Branched enzyme-cleavable linker-payload and method for efficiently producing same

Abstract

The present invention relates to a branched enzyme-cleavable linker-payload and a method for efficiently producing same.

Description

Branched enzyme-cleavable linker-payload and efficient method for preparing the same

[0001] The present invention relates to a branched enzyme-cleavable linker-payload and a manufacturing method for efficiently producing the same.

[0002]

[0003] Targeted drug delivery strategies, exemplified by antibody-drug conjugates (ADCs), have ushered in a new era of innovative targeted therapy for effective anticancer strategies. These strategies combine highly targeted delivery systems, such as antibodies, peptides, and aptamers, with drugs possessing potent physiological effects, such as potent apoptosis, to precisely and effectively eliminate cancer cells. Since their initial approval by the US Food and Drug Administration in 2000, 14 ADCs have been launched on the market, and over 800 have entered the clinical trial pipeline, demonstrating the rapid development of ADCs. Designed to leverage the therapeutic efficacy of drugs and the targeting specificity of antibodies, ADCs have emerged as a key class of pharmaceuticals. In addition to antibodies, the development of drug conjugates utilizing small delivery platforms, such as peptides and aptamers, with proven targeting capabilities, is also actively underway. These three main components of drug delivery systems—antibody / peptide / aptamer, linker, and payload—focused on improving the individual functions of the ADCs in their early stages. However, recent research has focused on developing linkers that enable more homogeneous ADC production and drug release in specific environments, as well as technologies that control the number of drugs conjugated to maximize their efficacy. These linker-payload conjugates can maximize the therapeutic efficacy of small-molecule drugs through selective targeting to target tissues and cells, while potentially minimizing dose-limiting toxicity by achieving desirable pharmacokinetic profiles. To meet these requirements, innovations in linker design are focused on issues ranging from serum stability to controlling the drug-to-antibody ratio (DAR) and its release mechanism.

[0004] Among the components described above, the dipeptide linker used in the present invention is designed to be cleavable by proteolytic enzymes. Upon entering cells, it is cleaved by lysosomes, releasing the drug. Unlike conventional disulfide linkers and pH-dependent cleavage linkers, this dipeptide linker stably delivers and releases drugs to target organs and cells without premature drug release, making it a widely used concept in the field of drug delivery system development. Various peptide linkers are currently being developed.

[0005] The present invention has been developed to increase the ratio of drugs bound to one aptamer by conjugating a single dipeptide linker-payload having such stable drug release characteristics to tris tri-acid to create a branched linker-payload, and further, has invented an effective manufacturing process and an increase in the production yield of the linker-payload using a commercialized intermediate material.

[0006] Accordingly, the present inventors intend to provide a linker-payload conjugate with improved yield and an efficient method for manufacturing the same in order to efficiently increase the drug-to-aptamer ratio (DApR) of an aptamer-drug conjugate (ApDC) that combines an aptamer and a drug instead of an antibody.

[0007]

[0008] The purpose of the present invention is to provide a manufacturing method capable of providing high purity and yield in manufacturing a branched linker-payload that can exhibit desirable pharmacodynamic characteristics and therapeutic effects by increasing the amount of drug that can be loaded into a drug delivery system such as a target-specific aptamer / antibody / peptide by manufacturing the linker-payload with a branched structure.

[0009]

[0010] The present invention provides a manufacturing method capable of providing high purity and yield in manufacturing a branched linker-payload that can exhibit desirable pharmacodynamic characteristics and therapeutic effects by increasing the amount of drug that can be loaded into a drug delivery system such as a target-specific aptamer / antibody / peptide by manufacturing the linker-payload with a branched structure.

[0011]

[0012] The method for manufacturing a linker-payload conjugate of the present invention can improve the existing method to simplify the synthesis process and provide a linker-payload conjugate to which one or more drugs are bound with high purity and higher yield.

[0013]

[0014] Each description and embodiment disclosed in this invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions described below.

[0015] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0016] Additionally, throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be further included, unless specifically stated otherwise.

[0017]

[0018] Hereinafter, the present invention will be described in more detail.

[0019]

[0020] As one embodiment for achieving the above object, the present invention comprises a first step of forming an amide bond by reacting a first reactant including an amine group at one terminal and a carboxyl group protected by a tert-butyl group at at least one other terminal with a second reactant including a maleimide group at one terminal and a carboxyl group at the other terminal in the presence of N-methylmorpholine (NMM) and HATU (HexafluorophosphateAzabenzotriazoleTetramethylUronium) in an organic solvent; a second step of deprotecting a product obtained from the first step in a mixed solvent of an organic solvent and trifluoroacetic acid (TFA) to convert a carboxyl group protected by the tert-butyl group into a carboxyl group; And the present invention provides a method for producing a linker-payload conjugate, comprising a third step of reacting a drug conjugated to an enzyme-cleavable linker having a reactive amine group at one terminal with a product obtained from the second step in an organic solvent in the presence of NMM and HATU.

[0021]

[0022] The present invention is characterized by discovering conditions that can provide a linker-payload conjugate with higher yield and purity by optimizing each step from a conventional linker-payload conjugate manufacturing method. Specifically, the conventional linker-payload conjugate manufacturing method, in the first and third steps, reacts a compound containing a maleimide group at one terminal and a carboxyl group at the other terminal and a compound containing a carboxyl group at the other terminal of the maleimide group obtained from the second step with NMM and HATU to introduce a triazolopyridinyl group at the carboxyl group, and then reacts with another reactant, whereas the manufacturing method of the present invention simultaneously mixes and reacts the two compounds with NMM and HATU instead of the conventional separate reactions, and in the similar third step reaction, similarly mixes NMM and HATU with the two compounds and reacts them in a single step, thereby ultimately maintaining a high yield of 92% or more, while increasing the yield by about 10 times compared to the conventional manufacturing method.

[0023]

[0024] For example, in the manufacturing method of the present invention, the reaction of the first step can be performed at 5 to 40°C, 10 to 35°C, 15 to 30°C or room temperature in the form of a solution dissolved in an organic solvent such as DMF (N,N-dimethylformamide). For example, the reaction can proceed for 3 to 48 hours, 3 to 24 hours, 6 to 18 hours or 9 to 18 hours, but is not limited thereto. At this time, the amounts of NMM and HATU used can be 1 to 2 equivalents and 1 to 1.5 equivalents, respectively, based on the reactants, but are not limited thereto.

[0025] Through the reaction of the first step above, an amide bond can be formed between the amine group of the first reactant and the carboxyl group of the second reactant.

[0026] For example, in the manufacturing method of the present invention, the reaction of the second step may use DCM (dichloromethane) as an organic solvent, but is not limited thereto. For example, the mixed solvent of the organic solvent and TFA may be a solvent mixed in a volume ratio of 3:7 to 7:3, but is not limited thereto. Furthermore, the reaction of the second step may be performed at 5 to 40°C, 10 to 35°C, 15 to 30°C, or room temperature, and may be performed for 30 minutes to 10 hours, 2 to 8 hours, or 3 to 7 hours, but is not limited thereto.

[0027] Through the reaction of the second step above, the terminal tert-butoxycarbonyl group can be converted into a carboxyl group.

[0028] For example, in the manufacturing method of the present invention, the reaction of the third step may be performed under similar conditions to the first step, but is not limited thereto. At this time, the amounts of NMM and HATU used may be 3 to 5 equivalents and 3 to 6 equivalents, respectively; or 3.5 to 4.5 equivalents and 4 to 5 equivalents, but are not limited thereto.

[0029] Through the reaction of the third step, an amide bond is formed between the amine group of the drug linked to the enzyme-cleavable linker including a reactive amine group at one end and the carboxyl group of the product obtained from the second step, thereby forming a linker-payload conjugate including a maleimide group capable of binding to a targeting unit at one end and linked in the order of the enzyme-cleavable linker-drug through an amide bond from the other end.

[0030] Furthermore, in the manufacturing method of the present invention, each step may additionally include one or more of the following steps: concentration, purification, washing, and / or drying after the reaction, but is not limited thereto. Alternatively, the obtained product may be used as a reactant in a subsequent step in the form of a crude product without further purification. Each of the above-mentioned steps can be performed without limitation using conventional methods known in the art.

[0031]

[0032] For example, a compound comprising an amine group at one terminal and a carboxyl group protected by a tert-butyl group at one or more other terminals may be, but is not limited to, a tripurcated Newkome-type monomer (TNM).

[0033] For example, the first reactant may be, but is not limited to, di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate.

[0034] For example, the second reactant may be, but is not limited to, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid.

[0035] For example, the product obtained from the first step is di-tert-butyl 3,3'-((2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propane-1,3-diyl)bis(oxy))dipropanoate, and the product obtained from the second step is 3,3'-((2-((2-carboxyethoxy)methyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propane-1,3-diyl)bis(oxy))dipropanoic acid), but is not limited thereto.

[0036] For example, the enzymatically cleavable linker may be, but is not limited to, a valine citrulline p-aminobenzylalcohol (VC-PAB) linker. The VC-PAB linker is a dipeptide linker cleavable by cathepsin B and is a representative linker used in antibody-drug conjugates (ADCs). Cathepsin B is an enzyme belonging to the lysosomal cysteine ​​protease family, plays an important role in intracellular proteolysis, and is known to be upregulated in cancer, pre-malignant lesions, and various pathological conditions. In addition, since cathepsin B tends to have an increased expression in carcinogenic cells, it is also used as a biomarker for various cancers.

[0037] Therefore, the linker-payload of the present invention including the VC-PAB linker can deliver multiple drugs to target tissues by combination with targeting factors such as antibodies, peptides or aptamers, and can be cleaved in cancer or other malignant lesions with increased cathepsin B expression to selectively release the drugs, thereby enabling more efficient treatment compared to existing linker-payload series drug delivery systems.

[0038] For example, the drug may be a cytotoxic drug selected from the group consisting of, but not limited to, tubulin-binding agents, tubulin-destructing agents, auristatins, DNA-binding agents, and DNA-alkylating agents.

[0039] The drug is selected from the group consisting of dolastatin; auristatin; epothilone; daunorubicin; doxorubicin; an alkylating agent such as thiotepa or cyclophosphamide (CYTOXAN™); an alkyl sulfonate such as busulfan, improsulfan or piposulfan; an aziridine such as benzodopa, carboquone, meturedopa or uredopa; an ethyleneimine and / or methylamelamine such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide or trimethylolomelamine; an acetogenin such as bullatacin or bullatacinone; a camptothecin such as the synthetic analogue topotecan; bryostatin; kallistatin; CC-1065 and / or its synthetic analogues adozelesin, caselesin or bizelesin; Cryptophycins, such as cryptophycin 1 or cryptophycin 8; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); eleuterobin; pancratistatin; sarcodictines; spongistatin; nitrogen mustards, such as chlorambucil, clomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibodies, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ1; dynemicins including dynemicin A; esperamicins; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycins, authramycins, azaserine, bleomycins, cactinomycins, carabicins, carminomycins, carzinophilin;Other doxorubicin derivatives including chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, celamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as prolic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; Bestrabucil; bisantrene; edatraxate; defopamine; demecolcine; diaziquone; elfomitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and N-glucosylmaytansinoid, ansamitocin, DM-1, DM-4; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; ®; razoxane; rhizoxine; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL ® , Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE ®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, carboplatin, and vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; Tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Pharestone); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; tubulysin; amanitins, such as α-amanitin; and pharmaceutically acceptable salts, acids; dolastatin 10 or any derivative thereof; dolastatin 15 or any derivative thereof; auristatin F or any derivative thereof; monomethyl and desmethyl dolastatins 10, 15, C, D, and H, monomethyl and desmethyl isodolastatin H, and analogs and derivatives thereof; Monomethyl and desmethyl auristatins E, F, EB, EFP, PY, PYE, PE, PHE, TP, 2-AQ and 6-AQ; maytansinoids; N-glucosylmaytansinoids; maytansine, ansamitocin, DM1 (also known as mertansine) or DM4 (also known as DM-4); other doxorubicin derivatives including daunorubicin, doxorubicin, detorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, rodorubicin, zorubicin and pirarubicin;Duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065; synthetic analogues of duocarmycins, such as adozelesin, bizelesin, carzelesin, KW-2189, and CBI-TMI; duocarmycin-saccharide conjugates; tubulysin; α-amanitin; cryptophycin; monomethyl auristatin E (MMAE); auristatin saccharide conjugates; MMAU; monomethyl auristatin F, W, or M; A cytotoxic drug selected from the group consisting of pyrrolobenzodiazepines (PBDs), abemycin, chikamycin, DC-81, mazethramycin, neothramycins A and B, prothramycin, protracarcin, sibiromycin, tomamycin, and PBD dimers; or analogs of any of the above. For example, the drug may be an antineoplastic agent. Specifically, the drug may be monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), cytarabine, gemcitabine, carboplatin, cisplatin, crizotinib, cyclophosphamide, docetaxel, doxorubicin, erlotinib, etoposide, fluorouracil, imatinib mesylate, irinotecan, methotrexate, paclitaxel, sorafinib, sunitinib, topotecan, trabectidine, vincristine, vinblastine, maytansine, DM1, DM4, calicheamicin and derivatives thereof, doxorubicin, duocarmycin and derivatives thereof, pyrrolobenzodiazepine (PBD), SN-38, α-amantine, or a tubulosin analog. Specifically, the drug may be, but is not limited to, monomethyl auristatin E.;

[0040]

[0041] In another aspect, the present invention provides a linker-payload represented by the following chemical formula 1:

[0042] [Chemical Formula 1]

[0043]

[0044] In the above chemical formula 1, D is a drug.

[0045] The above drug can be used without limitation with the above-mentioned drugs, and the linker-payload of the above-mentioned chemical formula 1 can be manufactured according to the above-mentioned method.

[0046] Specifically, the linker-payload conjugate synthesized according to the manufacturing method of the present invention may be a compound represented by the following chemical formula 2, which includes a drug monomethyl auristatin E linked to three molecules of valine citrulline PAB (VC-PAB) linker via a three-pronged Newcomb-type monomer (TNM), but is not limited thereto:

[0047] [Chemical Formula 2]

[0048] .

[0049]

[0050] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited by these examples.

[0051]

[0052] Example 1: Preparation of Maleimidocaproyl-tri(VC-PAB-MMAE) (Maleimidocaproyl-tri(VC-PAB-MMAE: Compound 5)

[0053]

[0054] Step 1

[0055]

[0056] Di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (compound 1, 15.0 g, 0.03 mol, 1.0 eq, purity: 99.9%) and 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (MC-COOH, 7.5 g, 0.04 mol, 1.2 eq) was added to DMF (N,N-dimethylformamide, 150 mL, 10 vol), and then NMM (N-methylmorpholine, 4.5 g, 0.04 mol, 1.5 eq) and HATU (HexafluorophosphateAzabenzotriazoleTetramethylUronium, 13.5 g, 0.04 mol, 1.2 eq) were added. The mixture was stirred at room temperature for 12 h, and the reaction was monitored by HPLC and TLC, and the completion of the reaction was confirmed by TLC (EtOAc / n-Hep = 2 / 1) (R for compound 1 f ~ 0.4, R for compound 2 fAfter verification by IPC, the reaction system was concentrated under reduced pressure at ~36°C for 2 h to remove DMF. The oily reaction mixture was diluted with EtOAc (150 mL), washed with H2O (150 mL) and brine (100 mL), and dried over Na2SO4. The solid was removed by filtration, and the organic phase was concentrated at ~36°C for about 2 hours to obtain the crude compound 2(di-tert-butyl 3,3'-((2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propane-1,3-diyl)bis(oxy))dipropanoate) as a pale yellow oil. The crude product was purified by column chromatography on silica gel. The residue was purified by chromatography. Qualified fractions were concentrated to obtain compound 2 (11.45 g, 0.016 mol, 98.65% purity, 56.2% yield) as a pale yellow oil. Column purification conditions (Table 1) and analytical data for identifying compound 2 are shown below.

[0057] Sample preparation: Dissolve the product in a total of 114 g of DCM and n-Hep. Experimental conditions: Column 500-800 mesh, Chromatography silica gel, Mobile phase An-Hep: DCM = 2:1 (v / v), Mobile phase BTHF, Flow rate 56 mL / min, Detection 210 nm, Monitor 254 nm, Injection 114 g line - Injection pressure 0.8 MPa, Equilibration 0% B balance / 10 min later - Injection pressure 0.9 MPa, Gradient time (min) 0 5 5 0 8 5 9 5 B (%) 0 0 1 7 1 7 (100) 100

[0058] IPC: CDo135013-01-ZZH-5125-17, R t=0.94min: DMF, R t =1.30min: HOAt, R t =3.70min: Mc-COOH,R t =8.65min: cpd#2A,HPLC: CDo135013-01-ZZH-5127-chun, R t =8.347 min, purity = 98.65%,

[0059] LCMS (ESI)calcd for C 35 H 58 N2O 12 [M +H] + m / z 699.4, found 699.6,

[0060] 1 H NMR(400 MHz, CDCl3): δ 1.26-1.34 (2H, m, CH2-CH2-CH2-CO-NH), 1.45 (27H, s, each of three -C(CH3)3), 1.56-1.65 (4H, m, CH2-CH2-CH2-CH2-CO-NH), 2.13-2.16 (2H, t, J=8 Hz, CH2-CH2-CH2-CO-NH), 2.43-2.47 (6H, t, J=8 Hz, each of three CH2-CH2-CO-O), 3.49-3.52 (2H, t, J=8 Hz, CH2-N), 3.63-3.66 (6H, t, J=8 Hz, each of three CH2-O-CH2-CH2), 3.70 (6H, s, each of three CH2-O-CH2-CH2), 6.06 (1H, s, NH), 6.68 (2H, s, CH=CH).

[0061]

[0062] 단계 2

[0063]

[0064] Compound 2 (11.2 g, 0.016 mol, 98.65% purity) obtained from the above step 1 was added to a mixture of DCM (dichloromethane, 34 mL) and TFA (trifloroacetic acid, 34 mL). The mixture was stirred at room temperature for 5 hours, and the completion of the reaction was confirmed by HPLC_IPC (AM-1001) (R t (2) = 8.347 min, R t (3) = 3.257 min). The reaction mixture was concentrated at ~36°C for about 2 h to remove TFA and DCM, and 3,3'-((2-((2-carboxyethoxy)methyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propane-1,3-diyl)bis(oxy))dipropanoic acid (compound 3, 14 g, crude) was obtained as a pale yellow oil.

[0065] IPC:CDo135013-01-ZZH-5133-10,R t =3.257min: cpd#3A (purity: 94.09%),

[0066] HPLC:CDo135013-01-ZZH-5133-20,R t =3.427 min, purity = 95.05%,

[0067] LCMS (ESI)calcd for C 23 H 34 N2O 12 [M +H] + m / z 531.2, found 531.3,

[0068] 1H NMR(400 MHz, CDCl3): δ 1.28-1.36 (2H, m, CH2-CH2-CH2-CO-NH), 1.59-1.66 (4H, m, CH2-CH2-CH2-CH2-CO-NH), 2.27-2.31 (2H, t, J=8 Hz, CH2-CH2-CH2-CO-NH), 2.63-2.66 (6H, t, J=8 Hz, each of three CH2-CH2-CO-O), 3.54-3.57 (2H, t, J=8 Hz, CH2-N), 3.73-3.76 (6H, t, J=8 Hz, each of three CH2-O-CH2-CH2), 3.80 (6H, s, each of three CH2-O-CH2-CH2), 6.66 (1H, s, NH), 6.74 (2H, s, CH=CH).

[0069] Q-NMR assay:59%

[0070]

[0071] 단계 3

[0072]

[0073] A solution of compound 3 (1.64 g, crude) obtained from step 2 above in DMF (32.8 mL, 20 vol) was added at room temperature to NMM (1.31 g, 12.9 mmol, 4.2 eq), HATU (5.29 g, 13.9 mmol, 4.5 eq) and 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (VC-PAB-MMAE, 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate, compound 4, 10.4 g, 9.3 mmol, 3 eq, purity: 98%) was added. The mixture was stirred at room temperature for 12 hours, and the completion of the reaction was confirmed by HPLC_IPC (AM-1001)(R t (3) = 3.257 min, R t (5: ASYM-135013) = 7.98 min). The mixture was concentrated under vacuum at ~35°C for approximately 2 h.

[0074] For purification, 158 mg each of compound 3 obtained from two other batches (01505136 and 01505138) were combined with this batch. The crude product was purified by preparative HPLC. Qualified fractions were collected and concentrated to 50% of the original volume at 35°C for 12 h to remove ACN. Lyophilization afforded maleimidocaproyl-tri(VC-PAB-MMAE) (compound 5, batch no. 135013, 7.6 g, 1.98 mmol, 98.24% purity, 90.8% yield) as a white solid. The column purification conditions (Table 2) and analytical data for the identification of compound 2 are shown below.

[0075] Sample preparation: Direct injection of sample into 45 mL DMF system. Experimental conditions: Column YMC AQ C18. Mobile phase A: 0.1% TFA in H2O. Mobile phase BACN. Flow rate: 500 mL / min. Detection: 210 nm. Monitor: 254 nm. Injection: 45 mL. Line - injection pressure: 2.45 MPa. Equilibration: 0% B balance / 10 min. After - injection pressure: 3.86 MPa. Gradient: T (min) 0 5 6 5 0 6 5 7 5 B (%) 1 0 1 0 3 0 7 5 7 5 (95) 95

[0076] IPC: R t =1.49min:HOAt,R t =5.00min:Cpd5A,R t =7.97min: 135013, HPLC: CDo135013-01-ZZH-5140-12, R t =8.3min, purity = 98.24%,

[0077] Tof-MS (ESI)calcd for C 197 H 310 N 32 O 45 [M / 3 +H] + m / z 1283.3, found 1283.4,

[0078] 1H NMR(400 MHz, DMSO_d6): δ 0.7-0.9 (68H, m), 0.9-1.1 (19H, m), 1.1-1.2 (2H, m), 1.2-1.6 (22H, m), 1.6-1.8 (11H, m), 1.8-2.2 (14H, m), 2.2-2.5 (10H, m), 2.7-2.9 (8H, m) 2.9-3.1 (11H, m), 3.1-3.3 (25H, m), 3.3-3.4 (4H, m), 3.4-3.7 (16H, m), 3.7-3.8 (3H, m), 3.8-4.6 (73H, m), 4.6-4.8 (4H, m), 4.9-5.2 (6H, m), 6.8-6.9 (1H, m), 6.9-7.0 (2H, m), 7.1-7.2 (3H, m), 7.2-7.4 (17H, m), 7.5-7.6 (5H, m), 7.6-7.7 (1H, m), 7.8-8.0 (4H, m), 8.0-8.2 (2H, m), 8.2-8.3 (3H, d), 8.3-8.4 (1H, d), 10.0-10.1 (3H, d),

[0079] 13C NMR (100 MHz, DMSO-d6): δ 172.84, 172.82, 171.70, 171.50, 171.02, 170.84, 170.34, 170.27, 169.22, 159.54, 159.10, 158.72, 156.65, 156.17, 144.10, 144.08, 139.02, 134.87, 132.24, 128.61, 128.25, 128.19, 127.17, 127.11, 126.9, 126.86, 119.44, 119.28, 117.2, 114.32, 85.86, 82.08, 78.14, 77.34, 75.25, 68.80, 67.90, 66.54, 63.73, 61.36, 60.73, 59.95, 59.12, 58.64, 57.80, 57.60, 57.56, 55.43, 54.58, 53.63, 50.21, 49.62, 47.68, 46.71, 44.21, 43.67, 37.58, 37.46, 36.27, 36.10, 35.55, 32.27, 31.99, 31.18, 30.14, 29.73, 28.29, 27.40, 27.15, 26.19, 25.78, 25.24, 24.80, 23.57, 19.63, 19.49, 19.34, 19.19, 18.98, 18.81, 18.57, 16.30, 16.06, 15.88, 15.72, 15.44, 10.83, 10.73

[0080]

[0081] Experimental Example 1: Qualitative and quantitative analysis of the products at each stage

[0082]

[0083] The reactants and products of each step were analyzed under the conditions of AM-1001, and the final product was analyzed under the conditions of AM-135013-04, and these conditions are shown in Tables 3 and 4 below, respectively. The qualitative and quantitative analysis results for the reactants and products of each step in the manufacturing method of Example 1 are summarized in Table 5 below.

[0084] ColumnZorbax Eclipse Plus C18, 4.6Х100 mm, 3.5 μm, P / N: 959961-902Flow rate1.5 mL / minColumn temperature40℃Detection210 nmInjection volume1-5 μLMobile phaseMobile phase A: 0.1% H3PO4 in water (v / v) / Mobile phase B: ACNGradient programTime(min)0912.012.115B(%)10951001010Dilution / needle washACN / H2O = 1 / 1Run time15 min

[0085] ColumnZorbax Eclipse Plus C18, 4.6Х100 mm, 3.5 μm, P / N: 959961-902Flow rate1.5 mL / minColumn temperature40℃Detection210 nmInjection volume3.0 μLMobile phaseMobile phase A: 0.1% H3PO4 in water (v / v) / Mobile phase B: ACNGradient programTime(min)07121515.118B(%)1075951001010Dilution / needle washACN / H2O = 4 / 1Run time18 min

[0086] Step ReactantProductReactantPurityIPC(Residual Reactant %)ConstantYieldProductPurity1 Compound 1 Compound 297%NDPale yellow oil55.3%98.54%(purified)2 Compound 2 Compound 398.65%(purified)0.15%Pale yellow oilND95.05%(crude product)3 Compound 3 Compound 595.05%NDWhite solid90.8%98.24%(purified)

[0087] As shown in Table 5 above, the manufacturing method consisting of a three-step process according to the present invention exhibited a high yield of 95% or more in each step, and was able to obtain a linker-payload conjugate with a high yield of 50% or more and a high purity of 92% or more throughout the entire process. Compared to the existing method, the linker-payload conjugate could be manufactured with a yield approximately 10 times higher (Jianjun Qi et al., Pharmaceuticals, 2022, 15(5): 558).

[0088] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A first step of forming an amide bond by reacting a first reactant including an amine group at one terminal and a carboxyl group protected with a tert-butyl group at one or more other terminals with a second reactant including a maleimide group at one terminal and a carboxyl group at the other terminal in the presence of N-methylmorpholine (NMM) and HATU (HexafluorophosphateAzabenzotriazoleTetramethylUronium) in an organic solvent; A second step of deprotecting the product obtained from the first step in a mixed solvent of an organic solvent and trifluoroacetic acid (TFA) to convert the carboxyl group protected by the tert-butyl group into a carboxyl group; and A method for producing a linker-payload conjugate, comprising a third step of reacting a drug conjugated to an enzymatically degraded linker having a reactive amine group at one terminal with a product obtained from the second step in the presence of NMM and HATU in an organic solvent.

2. In paragraph 1, A manufacturing method wherein the reaction of the first step is performed in a solution state dissolved in an organic solvent at 5 to 40°C and 10 to 35°C for 3 to 48 hours.

3. In paragraph 1, A manufacturing method wherein the amounts of NMM and HATU used in the reaction of the first step are 1 to 2 equivalents and 1 to 1.5 equivalents, respectively, based on the first reactant.

4. In paragraph 1, A manufacturing method, wherein in the reaction of the second step, the mixed solvent of the organic solvent and TFA is a solvent mixed in a volume ratio of 3:7 to 7:

3.

5. In paragraph 1, A manufacturing method wherein the above second step reaction is performed at 5 to 40°C for 30 minutes to 10 hours.

6. In paragraph 1, A manufacturing method wherein the reaction of the third step is performed at 5 to 40°C and 10 to 35°C for 3 to 48 hours in a solution state dissolved in an organic solvent.

7. In paragraph 1, A manufacturing method wherein the amounts of NMM and HATU used in the reaction of the first step are 3 to 5 equivalents and 3.5 to 4.5 equivalents, respectively, based on the product obtained from the second step as a reactant.

8. In paragraph 1, A manufacturing method, wherein the first reactant is a tripurcated Newkome-type monomer (TNM).

9. In paragraph 8, A manufacturing method, wherein the first reactant is di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate.

10. In paragraph 1, A manufacturing method, wherein the second reactant is 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid.

11. In paragraph 1, A manufacturing method, wherein the above enzyme-cleaved linker is a valine citrulline p-aminobenzylalcohol (VC-PAB) linker.

12. In paragraph 1, A method for manufacturing a drug, wherein the drug is a cytotoxic drug selected from the group consisting of a tubulin-binding agent, a tubulin-destructing agent, an auristatin, a DNA-binding agent and a DNA-alkylating agent.

13. Linker-payload represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, D is a drug.

14. In paragraph 13, A linker-payload, wherein the drug is a cytotoxic drug selected from the group consisting of tubulin-binding agents, tubulin-destructing agents, auristatins, DNA-binding agents and DNA-alkylating agents.

15. In paragraph 13, A linker-payload prepared by any one of the methods of claims 1 to 12.

Images