic interaction chromatography (HIC) mass spectrometry, ELISA test and HPLC.

[0137] The loading amount of the cytotoxic agent can be (1) controlling the molar ratio of the linking reagent to the monoclonal antibody, (2) controlling the reaction time and temperature, (3) selecting different reaction reagents, and can be controlled by non-limiting methods including.

[0138] The preparation of ordinary pharmaceutical compositions is shown in the Chinese Pharmacopoeia.

[0139] As used in the agents of the present disclosure, the term "vector" is a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and transport the drug to the target organ. By the release of the drug vector and the target system, the degradation and loss of the drug can be reduced, side effects can be decreased, and bioavailability can be improved. For example, due to its unique amphiphilic structure, a polymeric surfactant as a vector can self-assemble to form aggregates in various forms, and preferred examples include, for example, micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be good drug vectors.

[0140] "Administration" and "treatment", when applied to animals, humans, experimental subjects, cells, tissues, organs, and biological fluids, mean the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may, for example, mean therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of cells includes the contact between a reagent and the cells and the contact between the reagent and the fluid. "Administration" and "treatment" also mean, for example, treating cells in vitro and ex vivo with a reagent, diagnostic, binding composition, or through another type of cell. "Treatment" means therapeutic treatment, prevention or prophylactic measures, research, and diagnostic applications when applied to humans, veterinary medicine, or research subjects.

[0141] "Treatment" means, for example, administering to a patient an oral or topical therapeutic agent comprising a composition of any one of the binding compounds of the present disclosure, where the patient has one or more disease symptoms, and the known therapeutic agents have a therapeutic effect on these symptoms. Usually, for a patient or group receiving treatment, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms, thereby inducing the resolution of these symptoms or suppressing their progression to any clinically measurable degree. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as the "therapeutically effective amount") can vary depending on multiple factors such as the patient's disease state, age and weight, and the ability of the drug to produce the required therapeutic effect in the patient. The reduction of disease symptoms can be evaluated by any clinical detection method commonly used by a doctor or other professional healthcare provider to assess the severity or progression of the symptoms. Embodiments of the present disclosure (e.g., treatment methods or products) may be ineffective in alleviating each target disease symptom, but it has been confirmed that the target disease symptoms can be reduced in a statistically significant number of patients by any statistical testing method known in the art, such as the Student t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0142] "Effective amount" includes an amount sufficient for the improvement or prevention of symptoms or conditions of a medical disease. An effective amount further means an amount sufficient for the allowance or facilitation of a diagnosis. The effective amount used for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the route and dosage of administration, and the severity of side effects. The effective amount may be the maximum dosage or dosing schedule that avoids significant side effects or toxic effects.

[0143] "Replacement" refers to the replacement of the solvent system that dissolves the antibody protein. For example, the high-salt or high-osmotic pressure solvent system containing the antibody protein is replaced by a physical operation method in a buffer system that stabilizes the formulation so that the antibody protein is present in a stable formulation. The above physical operation methods include, but are not limited to, ultrafiltration, dialysis, or redissolution after centrifugation.

Brief Description of the Drawings

[0144]

Figure 1A

Figure 1B

Figure 1C

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Modes for Carrying Out the Invention

[0145] The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. In the examples of the present disclosure, experimental methods for which specific conditions are not specified generally follow normal conditions such as those published in the "Antibody Technology Experimental Manual" and "Molecular Cloning Manual" by Cold Spring Harbor Laboratory, or follow the conditions recommended by the raw material or product manufacturer. Reagents for which specific sources are not specified are common commercially available reagents.

[0146] I. Antibody-Drug Conjugate The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). For NMR measurements, a Bruker AVANCE-400 nuclear magnetic device was used, and the measurement solvents were deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), and deuterated methanol (CD 3 OD). The internal standard was tetramethylsilane (TMS), and the chemical shift was indicated in units of 10 -6 (ppm).

[0147] For MS measurements, a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model number: Finnigan LCQ advantage MAX) was used.

[0148] For UPLC measurements, a Waters Acquity UPLC SQD liquid chromatograph mass spectrometer was used.

[0149] For HPLC measurements, an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6 mm column) were used.

[0150] For UV-HPLC measurements, a Thermo nanodrop 2000 ultraviolet spectrophotometer was used.

[0151] Growth inhibition rate and IC 50 For the measurement of the value, a PHERA star FS microplate reader (BMG Labtech, Germany) was used.

[0152] As the silica gel plate for thin layer chromatography, Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate was used. The specification of the silica gel plate used for thin layer chromatography (TLC) is 0.15 mm - 0.2 mm, and the specification of the silica gel plate for separation and purification of products by thin layer chromatography is 0.4 mm - 0.5 mm.

[0153] Column chromatography generally used silica gel of 200 - 300 mesh from Yantai Huanghai as a vector.

[0154] The known starting materials according to the present disclosure may be synthesized by adopting or following the known methods in this field, or may be purchased from companies such as ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0155] In the examples, unless otherwise specified, all reactions were carried out in an argon or nitrogen atmosphere.

[0156] An argon or nitrogen atmosphere means that an argon or nitrogen balloon with a volume of about 1 L is connected to the reaction flask.

[0157] A hydrogen atmosphere means that a hydrogen balloon with a volume of about 1 L is connected to the reaction flask.

[0158] For the catalytic hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Qinglan QL - 500 hydrogen generator or an HC2 - SS hydrogenation apparatus were used.

[0159] The hydrogenation reaction usually involves repeating the operation of evacuating and filling with hydrogen three times.

[0160] For the microwave reaction, a CEM Discover-S 908860 type microwave reactor is used.

[0161] In the examples, unless otherwise specified, the solution during the reaction refers to an aqueous solution.

[0162] In the examples, unless otherwise specified, the reaction temperature is room temperature.

[0163] Room temperature is the optimal reaction temperature, and the temperature range is 20°C to 30°C.

[0164] Preparation of PBS buffer with pH = 6.5 in the examples: Take 8.5 g of KH 2 PO 4 , 8.56 g of K 2 HPO 4 .3H 2 O, 5.85 g of NaCl, 1.5 g of EDTA, put them into a flask, make up the volume to 2 L, dissolve it completely by ultrasonic wave, and obtain it after shaking well.

[0165] The eluent system of column chromatography and the developing solvent system of thin layer chromatography used for the purification of the compounds include system A: dichloromethane and isopropyl alcohol system, system B: dichloromethane and methanol system, and system C: petroleum ether and ethyl acetate system. The volume ratio of the solvents may be adjusted according to the polarity of the compound, or may be adjusted by adding a small amount of triethylamine and acidic or basic reagents, etc.

[0166] Some of the compounds of the present disclosure were characterized by Q-TOF LC / MS. For Q-TOF LC / MS, an Agilent 6530 accurate mass quadrupole-time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1×75 mm column) were used.

[0167] Example 1-1 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide 1

Chemical formula

[0168] 1 mL of N,N-dimethylformamide was added to exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in the patent application "EP0737686A1"), and the mixture was cooled to 0 - 5 °C in an ice-water bath. 1 drop of triethylamine was added dropwise, and the reaction solution was stirred until it became clear. 1-Hydroxycyclopropylformic acid 1a (1.4 mg, 3.7 μmol, prepared by a known method disclosed in "Tetrahedron Letters, 25(12), 1269 - 72, 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol) were sequentially added to the reaction solution. After the addition was completed, the reaction was carried out with stirring at 0 - 5 °C for 2 hours. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 1 (1.6 mg, yield: 82.1%). MS m / z (ESI): 520.2 [M+1] 1 H NMR (400 MHz, CDCl 3): δ 7.90 - 7.84 (m, 1H), 7.80 - 7.68 (m, 1H), 5.80 - 5.70 (m, 1H), 5.62 - 5.54 (m, 2H), 5.44 - 5.32 (m, 2H), 5.28 - 5.10 (m, 2H), 3.40 - 3.15 (m, 3H), 2.44 (s, 3H), 2.23 (t, 1H), 2.06 - 1.75 (m, 2H), 1.68 - 1.56 (m, 1H), 1.22 - 1.18 (m, 2H), 1.04 - 0.98 (m, 2H), 0.89 (t, 3H).

[0169] Example 1 - 2 (S)-2 - Cyclopropyl - N - ((1S,9S)-9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)-2 - hydroxyacetamide 2 - A (R)-2 - Cyclopropyl - N - ((1S,9S)-9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)-2 - hydroxyacetamide 2 - B [Chemical Structure]

[0170] To 1b (4 mg, 7.53 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The mixture was purged with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise. The reaction solution was stirred until it became clear. To the reaction solution were sequentially added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared by the method disclosed in the patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol). After the addition was complete, the reaction was carried out with stirring at 0 - 5 °C for 1 hour. The ice-water bath was removed, and the mixture was heated to 30 °C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude product, compound 2, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19×250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to obtain the title products (2-A: 1.5 mg, 2-B: 1.5 mg). MS m / z (ESI): 534.0 [M+1].

[0171] Compound 2-B with a single configuration (relatively short retention time): UPLC analysis: retention time: 1.06 minutes, purity: 88% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO-d 6): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58 - 5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32 - 5.29 (m, 2H), 3.60 (t, 1H), 3.19 - 3.13 (m, 1H), 2.38 (s, 3H), 2.20 - 2.14 (m, 1H), 1.98 (q, 2H), 1.87 - 1.83 (m, 1H), 1.50 - 1.40 (m, 1H), 1.34 - 1.28 (m, 1H), 0.86 (t, 3H), 0.50 - 0.39 (m, 4H).

[0172] Compound 2 - A in a single configuration (relatively long retention time): UPLC analysis: Retention time: 1.10 minutes, purity: 86% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6 ): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58 - 5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3.20 - 3.15 (m, 2H), 2.40 (s, 3H), 2.25 - 2.16 (m, 1H), 1.98 (q, 2H), 1.87 - 1.82 (m, 1H), 1.50 - 1.40 (m, 1H), 1.21 - 1.14 (m, 1H), 0.87 (t, 3H), 0.47 - 0.35 (m, 4H).

[0173] Examples 1 - 3 (S)-N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-A (R)-N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-B [Chemical Structure]

[0174] To compound 1b (5.0 mg, 9.41 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, and the mixture was cooled to 0 - 5 °C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise, and the reaction mixture was stirred until it became clear. To the reaction mixture were sequentially added 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier: Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol). After the addition was complete, the reaction was carried out with stirring at 0 - 5 °C for 10 minutes. The ice-water bath was removed, and the mixture was heated to 30 °C and stirred for 8 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 3, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding component was collected and concentrated under reduced pressure to obtain the title product (1.5 mg, 1.5 mg). MS m / z (ESI): 561.9 [M+1].

[0175] Compound in a single configuration (relatively short retention time): UPLC analysis: Retention time: 1.11 minutes, purity: 88% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6 ): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61 - 5.55 (m, 1H), 5.45 - 5.23 (m, 3H), 5.15 - 5.06 (m, 1H), 4.66 - 4.57 (m, 1H), 3.18 - 3.12 (m, 1H), 2.40 (s, 3H), 2.26 - 2.20 (m, 1H), 2.16 - 2.08 (m, 1H), 2.02 - 1.94 (m, 1H), 1.89 - 1.82 (m, 1H), 1.50 - 1.40 (m, 1H), 0.87 (t, 3H).

[0176] Compound in a single configuration (relatively long retention time): UPLC analysis: Retention time: 1.19 minutes, purity: 90% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63 - 5.55 (m, 1H), 5.45 - 5.20 (m, 3H), 5.16 - 5.07 (m, 1H), 4.66 - 4.57 (m, 1H), 3.18 - 3.12 (m, 1H), 2.40 (s, 3H), 2.22 - 2.14 (m, 1H), 2.04 - 1.95 (m, 2H), 1.89 - 1.82 (m, 1H), 1.50 - 1.40 (m, 1H), 0.87 (t, 3H).

[0177] Examples 1 - 4 N - ((1S,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl) - 1 - hydroxycyclopentane - 1 - carboxamide 4 [Chemical formula]

[0178] To 1 mL of N,N-dimethylformamide was added 3.0 mg (5.64 μmol) of Compound 1b, and the mixture was cooled to 0 - 5 °C in an ice-water bath. One drop of triethylamine was added dropwise, and the reaction solution was stirred until it became clear. To the reaction solution were sequentially added 4a (2.2 mg, 16.9 μmol, prepared by the method disclosed in the patent application "WO2013106717") of 1-hydroxy-cyclopentanecarboxylic acid and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol). After the addition was completed, the reaction was carried out with stirring at 0 - 5 °C for 1 hour. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%). MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, CDCl 3 ): δ 7.73 - 7.62 (m, 2H), 5.75 - 5.62 (m, 1H), 5.46 - 5.32 (m, 2H), 5.26 - 5.10 (m, 1H), 3.30 - 3.10 (m, 1H), 2.43 (s, 3H), 2.28 - 2.20 (m, 2H), 2.08 - 1.84 (m, 8H), 1.69 - 1.58 (m, 2H), 1.04 - 1.00 (m, 2H), 0.89 (t, 3H).

[0179] Examples 1 - 5 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

Chemical Structure

[0180] 1 mL of N,N-dimethylformamide was added to Compound 1b (2.0 mg, 3.76 μmol), and the mixture was cooled to 0 - 5 °C in an ice-water bath. 1 drop of triethylamine was added dropwise, and the reaction solution was stirred until it became clear. 1-(Hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared by the method disclosed in the patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) were successively added to the reaction solution. After the addition was completed, the reaction was carried out with stirring at 0 - 5 °C for 2 hours. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 5 (1.0 mg, yield: 50%). MS m / z (ESI): 533.9 [M+1]. 1 H NMR (400 MHz, CDCl 3 ): δ 8.07 (s, 1H), 7.23 - 7.18 (m, 2H), 6.71 - 6.64 (m, 1H), 6.55 - 6.51 (m, 1H), 5.36 - 5.27 (m, 2H), 4.67 - 4.61 (m, 2H), 3.53 - 3.48 (m, 1H), 3.30 - 3.22 (m, 2H), 3.18 - 3.13 (m, 1H), 2.71 - 2.61 (m, 2H), 2.35 - 2.28 (m, 1H), 2.04 - 1.91 (m, 4H), 1.53 - 1.40 (m, 3H), 0.91 - 0.75 (m, 4H).

[0181] Examples 1 - 6 N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

Chem.

[0182] 1 mL of N,N-dimethylformamide was added to Compound 1b (3.0 mg, 5.64 μmol), and the mixture was cooled to 0 - 5 °C in an ice-water bath. 1 drop of triethylamine was added dropwise, and the reaction solution was stirred until it became clear. 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol, prepared by the method disclosed in the literature “Journal of the American Chemical Society, 2014, vol.136, #22, p.8138 - 8142”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were sequentially added to the reaction solution. After the addition was completed, the reaction was carried out with stirring at 0 - 5 °C for 1 hour. 5 mL of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 6 (2.1 mg, yield: 67.9%). MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, DMSO-d 6): δ 7.85 - 7.62 (m, 1H), 6.88 (br, 1H), 5.87 - 5.48 (m, 2H), 5.47 - 5.33 (m, 1H), 5.31 - 5.06 (m, 1H), 4.25 - 3.91 (m, 2H), 3.25 (br, 1H), 2.60 - 2.32 (m, 3H), 2.23 (t, 1H), 2.15 - 1.95 (m, 3H), 1.70 - 1.56 (m, 2H), 1.41 - 1.17 (m, 9H), 1.03 (s, 1H), 0.95 - 0.80 (m, 2H).

[0183] Examples 1 - 7 N - ((1S,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl) - 1 - hydroxycyclobutane - 1 - carboxamide 7

Chemical Structure

[0184] To compound 1b (3.0 mg, 5.64 μmol), 2 mL of ethanol and 0.4 mL of N,N - dimethylformamide were added, and the mixture was cooled to 0 - 5 °C in an ice - water bath. 0.3 mL of N - methylmorpholine was added dropwise, and the reaction mixture was stirred until it became clear. To the reaction mixture, 1 - hydroxycyclobutane carboxylic acid 7a (2.0 mg, 17.22 μmol, supplier: Yakushi), 1 - hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) were added in sequence. After the addition was complete, the reaction was carried out with stirring at 0 - 5 °C for 10 minutes. The ice - water bath was removed, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by thin - layer chromatography with developing solvent system B to obtain the title product 7 (2.5 mg, yield: 83.1%). MS m / z (ESI): 534.0 [M + 1]. 11H NMR (400 MHz, DMSO-d 6 ): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59 - 5.51 (m, 1H), 5.41 (s, 2H), 5.20 - 5.01 (m, 2H), 3.27 - 3.17 (m, 1H), 3.15 - 3.05 (m, 1H), 2.71 - 2.63 (m, 1H), 2.37 (s, 3H), 2.12 - 2.05 (m, 1H), 2.03 - 1.94 (m, 2H), 1.92 - 1.78 (m, 4H), 1.50 - 1.42 (m, 1H), 0.90 - 0.83 (m, 4H).

[0185] Examples 1 - 8 1 - (((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8

Chemical Structure

[0186] Step 1 1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylic acid benzyl 8c

[0187] Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol, prepared by the method disclosed in patent application "US2005 / 20645") and ethyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methylacetate 8b (100 mg, 0.27 mmol, prepared by the method disclosed in patent application "CN105829346A") were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.6 mL of water was added, sodium hydrogen carbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing solvent system B to obtain the title product 8c (100 mg, yield: 73.6%). MS m / z (ESI): 501.0 [M+1].

[0188] Step 2 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylic acid 8d

[0189] Compound 8c (50 mg, 0.10 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium carbon (25 mg, content 10%) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated to obtain the title product 8d (41 mg, yield: 100%). MS m / z (ESI): 411.0 [M+1].

[0190] Step 3 (9H-Fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

[0191] Compound 1b (7 mg, 0.013 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, the mixture was replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, a 0.5 mL N,N-dimethylformamide solution of compound 8d (7 mg, 0.017 mmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol) was added, and the reaction was carried out with stirring in an ice bath for 35 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with a saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 8e (8.5 mg, yield: 78.0%). MS m / z (ESI): 828.0 [M+1].

[0192] Step 4 1-((2-Aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8f

[0193] Compound 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, 0.1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was decanted three times and concentrated under reduced pressure to obtain the crude title product 8f (2.9 mg), which was used in the next step reaction without purification. MS m / z (ESI): 606.0 [M+1].

[0194] Step 5 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8

[0195] The crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, and a 0.3 mL N,N-dimethylformamide solution of (S)-2-(-2-(-2-(6-(-2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetylamido)acetylamido)-3-phenylpropionic acid 8g (2.7 mg, 5.80 μmol, prepared by the method disclosed in the patent application "EP2907824") was added. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol) was added, and the reaction was carried out with stirring in an ice bath for 30 minutes. The ice bath was removed, the temperature was raised to room temperature, and stirring was continued for 15 minutes. The reaction solution was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding component was collected, concentrated under reduced pressure, and the title product 8 (2 mg, yield: 39.0%) was obtained. MS m / z (ESI): 1060.0 [M+1]. 1 H NMR (400 MHz, DMSO-d 6): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08 - 7.92 (m, 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24 - 7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5.12 (q, 2H), 4.62 (t, 1H), 4.52 (t, 1H), 4.40 - 4.32 (m, 1H), 3.73 - 3.47 (m, 8H), 3.16 - 3.04 (m, 2H), 2.89 (dd, 1H), 2.69 - 2.55 (m, 2H), 2.37 - 2.23 (m, 4H), 2.12 - 1.93 (m, 4H), 1.90 - 1.74 (m, 2H), 1.52 - 1.38 (m, 4H), 1.33 - 1.11 (m, 5H), 0.91 - 0.81 (m, 4H).

[0196] Examples 1 - 9 N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

Chem.

Chem.

[0197] Step 1 Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0198] Compound 2a (1.3 g, 11.2 mmol, prepared by the method disclosed in Patent Application “WO2013 / 106717”) was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, the filter cake was washed with ethyl acetate (10 mL), the filtrates were combined and concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 9a (2 g, yield: 86.9%).

[0199] Step 2 Benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundecane-11-carboxylate 9b

[0200] Compound 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, 4 mL of tetrahydrofuran was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice bath was removed, the temperature was raised to room temperature, and the mixture was stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 9b (48 mg, yield: 19%). MS m / z (ESI): 515.0 [M+1].

[0201] Step 3 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundecane-11-carboxylic acid 9c

[0202] Compound 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium carbon (12 mg, content 10%, dry) was added, and the mixture was replaced with hydrogen gas three times. The reaction was carried out with stirring at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 9c (13 mg), and the product was used directly in the next step reaction without purification. MS m / z (ESI): 424.9 [M+1].

[0203] Step 4 (9H-Fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

[0204] Compound 1b (10 mg, 18.8 μmol) was placed in a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, crude product 9c (13 mg, 30.6 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol) was added, and the reaction was carried out with stirring in an ice bath for 40 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 9d (19 mg, yield: 73.6%). MS m / z (ESI): 842.1 [M+1].

[0205] Step 5 2-((2-Aminoacetylamino)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide 9e

[0206] Compound 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the process of adding 1 mL of toluene and concentrating under reduced pressure was repeated twice. 3 mL of n-hexane was added to the residue and triturated. After standing, the supernatant was poured out, and the solid was retained. The solid residue was concentrated under reduced pressure and dried by an oil pump to obtain the title product 9e (17 mg) as a crude product, which was used directly in the next-step reaction without purification. MS m / z (ESI): 638.0[M+18].

[0207] Step 6 N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0208] The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, 8 g (21.2 mg, 44.8 μmol) of a 0.3 mL N,N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added, and the reaction was carried out with stirring in an ice bath for 10 minutes. The ice bath was removed, the temperature was raised to room temperature, and the reaction was carried out with stirring for 1 hour to produce compound 9. The reaction solution was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding components were collected, concentrated under reduced pressure to obtain the title product (9 - A: 2.4 mg, 9 - B: 1.7 mg). MS m / z (ESI): 1074.4 [M + 1].

[0209] Compound 9 - A of single configuration (relatively short retention time): UPLC analysis: retention time: 1.14 minutes, purity: 85% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6): δ 8.60 (t, 1H), 8.51 - 8.49 (d, 1H), 8.32 - 8.24 (m, 1H), 8.13 - 8.02 (m, 2H), 8.02 - 7.96 (m, 1H), 7.82 - 7.75 (m, 1H), 7.31 (s, 1H), 7.26 - 7.15 (m, 4H), 6.99 (s, 1H), 6.55 - 6.48 (m, 1H), 5.65 - 5.54 (m, 1H), 5.41 (s, 2H), 5.35 - 5.15 (m, 3H), 4.74 - 4.62 (m, 1H), 4.54 - 4.40 (m, 2H), 3.76 - 3.64 (m, 4H), 3.62 - 3.48 (m, 2H), 3.20 - 3.07 (m, 2H), 3.04 - 2.94 (m, 1H), 2.80 - 2.62 (m, 1H), 2.45 - 2.30 (m, 3H), 2.25 - 2.15 (m, 2H), 2.15 - 2.04 (m, 2H), 1.93 - 1.78 (m, 2H), 1.52 - 1.39 (m, 3H), 1.34 - 1.12 (m, 5H), 0.87 (t, 3H), 0.64 - 0.38 (m, 4H).

[0210] Compound 9 - B in a single configuration (relatively long retention time): UPLC analysis: Retention time: 1.16 minutes, purity: 89% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6): δ 8.68 - 8.60 (m, 1H), 8.58 - 8.50 (m, 1H), 8.32 - 8.24 (m, 1H), 8.13 - 8.02 (m, 2H), 8.02 - 7.94 (m, 1H), 7.82 - 7.75 (m, 1H), 7.31 (s, 1H), 7.26 - 7.13 (m, 3H), 6.99 (s, 1H), 6.55 - 6.48 (m, 1H), 5.60 - 5.50 (m, 1H), 5.41 (s, 2H), 5.35 - 5.15 (m, 2H), 4.78 - 4.68 (m, 1H), 4.60 - 4.40 (m, 2H), 3.76 - 3.58 (m, 4H), 3.58 - 3.48 (m, 1H), 3.20 - 3.10 (m, 2H), 3.08 - 2.97 (m, 2H), 2.80 - 2.72 (m, 2H), 2.45 - 2.30 (m, 3H), 2.25 - 2.13 (m, 2H), 2.13 - 2.04 (m, 2H), 2.03 - 1.94 (m, 2H), 1.91 - 1.78 (m, 2H), 1.52 - 1.39 (m, 3H), 1.34 - 1.12 (m, 4H), 0.91 - 0.79 (m, 3H), 0.53 - 0.34 (m, 4H).

[0211] Examples 1 - 10 N - ((2S,10S)-10 - benzyl - 2 - (((1S,9S)-9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)aminocarbonyl)-1,1,1 - trifluoro - 6,9,12,15 - tetraoxo - 3 - oxa - 5,8,11,14 - tetraazapentadecane - 16 - yl)-6 - (2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)hexanamide 10 - A N-((2R,10S)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

Chem.

Chem.

[0212] Step 1 Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

[0213] Compound 3a (1.80 g, 12.5 mmol) was dissolved in 100 mL of acetonitrile, and potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 mL, 37.5 mmol) and tetrabutylammonium iodide (231 mg, 0.63 mmol) were added in sequence. The reaction solution was heated to 60 °C and stirred for 5 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 10a (980 mg, yield: 33.5%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.43-7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H).

[0214] Step 2 Benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazoundecane-11-carboxylate 10b

[0215] Compound 8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to a reaction flask, 3 mL of tetrahydrofuran was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (38 mg, 0.34 mmol) was added, the ice bath was removed, the temperature was raised to room temperature, and the mixture was stirred for 20 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The combined organic phases were concentrated, the obtained residue was dissolved in 2 mL of dioxane, 0.4 mL of water was added, sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorenylmethyl chloroformate (49 mg, 0.19 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 10b (51 mg, yield: 55.3%). MS m / z (ESI): 559.9 [M+18].

[0216] Step 3 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazoundecane-11-carboxylic acid 10c

[0217] Compound 10b (15 mg, 0.28 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium carbon (15 mg, content 10%) was added, and the mixture was replaced with hydrogen gas three times. The reaction was carried out with stirring at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the crude title product 10c (13 mg). MS m / z (ESI): 452.9 [M+1].

[0218] Step 4 (9H-Fluoren-9-yl)methyl (2-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,1,1-trifluoro-3-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 10d

[0219] Compound 1b (10 mg, 18.8 μmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, a 0.5 mL N,N-dimethylformamide solution of 10c (13 mg, 28.7 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (11 mg, 39.7 μmol) was added, and the reaction was carried out with stirring in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, the organic phase was washed with saturated sodium chloride solution (10 mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 10d (16 mg, yield: 97.8%). MS m / z (ESI): 870.0[M+1].

[0220] Step 5 2-((2-Aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoropropanamide 10e

[0221] Compound 10d (16 mg, 18.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the addition of 2 mL of toluene followed by concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added to the residue and triturated, and after standing, the supernatant was poured off, and the solid was retained. This operation was repeated three times. The solid residue was concentrated under reduced pressure and dried by an oil pump to obtain the title product 10e (12 mg) as a crude product, which was used in the next step reaction without purification. MS m / z (ESI): 647.9 [M+1].

[0222] Step 6 N-((2S,10S)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A N-((2R,10S)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0223] The crude product 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide, replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, 8 g (14 mg, 29.6 μmol) of its 0.3 mL N,N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (15 mg, 54.2 μmol) was added, and the reaction was carried out with stirring in an ice bath for 30 minutes. Then the ice bath was removed, the temperature was raised to room temperature, and the reaction was continued with stirring for 1 hour to produce compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding component was collected, concentrated under reduced pressure, and the title product (2.7 mg, 2.6 mg) was obtained. MS m / z (ESI): 1102.0 [M+1].

[0224] Compound with single configuration (relatively short retention time): UPLC analysis: retention time: 1.18 minutes, purity: 91% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO-d 6): δ 8.97 (d, 1H), 8.85 - 8.76 (m, 1H), 8.37 - 8.27 (m, 1H), 8.12 - 8.02 (m, 1H), 8.02 - 7.95 (m, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.26 - 7.10 (m, 4H), 6.99 (s, 1H), 6.66 (br, 1H), 6.52 (s, 1H), 5.65 - 5.54 (m, 1H), 5.41 (s, 1H), 5.37 - 5.25 (m, 3H), 5.23 - 5.13 (m, 1H), 4.81 - 4.68 (m, 2H), 4.51 - 4.41 (m, 1H), 3.78 - 3.45 (m, 6H), 3.21 - 3.13 (m, 1H), 3.02 - 2.93 (m, 1H), 2.77 - 2.63 (m, 2H), 2.45 - 2.29 (m, 3H), 2.24 - 2.05 (m, 3H), 2.04 - 1.93 (m, 5H), 1.90 - 1.75 (m, 2H), 1.52 - 1.38 (m, 4H), 0.90 - 0.78 (m, 5H).

[0225] Compound in a single configuration (relatively long retention time): UPLC analysis: Retention time: 1.23 minutes, purity: 90% (column: ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6): δ 9.05 (d, 1H), 8.97 - 8.88 (m, 1H), 8.35 - 8.27 (m, 1H), 8.11 - 8.03 (m, 1H), 8.02 - 7.95 (m, 1H), 7.80 (d, 1H), 7.34 (s, 1H), 7.29 - 7.13 (m, 4H), 6.99 (s, 1H), 6.66 (br, 1H), 6.54 (s, 1H), 5.64 - 5.55 (m, 1H), 5.43 (s, 1H), 5.36 - 5.20 (m, 3H), 4.92 - 4.85 (m, 1H), 4.82 - 4.72 (m, 2H), 4.52 - 4.42 (m, 1H), 3.77 - 3.48 (m, 6H), 3.21 - 3.14 (m, 1H), 3.03 - 2.95 (m, 1H), 2.79 - 2.65 (m, 2H), 2.47 - 2.28 (m, 3H), 2.25 - 2.05 (m, 3H), 2.05 - 1.94 (m, 5H), 1.91 - 1.76 (m, 2H), 1.52 - 1.37 (m, 4H), 0.92 - 0.77 (m, 5H).

[0226] Examples 1 - 11 1 - (((S) - 7 - benzyl - 20 - (2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl) - 3,6,9,12,15 - pentaoxo - 2,5,8,11,14 - pentaazicosyl)oxy) - N - ((1S,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)cyclobutane - 1 - carboxamide 11

Chemical Structure

[0227] Step 1 1 - ((2 - ((((9H - Fluoren - 9 - yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane - 1 - carboxylic acid benzyl 11b

[0228] Benzyl 1-hydroxycyclobutane-carboxylate 11a (167 mg, 0.81 mmol, prepared by the method disclosed in the literature "Journal of Medicinal Chemistry, 2013, vol. 56, # 13, p. 5541 - 5552") and 8b (150 mg, 0.41 mmol) were placed in a reaction flask, 5 mL of tetrahydrofuran was added, replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (92 mg, 0.82 mmol) was added, the ice bath was removed, warmed to room temperature and stirred for 10 minutes, 20 mL of ice water was added, extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5), the organic phases were combined and concentrated, the obtained residue was dissolved in 3 mL of dioxane, 0.6 mL of water was added, sodium hydrogen carbonate (41 mg, 0.48 mmol) and 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol) were added, and stirred at room temperature for 1 hour. 20 mL of water was added, extracted with ethyl acetate (8 mL × 3), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 11b (37 mg, yield: 17.6%). MS m / z (ESI): 514.6 [M+1].

[0229] Step 2 1-(((2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylic acid 11c

[0230] Compound 11b (37 mg, 71.9 μmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium carbon (15 mg, content 10%) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, the filtrate was concentrated to obtain the title product 11c (35 mg, yield: 82%), and it was directly advanced to the next step.

[0231] Step 3 (9H-Fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 11d

[0232] Compound 1b (10 mg, 0.018 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, the mixture was replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, a 0.5 mL N,N-dimethylformamide solution of compound 11c (13 mg, 0.031 mmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol) was added, and the reaction was carried out with stirring in an ice bath for 40 minutes. 8 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with a saturated sodium chloride solution (8 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin-layer chromatography with developing solvent system A to obtain the title product 11d (19 mg, yield: 73.9%). MS m / z (ESI): 842.3 [M+1].

[0233] Step 4 1-((2-Aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 11e

[0234] Compound 11d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and the concentration under reduced pressure was repeated twice. 4 mL of n-hexane was added and triturated, and the upper n-hexane layer was decanted three times. The mixture was concentrated under reduced pressure to obtain the crude title product 11e (15 mg), which was used directly in the next step reaction without purification.

[0235] Step 5 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 11

[0236] The crude product 11e (2 mg, 3.22 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, replaced with argon gas three times, cooled to 0-5 °C in an ice-water bath, 8 g (1.5 mg, 3.17 μmol) of a 0.3 mL N,N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was rotary dried with an oil pump to remove DMF, the residue was dissolved in DCM, and then directly purified twice by thin-layer chromatography (polarity of the developing solvent: DCM / MeOH = 10 / 1) to obtain the title product 11 (1 mg, yield: 28.8%). MS m / z (ESI): 1073.6 [M+1]. 1 H NMR (400 MHz, CDCl 3 ): δ 8.70-8.60 (m, 1H), 8.28-8.19 (m, 1H), 8.13-7.91 (m, 3H), 7.79-7.71 (d, 1H), 7.29 (s, 1H), 7.25-7.09 (m, 4H), 6.98 (s, 1H), 6.71-6.62 (m, 1H), 6.55-6.47 (m, 1H), 5.64-5.54 (m, 2H), 5.40 (s, 1H), 5.35-5.27 (t, 2H), 5.17-5.10 (m, 2H), 4.60-4.51 (m, 1H), 4.51-4.35 (m, 2H), 3.93-3.78 (m, 3H), 3.71-3.59 (m, 3H), 3.01-2.88 (m, 3H), 2.70-2.64 (m, 2H), 2.44-2.30 (m, 3H), 2.28-2.14 (m, 3H), 2.11-1.92 (m, 6H), 1.90-1.76 (m, 3H), 1.51-1.39 (m, 4H), 0.92-0.75 (m, 6H).

[0237] Examples 1-12 (S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-A (R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-B [Chemical Structure]

[0238] Step 1 3-Cyclopropyl-2-hydroxypropionic acid 12b

[0239] Compound 12a (0.5 g, 3.87 mmol, supplier: Adamas) was dissolved in 35 mL of a mixed solvent of water and acetic acid (V:V = 4:1). The temperature was lowered to 0 - 5 °C in an ice-water bath, and a 2 M aqueous solution of sodium nitrite (0.53 g, 7.74 mmol) was added dropwise. The temperature was then raised to room temperature and the reaction was carried out with stirring for 3 hours. Solid sodium chloride was added to the reaction solution to saturate the aqueous phase, and the mixture was extracted with ethyl acetate (8 mL × 8), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title product 12b (0.45 g, yield: 89.3%).

[0240] Step 2 (S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-A (R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-B

[0241] To compound 1b (45 mg, 0.085 mmol), 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide were added. The mixture was replaced with argon gas three times, 0.1 mL of N-methylmorpholine was added dropwise, and the reaction solution was stirred until it became clear. To the reaction solution, compound 12b (90 mg, 0.691 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.256 mmol) were added in sequence. After the addition was completed, the reaction was carried out with stirring at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude product, compound 12, was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T C18 5 μm 21.2*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), and the title product (7 mg, 15 mg) was obtained. MS m / z (ESI): 547.9 [M+1].

[0242] Compound with single configuration (relatively short retention time): UPLC analysis: retention time: 1.345 minutes, purity: 72% (column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO-d 6): δ 8.42 (d, 1H), 7.78 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.60 - 5.50 (m, 2H), 5.42 (s, 1H), 5.19 (q, 2H), 4.02 - 4.00 (m, 1H), 3.21 - 3.11 (m, 2H), 2.39 (s, 3H), 2.21 - 2.07 (m, 2H), 2.05 - 1.95 (m, 1H), 1.92 - 1.68 (m, 4H), 1.53 - 1.41 (m, 1H), 0.87 (t, 3H), 0.48 - 0.34 (m, 2H), 0.14 - 0.01 (m, 2H).

[0243] Compound in a single configuration (relatively long retention time): UPLC analysis: Retention time: 1.399 minutes, purity: 88% (Column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1 * 50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO - d 6 ): δ 8.36 (d, 1H), 7.77 (d, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.58 - 5.51 (m, 1H), 5.48 (d, 1H), 5.42 (s, 1H), 5.20 (q, 2H), 4.09 - 4.02 (m, 1H), 3.22 - 3.11 (m, 2H), 2.39 (s, 3H), 2.27 - 2.06 (m, 2H), 2.05 - 1.95 (m, 1H), 1.93 - 1.81 (m, 2H), 1.65 - 1.43 (m, 2H), 1.32 - 1.21 (m, 1H), 0.87 (t, 3H), 0.48 - 0.33 (m, 2H), 0.14 - 0.01 (m, 2H).

[0244] Examples 1 - 13 (Reference Examples) N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

Chem.

[0245] The title compound 13 was prepared by the method disclosed in the patent “Example 76 on page 147 of the specification of EP2907824A1”.

[0246] Examples 1-14 N-((2R,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A N-((2S,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

Chem.

Chem.

[0247] Step 1 Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

[0248] Compound 12b (200 mg, 1.54 mmol) was dissolved in 20 mL of acetonitrile, and potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 mL, 1.34 mmol), and tetrabutylammonium iodide (28 mg, 0.07 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, the filter cake was washed with ethyl acetate (10 mL), the filtrates were combined and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 14a (140 mg, yield: 41.3%).

[0249] Step 2 Benzyl 10-(cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundecane-11-carboxylate 14b

[0250] Compound 14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) were added to a reaction flask, 10 mL of tetrahydrofuran was added, the mixture was replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (79 mg, 0.704 mmol) was added, the ice-water bath was removed, the temperature was raised to room temperature, stirred for 10 minutes, 20 mL of ice water was added, extracted with ethyl acetate (10 mL × 4), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 14b (50 mg, yield: 26.8%). MS m / z (ESI): 529.2 [M+1].

[0251] Step 3 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-oic acid 14c

[0252] Compound 14b (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate, palladium carbon (7 mg, content 10%, dry) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 14c (23 mg), and the next step reaction was carried out without purifying the product. MS m / z (ESI): 439.1 [M+1].

[0253] Step 4 (9H-Fluoren-9-yl)methyl(2-((((3-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 14d

[0254] Compound 1b (22 mg, 42.38 μmol) was placed in a reaction flask, 3 mL of N,N-dimethylformamide was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, triethylamine (4.3 mg, 42.49 μmol) was added dropwise, crude product 14c (23 mg, 51.1 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol) was added, and the reaction was carried out with stirring in an ice-water bath for 40 minutes. 15 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3), and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 14d (29 mg, yield: 79.9%). MS m / z (ESI): 856.1[M+1].

[0255] Step 5 2-((2-Aminoacetylamino)methoxy)-3-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)propanamide 14e

[0256] Compound 14d (29 mg, 33.9 μmol) was dissolved in 0.8 mL of dichloromethane, 0.4 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and the addition of 1 mL of toluene followed by concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added to the residue and triturated, and the supernatant was decanted after standing, which was repeated three times. The residue was concentrated under reduced pressure and dried with an oil pump to obtain the crude title product 14e (22 mg), which was used directly in the next-step reaction without purification. MS m / z (ESI): 634.1[M+1].

[0257] Step 6 N-((2R,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A N-((2S,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0258] The crude product 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide, replaced with argon gas three times, cooled to 0-5 °C in an ice-water bath, 8 g (24 mg, 50.8 μmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (14 mg, 50.6 μmol) were added in sequence, the ice bath was removed, the temperature was raised to room temperature, and the mixture was stirred for 1 hour to react to form compound 14. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), and the title product (2 mg, 2 mg) was obtained. MS m / z (ESI): 1088.4 [M+1].

[0259] Compound in a single configuration (relatively short retention time): UPLC analysis: Retention time: 1.18 minutes, purity: 88% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile).

[0260] Compound in a single configuration (relatively long retention time): UPLC analysis: Retention time: 1.23 minutes, purity: 96% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A - water (5 mmol NH 4 OAc), B - acetonitrile).

[0261] Example 1 - 15 1 - ((S) - 9 - benzyl - 22 - (2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl) - 5,8,11,14,17 - pentaoxo - 2 - oxa - 4,7,10,13,16 - pentaazadocosane) - N - ((1S,9S) - 9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)cyclopropane - 1 - carboxamide 15

Chemical formula

[0262] Step 1 Benzyl 1 - (10 - (9H - fluoren - 9 - yl) - 5,8 - dioxo - 2,9 - dioxo - 4,7 - diazadecyl)cyclopropane - 1 - carboxylate 15b

[0263] Compound 8b (500 mg, 1.35 mmol) was placed in a reaction flask, 6 mL of tetrahydrofuran was added, benzyl 1-hydroxymethylcyclopropane-1-carboxylate 15a (233 mg, 1.13 mmol, prepared by the method disclosed in Example 22-2 on page 262 of the specification of patent application "EP2862856A1") was added to the flask, the flask was purged with argon gas three times, cooled to 0-5 °C in an ice-water bath, sodium hydride (54 mg, 1.35 mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 40 minutes, cooled to zero degree, 20 mL of ice water was added, extracted with ethyl acetate (5 mL×2) and chloroform (5 mL×5), the organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing solvent system B to obtain the title product 15b (15 mg, yield: 2.5%). MS m / z (ESI): 515.2 [M+1].

[0264] Step 2 1-(10-(9H-Fluoren-9-yl)-5,8-dioxo-2,9-dioxo-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15c

[0265] Compound 15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate, palladium carbon (3 mg, content 10%, dry) was added, the flask was purged with hydrogen gas three times, and reacted with stirring at room temperature for 4.5 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the title product 15c (11 mg, yield: 89%). MS m / z (ESI): 425.2 [M+1].

[0266] Step 3 (9H-Fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyl)methoxy)methyl)amino)2-oxoethyl)carbamate 15d

[0267] Compound 1b (10 mg, 0.021 μmol) was placed in a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, compound 15c (11 mg, 0.026 mmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10.7 mg, 0.039 mmol) was added. After the addition was complete, the reaction was carried out with stirring for 60 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with a saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 15d (19 mg, yield: 87.0%). MS m / z (ESI): 842.2 [M+1].

[0268] Step 4 1-((2-Aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15e

[0269] Compound 15d (19 mg, 22.56 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure at 0 °C, 1 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper n-hexane layer was decanted three times. The mixture was concentrated under reduced pressure to obtain the title product 15e (13.9 mg) as a crude product, which was used directly in the next step reaction without purification. MS m / z (ESI): 620.1 [M+1].

[0270] Step 5 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazadocosane)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15

[0271] The crude product 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide, replaced with argon gas three times, cooled to 0-5 °C in an ice-water bath, 8g (15.8 mg, 33.4 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 μmol) was added, and the temperature was raised to room temperature and the reaction was carried out with stirring for 60 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding component was collected, concentrated under reduced pressure, and the title product 15 (2.5 mg, yield: 10.3%) was obtained. MS m / z (ESI): 1074.2 [M+1]. 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.51-8.37 (m, 1H), 8.22 (t, 1H), 8.14-8.02 (m, 2H), 8.011-7.94 (m, 1H), 7.82-7.73 (m, 1H), 7.29 (s, 1H), 7.26-7.10 (m, 3H), 6.98 (s, 1H), 6.53-6.47 (m, 1H), 5.62-5.50 (m, 1H), 5.45-5.36 (m, 1H), 5.35-5.23 (m, 2H), 5.13-5.02 (m, 2H), 4.61-4.50 (m, 2H), 4.42-4.28 (m, 2H), 3.76-3.61 (m, 3H), 3.60-3.45 (m, 3H), 3.27-3.23 (m, 1H), 3.20-2.81 (m,7H), 2.75-2.61 (m, 3H), 241-2.28 (m, 3H), 2.23-2.13 (m, 2H), 2.11-2.01 (m, 1H), 2.03-1.94 (m, 1H), 1.90 (s, 1H), 1.87-1.74 (m, 2H), 1.53-1.36 (m, 3H), 1.29-1.08 (m, 4H), 0.90-0.68 (m, 4H).

[0272] Example 1-16 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazadocosane)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16

Chemical formula

[0273] Step 1 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 16b

[0274] Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplier: Alfa) was dissolved in methanol (2 mL) and water (1 mL), sodium hydroxide (126 mg, 3.15 mmol) was added, the temperature was raised to 40 °C, and the reaction was carried out with stirring for 3 hours. After cooling to room temperature, it was concentrated under reduced pressure to remove the organic solvent, back-extracted with ether (10 mL), and the aqueous phase was collected. The aqueous phase was adjusted to pH 3 - 4 with 6 N hydrochloric acid aqueous solution and concentrated under reduced pressure to obtain a solid. 3 mL of toluene was added, and the concentration under reduced pressure and rotary drying were repeated three times. It was dried with an oil pump to obtain the crude title product 16b (206 mg), and the product was used directly in the next step reaction without purification. MS m / z (ESI,NEG): 129.2 [M - 1].

[0275] Step 2 Benzyl 1-(hydroxymethyl)cyclobutane-1-carboxylate 16c

[0276] The crude product 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 mL), potassium carbonate anhydrous (1.09 g, 7.90 mmol) and tetrabutylammonium iodide (29 mg, 78.51 μmol) were added, benzyl bromide (216 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature overnight. It was filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 16c (112 mg, yield: 32.1%). MS m / z (ESI): 221.1 [M + 1].

[0277] Step 3 Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16d

[0278] Compound 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to a reaction flask, 3 mL of tetrahydrofuran was added, the mixture was replaced with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, and the mixture was stirred in the ice bath for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL) and chloroform (5 mL × 5). The combined organic phases were concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.5 mL of water was added, sodium hydrogen carbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (71 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 16d (24 mg, yield: 16.7%). MS m / z (ESI): 551.3 [M+23].

[0279] Step 4 Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16e

[0280] Compound 16d (12 mg, 22.7 μmol) was dissolved in 1.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium carbon (5 mg, content 10%) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain the crude title product 16e (10 mg), which was used directly in the next step reaction without purification.

[0281] Step 5 (9H-Fluoren-9-yl)methyl (2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclobutyl)methoxy)methyl)amino)-2-oxoethyl)carbamate 16f

[0282] Compound 1b (7.5 mg, 0.014 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, a 0.5 mL N,N-dimethylformamide solution of crude product 16e (10 mg) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6 mg, 0.026 mmol) was added, and the reaction was carried out with stirring in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with a saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by thin-layer chromatography with developing solvent system B to obtain the title product 16f (10.6 mg, yield: 87.8%). MS m / z (ESI): 856.2 [M+1].

[0283] Step 6 1-(((2-aminoacetylamino)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16g

[0284] Compound 16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was decanted three times. It was concentrated under reduced pressure to obtain the title product 16g (8 mg) as a crude product, and the product was used directly in the next step reaction without purification. MS m / z (ESI): 634.1 [M+1].

[0285] Step 7 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazadocosane)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16

[0286] The crude product 16g (8 mg) was dissolved in 1 mL of N,N-dimethylformamide, 8g (8.8 mg, 18.6 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (5.2 mg, 18.8 μmol) was added, and the reaction was carried out with stirring at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), and the title product 16 (1.0 mg, yield: 7.2%) was obtained. MS m / z (ESI): 1088.0 [M+1].

[0287] Example 1 - 17 (1r,4r)-N-((S)-7-Benzyl-1-(1-(((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatritetratetracontan-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

Chem.

Chem.

[0288] Step 1 tert-Butyl 1-phenyl-2,5,8,11,14,17,20,23,26,29-decaoxahentriacontane-31-carboxylate 17b

[0289] 1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacosane-28-ol 17a (0.34 g, 0.67 mmol, supplier: Bide) was dissolved in 10 mL of dichloromethane, and silver oxide (0.24 g, 1.01 mmol), tert-butyl bromoacetate (0.16 g, 0.81 mmol), and potassium iodide (0.07 g, 0.40 mmol) were added in sequence, and the reaction was carried out with stirring at room temperature for 3 hours. After filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography with developing solvent system B to obtain the title product 17b (0.42 g, yield: 100%). MS m / z (ESI): 636.3 [M+18].

[0290] Step 2 tert-Butyl 29-hydroxy-3,6,9,12,15,18,21,24,27-nonaoxamontan-1-oate 17c

[0291] Compound 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran, palladium on carbon (110 mg, 10% content, dry) was added, the mixture was purged with hydrogen gas three times, heated to 60 °C and reacted with stirring for 3 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, the filtrate was concentrated to obtain the crude title product 17c (357 mg), and the product was used in the next step reaction without purification. MS m / z (ESI): 546.2 [M+18].

[0292] Step 3 tert-Butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxamontan-1-oate 17d

[0293] Compound 17c (357 mg, 0.675 mmol) was dissolved in 10 mL of toluene, diphenylphosphoric azide (279 mg, 1.014 mmol) and 1,8-diazabicycloundec-7-ene (206 mg, 1.353 mmol) were added, the mixture was purged with argon gas three times, reacted with stirring at room temperature for 2 hours, and then heated to 105 °C and reacted for 19 hours. The reaction mixture was cooled to room temperature, concentrated, 20 mL of water was added, extracted with ethyl acetate (10 mL×4), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing solvent system B to obtain the crude title product 17d (412 mg). MS m / z (ESI): 571.3 [M+18].

[0294] Step 4 tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxamontan-1-oate 17e

[0295] Compound 17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran, palladium carbon (58 mg, content 10%, dry) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, the filtrate was concentrated to obtain the crude title product 17e (220 mg), and the next step reaction was carried out without purifying the product. MS m / z (ESI): 528.2 [M+1].

[0296] Step 5 tert-Butyl 1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azapentatriacontan-31-oate 17f

[0297] (1r,4r)-4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) was dissolved in 10 mL of dichloromethane, 2-(7-benzotriazol-1-yloxy)-N,N,N',N'-tetramethyluronium hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol) were added, the mixture was replaced with argon gas three times, crude 17e (220 mg, 0.417 mmol) was added, and the reaction was carried out with stirring at room temperature for 1 hour. 15 mL of water was added, and the mixture was extracted with dichloromethane (8 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing solvent system B to obtain the title product 17f (122 mg, yield: 39.2%). MS m / z (ESI): 747.2[M+1].

[0298] Step 6 1-((1R,4R)-4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-aza-31-oic acid 17g

[0299] Compound 17f (122 mg, 0.163 μmol) was dissolved in 0.8 mL of dichloromethane, 0.4 mL of trifluoroacetic acid was added, and the reaction was carried out with stirring at room temperature for 1 hour. 15 mL of dichloromethane was added for dilution, concentrated under reduced pressure, the operation of adding 10 mL of n-hexane and concentrating under reduced pressure was repeated twice, then 10 mL of toluene was added and concentrated under reduced pressure, and the mixture was triturated three times with 10 mL of a mixed solvent of n-hexane:ether = 5:1 until the pH was close to 7, concentrated, and dried with an oil pump to obtain the title product 17g (98 mg, yield: 86.8%). MS m / z (ESI): 691.2[M+1].

[0300] Step 7 2,4-Dimethoxybenzyl-1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropyl-1-carboxylate 17h

[0301] Compound 8d (164 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL), and 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide dihydrochloride (115 mg, 0.60 mmol), and 4-dimethylaminopyridine (5 mg, 0.041 mmol) were added in sequence. After the addition was complete, the reaction was carried out with stirring at room temperature for 1 hour. 20 mL of water was added, and after shaking, the layers were separated. The aqueous phase was extracted with dichloromethane (8 mL×3), and the organic phases were combined. The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 17h (124 mg, yield: 55.4%). MS m / z (ESI): 583.1[M+23].

[0302] Step 8 2,4-Dimethoxybenzyl (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazapentadecane-17-yl)oxy)cyclopropyl-1-carboxylate 17j

[0303] Compound 17h (39 mg, 69.6 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was decanted three times and concentrated under reduced pressure. The obtained crude product was dissolved in 2 mL of N,N-dimethylformamide, (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared by the method disclosed in Examples 7 to 12 on page 13 of the specification of Patent Application "CN108853514A") was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83.1 μmol) was added, and the mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 17j (48 mg, yield: 83.9%). MS m / z (ESI): 822.0[M+1].

[0304] Step 9 (S)-1-((11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazapentadecan-17-yl)oxy)cyclopropane-1-carboxylic acid 17k

[0305] Compound 17j (48 mg, 58.4 μmol) was dissolved in 1.4 mL of a dichloromethane solution of 3% (v / v) dichloroacetic acid, cooled to 0 - 5 °C in an ice - water bath, triethylsilane (21 mg, 180.6 μmol) was added, and the reaction was carried out with stirring in the ice - water bath for 3 hours. It was concentrated under reduced pressure in the ice - water bath to remove half of the organic solvent, 5 mL of ether was added, it was allowed to warm naturally to room temperature and triturated, a white solid was precipitated, filtered, the filter cake was collected, and dried with an oil pump to obtain the title product 17k (33 mg, yield: 84.1%).

[0306] Step 10 (9H - Fluoren - 9 - yl)methyl ((S)-7 - benzyl - 1 - (1 - (((1S,9S)-9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12 - tetraoxo - 2,5,8,11 - tetraazatridecan - 13 - yl)carbamate 17l

[0307] Compound 1b (20 mg, 42.4 μmol) was placed in a reaction flask, 1 mL of a 10% (v / v) methanol dichloromethane solution was added, and the mixture was replaced with argon gas three times. It was cooled to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, and the mixture was stirred until compound 1b was dissolved. Compound 17k (33 mg, 49.1 μmol) was dissolved in 1 mL of a 10% (v / v) methanol dichloromethane solution and added dropwise to the above reaction solution. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol) was added. The temperature was raised to room temperature and the reaction was carried out with stirring for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, the mixture was stirred for 5 minutes, allowed to stand for phase separation, the organic phase was collected, the aqueous phase was extracted with dichloromethane (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 17l (37 mg, yield: 80.2%). MS m / z (ESI): 1090.1[M+1].

[0308] Step 11 (1r,4r)-N-((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatetratetracontan-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0309] Compound 17l (15.5 mg, 14.23 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was decanted three times. It was concentrated under reduced pressure and dried with an oil pump. The obtained crude product was dissolved in 1 mL of N,N-dimethylformamide, compound 17g (11 mg, 15.92 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6.0 mg, 21.68 μmol) was added, and the atmosphere was replaced with argon gas three times, and the reaction was carried out with stirring at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding component was collected, concentrated under reduced pressure, and the title product 17 (6 mg, yield: 27.4%) was obtained. MS m / z (ESI): 1556.4 [M+18]. 1 H NMR (400 MHz, DMSO-d 6): δ 8.98 (d, 1H), 8.76 (s, 1H), 8.20 (br, 1H), 8.12 - 7.95 (m, 3H), 7.93 - 7.76 (m, 2H), 7.75 - 7.66 (m, 2H), 7.24 (s, 1H), 7.20 - 7.05 (m, 6H), 6.97 (s, 1H), 6.64 (br, 1H), 6.55 (d, 1H), 6.47 (s, 1H), 5.61 - 5.52 (m, 2H), 5.37 (s, 1H), 5.33 - 5.23 (m, 2H), 5.18 (s, 1H), 5.13 (s, 1H), 5.05 (s, 1H), 5.00 (s, 1H), 4.65 - 4.55 (m, 2H), 4.53 - 4.45 (m, 1H), 4.38 - 4.28 (m, 2H), 3.84 (s, 2H), 3.67 (d, 3H), 3.60 - 3.40 (m, 33H), 3.18 (d, 1H), 3.15 - 3.08 (m, 3H), 2.28 (s, 3H), 2.00 - 1.92 (m, 3H), 1.85 (s, 2H), 1.82 - 1.73 (m, 2H), 1.68 - 1.52 (m, 4H), 1.29 - 1.15 (m, 3H), 0.86 - 0.76 (m, 5H).

[0310] Examples 1 - 18 (1r,4r)-N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxo-5,8,11,14,17-pentaazatetrahexadecane-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18 [Chemical formula] [Chemical]

[0311] Step 1 (R)-Benzyl 2-cyclopropyl-2-hydroxyacetate 18a (S)-Benzyl 2-cyclopropyl-2-hydroxyacetate 18b

[0312] Compound 2a (7.4 g, 63.7 mmol) was dissolved in 200 mL of acetonitrile, and potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol) and tetrabutylammonium iodide (500 mg, 1.36 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 16 hours, filtered through diatomaceous earth, the filter cake was washed with ethyl acetate (10 mL), the filtrates were combined and concentrated under reduced pressure, and the resulting residue (4.1 g) was purified by silica gel column chromatography using developing solvent system C and further purified by chiral resolution to obtain the title products 18a (1.1 g) and 18b (1.2 g).

[0313] Step 2 (R)-Benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundecane-11-carboxylate 18c

[0314] Compound 8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL), compound 18a (2.0 g, 9.70 mmol) was added, the mixture was cooled to 0 - 5 °C in an ice - water bath, potassium tert - butoxide (1.89 g, 16.84 mmol) was added, and the mixture was stirred in the ice - water bath for 10 minutes. Ethyl acetate (30 mL) and water (20 mL) were added, and the mixture was allowed to stand for phase separation. The aqueous phase was extracted with chloroform (30 mL × 5), and the organic phases were combined. The organic phase was concentrated under reduced pressure, and the obtained residue was dissolved in 1,4 - dioxane (32 mL) and water (8 mL). Sodium carbonate (1.78 g, 16.79 mmol) and 9 - fluorenylmethyl chloroformate (2.18 g, 8.42 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by column chromatography with developing solvent system C to obtain the title product 18c (1.3 g, yield: 30.0%). MS m / z (ESI): 515.2[M + 1].

[0315] Step 3 (R)-10 - Cyclopropyl - 1-(9H - fluoren - 9 - yl)-3,6 - dioxo - 2,9 - dioxo - 4,7 - diazaundecane - 11 - acid 18d

[0316] Compound 18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL), palladium carbon (260 mg, content 10%, dry) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 5 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate (20 mL) and methanol (20 mL), the filtrate was concentrated to obtain the crude title product 18d (980 mg), and the product was used in the next - step reaction without purification. MS m / z (ESI): 425.1 [M + 1].

[0317] Step 4 2,4-Dimethoxybenzyl (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-ester 18e

[0318] The crude product 18d (980 mg, 2.31 mmol) was dissolved in dichloromethane (15 mL), 2,4-dimethoxybenzyl alcohol (777 mg, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide dihydrochloride (664 mg, 3.46 mmol) and 4-dimethylaminopyridine (28 mg, 0.23 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to remove the organic solvent, 20 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by column chromatography with developing solvent system C to obtain the title product 18e (810 mg, yield: 61.1%). MS m / z (ESI): 575.0 [M+1].

[0319] Step 5 2,4-Dimethoxybenzyl (R)-2-((2-aminoacetylamino)methoxy)-2-cyclopropylacetate 18f

[0320] Compound 18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and pouring out the upper n-hexane layer was repeated three times. The mixture was concentrated under reduced pressure to obtain the crude title product 18f (21 mg), which was used in the next step reaction without purification.

[0321] Step 6

[0322] 2,4-Dimethoxybenzyl (11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosan-20-oic acid ester 18 g

[0323] The crude product 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, compound 17i (29 mg, 57.8 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19 mg, 68.7 μmol) was added, and the mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 18g (37 mg, yield: 77.1%). MS m / z (ESI): 853.0 [M+18].

[0324] Step 7 (11S,19R)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosan-20-oic acid 18h

[0325] Compound 18g (37 mg, 44.3 μmol) was dissolved in 1.4 mL of a 3% (v / v) dichloroacetic acid dichloromethane solution, cooled to 0-5 °C in an ice-water bath, triethylsilane (15.4 mg, 132.4 μmol) was added, and the reaction was carried out with stirring in the ice-water bath for 3 hours. Concentrate under reduced pressure in an ice bath to remove half of the organic solvent, add 5 mL of ether, allow to warm to room temperature naturally and triturate to precipitate a white solid, filter, collect the filter cake, and dry with an oil pump to obtain the title product 18h (24 mg, yield: 79.1%). MS m / z (ESI): 708.2 [M+23].

[0326] Step 8 (9H-Fluoren-9-yl)methyl ((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazapentadecane-16-yl)carbamate 18i

[0327] Compound 1b (30 mg, 63.6 μmol) was placed in a reaction flask, 1 mL of a 10% (v / v) methanol dichloromethane solution was added, and the mixture was replaced with argon gas three times. It was cooled to 0 - 5 °C in an ice water bath, 1 drop of triethylamine was added dropwise, and the mixture was stirred until compound 1b was dissolved. Compound 18h (65 mg, 94.8 μmol) was dissolved in 1 mL of a 10% (v / v) methanol dichloromethane solution and added dropwise to the above reaction solution. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 μmol) was added. The temperature was raised to room temperature and the reaction was carried out with stirring for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, the mixture was stirred for 5 minutes, allowed to stand for phase separation, the organic phase was collected, the aqueous phase was extracted with dichloromethane (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 18i (25 mg, yield: 35.6%). MS m / z (ESI): 1104.4[M+1].

[0328] Step 9 (S)-2-(2-(2-Aminoacetylamino)acetylamino)-N-(2-((((R)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropanamide 18j

[0329] Compound 18i (12 mg, 10.9 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was decanted three times and concentrated under reduced pressure to obtain the crude title product 18j (10 mg), which was used directly in the next step reaction without purification. MS m / z (ESI): 881.0 [M+1].

[0330] Step 10 (1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxo-5,8,11,14,17-pentaazatetrahexadecane-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0331] The crude product 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, compound 17g (8.5 mg, 12.3 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19×250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding component was collected and concentrated under reduced pressure to obtain the title product 18 (9.5 mg, yield: 56.2%). MS m / z (ESI): 1570.2 [M+18]. 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.77 (d, 1H), 8.59 - 8.55 (m, 1H), 8.42 (d, 1H), 8.37 - 8.28 (m, 1H), 8.25 - 8.06 (m, 2H), 7.96 - 7.86 (m, 1H), 7.86 - 7.70 (m, 2H), 7.32 - 7.28 (m, 1H), 7.25 - 7.14 (m, 3H), 6.67 (m, 1H), 5.96 (s, 1H), 5.80 - 5.72 (m, 1H), 5.62 - 5.52 (m, 2H), 5.43 - 5.30 (m, 3H), 5.28 - 5.17 (m, 2H), 5.12 - 5.08 (m, 1H), 4.72 - 4.35 (m, 8H), 3.95 - 3.70 (m, 13H), 3.35 - 3.22 (m, 14H), 2.42 - 2.32 (m, 3H), 2.05 - 1.98 (m, 4H), 1.88 - 1.82 (m, 12H), 1.47 - 1.39 (m, 3H), 1.32 - 1.18 (m, 11H), 0.90 - 0.80 (m, 4H), 0.52 - 0.37 (m, 3H), 0.32 - 0.18 (m, 2H).

[0332] Example 1 - 19 (1r,4r)-N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxo-5,8,11,14,17-pentaazatetradecan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

Chem.

Chem.

[0333] Step 1 (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxo-4,7-diazaundecane-11-oic acid benzyl 19a

[0334] Compound 18b (252 mg, 1.22 mmol) was placed in a reaction flask, 4 mL of dichloromethane was added, and the mixture was replaced with argon gas three times. The temperature was lowered to 0 - 5 °C in an ice-water bath, lithium tert-butoxide (98 mg, 1.22 mmol) was added, and the reaction was carried out with stirring for 15 minutes in the ice-water bath. When it became clear, 8b (300 mg, 814.3 μmol) was added, and the mixture was stirred for 2.5 hours in the ice-water bath. Water (10 mL) was added, and the layers were separated. The aqueous phase was extracted with dichloromethane (8 mL × 2). After combining the organic phases, they were washed with water (10 mL × 1) and saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The obtained residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 19a (282 mg, yield: 67.2%).

[0335] Step 2 (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-oic acid 19b

[0336] Compound 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate, palladium carbon (84 mg, content 10%, dry) was added, the mixture was replaced with hydrogen gas three times, and the reaction was carried out with stirring at room temperature for 3 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 19b (230 mg). The product was used in the next step reaction without purification.

[0337] Step 3 2,4-Dimethoxybenzyl (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundec-11-oate 19c

[0338] The crude product 19b (230 mg, 541.8 μmol) was dissolved in 7 mL of dichloromethane, 2,4-dimethoxybenzyl alcohol (136.7 mg, 812.7 μmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (155 mg, 808.5 μmol) and 4-dimethylaminopyridine (6.6 mg, 53.5 μmol) were added in sequence, and the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with 10 mL of dichloromethane, washed with water (10 mL×1), washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The obtained residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 19c (159 mg, yield: 51.0%).

[0339] Step 4 2,4-Dimethoxybenzyl (S)-2-((2-aminoacetylamino)methoxy)-2-cyclopropylacetate 19d

[0340] Compound 19c (60 mg, 104.4 μmol) was dissolved in 1 mL of dichloromethane, 0.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper n-hexane layer was decanted three times. The mixture was concentrated under reduced pressure to obtain the crude title product 19d (21 mg), which was used in the next step reaction without purification.

[0341] Step 5 2,4-Dimethoxybenzyl (11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosan-20-oate 19e

[0342] The crude product 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, compound 17i (52 mg, 103.6 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (34.6 mg, 125.0 μmol) was added, and the mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 19e (70 mg, yield: 80.2%).

[0343] Step 6 (11S,19S)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosanoic acid 19f

[0344] Compound 19e (70 mg, 83.7 μmol) was dissolved in 2.5 mL of a dichloromethane solution of 3% (v / v) dichloroacetic acid, cooled to 0 - 5 °C in an ice - water bath, triethylsilane (29 mg, 249.4 μmol) was added, and the reaction was carried out with stirring in the ice - water bath for 3 hours. The reaction mixture was concentrated under reduced pressure in the ice - water bath to remove half of the organic solvent, 5 mL of ether was added, the temperature was allowed to rise to room temperature naturally and shaken, a white solid was precipitated, filtered, the filter cake was collected, and dried with an oil pump to obtain the title product 19f (57 mg, yield: 99.2%).

[0345] Step 7 (9H - Fluoren - 9 - yl)methyl ((2S,10S)-10 - benzyl - 2 - cyclopropyl - 1 - (((1S,9S)-9 - ethyl - 5 - fluoro - 9 - hydroxy - 4 - methyl - 10,13 - dioxo - 2,3,9,10,13,15 - hexahydro - 1H,12H - benzo[de]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)amino - 1,6,9,12,15 - pentaoxo - 3 - oxa - 5,8,11,14 - tetraazapentadecane - 16 - yl)carbamate 19g

[0346] Compound 1b (30 mg, 63.6 μmol) was placed in a reaction flask, 1 mL of a 10% (v / v) methanol dichloromethane solution was added, the mixture was purged with argon gas three times, cooled to 0 - 5 °C in an ice-water bath, 1 drop of triethylamine was added dropwise, and the mixture was stirred until compound 1b was dissolved. Compound 19f (57 mg, 83.1 μmol) was dissolved in 1 mL of a 10% (v / v) methanol dichloromethane solution and added dropwise to the above reaction solution, and then 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol) was added. The temperature was raised to room temperature and the reaction was carried out with stirring for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, the mixture was stirred for 5 minutes, allowed to stand for phase separation, the organic phase was collected, the aqueous phase was extracted with dichloromethane (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 19g (56 mg, yield: 79.8%). MS m / z (ESI): 1103.1[M+1].

[0347] Step 8 (S)-2-(2-(2-Aminoacetylamino)acetylamino)-N-(2-((((S)-1-cyclopropyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide 19h

[0348] Compound 19g (4.6 mg, 4.16 μmol) was dissolved in 1.5 mL of dichloromethane, 0.75 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.6 hours. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and the concentration under reduced pressure was repeated twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was decanted three times. Then it was concentrated under reduced pressure to obtain the crude title product 19h (4.0 mg), which was used directly in the next step reaction without purification.

[0349] Step 9 (1r,4r)-N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxo-5,8,11,14,17-pentaazatetrahexadecane-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0350] The crude product 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, 17g (2.9 mg, 4.2 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 μmol) was added, and the mixture was stirred at room temperature for 40 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19×250 mm, mobile phase: A - water (10 mmol NH 4 OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding component was collected and concentrated under reduced pressure to obtain the title product 19 (2.1 mg, yield: 32.4%). 11H NMR (400 MHz, DMSO-d 6 ): δ 8.71 - 8.62 (m, 1H), 8.59 - 8.51 (m, 1H), 8.34 - 8.26 (m, 1H), 8.14 - 8.02 (m, 2H), 7.95 - 7.86 (m, 1H), 7.83 - 7.69 (m, 2H), 7.35 - 7.31 (m, 1H), 7.29 - 7.11 (m, 3H), 7.01 (s, 1H), 6.72 - 6.50 (m, 3H), 5.59 - 5.50 (m, 2H), 5.42 (s, 2H), 5.38 - 5.18 (m, 3H), 4.79 - 4.69 (m, 2H), 4.61 - 4.42 (m, 3H), 3.91 (s, 2H), 3.79 - 3.65 (m, 4H), 3.63 - 3.44 (m, 13H), 3.41 - 3.30 (m, 2H), 3.26 - 3.09 (m, 5H), 3.08 - 2.84 (m, 4H), 2.81 - 2.64 (m, 3H), 2.42 - 2.28 (m, 3H), 2.24 - 2.12 (m, 2H), 2.05 - 1.93 (m, 4H), 1.89 - 1.77 (m, 2H), 1.72 - 1.56 (m, 3H), 1.53 - 1.38 (m, 3H), 1.34 - 1.10 (m, 11H), 0.94 - 0.78 (m, 5H), 0.52 - 0.35 (m, 3H).

[0351] Examples 1 - 20 (Reference Examples)

Chemical Structure

[0352] Title Compound 20 was synthesized by the method provided in Example 58 on page 163 of the specification of Patent "CN104755494A".

[0353] The following antibodies were prepared by the usual methods for antibodies. For example, after constructing a vector, eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019) may be transfected, expressed, and purified to obtain them.

[0354] The following is the sequence of Trastuzumab. Light chain

Chem.

Chem.

[0355] The following is the sequence of Pertuzumab. Light chain

Chem.

Chem.

[0356] The following is the sequence of B7H3 antibody 1F9DS. Light chain

Chem.

[0357] Examples 1 - 21 ADC - 1

Chem.

[0358] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to the PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5, 2.5 mL, 9.96 mg / mL, 0.168 μmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was taken out for continuous reaction.

[0359] Compound 10 - a compound with a relatively short retention time (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, added to the above 2.0 mL of the solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours. The reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH = 6.5 containing 0.001 M EDTA), and an exemplary product ADC-1 of the general formula of FADC-1 in PBS buffer (5.0 mg / mL, 1.1 mL) was obtained and stored at 4 °C.

[0360] The average value was calculated by UV-HPLC: n = 5.09.

[0361] Example 1 - 22 ADC-2

Chemical formula

[0362] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to the PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5, 2.5 mL, 9.96 mg / mL, 0.168 μmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was taken out for continuous reaction.

[0363] Compound 10 - A compound with a relatively long retention time (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, added to the above 2.0 mL solution, placed in a water bath shaker, and reacted with shaking at 25°C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (4.95 mg / mL, 1.1 mL) of the exemplary product ADC-2 of the general formula of FADC-1 was obtained and stored at 4°C.

[0364] The average value was calculated by UV-HPLC: n = 7.39.

[0365] Example 1-23 ADC-3

Chemical formula

[0366] Under the condition of 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5, 2.5 mL, 9.96 mg / mL, 0.168 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was taken out and reacted continuously.

[0367] Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, added to the above 2.0 mL solution, placed in a water bath shaker, and reacted with shaking at 25°C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (5.24 mg / mL, 1.1 mL) of the exemplary product ADC-3 of the general formula of FADC-3 was obtained and stored at 4°C.

[0368] The average value was calculated by UV-HPLC: n = 7.36.

[0369] Example 1-24 ADC-4

Chemical formula

[0370] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) incorporated was added to an aqueous PBS buffer solution (0.05 M PBS buffer solution at pH = 6.5, 3.74 mL, 13.38 mg / mL, 0.338 μmol) of the antibody Trastuzumab, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, the reaction solution was cooled to 25 °C in a water bath, diluted to 6.7 mg / mL, and 1.3 mL of the solution was taken out and continuously reacted.

[0371] Compound 9 - Compound 9-A with a relatively short retention time (1.0 mg, 0.93 μmol) was dissolved in 0.10 mL of DMSO, added to the above 1.3 mL of the solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours. The reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA), and an exemplary product ADC-4 of the general formula of FADC-4A in PBS buffer (1.72 mg / mL, 2.36 mL) was obtained and stored at 4 °C.

[0372] The average value was calculated by UV-HPLC: n = 7.39.

[0373] Example 1-25 ADC-5

Chemical formula

[0374] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) formulated was added to the PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution at pH = 6.5, 3.0 mL, 6.70 mg / mL, 0.136 μmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25 °C in a water bath. 0.614 mL of the solution was taken out and the reaction was continued.

[0375] Compound 9 - Comparatively short retention time compound 9-A (0.5 mg, 0.42 μmol) was dissolved in 0.031 mL of DMSO, added to the above 0.614 mL of the solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours. The reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5 containing 0.001 M EDTA), and an exemplary product ADC-5 of the general formula of FADC-4A in PBS buffer (3.08 mg / mL, 0.82 mL) was obtained and stored at 4 °C.

[0376] The average value was calculated by UV-HPLC: n = 3.16.

[0377] Example 1-26 ADC-6

Chemical formula

[0378] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) formulated was added to the PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution at pH = 6.5, 3.74 mL, 13.38 mg / mL, 0.338 μmol). It was placed in a water bath shaker and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25 °C in a water bath and diluted to 6.7 mg / mL. 0.75 mL of the solution was taken out and the reaction was continued.

[0379] Compound 9 - A compound 9 - B with a relatively long retention time (0.68 mg, 0.63 μmol) was dissolved in 0.10 mL of DMSO, added to the above 0.75 mL of solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.78 mg / mL, 1.78 mL) of an exemplary product ADC - 6 of the general formula of FADC - 4B was obtained and stored at 4 °C.

[0380] The average value was calculated by UV - HPLC: n = 3.94.

[0381] Example 1 - 27 ADC - 7

Chemical formula

[0382] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution of antibody Pertuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5, 5.0 mL, 10 mg / mL, 0.338 μmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 1.0 mL of the solution was taken out for continuous reaction.

[0383] Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 mL of DMSO, added to the above 1.0 mL of solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.42 mg / mL, 2.15 mL) of an exemplary product ADC - 7 of the general formula of FADC - 7 was obtained and stored at 4 °C.

[0384] The average value was calculated by UV-HPLC: n = 6.91.

[0385] Example 1-28 ADC-8

Chemical formula

[0386] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) formulated was added to an aqueous PBS buffer solution of antibody Pertuzumab (0.05 M PBS buffer solution with pH = 6.5, 5.0 mL, 10 mg / mL, 0.338 μmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, the reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 1.6 mL of the solution was taken out and continuously reacted.

[0387] Compound 10 - a compound with a relatively short retention time (1.04 mg, 1.0 μmol) was dissolved in 0.1 mL of DMSO, added to the above 1.6 mL of the solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours. The reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with pH = 6.5 containing 0.001 M EDTA), and a PBS buffer solution of an exemplary product ADC-8 of the general formula of FADC-8 (2.14 mg / mL, 2.31 mL) was obtained and stored at 4 °C.

[0388] The average value was calculated by UV-HPLC: n = 6.58.

[0389] Example 1-29 ADC-9

Chemical formula

[0390] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to the PBS buffer aqueous solution of antibody Pertuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5, 5.0 mL, 10 mg / mL, 0.338 μmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours. The reaction was stopped, the reaction solution was cooled to 25 °C in a water bath, diluted to 5.0 mg / mL, and 0.8 mL of the solution was taken out and reacted continuously.

[0391] Compound 9 - relatively short retention time compound 9 - A (0.55 mg, 0.5 μmol) was dissolved in 0.1 mL of DMSO, added to the above 0.8 mL of the solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours. The reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH = 6.5 containing 0.001 M EDTA), and an exemplary product ADC - 9 of the general formula of FADC - 9A in PBS buffer (2.27 mg / mL, 1.11 mL) was obtained and stored at 4 °C.

[0392] The average value was calculated by UV - HPLC: n = 3.16.

[0393] Example 1 - 30 ADC - 10

Chemical formula

[0394] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) was added to the PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution with pH = 6.5, 10.0 mg / mL, 0.574 mL, 38.78 nmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours. The reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0395] Compound 14 - A compound with a relatively short retention time (0.64 mg, 588 nmol) was dissolved in 40 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (5.48 mg / mL, 1.03 mL) of the exemplary product ADC - 10 of the general formula of FADC - 10 was obtained and stored at 4 °C.

[0396] The average value was calculated by UV - Vis: n = 6.25.

[0397] Example 1 - 31 ADC - 11

Chemical formula

[0398] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) was added to an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer aqueous solution with a pH of 6.5, 10.0 mg / mL, 0.646 mL, 43.64 nmol), placed in a water bath shaker, and shaken at 37 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0399] Compound 14 - A compound with a relatively long retention time (0.72 mg, 662 nmol) was dissolved in 40 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (2.13 mg / mL, 1.87 mL) of the exemplary product ADC - 11 of the general formula of FADC - 10 was obtained and stored at 4 °C.

[0400] The average value was calculated by UV - Vis: n = 7.03.

[0401] Examples 1 - 32 ADC - 12

Chemical formula

[0402] Under the condition of 37°C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol) formulated in an aqueous PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 0.726 mL, 49.05 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0403] Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution of an exemplary product ADC - 12 of the general formula of FADC - 12 (3.34 mg / mL, 1.45 mL) was obtained and stored at 4°C.

[0404] The average value was calculated by UV - Vis: n = 6.93.

[0405] Examples 1 - 33 ADC - 13

Chemical formula

[0406] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol) incorporated in a PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution at pH = 6.5, 10.0 mg / mL, 0.287 mL, 19.39 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0407] Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and an example product ADC-13 of the general formula of FADC-13 in PBS buffer (2.37 mg / mL, 0.88 mL) was obtained and stored at 4 °C.

[0408] The average value was calculated by UV-Vis: n = 6.53.

[0409] Example 1-34 ADC-14

Chemical formula

[0410] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) incorporated in a PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution at pH = 6.5, 10.0 mg / mL, 0.592 mL, 40.0 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0411] Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 °C for 3 hours to carry out a reaction, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (0.30 mg / mL, 12.0 mL) of the exemplary product ADC-14 of the general formula of FADC-14 was obtained and stored at 4 °C.

[0412] The average value was calculated by UV-Vis: n = 7.61.

[0413] Example 1-35 ADC-15

Chemical formula

[0414] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer aqueous solution with a pH of 6.5, 10.0 mg / mL, 0.592 mL, 40.0 nmol), placed in a water bath shaker, and shaken at 37 °C for 3 hours to carry out a reaction, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0415] Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 °C for 3 hours to carry out a reaction, and then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (0.32 mg / mL, 11.8 mL) of the exemplary product ADC-15 of the general formula of FADC-15 was obtained and stored at 4 °C.

[0416] The average value was calculated by UV-Vis: n = 7.89.

[0417] Example 1-36 ADC-16

Chem.

[0418] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol) formulated in a PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution at pH = 6.5, 10.0 mg / mL, 0.53 mL, 35.8 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0419] Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution of an exemplary product ADC-16 of the general formula of FADC-16 (0.32 mg / mL, 12.0 mL) was obtained and stored at 4 °C.

[0420] The average value was calculated by UV-Vis: n = 7.43.

[0421] Example 1-37 ADC-17

Chem.

[0422] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol) formulated in a PBS buffer aqueous solution of antibody Trastuzumab (0.05 M PBS buffer aqueous solution at pH = 6.5, 10.0 mg / mL, 2.0 mL, 135.12 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0423] Compound 9 - Compound 9 - A with a relatively short retention time (2.22 mg, 2067 nmol) was dissolved in 175 μL of DMSO, added to the above reaction solution, placed on a water bath shaker, and shaken at 25 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.32 mg / mL, 12.0 mL) of the exemplary product ADC - 17 of the general formula of FADC - 4A was obtained and stored at 4 °C.

[0424] The average value was calculated by UV - Vis: n = 5.42.

[0425] Example 1 - 38 ADC - 18 (Reference Example)

Chemical Formula

[0426] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) prepared was added to an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer aqueous solution with a pH of 6.5, 10.0 mg / mL, 1.5 mL, 101.3 nmol), placed on a water bath oscillator, and oscillated at 37 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0427] Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed on a water bath shaker, and shaken at 25 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (0.79 mg / mL, 13.0 mL) of the exemplary product ADC - 18 of the general formula of FADC - 18 was obtained and stored at 4 °C.

[0428] The average value was calculated by UV-Vis: n = 7.23.

[0429] Examples 1-39 ADC-19

Chemical formula

[0430] Under the condition of 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol) was added to an aqueous PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 1.36 mL, 91.9 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0431] Compound 9 - Comparatively short retention time compound 9-A (2.0 mg, 1862 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffer solution (0.73 mg / mL, 13.0 mL) of the exemplary product ADC-19 of the general formula of FADC-4A, and stored at 4°C.

[0432] The average value was calculated by UV-Vis: n = 6.26.

[0433] Examples 1-40 ADC-20

Chemical formula

[0434] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) prepared was added to an aqueous PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 1.5 mL, 101.3 nmol), placed in a water bath shaker, and subjected to an oscillating reaction at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0435] Compound 10 - a compound with a relatively long retention time (2.0 mg, 1815 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and subjected to an oscillating reaction at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and an exemplary product ADC-20 of the general formula of FADC-1 in PBS buffer solution (0.73 mg / mL, 13.0 mL) was obtained and stored at 4 °C.

[0436] The average value was calculated by UV-Vis: n = 7.43.

[0437] Example 1-41 ADC-21 (Reference Example)

Chemical formula

[0438] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) formulated was added to an aqueous PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 1.86 mL, 125.4 nmol), placed in a water bath shaker, and subjected to an oscillating reaction at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0439] Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, shaken and reacted at 25 °C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (2.91 mg / mL, 4.44 mL) of the exemplary product ADC-21 of the general formula of FADC-18 was obtained and stored at 4 °C.

[0440] The average value was calculated by UV-Vis: n = 7.23.

[0441] Example 1-42 ADC-22

Chemical formula

[0442] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol) was added to an aqueous PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer aqueous solution with a pH of 6.5, 10.0 mg / mL, 1.88 mL, 127.2 nmol), placed in a water bath shaker, shaken and reacted at 37 °C for 3 hours, and the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0443] Compound 9 - relatively short retention time compound 9-A (2.1 mg, 1955 nmol) was dissolved in 150 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, shaken and reacted at 25 °C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (3.56 mg / mL, 3.98 mL) of the exemplary product ADC-22 of the general formula of FADC-4A was obtained and stored at 4 °C.

[0444] The average value was calculated by UV-Vis: n = 6.79.

[0445] Example 1 - 43 ADC - 23 (Reference Example)

Chemical Structure

[0446] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μmol) was added to the PBS - buffered aqueous solution of antibody Trastuzumab (0.05 M PBS - buffered aqueous solution with pH = 6.5, 10.0 mg / mL, 345 mL, 23.31 μmol). It was placed in a water - bath shaker and reacted with shaking at 37 °C for 3.5 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water - bath.

[0447] Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL of MeCN and 3.56 mL of DMSO, added to the above reaction solution, placed in a water - bath shaker, and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was passed through an ultrafiltration membrane bag and desalted and purified successively with a PBS - buffered aqueous solution (0.05 M PBS - buffered aqueous solution with pH = 6.5) containing 2% (v / v) MeCN and 1% (v / v) DMSO, and a succinic acid - buffered aqueous solution (0.01 M succinic acid - buffered aqueous solution with pH = 5.3). Then, sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL. It was put into a flask, freeze - dried, and a freeze - dried powder sample of the exemplary product ADC - 23 of the general formula of FADC - 18 was obtained and stored at 4 °C.

[0448] The average value was calculated by UV - Vis: n = 7.05.

[0449] Example 1 - 44 ADC - 24

Chemical Structure

[0450] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol) was added to an aqueous PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 332 mL, 22.43 μmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3.5 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0451] Compound 9 - Relatively short retention time compound 9 - A (241 mg, 224 μmol) was dissolved in 13.76 mL of MeCN and 6.88 mL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was passed through an ultrafiltration membrane bag and sequentially desalted and purified with a PBS buffer aqueous solution (0.05 M PBS buffer aqueous solution at pH = 6.5) containing 4% (v / v) MeCN and 2% (v / v) DMSO, and a succinic acid buffer aqueous solution (0.01 M succinic acid buffer aqueous solution at pH = 5.3). Then, sucrose was added to 60 mg / mL and Tween 20 was added to 0.2 mg / mL, placed in a flask, freeze-dried, and a freeze-dried powder sample of the exemplary product ADC - 24 of the general formula of FADC - 4A was obtained and stored at 4 °C.

[0452] The average value was calculated by UV-Vis: n = 7.07.

[0453] Example 1 - 45 ADC - 25

Chemical formula

[0454] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol) was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 2.14 mL, 144.60 nmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0455] Compound 9 - A compound 9 with a relatively short retention time (3.0 mg, 2793 nmol) was dissolved in 150 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, shaken and reacted at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.28 mg / mL, 13.0 mL) of an exemplary product ADC - 25 of the general formula of FADC - 25 was obtained and stored at 4°C.

[0456] The average value was calculated by UV - Vis: n = 6.87.

[0457] Example 1 - 46 ADC - 26 (Reference Example) [Chemical formula]

[0458] Under the condition of 37°C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with a pH of 6.5, 10.0 mg / mL, 0.89 mL, 60.14 nmol), placed in a water bath shaker, shaken and reacted at 37°C for 3 hours, and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0459] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, shaken and reacted at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.61 mg / mL, 4.0 mL) of an exemplary product ADC - 26 of the general formula of FADC - 26 was obtained and stored at 4°C.

[0460] The average value was calculated by UV-Vis: n = 6.15.

[0461] Examples 1-47 ADC-27

Chemical formula

[0462] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) incorporated into an aqueous PBS buffer solution (0.05 M PBS buffer solution with pH = 6.5, 10.0 mg / mL, 0.89 mL, 60.14 nmol) of antibody B7H3 antibody 1F9DS was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0463] Compound 9 - relatively short retention time compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.94 mg / mL, 3.5 mL) of an exemplary product ADC-27 of the general formula of FADC-25 was obtained and stored at 4 °C.

[0464] The average value was calculated by UV-Vis: n = 6.11.

[0465] Example 1-48 ADC-28 (reference example)

Chemical formula

[0466] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol) formulated in a PBS buffer aqueous solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with pH = 6.5, 10.0 mg / mL, 2.36 mL, 159.47 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0467] Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours to stop the reaction. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with pH = 6.5, containing 0.001 M EDTA), and a PBS buffer solution of an exemplary product ADC-28 of the general formula of FADC-26 (1.29 mg / mL, 13.0 mL) was obtained and stored at 4 °C.

[0468] The average value was calculated by UV-Vis: n = 7.46.

[0469] Example 1-49 ADC-29

Chemical formula

[0470] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 290 nmol) formulated in a PBS buffer aqueous solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with pH = 6.5, 10.0 mg / mL, 0.80 mL, 50.06 nmol) was added, placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours to stop the reaction. The reaction solution was cooled to 25 °C in a water bath.

[0471] Compound 9 - Compound 9 - A with a relatively short retention time (1.29 mg, 1201 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (2.63 mg / mL, 2.4 mL) of the exemplary product ADC - 29 of the general formula of FADC - 25 was obtained and stored at 4 °C.

[0472] The average value was calculated by UV - Vis: n = 7.24.

[0473] Example 1 - 50 ADC - 30 (Reference Example)

Chemical formula

[0474] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol) was added to an aqueous PBS buffer solution of antibody B7H3 antibody 1F9DS (0.05 M PBS buffer aqueous solution with a pH of 6.5, 10.0 mg / mL, 0.86 mL, 58.4 nmol), placed in a water bath shaker, and shaken at 37 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0475] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 °C for 3 hours for a reaction, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer aqueous solution with a pH of 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.61 mg / mL, 4.0 mL) of the exemplary product ADC - 30 of the general formula of FADC - 26 was obtained and stored at 4 °C.

[0476] The average value was calculated by UV - Vis: n = 6.15.

[0477] Examples 1 - 51 ADC - 31

Chem.

[0478] Under the condition of 37 °C, an aqueous solution of tris(2 - carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) formulated in an aqueous PBS buffer solution of anti - body B7H3 antibody 1F9DS (0.05 M PBS buffer solution with pH = 6.5, 10.0 mg / mL, 0.89 mL, 60.14 nmol) was added. It was placed in a water - bath shaker and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0479] Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μL of DMSO, added to the above reaction solution, placed in a water - bath shaker, and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with pH = 6.5, containing 0.001 M EDTA), and a PBS buffer solution of an exemplary product ADC - 31 of the general formula of FADC - 31 (1.47 mg / mL, 4.5 mL) was obtained and stored at 4 °C.

[0480] The average value was calculated by UV - Vis: n = 6.33.

[0481] Examples 1 - 52 ADC - 32

Chem.

[0482] Under the condition of 25 °C, a buffer solution (pH 5.6) containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA, 5.0 g of trastuzumab stock solution (34.44 μmol, trastuzumab was diluted to an antibody final concentration of 15 mg / mL with 20 mM histidine-hydrochloric acid buffer solution), and 34.64 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 120.84 μmol) were reacted with stirring in a constant temperature water bath for 3 hours to generate an intermediate I solution.

[0483] The relatively short retention time compound 9-A (406.2 mg, 378.17 μmol) of compound 9 was dissolved in 9.98 mL of DMSO to generate a DMSO solution of compound 9-A. 23.42 mL of DMSO was added to the above intermediate I solution in advance, and then the DMSO solution of compound 9-A was added to the intermediate I solution with DMSO added in advance. The reaction was carried out with stirring at 25 °C in a water bath for 1 hour. Cysteine was added to quench the reaction, and then filtration was performed. At a temperature of 25 °C, the reaction solution was passed through an ultrafiltration membrane package (30 kd) and sequentially ultrafiltered with 20 mM histidine-hydrochloric acid containing 10% (v / v) DMSO, 2.5 mM EDTA buffer aqueous solution (pH = 6.0), and 10 mM histidine-hydrochloric acid buffer aqueous solution (pH = 5.5) with volumes 10 times and 16 times the volume for equal-volume ultrafiltration liquid replacement to remove small molecules and residual solvents, and an exemplary product ADC-32 of the general formula of FADC-4A was obtained.

[0484] The average value was calculated by the HIC method: n = 6.0.

[0485] Example 1-53 ADC-33

Chemical formula

[0486] Under the condition of 37 °C, a buffer solution (pH 6.0) containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA, 8.0 g of trastuzumab stock solution (55.11 μmol, trastuzumab was diluted to an antibody final concentration of 15 mg / mL with 20 mM histidine buffer solution), and 123.95 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 432.41 μmol) were reacted with stirring in a constant temperature water bath for 5 hours to generate an intermediate I solution.

[0487] The relatively short retention time compound 9-A (754.7 mg, 702.63 μmol) of compound 9 was dissolved in 15.99 mL of DMSO to generate a DMSO solution of compound 9-A. 37.34 mL of DMSO was added to the above intermediate I solution in advance, and then the DMSO solution of compound 9-A was added to the intermediate I solution with DMSO added in advance, and the reaction was carried out with stirring at 37 °C in a water bath for 1 hour, and the reaction was stopped and filtered. At a temperature of 25 °C, the reaction solution was passed through an ultrafiltration membrane package (30 kd) and sequentially subjected to ultrafiltration liquid replacement with 8 times and 16 times the volume of 20 mM histidine-hydrochloric acid containing 10% (v / v) DMSO, 2.5 mM EDTA buffer aqueous solution (pH = 6.0), and 10 mM histidine-hydrochloric acid buffer aqueous solution (pH = 6.0) to remove small molecules and residual solvents, and an exemplary product ADC-33 of the general formula of FADC-4A was obtained.

[0488] The average value was calculated by the HIC method: n = 7.15. Analysis of the drug loading of the ADC stock solution

[0489] Purpose and principle of the experiment ADC is an antibody conjugate drug, and its mechanism of treating diseases is to kill cells by transporting toxin molecules into cells through the target-directed property of the antibody. The drug loading plays a decisive role in the drug efficacy. The drug loading of the ADC stock solution was measured by ultraviolet-visible spectrophotometry (UV-Vis) or hydrophobic chromatography (HIC).

[0490] I. Ultraviolet-visible spectrophotometry A cuvette containing sodium succinate buffer was placed in each of the reference absorption cell and the sample measurement absorption cell. After subtracting the solvent blank, a cuvette containing the test solution was placed in the sample measurement absorption cell, and the absorbances at 280 nm and 370 nm were measured.

[0491] Calculation of results: The ADC stock solution loading amount was measured by ultraviolet spectrophotometry (equipment used: Thermo nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance value of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody at that wavelength. That is, (1) A 280nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 : The average molar extinction coefficient of the drug at 280 nm is 5100, C Drug : The concentration of the drug, ε mab-280 : The average molar extinction coefficient of the trastuzumab stock solution or pertuzumab stock solution at 280 nm is 214600, C mab : The concentration of the trastuzumab stock solution or pertuzumab stock solution, b: The optical path length is 1 cm. Similarly, the total absorbance value equation of the sample at 370 nm can be obtained, that is, (2) A 370nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 : The average molar extinction coefficient of the drug at 370 nm is 19000, C Drug: The concentration of the drug, ε mab-370 : The extinction coefficient of the trastuzumab stock solution or pertuzumab stock solution at 370 nm is 0, Cmab: Concentration of the trastuzumab stock solution, b: The optical path length is 1 cm.

[0492] (1) and (2) The drug loading can be calculated by combining the data of the extinction coefficients and concentrations at two detection wavelengths of the monoclonal antibody and the drug. Drug loading n = CDrug / Cmab.

[0493] II. Hydrophobic chromatography 2.1 HPLC analysis was performed under the following measurement conditions. HPLC system: Agilent high-performance liquid chromatography HPLC system Detector: DAD detector (measurement wavelength: 280 nm) Column: TSKgel Butyl-NPR (4.6 mm × 100 mm, 2.5 μm) Column temperature: 30 °C Flow rate: 0.5 mL / min Temperature of the sample chamber: 4 °C Mobile phase A: Aqueous solution of pH 7.00 containing 1.5 M ammonium sulfate ((NH 4 ) 2 SO 4 ) and 20 mM disodium hydrogen phosphate (Na2HPO4). Mobile phase B: A mixed solution of pH 7.00 containing 75% 20 mM disodium hydrogen phosphate (Na2HPO4) and 25% isopropanol. Gradient program: 0.0%B~0.0%B (0 min~3.0 min), 0.0%B~100.0%B (3.0 min~30.0 min), 100.0%B~ 100.0%B (30.0 min~33.0 min), 0.0%B~0.0%B (33.1 min~40.0 min), Injected sample volume: 50 μg

[0494] 2.2 Data analysis Based on the current data, due to the characteristics of the column, the antibody-drug conjugates were eluted in ascending order of the number of bound drugs based on the difference in salt concentration, and thus the distribution of the number of conjugated drugs was obtained by measuring each peak area value. The peaks were eluted in the order of D0 (antibody not bound to the drug linker), D2, D4, D6, and D8, and the calculation formula for the drug loading amount DAR(n) is as follows.

[0495]

Table 4

[0496] Biological evaluation Test Example 1-1: In vitro growth inhibition test of the compound of the present disclosure on tumor cells I. Purpose of the test This experiment aims to detect the in vitro growth inhibitory activity of the drug compound of the present disclosure on U87MG cells (Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, product number HTB-30). Cells were treated in vitro with different concentrations of the compound and cultured for 6 days, and then the growth of the cells was detected using CTG (CellTiter-Glo (R) Luminescent Cell Viability Assay, Promega, product number: G7573) reagent, and the in vitro activity of the compound was evaluated based on the IC 50 value.

[0497] II. Experimental method Hereinafter, taking the method of the in vitro growth inhibition test on U87MG cells as an example, the method for conducting the in vitro growth inhibition activity test of the compound of the present disclosure in the present disclosure on tumor cells will be exemplarily described. This method is similarly applicable to, but not limited to, the in vitro growth inhibition activity test on other tumor cells. 1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, product number SH30024.01) containing 10% FBS and McCoy's 5A medium (Gibco, product number 16600-108) containing 10% FBS, respectively. 2. Preparation of cells: U87MG and SK-BR-3 cells in the logarithmic growth phase were taken, washed once with PBS (phosphate buffer solution, Shanghai Yuanpei Biotechnology Co., Ltd.), then 2 - 3 mL of trypsin (0.25% Trypsin-EDTA(1x), Gibico, Life Technologies) was added and digested for 2 - 3 minutes. After the cells were completely digested, 10 - 15 mL of cell culture medium was added to elute the digested cells, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and then 10 - 20 mL of cell culture medium was added to resuspend the cells to prepare a single-cell suspension. 3. Seeding of cells: The single-cell suspensions of U87MG and SK-BR-3 were uniformly mixed, and the viable cell density was adjusted to 2.75×10 3 cells / mL and 8.25×10 3 cells / mL respectively with cell culture medium. The cell suspensions with adjusted density were uniformly mixed and placed into a 96-well cell culture plate at 180 μL / well. Only 200 μL of medium was added to the outer wells of the 96-well plate. The culture plate was incubated in an incubator for 24 hours (37°C, 5% CO 2 ₂). 4. Preparation of compounds: The compounds were dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a stock solution with an initial concentration of 10 mM. The initial concentration of the small molecule compound was 500 nM, and the drug preparation method was as follows. 30 μL of different samples to be tested were added to each well in the first column of a 96-well U-bottom compounding plate, with the sample concentration set to 100 μm, and 20 μL of DMSO was added to each well in the second to eleventh columns. 10 μL of the sample from the first column was taken and added to the 20 μL of DMSO in the second column, mixed uniformly, 10 μL was taken and added to the third column, and so on until the tenth column. 5 μL of the drug in the compounding plate was taken for each well and added to 95 μL of EMEM medium, mixed uniformly for use. The initial concentration of the ADC was 10 nM or 500 nM, and the drug preparation method was as follows. In the first column of a 96-well plate, 100 μL of different samples to be tested were added respectively, with the sample concentration being 100 nM or 5 μM, and 100 μL of PBS was added to each well of columns 2 to 11. 50 μL of the sample in column 1 was taken and added to 100 μL of PBS in column 2, mixed evenly, and 50 μL was taken and added to column 3, and thus, the samples were diluted 3-fold by analogy up to column 10. 5. Sample addition: 20 μL of samples to be measured, formulated at different concentrations, were added to the culture plate so that there were two duplicate wells per sample. The culture plate was then incubated in an incubator (37°C, 5% CO) for 6 days. 2 ) incubated. 6. Color development procedure: Take out a 96-well cell culture plate, add 90 μL of CTG solution to each well, and incubate at room temperature for 10 minutes. 7. Plate reading operation: The 96-well cell culture plate was taken out and placed on a microplate reader (BMG labtech, PHERAstar FS), and the chemiluminescence was measured by the microplate reader.

[0498] 3. Data Analysis Data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in Table 2 below.

[0499] [Table 5]

[0500] Conclusion: The small molecule fragments in this disclosure have significant growth inhibitory activity against SK-BR-3 and U87 cells, and the chiral center has a certain effect on the inhibitory activity of the compounds.

[0501] Test Example 1-2: In vitro growth inhibition test of HER2-targeted tumor cells by the antibody-drug conjugate of the present disclosure This experiment aims to detect the in vitro growth inhibitory activity of the antibody-drug conjugate of the present disclosure targeting HER2 against SK-BR-3 (human breast cancer cells, ATCC, product number HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, product number HTB-132). Cells were treated in vitro with compounds at different concentrations, cultured for 6 days, and then the CTG reagent was used to detect cell growth, and the in vitro activity of the compound was evaluated based on the IC 50 value.

[0502] By the test method of Test Example 1, the test cells were SK-BR-3 and MDA-MB-468, and the cell culture media were McCoy's 5A medium (Gibco, product number 16600-108) containing 10% FBS, EMEM medium (GE, product number SH30024.01) containing 10% FBS, and L-15 medium (ThermoFisher, product number 11415-114) containing 10% FBS, respectively. The three cell lines were adjusted with the cell culture media to a viable cell density of 8.33×10 3 cells / mL, 8.33×10 3 cells / mL and 1.39×10^4 cells / mL, respectively. The cell suspensions with adjusted densities were uniformly mixed and placed in a 96-well cell culture plate at 180 μL / well. Related compounds were tested, and the results obtained are shown in Table 3 below.

[0503]

Table 6

[0504] Conclusion: The antibody-drug conjugate of the present disclosure targeting HER2 has obvious growth inhibitory activity against HER2-positive cells SK-BR-3, weak growth inhibitory activity against HER2-negative cells MDA-MB-468, and good selectivity.

[0505] Test Examples 1-3: Plasma Stability Experiment of Her2-ADC The ADC-19 sample, ADC-18 sample, ADC-20 sample, human plasma, monkey plasma (Shanghai Medicilon Inc.), and 1% BSA (Sigma) PBS solution (Shanghai Sangon Biotech) were each filtered through a 0.22 μm filter to sterilize. ADC-19, ADC-18, and ADC-20 were each added to the above sterilized plasma or 1% BSA PBS solution at a final concentration of 200 μg / mL, placed in a cell incubator at 37°C for incubation, and the incubation day was marked as day 0. Thereafter, samples were taken out on days 7, 14, and 21 respectively, and free toxin detection was performed.

[0506] 25 μL of the sample was taken and placed in a 96-well plate, 50 μL of the internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μL of acetonitrile were added, mixed by vortex for 5 minutes, centrifuged at 4000 rpm for 10 minutes, and 5 μL was analyzed by LC / MS / MS (Applied Biosystems, USA).

[0507] As a result, as shown in Figure 1A, ADC-19 was very stable in both human and monkey plasma and 1% BSA PBS solution, the release rate of free toxin did not exceed 2.1% at most, and it showed a tendency to be stable on day 14.

[0508] ADC-18 had low stability in human and monkey plasma, and the release rates of free toxin were 14.5% and 8.10% at most respectively. As shown in Figure 1B, it was relatively stable in 1% BSA PBS solution.

[0509] ADC-20 had low stability in human plasma, monkey plasma, and 1% BSA PBS solution, and the release rates of free toxin were 21.7%, 29.7%, and 21.7% at most respectively. Also, as shown in Figure 1C, it was always in a decomposed state in 1% BSA PBS solution.

[0510] Test Example 1-4: Evaluation of the drug efficacy on JIMT-1 tumor-bearing mice I. Purpose of the test Using nude mice as test animals, the therapeutic effects of Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 were evaluated after intraperitoneal injection into nude mice bearing transplanted tumors of human breast cancer cell trastuzumab drug-resistant strain (Herceptin) JIMT-1.

[0511] II. Test Drugs and Materials 1. Test Drugs T-DM1 (prepared with reference to US20050169933) ADC-21: 3 mg / kg ADC-21: 10 mg / kg ADC-24: 3 mg / kg ADC-24: 10 mg / kg Blank control (Blank): PBS 2. Preparation method: All were prepared by diluting with PBS. 3. Test Animals Nude mice purchased from Beijing Vital River.

[0512] III. Test Methods JIMT-1 cells (Nanjing Kebai) (5×10 6 / mouse, with 50% Matrigel) were inoculated subcutaneously on the right rib of the mice, and the tumors were allowed to grow for 8 days until they reached 203.09±11.94 mm 3 . Then, the animals were randomly grouped into a total of 6 groups with 8 animals per group (d1). A total of 2 administrations were given by intraperitoneal injection. The tumor volume and body weight were measured twice a week, and the data were recorded. The data were statistically analyzed using Excel 2003 statistical software. The average value was calculated as avg, the SD value was calculated as STDEV, the SEM value was calculated as STDEV / SQRT, and the between-group difference P value was calculated as TTEST. Calculation formula for tumor volume (V): V = 1 / 2 × L 長 × L 短 2 Relative volume (RTV) = V T / V 0 Tumor inhibition rate (%) = (C RTV - T RTV) / C RTV (%) Among them, V 0 and V T are the tumor volumes at the start and end of the experiment, respectively. C RTV , T RTV are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.

[0513] IV. Results of the Test As shown in Figure 2, the results of the experiment were that the experiment was terminated when the injection was administered intraperitoneally twice and observed until the 34th day. T-DM1 (10 mg / kg) had no inhibitory effect on the tumor. For ADC-21, the tumor inhibition rate was 46.22% (P<0.01) at 3 mg / kg and 56.77% (P<0.001) at 10 mg / kg. For ADC-24, the tumor inhibition rate was 62.77% (P<0.001) at 3 mg / kg and 76.32% (P<0.001) at 10 mg / kg. When the dosages were the same, the tumor inhibition effect of ADC-24 was significantly better than that of ADC-21.

[0514] Test Example 1-5: Evaluation of the Efficacy on SK-BR-3 Tumor-Bearing Mice I. Purpose of the Test Using nunu nude mice as test animals, the therapeutic effects of Her2-ADC antibodies ADC-21 and ADC-22 on nude mice with transplanted tumors of human breast cancer cell line SK-BR-3 were evaluated after intraperitoneal injection.

[0515] II. Test Articles and Materials 1. Test Articles ADC-21: 1 mg / kg ADC-21: 6 mg / kg ADC-22: 1 mg / kg ADC-22: 6 mg / kg Blank control: PBS. 2. Preparation Method: All were prepared by diluting with PBS. 3. Test Animals Nunu nude mice purchased from Beijing Vital River Laboratories.

[0516] III. Test method SK-BR-3 cells (ATCC) (5×10 6 / mouse, with 50% Matrigel) were inoculated subcutaneously into the right rib region of mice, and the tumors were allowed to grow for 20 days. After reaching 153.34±11.73 mm 3 , the animals were randomly grouped into a total of 5 groups with 8 animals / group (d0). They were administered once by intraperitoneal injection. The tumor volume and body weight were measured twice a week, and the data were recorded. The data were statistically analyzed using Excel 2003 statistical software. The average value was calculated as avg, the SD value was calculated as STDEV, the SEM value was calculated as STDEV / SQRT, and the inter-group difference P value was calculated as TTEST. Calculation formula for tumor volume (V): V = 1 / 2 × L 長 × L 短 2 Relative volume (RTV) = V T / V 0 Tumor inhibition rate (%) = (C RTV − T RTV ) / C RTV (%) Among them, V 0 , V T are the tumor volumes at the start and end of the experiment, respectively. C RTV , T RTV are the relative tumor volumes of the blank control and the experimental group at the end of the experiment, respectively.

[0517] IV. Test results As shown in Figure 3, the results of the experiment were as follows: When administered by a single intraperitoneal injection and observed until the 28th day to end the experiment, for ADC-21, the tumor inhibition rate was 15.01% at 1 mg / kg and 77.4% at 6 mg / kg, showing a significant difference compared to the blank control (P < 0.001). For ADC-22, the tumor inhibition rate was 19.82% at 1 mg / kg and 98.38% at 6 mg / kg (P < 0.001). When the dose was the same at 6 mg / kg, the tumor inhibition effect of ADC-22 was also significantly better than that of ADC-21.

[0518] Test Examples 1-6: Plasma Stability Sample ADC-25 was uniformly mixed with human plasma, monkey plasma, and 1% BSA PBS solution at a final concentration of 100 μg / mL, respectively. After filtration and sterilization, it was placed in a water bath at 37°C for incubation. The incubation day was marked as day 0. Then, samples were taken out on day 7, day 14, and day 21, respectively, and free toxin was detected.

[0519] After taking out samples at different time points, they were left at room temperature, mixed uniformly by vortex, 25 μL of the sample was taken and put into a 96-well plate, 50 μL of internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μL of acetonitrile were added, mixed by vortex for 5 minutes, centrifuged at 4000 rpm for 10 minutes, and 5 μL of the supernatant was taken for LC / MS / MS analysis.

[0520] As a result, as shown in Figure 4, ADC-25 was very stable in both human and monkey plasma and 1% BSA PBS solution, the release rate of free toxin did not exceed 2% at most, and there was a tendency to be stable on day 14.

[0521] Test Example 1-7: Evaluation of the Therapeutic Effect of ADC on Human Glioblastoma U87MG Nude Mouse Transplanted Tumors I. Purpose of the Test In this experiment, BALB / cA-nude nude mice were used as test animals to evaluate the therapeutic effect of the ADC compound of the present disclosure on human glioblastoma U87MG nude mouse transplanted tumors.

[0522] II. Test Drug and Materials 1. Test Drug ADC-27 (3 mg / kg) ADC-26 (3 mg / kg) Blank control (Blank): PBS buffer solution with pH 7.4. 2. Preparation method: PBS buffer solution with pH 7.4. 3. Test Animals BALB / cA-nude nude mice: Purchased from Shanghai Jieshijie Laboratory Animal Co., Ltd.

[0523] III. Test methods Female experimental BALB / cA-nude nude mice at 6 - 7 weeks of age were subcutaneously inoculated with human glioblastoma U87MG cells (human glioblastoma, Chinese Academy of Sciences Cell Bank, Catalog # TCHu138). On the 10th day after cell inoculation, the animals were randomly divided into groups of 8 per group (D0), and began to be administered by intraperitoneal injection once a week for a total of 3 administrations. The tumor volume and body weight were measured 2 - 3 times per week, and the data were recorded. The calculation formula for tumor volume (V) is as follows. V = 1 / 2 × a × b 2 Among them, a and b represent length and width respectively. Relative tumor volume (RTV) = V T / V 0 Tumor inhibition rate (%) = (C RTV − T RTV ) / C RTV (%) Among them, V 0 , V T are the tumor volumes at the start and end of the experiment respectively. C RTV , T RTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment respectively.

[0524] IV. Test results When administered by intraperitoneal injection (i.p.) once a week for a total of 3 administrations and observed until the 22nd day, the tumor inhibition rate of 3 mg / kg by ADC-27 was 63.3% (P < 0.0001), and the inhibition rate of 3 mg / kg by ADC-26 reached 49.1%. ADC-27 showed a stronger antitumor effect than ADC-26. During the administration, the body weights of the animals in each group were normal, indicating that there were no obvious toxicities or side effects of ADC. The detection results were as shown in Table 4 and Figure 5. The detected antibodies could effectively inhibit the growth of U87MG transplanted tumors in tumor-bearing nude mice and showed dose-dependence.

[0525]

Table 7

[0526] Test Examples 1 - 8: Evaluation of the Therapeutic Effect of ADC on the Xenograft Tumor of Human Pharyngeal Cancer Pleural Effusion Metastatic Cells Detroit 562 in Nude Mice

[0527] I. Purpose of the Test In this experiment, using BALB / cA-nude nude mice as test animals, the therapeutic effect of the ADC compound of the present disclosure on the xenograft tumor of human pharyngeal cancer pleural effusion metastatic cells Detroit 562 in nude mice was evaluated.

[0528] II. Test Drugs and Materials 1. Test Drugs ADC-29 (3 mg / kg) ADC-28 (3 mg / kg) Negative control ADC (3 mg / kg): An antibody-drug conjugate formed by conjugating a non-B7H3 targeting antibody and compound 20. 2. Preparation Method: All were prepared by diluting with PBS. 3. Test Animals BALB / cA-nude nude mice: Purchased from TIFF0007689979000103.tif666.

[0529] III. Test Method Female experimental BALB / cA-nude nude mice at 6 - 7 weeks of age were subcutaneously inoculated with human pharyngeal cancer pleural effusion metastatic cells Detroit 562 cells (ATCC, Catalog #ATCC (R) CCL-138 TM ). On the 10th day after cell inoculation, the animals were randomly divided into groups of 8 per group (D0), and began to be administered by intraperitoneal injection once a week for a total of 3 administrations. The tumor volume and body weight were measured 2 - 3 times per week, and the data were recorded. The calculation formula for the tumor volume (V) is as follows. V = 1 / 2 × a × b 2 Among them, a and b represent the length and width respectively. Relative Tumor Volume (RTV) = V T / V 0 Tumor Inhibition Rate (%) = (C RTV −T RTV ) / C RTV (%) Among them, V 0 , V T are the tumor volumes at the start and end of the experiment, respectively. C RTV , T RTV are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.

[0530] IV. Test Results When administered intraperitoneally once a week for a total of 3 times and observed until day 28, the tumor inhibition rates of the test ADCs were as follows: the tumor inhibition rate of 3 mg / kg (3 mpk) by ADC-29 reached 72.27% (P < 0.001), and the tumor inhibition rate of 3 mg / kg (3 mpk) by ADC-28 reached 56.2% (P < 0.001). ADC-29 showed a stronger antitumor effect than ADC-28 in all cases. During the administration, the body weights of the animals in each group were normal, indicating that there were no obvious toxicities or side effects of the ADCs. The detection results were as shown in Table 5 and Figure 6. The detected antibodies could effectively inhibit the growth of Detroit 562 xenograft tumors in tumor-bearing nude mice and showed dose-dependence.

[0531]

Table 8

[0532] Test Example 1-9: Evaluation of Efficacy on U87-MG Tumor-Bearing Mice I. Test Objectives Using Balb / c nude mice as test animals, the therapeutic effect after intraperitoneal injection of B7H3-antibody drug conjugate in its human glioma cell U87MG xenograft tumor model was evaluated.

[0533] II. Test Drugs and Materials 1. Test Drugs ADC-30 at 1 mg / kg ADC-30 at 3 mg / kg ADC-31 at 1 mg / kg ADC-31 at 3 mg / kg Blank control: PBS 2. Preparation method: All were prepared by diluting with PBS. 3. Test animals BALB / cA-nude mice: Purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0534] III. Test method U87MG cells (human glioblastoma, Chinese Academy of Sciences Cell Bank, Catalog # TCHu138) (2.5×10 6 / mouse) were inoculated subcutaneously into the right rib area of the mice, and the tumors were allowed to grow for 14 days. After reaching 167.49 mm 3 , the animals were randomly grouped into a total of 5 groups with 8 animals / group (d1). Administration was performed by intraperitoneal injection once a week for a total of 3 times. The tumor volume and body weight were measured twice a week, and the data were recorded. Data statistics were performed using Excel 2003 statistical software. The average value was calculated as avg, the SD value was calculated as STDEV, the SEM value was calculated as STDEV / SQRT, and the P value of the difference between groups was calculated as TTEST. Calculation formula for tumor volume (V): V = 1 / 2 × L 長 × L 短 2 Relative volume (RTV) = V T / V 0 Tumor inhibition rate (%) = (C RTV − T RTV ) / C RTV (%) Among them, V 0 , V T are the tumor volumes at the start and end of the experiment, respectively. C RTV , T RTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively.

[0535] IV. Test Results As shown in Figure 7, when the test ADC was administered by intraperitoneal injection once a week for a total of 3 times and observed until the 18th day, the tumor inhibition rates of the test ADC were as follows: the tumor inhibition rate of 1 mg / kg by ADC-30 was 0.31%, the tumor inhibition rate of 3 mg / kg by ADC-30 was 45.23% (P<0.0001), the tumor inhibition rate of 1 mg / kg by ADC-31 was 39.22% (P<0.01), and the tumor inhibition rate of 3 mg / kg by ADC-31 reached 80.24% (P<0.0001). When the dosages were the same, the tumor inhibition effect of ADC-31 was significantly better than that of ADC-30.

[0536] II. Formulation The equipment used in the preparation and detection of the formulation and the calculation method of the results were as follows. SEC Size Exclusion Chromatography: It was an analytical method for separating solutes based on the correlation between the pore size of the gel pores and the coil size of the polymer sample molecules. SEC% (content percentage of SEC alone) = A monomer / A total * 100% (A monomer is the peak area of the main peak monomer in the sample, and A total is the sum of all peak areas.) Equipment for SEC measurement: Agilent 1260, Column: waters, XBrige BEH200Å SEC (300×7.8 mm 3.5 μm)

[0537] CE Capillary Gel Electrophoresis: It was an electrophoresis performed by moving the gel as a supporting medium in a capillary, and it was a method of separating samples according to the molecular weight at a certain voltage. Purity percentage of reduced CE = A main peak / A total * 100% (A main peak is the peak area of the light chain main peak + heavy chain main peak in the sample, and A total is the sum of all peak areas.) Equipment for CE measurement: Beckman model number plus800

[0538] Measurement of turbidity: It is the degree of obstruction when light passes through the aqueous layer, indicating the ability of the aqueous layer to scatter and absorb light, which is related not only to the content of suspended substances but also to the components, sizes, shapes of the particles and the reflection characteristics of their surfaces. By comparing the absorption values of the same concentration and the same wavelength (near-ultraviolet and visible light wavelength regions) of the same protein sample, it was shown that the greater the absorption value, the greater the turbidity and the more obvious the aggregation tendency of protein molecules in the sample. The measuring instrument was a multifunctional microplate reader (Molecular Devices M5), and samples of the same volume were placed in a 96-well plate to read the absorbance values.

[0539] Measurement of osmotic pressure: The osmotic pressure was measured by the freezing point method. Based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution, a high-sensitivity temperature measuring element was used to measure the freezing point of the solution and convert it into osmotic pressure in terms of electricity. Instrument manufacturer: Loser, model number: OM815.

[0540] Measurement of protein concentration: Since the toxin in the antibody-drug conjugate absorbs at 280 nm, the protein concentration was corrected by the following formula, A280 = Cd * ε280d + Cmab * ε280mab A370 = Cd * ε370d Cd represents the concentration of the toxin, Cmab represents the concentration of the protein, ε280d represents the extinction coefficient of the toxin at 280 nm, ε280mab represents the extinction coefficient of the protein at 280 nm, ε370d represents the extinction coefficient of the toxin at 370 nm, ε280mab = 1.49 mg - 1*cm - 1*mL, ε280d = 5000 (molar extinction coefficient of the toxin at 280 nm) / 1074.13 (toxin molecular weight) = 4.65 mg - 1*cm - 1*mL, ε370d = 19000 (molar extinction coefficient of the toxin at 370 nm) / 1074.13 (toxin molecular weight) = 17.69 mg - 1*cm - 1*mL. The above extinction coefficients were mass extinction coefficients. The measuring instrument for protein concentration was an ultraviolet-visible spectrophotometer, model number: Nano Drop oneC, with an optical path of 1 mm. For example, the molecular weight of the naked antibody is 145.181 kDa, the molecular weight of the toxin is 1074 Da, and when calculated with an average DAR value of 5.7, the molecular weight of the ADC is 151.317 kDa, which is 1.042 times that of the naked antibody. For example, when the ADC DAR value is 5.7 and the concentration of the naked antibody protein is 20.00 mg / mL, the concentration of the ADC is 20.84 mg / mL.

[0541] Example 2-1. Selection of formulation buffer system and pH value The following different buffer systems were used to prepare formulations with an ADC-33 protein concentration of 20 mg / mL (in terms of the concentration of the naked antibody) (except for the following buffer systems, the formulations do not contain sugar, surfactant, and other buffers). 1) 10 mM histidine-sodium acetate, pH 5.0 2) 10 mM histidine-sodium acetate, pH 5.5 3) 10 mM histidine-sodium acetate, pH 6.0 4) 10 mM histidine-sodium acetate, pH 6.5 5) 10 mM histidine-histidine hydrochloride, pH 5.5 6) 10 mM succinic acid-sodium succinate, pH 5.5 7) 10 mM citric acid-sodium citrate, pH 5.5

[0542] Samples were taken for high-temperature stability studies (40 °C) and shaking studies (25 °C, 300 rpm). Among them, the shaking samples contained 0.1 mg / mL of polysorbate 80 (PS80), and the other samples contained 0.4 mg / mL of PS80. Each formulation was filtered, filled into a container, plugged, and capped. The above forced degradation experiments were performed on the samples, and the appearance, SEC, and turbidity were examined.

[0543] The results are shown in Table 6. As is clear from the results, when histidine-sodium acetate was used as a buffer and in the pH range of 5.0 to 6.5, as the pH increased, the appearance of the sample gradually deteriorated, the turbidity increased, and the ratio of the SEC alone decreased. Under the condition of pH 5.5, the samples using 10 mM histidine-sodium acetate and 10 mM histidine hydrochloride had an appearance close to the SEC%, and were superior to 10 mM succinic acid-sodium succinate and 10 mM citric acid-sodium citrate in order. Surprisingly, the formulation containing 10 mM succinic acid-sodium succinate had significantly better turbidity than the formulations of other groups after the shaking study was conducted.

[0544]

Table 9

[0545] Example 2-2 Selection of Surfactant Types Formulations containing different polysorbate surfactants and all containing 10 mM succinic acid-sodium succinate (pH 5.0) buffer, 80 mg / mL sucrose, and ADC-32 with a protein concentration of 20 mg / mL (at the concentration of the naked antibody) were prepared. Each formulation was filtered, filled into a container, plugged, and capped. High-temperature stability studies (40 °C) and shaking studies (25 °C, 300 rpm) were conducted on the samples, and the appearance, SEC, and reduced CE were examined.

[0546] The results are shown in Table 7. It was revealed that the formulations containing PS20 (polysorbate 20) and PS80 had no significant differences in appearance, SEC, and reduced CE under different conditions, and both PS20 and PS80 could effectively stabilize ADC-32.

[0547]

Table 10

[0548] Example 2-3 Selection of Surfactant Concentrations Samples were prepared with the concentration of PS80 being 0, 0.1, 0.2, 0.4, and 0.6 mg / mL in sequence. Each sample further contained 10 mM succinic acid - sodium succinate (pH 5.0), 80 mg / mL sucrose, and ADC - 32 with a protein concentration of 20 mg / mL (the concentration of the naked antibody). They were sterilized by filtration to remove particles, diluted with 0.9% NaCl, and the concentration of ADC - 32 after dilution was 0.2 mg / mL. The appearance and insoluble microparticles of the diluted chemical solution were examined.

[0549] The results are shown in Table 8. When the PS80 concentration in the formulation is 0 - 0.2 mg / mL, with the increase in concentration, the amplification of insoluble microparticles in the dilution is reduced, the visible particles decrease, and when the concentration is 0.2 - 0.6 mg / mL, there is no significant difference in the insoluble microparticles in the dilution, and the appearance is good.

[0550]

Table 11

[0551] Example 2 - 4 Selection of Sugar Types Different samples containing 80 mg / mL sucrose, trehalose, and mannitol were prepared. Each sample further contained 10 mM succinic acid - sodium succinate (pH 5.0), 0.2 mg / mL PS80, and ADC - 32 with a protein concentration of 20 mg / mL (the concentration of the naked antibody). They were sterilized by filtration to remove particles, each formulation was filtered, filled into containers, plugged, and capped. High - temperature stability studies (40°C) and freeze - thaw cycle studies at - 35°C / 4°C were conducted on the samples, and the appearance, SEC, and reduced CE were examined.

[0552] The results are shown in Table 9. Under the freeze - thaw conditions, the appearance of sucrose is superior to that of trehalose and mannitol. From the SEC results, it is shown that sucrose and trehalose are superior to mannitol. Under the condition of 40°C, from the results of reduced CE, it is shown that sucrose is slightly superior to trehalose.

[0553]

Table 12

[0554] Example 2-5 Screening Experiment on pH, Concentration of Antibody-Drug Conjugate, and Polysorbate As shown in Table 10, with 10 mM succinic acid-sodium succinate as the buffer and 80 mg / mL sucrose as the stabilizer, formulations were designed for pH, protein concentration of ADC-32 (naked antibody), and polysorbate concentration. High-temperature stability study (40 °C), light exposure study (4 °C, 4500 Lx), and freeze-thaw cycle study at -35 °C / 4 °C were carried out. Using SEC and reduced CE as evaluation indicators, the results were statistically analyzed by the least squares method.

[0555] The results are shown in Table 11 and Figure 8. The formulations of the present disclosure show high pH-dependence. Under the conditions of light exposure, shaking, and 40 °C, as the pH increases, the SEC drop value increases. Similarly, under the light exposure condition, for reduced CE, as the pH increases, the monomer peak drop value increases, suggesting that the formulations of the present disclosure should adopt low pH conditions to improve stability. Under the shaking condition, as the concentration of PS80 increases, the SEC drop value tends to decrease. Under other conditions, there is no difference in the influence of PS80 on SEC and reduced CE. As the protein concentration of ADC-32 increases, under the condition of 40 °C, the SEC monomer peak drop increases. Under other conditions, there is no obvious tendency in the influence of the protein concentration of ADC-32 on SEC and reduced CE.

[0556]

Table 13

[0557]

Table 14

[0558] Example 2-6 Development Experiment on Freeze-Drying Process The stock solution was prepared with 10 mM succinic acid - sodium succinate at pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration, naked antibody) of ADC - 33. After filtration and filling into containers, it was lyophilized according to the lyophilization process parameters 1 (see Table 12). The lyophilized sample was a flat white solid powder with a slight depression in the center of the bottom. The water content was 1.05%. After redissolving with water for injection and measuring the pH, the results are shown in Table 13. At an ionic strength ≥ 10 mM, all can meet the buffering effect. The sucrose in this formulation is 80 mg / mL and can meet the isotonicity requirement.

[0559]

Table 15

[0560]

Table 16

[0561] The stock solution was prepared with 10 mM succinic acid - sodium succinate at pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration, naked antibody) of ADC - 33. After filtration and filling into containers, it was lyophilized according to the lyophilization process parameters 2 (see Table 14). The surface of the solid powder was smooth without wrinkles and depressions, and the water content was 0.89%. This lyophilization process can meet the quality requirements of the product.

[0562]

Table 17

[0563] The stock solution was prepared with 10 mM succinic acid - sodium succinate at pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and ADC - 32 at 20 mg / mL (protein concentration, naked antibody), filtered, filled into containers, and then lyophilized according to the lyophilization process parameters 3 (see Table 15). The surface of the solid powder was flat without depressions, the bottom edge of the solid powder was slightly shrunk, the water content was 1.17%, and the lyophilization process basically met the quality requirements of the product.

[0564]

Table 18

[0565] Example 2 - 7 Long - term stability data The stock solution was prepared with 10 mM succinic acid - sodium succinate at pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and ADC - 32 with a protein concentration of 20 mg / mL (naked antibody). After filtration and filling into containers, the finished product of the pilot experiment lot of the stock solution was prepared according to the lyophilization process parameters 2, stored long - term at 2 - 8°C, detected after re - dissolution, and the results are shown in Table 16. At 2 - 8°C, for M3, compared with D0, SEC, reduced CE, and free toxin were all within the acceptable range (SEC%≥93%, reduced CE%≥95%, free toxin≤330 ppm). Except that SEC was slightly 0.5% lower, reduced CE and free toxin did not change.

[0566]

Table 19

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has the general formula (Pc-L-Y-D), that is, 【Chemical 1】 has the structure represented by Among them, Y is -O-(CR ａ R ｂ ) ｍ -CR １ R ２ -C(O)-, R ａ and R ｂ are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen and a C1-6 alkyl group, R1 is selected from a C3-7 cycloalkyl group and a C3-7 cycloalkyl C1-6 alkyl group, R ２ is a hydrogen atom, Alternatively, R １ and R ２ together with the carbon atoms to which they are attached form a C3-7 cycloalkyl group, m is 0 or 1, n is from 1 to 10, and n is a decimal or an integer, L is a linker unit -L1-L2-L3-L4-, L1 is 【Chemical 2】 where s1 is an integer from 2 to 8, L2 is a chemical bond, L3 is a tetrapeptide residue, L4 is -NR5(CR6R7)t-, R5 is a hydrogen atom or a C1-6 alkyl group, R6 and R7 are the same or different and are each independently a hydrogen atom or a C1-6 alkyl group, and t is 1 or 2, Pc is trastuzumab, The pharmaceutical composition has a pH of 4.5 to 5.2, preferably a pH of 4.8 to 5.2, more preferably a pH of 5.0 to 5.1, The buffer is a succinate buffer, Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, further comprising a surfactant, wherein the surfactant is preferably polysorbate, more preferably polysorbate 80 or polysorbate 20, and most preferably polysorbate 80.

3. The pharmaceutical composition according to claim 2, wherein the concentration of the surfactant is about 0.01 mg / mL to about 1.0 mg / mL, preferably about 0.05 mg / mL to about 0.5 mg / mL, more preferably about 0.2 mg / mL.

4. The pharmaceutical composition according to any one of claims 1 to 3, further comprising a sugar, wherein the sugar is preferably selected from sucrose, mannitol, sorbitol and trehalose, and more preferably sucrose.

5. The pharmaceutical composition according to claim 4, wherein the concentration of the sugar is about 60 mg / mL to about 90 mg / mL, preferably about 80 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the concentration of the antibody-drug conjugate is about 1 mg / mL to about 100 mg / mL, preferably about 10 mg / mL to about 30 mg / mL, more preferably about 20 mg / mL to about 22 mg / mL.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the succinate buffer is a succinic acid-sodium succinate buffer.

8. The concentration of the buffer is about 5 mM to about 50 mM, preferably about 5 mM to about 20 mM, more preferably about 10 mM, and the pharmaceutical composition according to any one of claims 1 to 7.

9. The following components, namely, (a) about 10 mg / mL to about 30 mg / mL of the antibody-drug conjugate, (b) about 0.05 mg / mL to about 0.5 mg / mL of polysorbate, (c) about 60 mg / mL to about 90 mg / mL of sugar, and (d) about 5 mM to about 20 mM of a buffer, with a pH of 4.8 to 5.2, Preferably, the following components, namely, (a) about 20 mg / mL to about 22 mg / mL of the antibody-drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, and (d) about 10 mM of succinate buffer, with a pH of 5.0 to 5.1, The pharmaceutical composition according to any one of claims 1 to 8.

10. A pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has the following formula, namely, 【Chemical Formula 2】 has a structure represented by, Among them, n is 3 to 8, and n is a decimal or an integer, Pc is an antibody or an antigen-binding fragment thereof, The pharmaceutical composition has a pH of 4.5 to 5.2, preferably a pH of 4.8 to 5.2, more preferably a pH of 5.0 to 5.

1. Pharmaceutical composition.

11. The antibody or an antigen-binding fragment thereof is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 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-SLC44A4 antibody, and an anti-Mesothelin antibody, or an antigen-binding fragment thereof. Preferably, the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glemamab, or its antigen-binding fragment. The pharmaceutical composition according to any one of claims 1 to 10.

12. The antibody complex has the following formula, namely, 【Chemical Formula 3】 has a structure represented by wherein n is from 3 to 8, and n is a decimal or an integer. The pharmaceutical composition according to any one of claims 1 to 11.

13. The following formula, namely, 【Chemical Formula 4】 contains an antibody-drug conjugate having a structure represented by wherein n is from 3 to 8, and n is a decimal or an integer, and (a) about 10 mg / mL to about 30 mg / mL of the antibody-drug conjugate, (b) about 0.05 mg / mL to about 0.5 mg / mL of polysorbate, (c) about 60 mg / mL to about 90 mg / mL of sugar, and (d) about 5 mM to about 20 mM of a buffer, and the pH is 4.8 to 5.

2. Preferably, it contains (a) about 20 mg / mL to about 22 mg / mL of the antibody-drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, and (d) about 10 mM of succinate buffer, and the pH is 5.0 to 5.

1. Pharmaceutical composition.

14. A lyophilized preparation containing an antibody-drug conjugate, which can form the pharmaceutical composition according to any one of claims 1 to 13 after reconstitution.

15. A method for preparing a lyophilized preparation containing an antibody-drug conjugate, which includes the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 13.

16. A lyophilized preparation containing an antibody-drug conjugate, which is obtained by lyophilizing the pharmaceutical composition according to any one of claims 1 to 13.

17. A reconstitution solution containing an antibody-drug conjugate, which is obtained by reconstituting the lyophilized preparation according to claim 14 or 16. Preferably, the following components, namely, (a) said antibody-drug conjugate at about 10 mg / mL to about 30 mg / mL, (b) polysorbate at about 0.05 mg / mL to about 0.5 mg / mL, (c) sugar at about 60 mg / mL to about 90 mg / mL, and (d) buffer at about 5 mM to about 20 mM, with a pH of 4.8 to 5.2, More preferably, the following components, namely, (a) an antibody-drug conjugate at about 20 mg / mL to about 22 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 80 mg / mL, and (d) succinate buffer at about 10 mM, with a pH of 5.0 to 5.1, A reconstitution solution, characterized in that.

18. A product comprising a pharmaceutical composition according to any one of claims 1 to 13, a lyophilized preparation according to claim 14 or 16, or a container containing the reconstitution solution according to claim 17.

19. A lyophilized preparation according to claim 14, a reconstitution solution according to claim 17, or a product according to claim 18 for use as a medicament for treating cancer associated with HER2 expression, preferably cancer selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

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