Drug conjugate or salt thereof

Abstract

The purpose of the present invention is to provide a (PSMA ligand)-(low-molecular-weight drug) conjugate which has excellent affinity for PSMAs and enables selective anticancer agent treatment that targets cancer lesions.　Provided is a drug conjugate represented by formula (1) [wherein X represents -(CH2)n-CO- or -(CH2)n-NRCO- (wherein R represents a hydrogen atom, a lower alkyl group, a cyclo-lower-alkyl group, an aryl group which may have a substituent, or an aralkyl group which may have a substituent; and n represents an integer of 9-17); L represents a peptide linker; and D represents an antitumor drug moiety], or a salt thereof.

Description

Drug conjugate or its salt

[0001] The present invention relates to a drug conjugate or a salt thereof that targets prostate-specific membrane antigen.

[0002] Prostate cancer is the second most common cancer in men and the fifth leading cause of cancer-related deaths, with an estimated 1.4 million new cases and 375,000 deaths worldwide in 2020. Prostate cancer can be managed with surgery, radical prostatectomy, and radiation therapy as long as the cancer is localized to the prostate. However, metastatic castration-resistant prostate cancer (mCRPC) is associated with high mortality. Patients with mCRPC are treated with conventional chemotherapy drugs such as docetaxel, but these cytotoxic agents can cause multidrug resistance and undesirable side effects, necessitating the development of more specific and safer chemotherapy agents.

[0003] Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells compared with normal prostate cells, and the expression level of PSMA on cancer cells has been reported to increase with tumor aggressiveness.Therefore, PSMA is an attractive target for imaging diagnosis and treatment of prostate cancer.For example, a promising method using PSMA ligands is radiopharmaceutical therapy (RPT), which includes therapeutic radionuclides that emit β particles. 177 Lu-PSMA-617 has been approved by the U.S. Food and Drug Administration for the treatment of metastatic prostate cancer. However, RPT requires specialized facilities to produce therapeutic radionuclides. Therefore, the development of PSMA-targeted drug conjugates in which small molecule cytotoxic drugs are linked to PSMA ligands is desirable.

[0004] Compared with indiscriminate chemotherapy, targeted therapy has the advantage of selectively delivering anticancer drugs to cancer cells, while drugs bound to PSMA ligands have the advantage that PSMA ligands can enter PSMA-expressing cells through endocytosis, thereby releasing the drug intracellularly.Therefore, PSMA-targeted drug conjugates incorporate cleavable linkers for efficient intracellular drug release.In recent years, as PSMA-targeted drug conjugates, in addition to PSMA antibody-drug conjugates (ADCs) that combine PSMA antibodies with drugs, PSMA ligand-small molecule drug conjugates (SMDCs) that combine small molecule drugs with PSMA ligands have been proposed, which are non-immunogenic, have high tissue permeability, and can be produced at low cost (e.g., Patent Document 1, Patent Document 2).

[0005] US Patent Application Publication No. 2021 / 60173 International Patent Application Publication No. 2022 / 108992

[0006] The present invention relates to providing a PSMA ligand-low-molecular-weight drug conjugate that has excellent affinity for PSMA and enables selective anticancer drug therapy targeting cancer lesions.

[0007] In light of the above-mentioned problems, the inventors conducted extensive research and found that a low molecular weight drug conjugate in which a spacer containing a long-chain hydrocarbon group is attached to a PSMA ligand has excellent affinity for PSMA and can exert selective cytotoxicity on cancer cells.

[0008] That is, the present invention relates to the following 1) to 11): 1) A compound represented by the following formula (1):

[0009]

[0010] [Wherein X is —(CH2) n -CO- or -(CH2) n -NRCO- (wherein R represents a hydrogen atom, a lower alkyl group, a cyclo-lower alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group, and n represents an integer of 9 to 17), L represents a peptide linker, and D represents an antitumor drug moiety. 2) A drug conjugate represented by the formula (1) or a salt thereof, wherein X is -(CH2) n1) The drug conjugate or salt thereof of 1), wherein n is —CO—. 3) The drug conjugate or salt thereof of 1), wherein n is 9 to 15. 4) L is —[AA]. m -or- [AA] m -B-, wherein AA is a residue of an amino acid selected from Gly, Ala, Val, Phe, Asn, Lys, Glu, and Cit, B is p-aminobenzyloxycarbonyl, and m is 2 to 4. 5) The drug conjugate or salt thereof of 1), wherein L is -Val-Cit-, -Val-Cit-Phe-, or -Glu-Val-Cit-p-aminobenzyloxycarbonyl. 6) The drug conjugate or salt thereof of 1), wherein the antitumor drug is exatecan, monomethyl auristatin F, or monomethyl auristatin E. 7) An anticancer agent comprising the drug conjugate or salt thereof of 1) as an active ingredient. 8) The anticancer agent of 7), wherein the cancer is prostate cancer. 9) Use of the drug conjugate or salt thereof of 1) for the manufacture of an anticancer agent. 10) The drug conjugate or salt thereof according to 1) for preventing or treating cancer. 11) A method for preventing or treating cancer, comprising administering to a patient an effective amount of the drug conjugate or salt thereof according to 1).

[0011] The drug conjugate or a salt thereof of the present invention makes it possible to provide locally selective anticancer drug therapy that targets cancer lesions.

[0012] Cleavage test of compound 4 by cathepsin B Antitumor activity test of compound 4 (excised tumor size) Antitumor activity test of compound 4 (excised tumor size and weight) Antitumor activity test of compound 4 (external appearance 30 days after administration) Antitumor activity test of compound 4 (mouse weight 30 days after administration)

[0013] In this specification, unless otherwise specified, the term "lower" means that the number of carbon atoms in the hydrocarbon portion of the group to which this term is attached is 1 to 12 if the hydrocarbon portion is linear, and 3 to 12 if the hydrocarbon portion is cyclic, and the linear hydrocarbon portion may be either a straight chain or a branched chain. In this specification, the number of carbon atoms in the hydrocarbon portion (x to y) is not limited to "C x-y"Optionally substituted" means that a hydrogen atom of the target group may be substituted with another group, and the number of the substituents may be one or more, and when there are two or more substituents, the substituents may be the same or different.

[0014] In formula (1) of the present invention, the following partial structure is a PSMA ligand moiety: The drug conjugate of the present invention binds to prostate-specific membrane antigen (PSMA) via this moiety.

[0015] (wherein * indicates the point of attachment to X.)

[0016] In the formula (1) of the present invention, X is a spacer interposed between the PSMA ligand and the peptide linker. X is —(CH) n -CO- or -(CH2) n -NRCO-, where n represents an integer of 9 to 17. From the viewpoints of affinity to PSMA and cytotoxic selectivity, n is preferably 10 or more, more preferably 11 or more, and is preferably 9 to 15, more preferably 11 to 13.

[0017] -(CH2) represented by X n In —NRCO—, R may be a hydrogen atom, a lower alkyl group, a cyclo-lower alkyl group, an aryl group which may have a substituent, or an aralkyl group which may have a substituent. As the lower alkyl group, 1-7 An alkyl group is preferred, and a C 1 group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, or a tert-butyl group is more preferred. 1-4 Examples of the aryl group include alkyl groups, more preferably methyl groups, ethyl groups, isopropyl groups, and butyl groups. Examples of the aryl group include C aryl groups such as phenyl groups and naphthyl groups. 6-10 Examples of groups that can be substituted on the aryl group include a halogen atom (e.g., a fluorine atom), a hydroxy group, a C 1-12 Alkyl groups (e.g., methyl, ethyl, isopropyl, butyl, etc.), haloC 1-3Alkyl groups (e.g., difluoromethyl groups, trifluoromethyl groups, 2,2,2-trifluoroethyl groups, etc.), C 1-12 Examples include alkoxy groups (for example, methoxy, ethoxy, isopropoxy, butoxy, etc.), and nitro groups.

[0018] The aralkyl group is a phenyl C group in which the aryl moiety is a benzene ring or a naphthalene ring and the alkyl moiety is an alkyl group having 1 to 3 carbon atoms. 1-3 Alkyl group, naphthyl C 1-3 An alkyl group is preferred, and examples thereof include a benzyl group, a 1-phenylethyl group, a 2-phenylethyl group, a 1-naphthylmethyl group, and a 1-naphthylethyl group, and more preferred are a benzyl group and a 1-naphthylmethyl group. Examples of groups that can be substituted on the aralkyl group include groups that can be substituted on the aryl moiety of an aralkyl group, and examples thereof include a halogen atom (e.g., a fluorine atom), a C 1-4 Alkyl groups (e.g., methyl, ethyl, isopropyl, butyl, etc.), haloC 1-3 Examples include alkyl groups (for example, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, etc.).

[0019] X is -(CH2) n -CO- or -(CH2) n -NRCO-, where R is a hydrogen atom or C 1-4 Preferably, X is an alkyl group. More preferably, X is —(CH2) n In —CO—, n is 9 to 15, more preferably 11 to 13.

[0020] In formula (1) of the present invention, the peptide linker represented by L means a linker that can be cleaved to release a drug after the drug conjugate represented by formula (1) enters a target cell, and more specifically, includes peptides that can be cleaved by an in vivo enzyme, such as a protease or a cathepsin.

[0021] The peptide linker is -[AA] m - (where AA is an amino acid residue), but may be represented as -[AA] mAs in -B-, a self-immolative group represented by B can be present on the bond side to D (the antitumor drug moiety), where AA represents an amino acid residue selected from valine (Val), citrulline (Cit), phenylalanine (Phe), lysine (Lys), glycine (Gly), glutamic acid (Glu), alanine (Ala), and asparagine (Asn). The self-immolative group represented by B is -[AA] m - is a group that can be spontaneously cleaved after enzymatic cleavage to release the antitumor drug D. B is bonded to -[AA] by an amide bond. m -, and at the other end can be covalently bonded to a reactive functional group derived from an antitumor drug, and examples thereof include p-aminobenzyloxycarbonyl (PABC), p-aminobenzyl ether (PABE), methylated ethylenediamine (MED), etc. m is an integer of 2 to 4.

[0022] - [AA] m Specifically, the peptide is represented by the following formula:

[0023] In the formula, Y is a hydrogen atom, a methyl group, an isopropyl group, a benzyl group, -CHCONH, -(CH)NH, -CHCHCOOH, or -(CH)NHCONH, and the corresponding amino acid residues are Gly, Ala, Val, Phe, Asn, Lys, Glu, and Cit, respectively. m - is preferably -Val-Cit-, -Val-Cit-Phe-, -Glu-Val-Cit-, -Val-Glu-, -Phe-Lys-, -Val-Ala-, -Val-Gly-, -Gly-Gly-, -Ala-Phe-, -Gly-Gly-Gly-, -Gly-Gly-Phe-Gly-, -Ala-Ala-Asn-, -Val-Cit-, -Val-Cit-Phe-, -Gly-Gly-Phe-Gly- are more preferred, and -Val-Cit- is even more preferred. m Preferred examples of -B- include -Val-Cit-PABC and -Glu-Val-Cit-PABC, and -Val-Cit-PABC is more preferred.

[0024] In the drug conjugate represented by formula (1), a suitable "-XL-" is -(CH2) n -CO-Val-Cit- (where n is 11 to 13, more preferably 11), or -(CH) n -CO-Val-Cit-PABC (where n is 11 to 13, more preferably 11).

[0025] In formula (1) of the present invention, the drug used in the antitumor drug moiety represented by D is not particularly limited as long as it is a compound having a substituent (e.g., a primary amine group, a secondary amine group, a hydroxyl group, a sulfhydryl group, a carboxyl group, etc.) or a partial structure that can be bonded to the linker L. In the drug conjugate or a salt thereof of the present invention, the linker is partially or completely cleaved in tumor cells, liberating the antitumor drug moiety and thereby exerting an antitumor effect. Antitumor drugs refer to drugs that have a cell proliferation inhibitory effect and are useful for treating or preventing cancer or neovascularization, and include topoisomerase I inhibitors (e.g., topotecan, gimatecan, irinotecan, camptothecin and its derivatives (e.g., exatecan)), topoisomerase II inhibitors (e.g., doxorubicin, doxorubicin derivatives, anthracyclines (daunorubicin, epirubicin, idarubicin, nemorubicin)), anthraquinones (e.g., mitoxantrone, losoxantrone), podophyllotoxins (e.g., etoposide, teniposide), antiandrogens (e.g., bicalutamide), microtubule activators (e.g., taxanes (paclitaxel, docetaxel, etc.)), vinca alkaloids (e.g., vinblastine, vincristine, vinflunine, vinorelbine), discodermolide, colchicine, epothilone and its derivatives, etc.), alkylating agents (e.g., melphalan, cyclophosphamide, nimustine, buzulfan), antimetabolites (e.g., methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside), antibiotics (e.g., actinomycin, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, calicheamicin, esperamicin, etc.), antimitotic agents (e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF)), and the like.

[0026] The salt of the drug conjugate may be a pharmaceutically acceptable salt, and may be an acid or base addition salt. The acid addition salt may be an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, or phosphoric acid. Examples of organic acids include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid.

[0027] Base addition salts include alkali metal salts, alkaline earth metal salts, transition metal salts, including, for example, calcium, magnesium, potassium, sodium, and zinc salts, or organic salts prepared from basic amines, such as N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.

[0028] The drug conjugate of formula (1) of the present invention can exist not only in an unsolvated form but also as a hydrate or solvate, and therefore all crystalline forms and hydrates or solvates thereof are included as part of the present invention.

[0029] The drug conjugates of the present invention represented by formula (1) may exist in their tautomeric forms (e.g., keto-enol tautomers, etc.), and all tautomeric forms are encompassed in the drug conjugates of the present invention.

[0030] The drug conjugate of the present invention represented by formula (1) can exist as stereoisomers (enantiomers, diastereomers, etc.). Accordingly, all stereoisomers are encompassed as part of the present invention. Furthermore, each stereoisomer of the drug conjugate of the present invention can be substantially pure, e.g., free from other isomers, or can exist as a mixture, e.g., a racemate.

[0031] The drug conjugate of the present invention represented by formula (1) can exist as a geometric isomer (E or Z), and both geometric isomers are encompassed in the drug conjugate of the present invention.

[0032] The drug conjugate represented by formula (1) of the present invention can be produced by using established organic chemical synthesis methods from commercially available reagents. For example, as shown in Scheme A below, a PSMA ligand having a protected carboxyl group is linked to a spacer X 1 The compound I into which the formula (I) is introduced is condensed with a peptide linker compound to form a linker peptide L 1 is attached via its N-terminus to form compound II, and then the antitumor drug D is attached to the linker peptide L 1 can be produced by binding to the other end of

[0033]

[0034] [In the formula, X 1 is -(CH2) n -COOH or -(CH2) n -NHR (n and R are the same as above), L 1 Ha-[AA] m (-COOH) or -[AA] m -B(-OH) (AA, m, and B are the same as above), Z is a carboxy-protecting group, and X, L, and D are the same as above.

[0035] In one embodiment, X is —(CH) n -CO- and L is -[AA] m Compound (1a), which is -B- (B: p-aminobenzyloxycarbonyl (PABC)), can be produced by the method shown in the following scheme B. That is, a) a carboxylic acid compound (X) represented by compound I-a 1a : - (CH2)n -COOH) and a peptide linker compound ((HN-)[AA] m -PAB-OH) is condensed in a suitable solvent using a condensing agent used in amide synthesis (e.g., HBTU, HATU, TATU, EDCI, DCC, DIC, DMT-MM, BOP, PyBOP, COMU, etc.) in the presence of a base (e.g., diisopropylethylamine, triethylamine, etc.) to give compound II-a. b) Next, compound II-a is reacted with an activated esterification reagent (e.g., bis 4-nitrophenyl carbonate, 4-nitrophenyl chloroformate, etc.) in a suitable solvent in the presence of a base (e.g., diisopropylethylamine, triethylamine, etc.) to convert the terminal hydroxy group of compound II-a to an activated carbonate ester, giving compound III-a. c) Next, compound III-a is condensed with an antitumor drug using a functional group (e.g., amine, hydroxy, etc.) on the antitumor drug, and the protecting group is removed to give compound (1a).

[0036]

[0037] [In the formula, X 1a Ha-(CH2) n -COOH (n is the same as above), and X a Ha-(CH2) n -CO- (n is the same as above), and Y, m, Z, and D are the same as above.

[0038] In another embodiment, for example, X is —(CH2) n -NRCO-, L is -[AA] m Compound (1b), which is -B- (B: p-aminobenzyloxycarbonyl (PABC)), can be produced as shown in the following scheme C. That is, a) a carboxylic acid represented by compound I-a is subjected to a Curtius rearrangement reaction to produce amine compound I-b (X 1b-1 : - (CH2) n If necessary, this may be subjected to alkylation to give a substituted amine compound I-b' (X 1b-2 : - (CH2) n b) Then, compound I-b is reacted with a peptide linker compound ((HN-)[AA] mc) Compound (1b) is obtained from compound II-b by a method similar to that in Scheme B.

[0039]

[0040] [In the formula, X 1a Ha-(CH2) n -COOH (n is the same as above), and X 1b-1 Ha-(CH2) n -NH2 (n is the same as above), and X 1b-2 Ha-(CH2) n -NHR' (R' represents a lower alkyl group, a cyclo-lower alkyl group, an aryl group which may have a substituent, or an aralkyl group which may have a substituent, and n is the same as above), X b Ha-(CH2) n -NRCO- (R and n are the same as above), and Y, m, Z, and D are the same as above).

[0041] The above compound Ia can be produced, for example, by the following scheme D.

[0042]

[0043] (wherein Z is the same as defined above).

[0044] That is, a) a commonly available bromoalcohol 1 is converted to aldehyde 2 by oxidation using an oxidizing agent (e.g., 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)) in a suitable solvent. b) The carbonyl group of aldehyde 2 is then acetal-protected using ethylene glycol in a suitable solvent to produce acetal 3. c) This is then subjected to dehydrohalogenation using a nucleophile such as sodium iodide and a base (e.g., potassium tert-butoxide) to produce compound 4 having a terminal double bond. d) Separately, commercially available L-2-allylglycine (5) is reacted with a carbonyl-protecting agent such as tert-butyl ester and an amino-protecting agent such as benzyl chloroformate to sequentially protect the carbonyl and amino groups, producing protected compound 6. e) Compound 4 and protected compound 6 are converted to compound 7 by olefin metathesis using a HOVEYDA-GRUBBS catalyst. f) Compound 7 is then subjected to catalytic hydrogen reduction using a palladium carbon catalyst to reduce the double bond and deprotect the amino group, yielding compound 8. g) Compound 8 is then reacted with a protected glutamic acid using triphosgene to yield urea 9. h) The acetal of urea 9 is then deprotected in an acidic solution to yield aldehyde 10. i) Aldehyde 10 is then oxidized by an oxidation method using 2-methyl-2-butene, sodium hypochlorite, and sodium dihydrogen phosphate dihydrate, or the like, to yield compound I-a.

[0045] As shown in the test examples described below, the drug conjugate or salt thereof of the present invention thus obtained has excellent binding activity to PSMA and exhibits excellent cytotoxicity selectively toward PSMA-positive cancer cells. Therefore, the drug conjugate or salt thereof of the present invention can be a highly safe anticancer agent useful for the prevention or treatment (also referred to as anticancer therapy) of cancers expressing PSMA, and can be used to manufacture such anticancer agents. PSMA is overexpressed in prostate cancer cells, but has been reported to be specifically expressed in the neovascularization of many solid cancers. Therefore, cancers that can be treated or prevented by the antitumor agent of the present invention are not limited to prostate cancer, but may also include, for example, kidney cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, brain tumor, melanoma, neuroendocrine tumor, ovarian cancer, or sarcoma, with prostate cancer being preferred. Prostate cancer includes metastatic or non-metastatic, castration-resistant or castration-sensitive prostate cancer, and any of these may be used, but is preferably metastatic prostate cancer or metastatic castration-resistant prostate cancer.

[0046] When the drug conjugate of the present invention or a salt thereof is used as an anticancer agent (pharmaceutical composition), it can be formulated as a composition with a pharmaceutically acceptable carrier for parenteral administration such as injection or rectal administration, or oral administration in solid, semi-solid, or liquid form. Composition forms for injection include pharmaceutically acceptable sterile aqueous, non-aqueous solutions, suspensions, or emulsions. Examples of suitable non-aqueous carriers, diluents, solvents, or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Such compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, and dispersing agents. These compositions can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use.

[0047] Solid preparations for oral administration include capsules, tablets, pills, lozenges, powders, granules, etc. In preparing such solid preparations, the drug conjugate of the present invention or a salt thereof is generally mixed with at least one inert diluent, such as sucrose, lactose, starch, etc. In the usual formulations, such preparations may contain additional substances other than inert diluents, such as lubricants (e.g., magnesium stearate, etc.). In the case of capsules, tablets, and pills, a buffering agent may also be contained. Tablets and pills may further be provided with an enteric coating.

[0048] Liquid preparations for oral administration include inert diluents commonly used by those skilled in the art, such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc. containing water. In addition to such inert diluents, the compositions may also contain auxiliary substances such as wetting agents, emulsifiers, suspending agents, sweeteners, seasonings, and flavoring agents. Preparations for rectal administration preferably contain excipients such as cocoa butter, suppository wax, etc. in addition to the compound of the present invention.

[0049] The dose of the drug conjugate or salt thereof of the present invention administered to a subject patient can be varied as appropriate depending on the properties of the compound to be administered, the route of administration, the desired treatment period, and other factors, but may be, for example, in the range of 0.01 mg / kg to 100 mg / kg per dose, preferably 0.01 mg / kg to 10 mg / kg, and more preferably 0.05 mg / kg to 5 mg / kg. The administration interval may be once per week (q1w), once per two weeks (q2w), once per three weeks (q3w), or once per four weeks (q4w), etc.

[0050] The present invention will be described in more detail below with reference to examples and comparative examples. Preparation Example 1 Preparation of drug conjugate (compound 1) (1) (((9-bromononyl)oxy)methyl)benzene (1-a)

[0051] A recovery flask was charged with 9-bromo-1-nonanol (4.817 g, 21.6 mmol), tetrahydrofuran (45 mL), sodium hydride (60% in oil, 2.172 g, 54.3 mmol), and benzyl bromide (3.9 mL, 32.6 mmol). After stirring at room temperature for 68 hours, the temperature was lowered to 0°C, and saturated aqueous sodium bicarbonate was added until the reaction solution changed from gray to transparent, quenching the reaction. Water was then added, and the mixture was extracted with diethyl ether. The combined organic layer was washed with saturated brine and dried over sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using dichloromethane and hexane (concentration gradient: 16%-100%) as an eluent to obtain the title compound 1-a (6.607 g, 98%) as a colorless oil.

[0052] HRMS m / z calculation for C 16 H 26 BrO + [M + H] + 313.1162, found 313.1164.

[0053] 1 H NMR (500 MHz, CDCl3) δ 7.34 (d, J= 3.8 Hz, 4H), 7.31 - 7.25 (m, 1H), 4.50 (s, 2H), 3.46 (t, J = 6.6 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 1.89 - 1.80 (m, 2H), 1.66 - 1.56 (m, 2H), 1.45 - 1.28 (m, 10H).

[0054] (2) 5-((9-(benzyloxy)nonyl)thio)-1-phenyl-1H-tetrazole (1-b)

[0055] Compound 1-a (5.756 g, 18.4 mmol), 5-mercapto-1-phenyltetrazole (3.284 g, 18.4 mmol), potassium carbonate (3.817 g, 27.6 mmol), and acetone (30 mL) were added to a recovery flask and stirred at 80°C for 4 hours. The reaction solution was returned to room temperature, insoluble matter was filtered off, and the mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 0%-100%) as an eluent to obtain the title compound 1-b (5.426 g, 72%) as a white solid.

[0056] HRMS m / z calculation for C 23 H 31 N4OS + [M + H] + 411.2213, found 411.2225.

[0057] 1 H NMR (500 MHz, CDCl3) δ 7.62 - 7.48 (m, 5H), 7.47 - 7.39 (m, 1H), 7.36 - 7.24 (m, 4H), 4.50 (s, 2H), 3.46 (t, J= 6.6 Hz, 2H), 3.42 - 3.35 (m, 2H), 1.86 - 1.76 (m, 2H), 1.65 - 1.51 (m, 2H), 1.47 - 1.39 (m, 2H), 1.38 - 1.25 (m, 8H).

[0058] (3) 5-((9-(benzyloxy)nonyl)sulfonyl)-1-phenyl-1H-tetrazole (1-c)

[0059] Compound 1-b (5.426 g, 13.2 mmol), hexaammonium heptamolybdate tetrahydrate (3.200 g, 2.59 mmol), 34.5% aqueous hydrogen peroxide (12 mL, 134 mmol), and ethanol (100 mL) were added to a recovery flask and stirred at room temperature for 8 hours. The reaction solution was concentrated under reduced pressure, followed by addition of water and extraction with dichloromethane. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 0%-5%) as the eluent to obtain the title compound 1-c (5.098 g, 87%) as a white solid.

[0060] HRMS m / z calculation for C 23 H 31 N4O3S + [M + H] + 443.2112, found 443.2111.

[0061] 1 H NMR (500 MHz, CDCl3) δ 7.73 - 7.67 (m, 2H), 7.65 - 7.57 (m, 3H), 7.38 - 7.31 (m, 3H), 7.32 - 7.25 (m, 2H), 4.50 (s, 2H), 3.76 - 3.69 (m, 2H), 3.46 (t, J = 6.6 Hz, 2H), 2.00 - 1.90 (m, 2H), 1.65 - 1.52 (m, 2H), 1.52 - 1.44 (m, 2H), 1.38 - 1.27 (m, 8H).

[0062] (4) tert-butyl (S)-13-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)tridec-4-enoate(1-d)

[0063] Compound 1-c (3.764 g, 8.50 mmol) and tetrahydrofuran (20 mL) were added to a two-necked eggplant-shaped flask and the temperature was adjusted to -40°C. Then, a tetrahydrofuran solution of lithium bis(trimethylsilyl)amide (1.3 M, 13.2 mL, 17.2 mmol) was added, and a solution of (S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-4-oxobutanoate (2.640 g, 8.59 mmol) dissolved in tetrahydrofuran (10 mL) was added, and the mixture was stirred at -40°C for 2 hours, then the temperature was raised to 0°C and the mixture was stirred for 1 hour. A saturated aqueous solution of ammonium chloride was added to the reaction solution to terminate the reaction, and the mixture was extracted with ethyl acetate. The combined organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using dichloromethane and toluene (concentration gradient: 33%-100%) as an eluent to give the title compound 1-d (1.058 g, 25%) as a pale yellow oil.

[0064] HRMS m / z calculation for C 32 H 45 NO5Na + [M + Na] + 546.3190, found 546.3180.

[0065] 1 H NMR (500 MHz, CDCl3) δ 7.38 - 7.24 (m, 10H), 5.58 - 5.46 (m, 1H), 5.32 - 5.23 (m, 2H), 5.10 (s, 2H), 4.50 (s, 2H), 4.37 - 4.24 (m, 1H), 3.46 (t, J = 6.7 Hz, 2H), 2.50 - 2.40 (m, 2H), 2.01 - 1.92 (m, 2H), 1.65 - 1.55 (m, 2H), 1.49 - 1.40 (m, 2H), 1.45 (s, 9H), 1.37 - 1.24 (m, 8H).

[0066] (5) tert-butyl (S)-2-amino-13-hydroxytridecanoate (1-e)

[0067] Compound 1-d (473.2 mg, 0.904 mmol), tert-butanol (5 mL), and palladium hydroxide on carbon (20% Pd, 50% water content, 251.7 mg, 0.179 mmol) were placed in a recovery flask, the mixture was purged with hydrogen, and the mixture was stirred at room temperature for 23 hours. The mixture was filtered through Celite using tert-butanol and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 0%-5%) as an eluent to give the title compound 1-e (222.4 mg, 82%) as a colorless oil.

[0068] 1 H NMR (500 MHz, CDCl3) δ 3.64 (t, J= 6.6 Hz, 2H), 3.30 (dd, J = 7.3, 5.5 Hz, 1H), 1.72 - 1.49 (m, 4H), 1.46 (s, 9H), 1.40 - 1.25 (m, 16H).

[0069] (6) di-tert-butyl (((S)-1-(tert-butoxy)-13-hydroxy-1-oxotridecan-2-yl)carbamoyl)-L-glutamate (1-f)

[0070] Di-tert-butyl L-glutamate hydrochloride (175.9 mg, 0.595 mmol) and dehydrated dichloromethane (2.0 mL) were added to a recovery flask and the temperature was adjusted to -78°C. Then, triphosgene (59.0 mg, 0.199 mmol) and diisopropylethylamine (260 μL, 1.49 mmol) were added and the mixture was stirred for 1 hour. The temperature was then raised to room temperature and the mixture was stirred for 15 minutes. After cooling to -78°C again, a solution of compound 1-e (148.4 mg, 0.492 mmol) dissolved in dehydrated dichloromethane (1.0 mL) and diisopropylethylamine (260 μL, 1.49 mmol) were added and the mixture was stirred at -78°C for 2 hours. The mixture was then warmed to room temperature and stirred for 1 hour. Water was added to the reaction solution to quench the reaction, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 33%-100%) as an eluent to give the title compound 1-f (175.9 mg, 61%) as a colorless oil.

[0071] HRMS m / z calculation for C 31 H 58 N2O8Na + [M + Na] + 609.4085, found 609.4087.

[0072] 1 H NMR (500 MHz, CDCl3) δ 4.95 (d, J= 7.9 Hz, 1H), 4.89 (d, J = 7.9 Hz, 1H), 4.38 - 4.28 (m, 2H), 3.64 (t, J= 6.6 Hz, 2H), 2.37 - 2.24 (m, 2H), 2.11 - 2.05 (m, 2H), 1.92 - 1.81 (m, 1H), 1.76 - 1.71 (m, 1H), 1.67 - 1.53 (m, 4H), 1.53 - 1.38 (m, 25H), 1.38 - 1.15 (m, 16H).

[0073] (7) di-tert-butyl (((S)-1-(tert-butoxy)-1,13-dioxotridecan-2-yl)carbamoyl)-L-glutamate (1-g)

[0074] 1-f (29.6 mg, 0.0506 mmol), Dess-Martin periodinane (33.2 mg, 0.0783 mmol), sodium bicarbonate (11.0 mg, 0.131 mmol), and anhydrous dichloromethane (3 mL) were added to a recovery flask and stirred at room temperature for 16 hours. A mixture of saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate was added to the reaction solution to terminate the reaction, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to yield the title compound 1-g (13.2 mg, 45%) as a colorless oil.

[0075] HRMS m / z calculation for C 31 H 57 N2O8 + [M + H] + 585.4102, found 585.4110.

[0076] 1 H NMR (500 MHz, CDCl3) δ 9.75 (t, J= 1.9 Hz, 1H), 5.04 (d, J = 8.0 Hz, 1H), 4.98 (d, J = 8.0 Hz, 1H), 4.37 - 4.26 (m, 2H), 2.46 - 2.40 (m, 2H), 2.12 - 2.02 (m, 1H), 1.92 - 1.80 (m, 1H), 1.78 - 1.69 (m, 2H), 1.67 - 1.55 (m, 4H), 1.48 - 1.38 (m, 23H), 1.38 - 1.20 (m, 18H).

[0077] (8) (S)-13-(tert-butoxy)-12-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-13-oxotridecanoic acid (1-h)

[0078] To a recovery flask were added 1-g (41.6 mg, 0.0714 mmol), tert-butanol (2.0 mL), water (0.4 mL), 2-methyl-2-butene (40 μL, 0.376 mmol), sodium hypochlorite (29.6 mg, 0.259 mmol), and sodium dihydrogen phosphate dihydrate (17.2 mg, 0.110 mmol), and the mixture was stirred at room temperature for 4.5 hours. The reaction solution was concentrated under reduced pressure and then extracted with ethyl acetate. The combined organic layer was dried over aqueous sodium sulfate, and the insoluble material was removed by filtration. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 50%-100%) as an eluent to give the title compound 1-h (24.3 mg, 57%) as a colorless oil.

[0079] HRMS m / z calculation for C 31 H 56 N2O9Na + [M + Na] + 623.3878, found 623.3877.

[0080] 1 H NMR (500 MHz, CDCl3) δ 5.43 - 5.32 (m, 2H), 4.39 - 4.31 (m, 1H), 4.29 - 4.25 (m, 1H), 2.37 - 2.25 (m, 4H), 2.11 - 2.02 (m, 2H), 1.89 - 1.55 (m, 4H), 1.50 - 1.40 (m, 27H), 1.34 - 1.21 (m, 14H).

[0081] (9) di-tert-butyl (((S)-1-(tert-butoxy)-13-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan- 2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1,13-dioxotridecan-2-yl)carbamoyl)-L-glutamate (1-i)

[0082] 1-h (45.2 mg, 0.0752 mmol), dimethylformamide (2 mL), diisopropylethylamine (50 μL, 0.287 mmol), and HATU (31.4 mg, 0.0827 mmol) were added to a recovery flask and stirred at room temperature. After 50 minutes, Val-Cit-PAB-OH (42.8 mg, 0.113 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution. Water and methanol were added, and the mixture was extracted with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 0%-14%) as an eluent to obtain the title compound 1-i (52.1 mg, 72%) as a white powder.

[0083] HRMS m / z calculation for C 49 H 84 N7O 12 + [M + H] + 962.6172, found 962.6177.

[0084] 1H NMR (500 MHz, MeOD) δ 8.02 - 7.97 (m, 1H), 7.90 (s, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 4.61 - 4.48 (m, 3H), 4.23 - 4.10 (m, 3H), 3.25 - 3.15 (m, 1H), 3.15 - 3.03 (m, 1H), 2.42 - 2.21 (m, 4H), 2.15 - 1.96 (m, 3H), 1.95 - 1.67 (m, 4H), 1.68 - 1.21 (m, 46H), 1.07 - 0.87 (m, 6H).

[0085] (10) di-tert-butyl (((S)-1-(tert-butoxy)-13-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)- 1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-1,13-dioxotridecan-2-yl)carbamoyl)-L-glutamate (1-j)

[0086] 1-i (52.1 mg, 0.0541 mmol), dimethylformamide (2 mL), diisopropylethylamine (30 μL, 0.172 mmol), and bis-4-nitrophenyl carbonate (32.9 mg, 0.108 mmol) were added to a recovery flask and stirred at room temperature for 16 hours. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and chloroform (concentration gradient: 0%-4%) as an eluent to give the title compound 1-j (46.0 mg, 75%) as a white solid.

[0087] HRMS m / z calculation for C 56 H 87 N8O 16 + [M + H]+ 1127.6235, found 1127.6239.

[0088] 1 H NMR (500 MHz, MeOD) δ 8.34 - 8.27 (m, 2H), 7.68 - 7.61 (m, 2H), 7.49 - 7.43 (m, 2H), 7.43 - 7.38 (m, 2H), 6.36 (dd, J= 8.4, 3.5 Hz, 1H), 5.26 (s, 2H), 4.52 (dd, J = 9.0, 5.1 Hz, 1H), 4.23 - 4.09 (m, 3H), 3.27 - 3.16 (m, 1H), 3.16 - 3.05 (m, 1H), 2.35 - 2.24 (m, 4H), 2.13 - 1.98 (m, 2H), 1.97 - 1.85 (m, 1H), 1.85 - 1.67 (m, 2H), 1.67 - 1.52 (m, 3H), 1.52 - 1.41 (m, 29H), 1.39 - 1.25 (m, 15H), 1.01- 0.95 (m, 6H).

[0089] (11)(((S)-1-carboxy-12-(((S)-1-(((S)-1-((4-(((((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)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-12-oxododecyl)carbamoyl)-L-glutamic acid(1)

[0090]

[0091] Compound 1-j (17.0 mg, 0.0151 mmol), dimethylformamide (1 mL), exatecan mesylate (8.0 mg, 0.0151 mmol), pyridine (40 μL, 0.497 mmol), and hydroxybenzotriazole (1.0 mg, 0.00740 mmol) were added to a recovery flask and stirred at room temperature for 48 hours. Water and methanol were added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and ethyl acetate (concentration gradient: 0%-15%) as the eluent. The resulting residue (13.8 mg), trifluoroacetic acid (1 mL), and dichloromethane (1 mL) were added to a recovery flask and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase high performance liquid chromatography to obtain the title compound 1 (4.8 mg, 25%) as a pale yellow powder.

[0092] HRMS m / z calculation for C 62 H 78 FN 10 O 17 - [M-H] - 1253.5536, found 1253.5525.

[0093] 1H NMR (500 MHz, DMSO) δ 10.00 (s, 1H), 8.11 - 8.05 (m, 2H), 7.85 - 7.76 (m, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.32 (s, 1H), 6.53 (s, 1H), 6.30 (dd, J = 15.0, 8.2 Hz, 1H), 6.00 (s, 1H), 5.48 - 5.42 (m, 4H), 5.35 - 5. 24 (m, 3H), 5.10 - 5.07 (m, 2H), 4.42 - 4.34 (m, 1H), 4.19 (dd, J = 8.6, 6.9 Hz, 1H), 4.13 - 4.01 (m, 1H), 3.26 - 2.83 (m, 3H), 2.46 - 2.35 (m, 3H), 2.31 - 2.05 (m, 6H), 2.03 - 1.79 (m, 5H), 1.79 - 1.13 (m, 24H), 0.92 - 0.81 (m, 9H).

[0094] Preparation Example 2 Preparation of Drug Conjugate (Compound 2) (1) 12-bromododecanal (2-a)

[0095] 12-Bromo-1-dodecanol (1.003 g, 3.78 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl (120.8 mg, 0.773 mmol), iodobenzene diacetate (1.628 g, 5.05 mmol), and dichloromethane (30 mL) were added to a recovery flask and stirred at room temperature for 2 hours. A mixture of saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate was added to the reaction solution to terminate the reaction, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Insoluble material was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 1%-100%) as the eluent to obtain the title compound 2-a (363.6 mg, 37%) as a colorless oil.

[0096] 1H NMR (500 MHz, CDCl3) δ 9.77 (t, J = 1.9 Hz, 1H), 3.41 (t, J = 6.9 Hz, 2H), 2.42 (td, J = 7.3, 1.9 Hz, 2H), 1.85 (tt, J = 8.7, 6.9 Hz, 2H), 1.63 (tt, J = 7.3, 7.3 Hz, 2H), 1.42 (tt, J = 7.2, 7.2 Hz, 2H), 1.35 - 1.22 (m, 12H).

[0097] (2) 2-(11-bromoundecyl)-1,3-dioxolane (2-b)

[0098] Compound 2-a (544.8 mg, 2.07 mmol), ethylene glycol (1.1 mL, 20.7 mmol), paratoluenesulfonic acid monohydrate (40.1 mg, 0.211 mmol), and cyclohexane (30 mL) were added to a recovery flask and stirred at 130°C. After 20 hours, the reaction solution was returned to room temperature, and the reaction was quenched by adding water and saturated aqueous sodium bicarbonate solution, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 1%-50%) as an eluent to obtain the title compound 2-b (547.9 mg, 86%) as a white powder.

[0099] 1 H NMR (500 MHz, CDCl3) δ 4.84 (t, J= 4.8 Hz, 1H), 4.01 - 3.92 (m, 2H), 3.89 - 3.81 (m, 2H), 3.41 (t, J = 6.9 Hz, 2H), 1.85 (tt, J = 7.0, 7.0 Hz, 2H), 1.69 - 1.60 (m, 2H), 1.48 - 1.35 (m, 2H), 1.31 - 1.23 (m, 14H).

[0100] (3) 2-(undec-10-en-1-yl)-1,3-dioxolane (2-c)

[0101] Compound 2-b (878.6 mg, 2.86 mmol), sodium iodide (1.534 g, 10.2 mmol), and acetone (30 mL) were added to a recovery flask and stirred at 80°C for 14 hours. The reaction solution was returned to room temperature, saturated saline was added to terminate the reaction, and then the solution was extracted with diethyl ether. The collected organic layer was dried over anhydrous sodium sulfate, and the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure. The residue (1.062 g), potassium tert-butoxide (739.2 mg, 6.59 mmol), and THF (30 mL) were added to a recovery flask and stirred at room temperature for 4 hours. Water was added to the reaction solution to terminate the reaction, and the solution was extracted with ethyl acetate. The collected organic layer was washed with saturated saline and then dried over anhydrous sodium sulfate. The insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-100%) as an eluent to give the title compound 2-c (284.1 mg, 44%) as a pale brown oil.

[0102] HRMS m / z calculation for C 14 H 27 O2 + [M + H] + 227.2006, found 227.2205.

[0103] 1 H NMR (500 MHz, CDCl3) δ 5.81 (dt, J= 16.9, 6.7 Hz, 1H), 4.99 (dd, J= 17.2, 1.6 Hz, 1H), 4.93 (dd, J= 10.2, 1.0 Hz, 1H), 4.84 (t, J = 4.8 Hz, 1H), 4.00 - 3.92 (m, 2H), 3.90 - 3.80 (m, 2H), 2.08 - 1.99 (m, 2H), 1.69 - 1.61 (m, 2H), 1.46 - 1.32 (m, 2H), 1.47 - 1.22 (m, 12H).

[0104] (4) tert-butyl (S)-2-((benzyloxy)carbonyl)amino)pent-4-enoate (2-d)

[0105] L-2-allylglycine (1.000 g, 8.69 mmol), 70% perchloric acid (2 mL, 23.3 mmol), and tert-butyl acetate (50 mL, 373 mmol) were added to a recovery flask and stirred at room temperature for 72 hours. Saturated aqueous sodium bicarbonate and sodium carbonate were added to the reaction solution to terminate the reaction, followed by extraction with ethyl acetate. The collected organic layer was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue (22.04 g), benzyl chloroformate (2 mL, 14.1 mmol), sodium carbonate (1.992 g, 18.8 mmol), and THF (20 mL) were added to a recovery flask and stirred at room temperature for 1 hour. Insoluble matter was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The collected organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 20%-100%) as an eluent to give the title compound 2-d (1.095 g, 42%) as a colorless oil.

[0106] HRMS m / z calculation for C 17 H 23 NO4Na + [M + Na] + 328.1519, found 328.1522.

[0107] 1 H NMR (500 MHz, CDCl3) δ 7.47 (s, 1H), 7.41 - 7.34 (m, 4H), 7.34 - 7.30 (m, 1H), 5.75 - 5.64 (m, 1H), 5.17 (d, J = 4.0 Hz, 1H), 5.10 (s, 2H), 4.71 (d, J = 5.9 Hz, 1H), 4.37 - 4.30 (m, 1H), 2.62 - 2.43 (m, 2H), 1.46 (s, 9H).

[0108] (5) tert-butyl (S)-2-((benzyloxy)carbonyl)amino)-14-(1,3-dioxolan-2-yl)tetradec-4-enoate (2-e)

[0109] Compound 2-d (40.5 mg, 0.133 mmol), toluene (3.0 mL), compound 2-c (150.0 mg, 0.663 mmol), and second-generation HOVEYDA-GRUBBS catalyst (8.3 mg, 0.0132 mmol) were added to a recovery flask and stirred at 50°C for 3 hours. The reaction solution was returned to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-25%) as an eluent to obtain the title compound 2-e (39.2 mg, 59%) as a colorless oil.

[0110] HRMS m / z calculation for C 29 H 45 NO6Na + [M + Na] + 526.3139, found 526.3140.

[0111] 1 H NMR (500 MHz, CDCl3) δ 7.47 (s, 1H), 7.37 - 7.33 (m, 4H), 7.33 - 7.29 (m, 1H), 5.51 (dt, J = 14.1, 6.7 Hz, 1H), 5.31 -5.21 (m, 1H), 5.10 (s, 2H), 4.84 (t, J = 4.9 Hz, 1H), 4.28 (dt, J = 6.5, 5.9 Hz, 1H), 4.00 - 3.91 (m, 2H), 3.90 - 3.81 (m, 2H), 2.57 -2.30 (m, 2H), 2.03 - 1.96 (m, 2H), 1.65 (dt, J = 8.9, 5.2 Hz, 2H), 1.46 (s, 9H), 1.44 - 1.36 (m, 6H), 1.36 - 1.18 (m, 8H).

[0112] (6) tert-butyl (S)-2-amino-14-(1,3-dioxolan-2-yl)tetradecanoate (2-f)

[0113] Compound 2-e (79.2 mg, 0.157 mmol), tert-butyl alcohol (4 mL), and palladium on carbon (Pd 10%, 20.2 mg, 0.0190 mmol) were added to a recovery flask and stirred at room temperature for 2 hours under a hydrogen gas atmosphere. The reaction solution was filtered through Celite to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 1%-5%) as an eluent to obtain the title compound 2-f (31.0 mg, 53%) as a colorless oil.

[0114] HRMS m / z calculation for C 21 H 41 NO4Na + [M + Na] + 394.2928, found 394.2929.

[0115] 1 H NMR (500 MHz, CDCl3) δ 4.84 (t, J = 4.9 Hz, 1H), 4.00 - 3.91 (m, 2H), 3.90 - 3.82 (m, 2H), 3.32 - 3.27 (m, 1H), 1.65 (dt, J = 14.3, 5.4 Hz, 2H), 1.46 (s, 9H), 1.37 - 1.15 (m, 22H).

[0116] (7) di-tert-butyl (((S)-1-(tert-butoxy)-14-(1,3-dioxolan-2-yl)-1-oxotetradecan-2-yl)carbamoyl)-L-glutamate (2-g)

[0117] According to a similar procedure to that of Production Example 1 (6), di-tert-butyl L-glutamate hydrochloride (132 mg, 0.466 mmol) and dichloromethane (1.5 mL) were added to a two-necked eggplant-shaped flask and cooled to -78°C. After that, triphosgene (51.9 mg, 0.175 mmol) and diisopropylethylamine (290 μL, 1.66 mmol) were added and stirred at -78°C for 1 hour. The reaction solution was warmed to room temperature and stirred for 15 minutes. The reaction solution was again cooled to -78°C, and then a dichloromethane solution (1.5 mL) of compound 2-f (123.4 mg, 0.332 mmol) and diisopropylethylamine (290 μL, 1.66 mmol) were added. The mixture was stirred at -78°C for 2 hours, then warmed to room temperature and stirred for 1 hour. Water was added to the reaction solution to terminate the reaction, and the mixture was extracted with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove insoluble material, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to give the title compound 2-g (190.4 mg, 87%) as a colorless oil.

[0118] HRMS m / z calculation for C 35 H 64 N2O9Na + [M + Na] + 679.4504, found 679.4509.

[0119] 1 H NMR (500 MHz, CDCl3) δ 5.01 (d, J= 7.8 Hz, 1H), 4.88 (d, J = 8.1 Hz, 1H), 4.84 (t, J = 4.9 Hz, 1H), 4.37 - 4.29 (m, 2H), 4.00 - 3.94 (m, 2H), 3.88 - 3.80 (m, 2H), 2.39 - 2.22 (m, 2H), 2.12 - 2.06 (m, 2H), 1.86 (dt, J= 9.9, 6.6 Hz, 2H), 1.69 - 1.59 (m, 2H), 1.49 - 1.42 (m, 27H), 1.30 - 1.21 (m, 20H).

[0120] (8) di-tert-butyl (((S)-1-(tert-butoxy)-1,15-dioxopentadecan-2-yl)carbamoyl)-L-glutamate (2-h)

[0121] Compound 2-g (49.5 mg, 0.0754 mmol), 2 M hydrochloric acid (1 mL), and tert-butyl alcohol (1.0 mL) were added to a recovery flask and stirred at room temperature for 3.5 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution to terminate the reaction, followed by addition of water and extraction with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the insoluble material was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 16%-50%) as the eluent to obtain the title compound 2-h (30.0 mg, 65%) as a colorless oil.

[0122] HRMS m / z calculation for C 33 H 60 N2O8Na + [M + Na] + 635.4242, found 635.4249.

[0123] 1 H NMR (500 MHz, CDCl3) δ 9.77 (t, J= 1.9 Hz, 1H), 5.00 (d, J = 7.9 Hz, 1H), 4.94 (d, J = 7.8 Hz, 1H), 4.37 - 4.28 (m, 2H), 2.42 (td, J= 7.4, 1.9 Hz, 2H), 2.38 - 2.22 (m, 2H), 2.13 - 2.02 (m, 2H), 1.94 - 1.81 (m, 2H), 1.62 (tt, J = 7.7, 7. 3 Hz, 2H), 1.49 - 1.42 (m, 27H), 1.31 - 1.22 (m, 18H).

[0124] (9) (S)-15-(tert-butoxy)-14-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-15-oxopentadecanoic acid (2-i)

[0125] According to a similar procedure to that of Preparation Example 1(8), compound 2-h (58.5 mg, 0.0955 mmol), tert-butyl alcohol (2 mL), water (0.4 mL), 2-methyl-2-butene (100 μL, 0.956 mmol), sodium hypochlorite (42.0 mg, 0.464 mmol), and sodium dihydrogen phosphate dihydrate (24.4 mg, 0.156 mmol) were added to a recovery flask and stirred at room temperature for 2 hours. The reaction solution was then concentrated under reduced pressure. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to obtain the title compound 2-i (28.8 mg, 48%) as a white powder.

[0126] HRMS m / z calculation for C 33 H 60 N2O9Na + [M + Na] + 651.4191, found 651.4195.

[0127] 1 H NMR (500 MHz, CDCl3) δ 5.43 (d, J= 8.6 Hz, 1H), 5.01 (d, J = 7.9 Hz, 1H), 4.40 - 4.28 (m, 2H), 2.41 - 2.23 (m, 2H), 2.34 - 2.23 (m, 2H), 2.11 - 2.03 (m, 2H), 1.86 (dt, J = 14.1, 8.8 Hz, 2H), 1.69 - 1.51 (m, 2H), 1.48 - 1.42 (m, 27H), 1. 39 - 1.17 (m, 18H).

[0128] (10) di-tert-butyl (((S)-1-(tert-butoxy)-15-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2 -yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1,15-dioxopentadecan-2-yl)carbamoyl)-L-glutamate (2-j)

[0129] Following a procedure similar to that of Preparation Example 1(9), compound 2-i (208.1 mg, 0.331 mmol), dimethylformamide (6 mL), diisopropylethylamine (280 μL, 1.61 mmol), and HATU (145.0 mg, 0.381 mmol) were added to a recovery flask and stirred at room temperature. After 50 minutes, Val-Cit-PAB-OH (115.3 mg, 0.304 mmol) was added, and the mixture was stirred at room temperature for 41 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution. Water and methanol were added, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 5%-12%) as an eluent to give the title compound 2-j (158.9 mg, 49%) as a white powder.

[0130] HRMS m / z calculation for C 51 H 87 N7O 12 Na + [M + Na] + 1012.6305, found 1012.6312.

[0131] 1H NMR (500 MHz, MeOD) δ 7.56 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 5.50 (s, 1H), 4.56 (s, 2H), 4.54 - 4.48 (m, 1H), 4.22 - 4.12 (m, 3H), 3.29 - 3.05 (m, 3H), 2.35 - 2.25 (m, 4H), 2.12 - 1.99 (m, 2H), 1.94 - 1.56 (m, 8H), 1.50 - 1.44 (m, 27H), 1.38 - 1.28 (m, 22H), 0.98 (t, J = 6.3 Hz, 6H).

[0132] (11) di-tert-butyl (((S)-1-(tert-butoxy)-15-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1 -oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-1,15-dioxopentadecan-2-yl)carbamoyl)-L-glutamate (2-k)

[0133] According to a similar procedure to that of Preparation Example 1 (10), compound 2-j (158.9 mg, 0.161 mmol), dimethylformamide (7 mL), diisopropylethylamine (110 μL, 0.632 mmol), and bis-4-nitrophenyl carbonate (142.9 mg, 0.470 mmol) were added to a recovery flask and stirred at room temperature for 16 hours. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and chloroform (concentration gradient: 0%-10%) as an eluent to obtain the title compound 2-k (183.5 mg, 99%) as a white solid.

[0134] HRMS m / z calculation for C 58 H 90 N8O16 Na + [M + Na] + 1177.6367, found 1177.6365.

[0135] 1 H NMR (500 MHz, MeOD) δ 8.35 - 8.30 (m, 2H), 7.68 - 7.62 (m, 2H), 7.50 - 7.44 (m, 2H), 7.44 - 7.38 (m, 2H), 6.39 - 6.33 (m, 1H), 5.27 (s, 2H), 4.61 (s, 1H), 4.52 (dd, J = 9.0, 5.1 Hz, 1H), 4.20 - 4.11 (m, 2H), 3.27 - 3.07 (m, 2H), 2.39 - 2.25 (m, 4H), 2.13 - 1.99 (m, 1H), 1.96 - 1.85 (m, 1H), 1.85 - 1.69 (m, 2H), 1.69 - 1.53 (m, 5H), 1.53 - 1.41 (m, 27H), 1.41 - 1.24 (m, 22H), 1.02 - 0.95 (m, 6H).

[0136] (12)(((S)-1-carboxy-14-(((S)-1-(((S)-1-((4-(((((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)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-14-oxotetradecyl)carbamoyl)-L-glutamic acid(2)

[0137] Following a procedure similar to that of Preparation Example 1 (11), compound 2-k (61.1 mg, 0.0529 mmol), dimethylformamide (4.5 mL), exatecan mesylate (35.6 mg, 0.0670 mmol), pyridine (150 μL, 1.86 mmol), and hydroxybenzotriazole (14.7 mg, 0.109 mmol) were added to an eggplant-shaped flask and stirred at room temperature for 40 hours. Water and methanol were added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and ethyl acetate (concentration gradient: 0%-15%) as the eluent. The resulting residue (45.1 mg), trifluoroacetic acid (1 mL), and dichloromethane (1 mL) were added to an eggplant-shaped flask and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase high performance liquid chromatography to give the title compound 2 (4.6 mg, 7%) as a pale yellow powder.

[0138] HRMS m / z calculation for C 64 H 82 FN 10 O 17 - [M-H] - 1281.5849, found 1281.5858.

[0139] 1H NMR (500 MHz, DMSO) δ 9.98 (s, 1H), 8.09 - 8.03 (m, 2H), 7.82 - 7.74 (m, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 7.31 (s, 1H), 6.52 (s, 1H), 6.31 (d, J = 8.5 Hz, 1H), 6.27 (d, J = 8.3 Hz, 1H), 5.98 (t, J = 5.9 Hz, 1H), 5.47 - 5.38 (m, 4H), 5.31 - 5.22 (m, 3H), 5.08 (s, 2H), 4.44 - 4.34 (m, 1H), 4.18 (dd, J = 8.6, 6.8 Hz, 1H), 4.14 - 4.00 (m, 2H), 3.27 - 2.87 (m, 3H), 2.41 - 2. 34 (m, 3H), 2.31 - 2.04 (m, 6H), 2.01 - 1.78 (m, 5H), 1.74 - 1.30 (m, 9H), 1.29 - 1.16 (m, 17H), 0.91 - 0.80 (m, 9H).

[0140] Preparation Example 3 Preparation of drug conjugate (compound 3) (((1S)-1-carboxy-14-(((2S)-1-(((2S)-1-((4-(((((2S)-1-(((2S)-1-(((4S,5R)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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)carbamoyl)oxy)methyl)phenyl )amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-14-oxotetradecyl)carbamoyl)-L-glutamic acid(3)

[0141] Following a procedure similar to that of Preparation Example 2(11), compound 2-k (40.8 mg, 0.0353 mmol), dimethylformamide (3 mL), monomethyl auristatin E (28.0 mg, 0.0390 mmol), pyridine (100 μL, 1.24 mmol), and hydroxybenzotriazole (10.0 mg, 0.0740 mmol) were added to an eggplant-shaped flask and stirred at room temperature for 26 hours. Water and methanol were added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and ethyl acetate (concentration gradient: 0%-15%) as the eluent. The resulting residue (15.6 mg), trifluoroacetic acid (1 mL), and dichloromethane (1 mL) were added to an eggplant-shaped flask and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase high performance liquid chromatography to give the title compound 3 (5.6 mg, 10%) as a white powder.

[0142] HRMS m / z calculation for C 79 H 129 N 12 O 20 + [M + H] + 1565.9441, found 1565.9474.

[0143] 1 H NMR (500 MHz, DMSO) δ 9.91 (s, 1H), 8.14 - 8.01 (m, 2H), 7.95 - 7.77 (m, 2H), 7.67 - 7.46 (m, 3H), 7.45 - 7.02 (m, 3H), 6.45 - 6.24 (m, 2H), 6.09 - 5.93 (m, 1H), 5.54 - 5.26 (m, 3H), 5.26 - 4.88 (m, 1H), 4.82 - 2.75 (m, 52H, merged in moisture peak), 2.35 - 2.07 (m, 4H), 2.07- 0.66 (m, 40H).

[0144] Preparation Example 4 Preparation of Drug Conjugate (Compound 4) (1) 14-bromotetradecanal (4-a)

[0145] Following a procedure similar to that of Preparation Example 2(1), 14-bromo-1-tetradecanol (3.380 g, 11.5 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl (168.7 mg, 1.08 mmol), iodobenzene diacetate (4.8350 g, 15.0 mmol), and dichloromethane (160 mL) were added to a recovery flask and stirred at room temperature for 2 hours. The reaction was terminated by adding a mixture of saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate to the reaction solution, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-20%) as the eluent to give the title compound 4-a (2.480 g, 73%) as a yellow oil.

[0146] 1 H NMR (500 MHz, CDCl3) δ 9.77 (t, J= 1.9 Hz, 1H), 3.41 (t, J = 6.9 Hz, 2H), 2.42 (td, J = 7.4, 1.9 Hz, 2H), 1.90 - 1.81 (m, 2H), 1.67 - 1.56 (m, 2H), 1.47 - 1.37 (m, 2H), 1.36 - 1.23 (m, 16H).

[0147] (2) 2-(13-bromotridecyl)-1,3-dioxolane (4-b)

[0148] Following a procedure similar to that of Preparation Example 2(2), compound 4-a (2.480 g, 8.52 mmol), ethylene glycol (2.1 mL, 37.7 mmol), paratoluenesulfonic acid monohydrate (18.0 mg, 0.0946 mmol), and cyclohexane (50 mL) were added to a recovery flask and stirred at 135°C. After 20 hours, the reaction solution was returned to room temperature, and the reaction was quenched by adding water and saturated aqueous sodium bicarbonate solution, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-50%) as an eluent to obtain the title compound 4-b (2.537 g, 89%) as a white powder.

[0149] HRMS m / z calculation for C 16 H 31 BrO2Na + [M + Na] + 357.1399, found 357.1397.

[0150] 1H NMR (500 MHz, CDCl3) δ 4.84 (t, J= 4.9 Hz, 1H), 4.01 - 3.92 (m, 2H), 3.90 - 3.82 (m, 2H), 3.41 (t, J = 6.9 Hz, 2H), 1.90 - 1.81 (m, 2H), 1.68 - 1.62 (m, 2H), 1.47 - 1.36 (m, 4H), 1.35 - 1.26 (m, 16H).

[0151] (3) 2-(tridec-12-en-1-yl)-1,3-dioxolane (4-c)

[0152] Following the same procedure as in Production Example 2(3), compound 4-b (2.537 g, 7.56 mmol), 4A molecular sieves (10.2 mg), sodium iodide (4.024 g, 26.9 mmol), and acetone (130 mL) were added to an eggplant-shaped flask and stirred at 80°C for 16 hours. The reaction solution was returned to room temperature, saturated saline was added to terminate the reaction, and the mixture was extracted with diethyl ether. The collected organic layer was dried over anhydrous sodium sulfate, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure. The residue (2.598 g), potassium tert-butoxide (1.726 g, 15.4 mmol), and THF (80 mL) were added to an eggplant-shaped flask and stirred at room temperature for 4 hours. Water was added to the reaction solution to terminate the reaction, and the mixture was extracted with ethyl acetate. The collected organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-13%) as an eluent to give the title compound 4-c (1.664 g, 86%) as a pale brown oil.

[0153] HRMS m / z calculation for C 16 H 31 O2 + [M + H] + 255.2319, found 255.2317.

[0154] 1H NMR (500 MHz, CDCl3) δ 5.87 - 5.76 (m, 1H), 5.03 - 4.96 (m, 1H), 4.95 - 4.90 (m, 1H), 4.84 (t, J = 4.8 Hz, 1H), 4.02 - 3.92 (m, 2H), 3.90 - 3.79 (m, 2H), 2. 08 - 1.99 (m, 2H), 1.69 - 1.61 (m, 2H), 1.48 - 1.34 (m, 2H), 1.34 - 1.26 (m, 16H).

[0155] (4) tert-butyl (S)-2-((benzyloxy)carbonyl)amino)-16-(1,3-dioxolan-2-yl)hexadec-4-enoate (4-d)

[0156] According to a similar procedure to that of Preparation Example 2(5), compound 2-d (24.0 mg, 0.0786 mmol), toluene (2.7 mL), compound 4-c (109.3 mg, 0.430 mmol), and second-generation HOVEYDA-GRUBBS catalyst (6.6 mg, 0.00777 mmol) were added to a recovery flask and stirred at 50°C for 3 hours. The reaction solution was returned to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-20%) as an eluent to obtain the title compound 4-d (27.1 mg, 65%) as a colorless oil.

[0157] HRMS m / z calculation for C 31 H 49 NO6Na + [M + Na] + 554.3452, found 554.3450.

[0158] 1H NMR (500 MHz, CDCl3) δ 7.40 - 7.28 (m, 5H), 5.55 - 5.46 (m, 1H), 5.31 - 5.22 (m, 2H), 5.15 - 5.08 (m, 2H), 4.84 (t, J= 4.9 Hz, 1H), 4.37 - 4.24 (m, 1H), 4.00 - 3.91 (m, 2H), 3.90 - 3.81 (m, 2H), 2.53 - 2.39 (m, 2H), 2.02 - 1.93 (m, 2H), 1.69 - 1.61 (m, 2H), 1.48 - 1.37 (m, 11H), 1.33 - 1.22 (m, 16H).

[0159] (5) tert-butyl (S)-2-amino-16-(1,3-dioxolan-2-yl)hexadecanoate (4-e)

[0160] According to a similar procedure to that of Preparation Example 2(6), compound 4-d (174.6 mg, 0.328 mmol), tert-butyl alcohol (5 mL), and palladium on carbon (Pd 10%, 69.9 mg, 0.0656 mmol) were added to a recovery flask and stirred at room temperature under a hydrogen gas atmosphere for 22.5 hours. The reaction solution was filtered through Celite to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 5%-10%) as an eluent to give the title compound 4-e (106.6 mg, 81%) as a colorless oil.

[0161] HRMS m / z calculation for C 23 H 46 No. 4 + [M + H] + 400.3422, found 400.3425.

[0162] 1H NMR (500 MHz, CDCl3) δ 4.84 (t, J= 4.9 Hz, 1H), 4.02 - 3.91 (m, 2H), 3.90 - 3.79 (m, 2H), 3.30 (dd, J = 7.2, 5.5 Hz, 1H), 1.72 - 1.61 (m, 2H), 1.59 - 1.51 (m, 4H), 1.46 (s, 9H), 1.42 - 1.23 (m, 22H).

[0163] (6) di-tert-butyl (((S)-1-(tert-butoxy)-16-(1,3-dioxolan-2-yl)-1-oxohexadecan-2-yl)carbamoyl)-L-glutamate (4-f)

[0164] According to a similar procedure to that of Production Example 1 (6), di-tert-butyl L-glutamate hydrochloride (20.3 mg, 0.0686 mmol) and dichloromethane (1 mL) were added to a two-necked eggplant-shaped flask and cooled to -78°C. After that, triphosgene (7.0 mg, 0.0236 mmol) and diisopropylethylamine (43 μL, 0.247 mmol) were added and stirred at -78°C for 30 minutes. The reaction solution was warmed to room temperature and stirred for 30 minutes. The reaction solution was cooled again to -78°C, and then a dichloromethane solution (1 mL) of compound 4-e (21.9 mg, 0.0548 mmol) and diisopropylethylamine (50 μL, 0.287 mmol) were added and stirred at -78°C for 2 hours, after which the temperature was warmed to room temperature and stirred for 1 hour. Water was added to the reaction solution to terminate the reaction, and the mixture was extracted with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove insoluble matter, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to give the title compound 4-f (30.9 mg, 82%) as a colorless oil.

[0165] HRMS m / z calculation for C 37 H 68 N2O9Na + [M + Na] +707.4817, found 707.4825.

[0166] 1 H NMR (500 MHz, CDCl3) δ 5.14 (d, J= 8.0 Hz, 1H), 5.09 (d, J = 8.0 Hz, 1H), 4.84 (t, J = 4.9 Hz, 1H), 4.39 - 4.28 (m, 2H), 4.01 - 3.91 (m, 2H), 3.91 - 3.82 (m, 2H), 2.39 - 2.22 (m, 2H), 2.14 - 2.01 (m, 1H), 1.92 - 1.80 (m, 1H), 1.78 - 1.69 (m, 1H), 1.69 - 1.62 (m, 3H), 1.48 - 1.36 (m, 27H), 1.34 - 1.22 (m, 24H).

[0167] (7) di-tert-butyl (((S)-1-(tert-butoxy)-1,17-dioxoheptadecan-2-yl)carbamoyl)-L-glutamate (4-g)

[0168] According to a similar procedure to that of Production Example 2(8), compound 4-f (125.1 mg, 0.183 mmol), 2 M hydrochloric acid (2.5 mL), and tert-butyl alcohol (2.5 mL) were added to a recovery flask and stirred at room temperature for 22 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution to terminate the reaction, followed by addition of water and extraction with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the insoluble material was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 16%-50%) as an eluent to obtain the title compound 4-g (106.2 mg, 91%) as a colorless oil.

[0169] HRMS m / z calculation for C 35 H 64 N2O8Na + [M + Na] + 663.4555, found 663.4561.

[0170] 1 H NMR (500 MHz, CDCl3) δ 9.77 (t, J= 1.9 Hz, 1H), 4.96 (d, J = 8.0 Hz, 1H), 4.90 (d, J = 7.9 Hz, 1H), 4.38 - 4.28 (m, 2H), 2.42 (td, J= 7.4, 1.9 Hz, 2H), 2.38 - 2.22 (m, 2H), 2.12 - 2.01 (m, 1H), 1.94 - 1.80 (m, 1H), 1.79 - 1.69 (m, 1H), 1.68 - 1.53 (m, 5H), 1.46 (s, 18H), 1.44 (s, 9H), 1.35 - 1.22 (m, 20H).

[0171] (8) (S)-17-(tert-butoxy)-16-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-17-oxoheptadecanoic acid (4-h)

[0172] According to a similar procedure to that of Production Example 1(8), compound 4-g (106.2 mg, 0.166 mmol), tert-butyl alcohol (1.7 mL), water (0.3 mL), 2-methyl-2-butene (90 μL, 0.847 mmol), sodium hypochlorite (67.1 mg, 0.586 mmol), and sodium dihydrogen phosphate dihydrate (40.4 mg, 0.259 mmol) were added to a recovery flask and stirred at room temperature for 8 hours. The reaction solution was then concentrated under reduced pressure. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 30%-50%) as an eluent to give the title compound 4-h (75.9 mg, 70%) as a white powder.

[0173] HRMS m / z calculation for C 35 H 64 N2O9Na + [M + Na] +679.4504, found 679.4501.

[0174] 1 H NMR (500 MHz, CDCl3) δ 5.31 (d, J= 8.0 Hz, 2H), 4.42 - 4.23 (m, 2H), 2.39 - 2.24 (m, 5H), 2.12 - 2.06 (m, 1H), 1.91 - 1.80 (m, 1H), 1.80 - 1.69 (m, 1H), 1.68 - 1.55 (m, 5H), 1.46 (s, 18H), 1.44 (s, 9H), 1.30 - 1.22 (m, 20H).

[0175] (9) di-tert-butyl (((S)-1-(tert-butoxy)-17-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2 -yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1,17-dioxoheptadecan-2-yl)carbamoyl)-L-glutamate (4-i)

[0176] Following a procedure similar to that of Preparation Example 1(9), compound 4-h (74.6 mg, 0.114 mmol), dimethylformamide (5 mL), diisopropylethylamine (100 μL, 0.574 mmol), and HATU (52.3 mg, 0.138 mmol) were added to a recovery flask and stirred at room temperature. After 30 minutes, Val-Cit-PAB-OH (65.6 mg, 0.173 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution. Water was added, and the mixture was extracted with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 5%-10%) as an eluent to give the title compound 4-i (65.4 mg, 57%) as a pale yellow solid.

[0177] HRMS m / z calculation for C 53H 91 N7O 12 Na + [M + Na] + 1040.6618, found 1040.6615.

[0178] 1 H NMR (500 MHz, MeOD) δ 7.59 - 7.52 (m, 3H), 7.32 - 7.27 (m, 3H), 6.38 - 6.32 (m, 2H), 4.61 - 4.54 (m, 4H), 4.50 (dd, J= 9.0, 5.2 Hz, 1H), 4.23 - 4.10 (m, 4H), 3.25 - 3.01 (m, 2H), 2.39 - 2.21 (m, 5H), 2.13 - 1.98 (m, 3H), 1.95 - 1.70 (m, 3H), 1.68 - 1.52 (m, 5H), 1.52 - 1.21 (m, 48H), 1.00 - 0.88 (m, 6H).

[0179] (10)di-tert-butyl (((S)-1-(tert-butoxy)-17-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-1,17-dioxoheptadecan-2-yl)carbamoyl)-L-glutamate(4-j)

[0180] According to a similar procedure to that of Preparation Example 1 (10), compound 4-i (64.7 mg, 0.0635 mmol), dimethylformamide (2 mL), N,N-diisopropylethylamine (45 μL, 0.258 mmol), and bis-4-nitrophenyl carbonate (54.1 mg, 0.178 mmol) were added to a recovery flask and stirred at room temperature for 20 hours. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 1%-3%) as an eluent to give the title compound (74.2 mg, 99%) as a pale yellow solid.

[0181] HRMS m / z calculation for C 60 H 94 N8O 16 Na + [M + Na] + 1205.6680, found 1205.6677.

[0182] 1 H NMR (500 MHz, MeOD) δ 8.34 - 8.27 (m, 2H), 7.67 - 7.61 (m, 2H), 7.50 - 7.39 (m, 4H), 6.38 - 6.31 (m, 2H), 5.26 (s, 2H), 4.61 - 4.58 (m, 2H), 4.55 - 4.46 (m, 1H), 4.22 - 4.07 (m, 4H), 3.25 - 3.00 (m, 3H), 2.35 - 2.23 (m, 4H), 2.14 - 1.96 (m, 3H), 1.95 - 1.67 (m, 5H), 1.67 - 1.19 (m, 48H), 1.01 - 0.94 (m, 6H)

[0183] (11)(((S)-1-carboxy-16-(((S)-1-(((S)-1-((4-((((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]ind olizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopenta n-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-16-oxohexadecyl)carbamoyl)-L-glutamic acid(4)

[0184] Following a procedure similar to that of Preparation Example 1 (11), compound 4-j (74.2 mg, 0.0627 mmol), dimethylformamide (3.0 mL), exatecan mesylate (47.1 mg, 0.0886 mmol), pyridine (170 μL, 2.11 mmol), and hydroxybenzotriazole (16.7 mg, 0.124 mmol) were added to a recovery flask and stirred at room temperature for 60 hours. Water and methanol were added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and ethyl acetate (concentration gradient: 0%-15%) as the eluent. The resulting residue (62.5 mg), trifluoroacetic acid (1 mL), and dichloromethane (1 mL) were added to a recovery flask and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase high performance liquid chromatography to give the title compound 4 (18.1 mg, 22%) as a pale brown powder.

[0185] HRMS m / z calculation for C 66 H 88 FN 10 O 17 + [M + H] +1311.6311, found 1311.6318.

[0186] 1 H NMR (500 MHz, DMSO) δ 9.99 (s, 1H), 8.11 - 8.06 (m, 2H), 7.84 - 7.76 (m, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.32 (s, 1H), 6.56 - 6.51 (m, 2H), 6.00 - 5.97 (m, 1H), 5.47 - 5.42 (m, 4H), 5.32 - 5.28 (m, 3H), 5.08 (s, 2H), 4.41 - 4.36 (m, 1H), 4.23 - 4.16 (m, 1H), 4.13 - 4.01 (m, 2H), 3.30 - 2.88 (m, 6H), 2.46 - 2.34 (m, 4H), 2.34 - 2.03 (m, 6H), 2.03 - 1.78 (m, 3H), 1.78 - 1.31 (m, 9H), 1.31 - 1.15 (m, 20H), 0.92 - 0.81 (m, 9H)

[0187] Production Example 5 Production of Drug Conjugate (Compound 5) (1) 16-bromohexadecanal (5-a)

[0188] According to a similar procedure to that of Preparation Example 2(1), 16-bromo-1-hexadecanol (310.8 mg, 0.967 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl (13.2 mg, 0.0845 mmol), iodobenzene diacetate (362.4 mg, 1.13 mmol), and anhydrous dichloromethane (4 mL) were added to a recovery flask and stirred at room temperature for 3 hours. A mixture of saturated aqueous sodium bicarbonate (4 mL) and saturated aqueous sodium thiosulfate (9 mL) was added to the reaction solution to terminate the reaction, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 1%-100%) as an eluent to obtain the title compound 5-a (249.5 mg, 81%) as a colorless oil.

[0189] 1 H NMR (500 MHz, CDCl3) δ 9.77 (d, J= 1.9 Hz, 1H), 3.41 (t, J = 6.8 Hz, 2H), 2.45 - 2.38 (m, 2H), 1.90 - 1.81(m, 2H), 1.69 - 1.58 (m, 2H), 1.47 - 1.37 (m, 2H), 1.37 - 1.19 (m, 22H).

[0190] (2) 2-(15-bromopentadecyl)-1,3-dioxolane (5-b)

[0191] According to a similar procedure to that of Preparation Example 2(2), compound 5-a (447.4 mg, 1.40 mmol), ethylene glycol (800 μL, 14.4 mmol), paratoluenesulfonic acid monohydrate (30.0 mg, 0.158 mmol), and cyclohexane (15 mL) were added to a recovery flask and stirred at 100°C. After 90 hours, the reaction solution was returned to room temperature, and the reaction was quenched by adding water and saturated aqueous sodium bicarbonate solution, followed by extraction with dichloromethane. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 1%-50%) as an eluent to obtain the title compound 5-b (420.0 mg, 83%) as a white powder.

[0192] 1 H NMR (500 MHz, CDCl3) δ 4.87 - 4.81 (m, 1H), 4.00 - 3.92 (m, 2H), 3.90 - 3.82 (m, 2H), 3.44 - 3.38 (m, 2H), 1.90 - 1.81 (m, 2H), 1.69 - 1.61 (m, 2H), 1.46 - 1.36 (m, 4H), 1.25 (m, 20H).

[0193] (3) 2-(pentadec-14-en-1-yl)-1,3-dioxolane (5-c)

[0194] Following the same procedure as in Production Example 2(3), compound 5-b (87.9 mg, 0.242 mmol), 4A molecular sieves (10.2 mg), sodium iodide (114.8 mg, 0.766 mmol), and acetone (2 mL) were added to a recovery flask and stirred at 80°C for 23 hours. The reaction solution was returned to room temperature, saturated saline was added to terminate the reaction, and the mixture was extracted with diethyl ether. The collected organic layer was dried over anhydrous sodium sulfate, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure. The residue (95.7 mg), potassium tert-butoxide (71.7 mg, 0.639 mmol), and THF (5 mL) were added to a 50 mL recovery flask and stirred at room temperature for 25 hours. Water was added to the reaction solution to terminate the reaction, and the mixture was extracted with ethyl acetate. The collected organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-100%) as an eluent to give the title compound 5-c (41.8 mg, 61%) as a pale brown oil.

[0195] HRMS m / z calculation for C 18 H 35 O9 + [M + H] + 283.2632, found 283.2630.

[0196] 1 H NMR (500 MHz, CDCl3) δ 5.82 (ddt, J= 16.9, 10.1, 6.6 Hz, 1H), 4.99 (dd, J= 17.2, 1.9 Hz, 1H), 4.93 (d, J= 10.1 Hz, 1H), 4.84 (t, J = 4.9 Hz, 1H), 4.02 - 3.91 (m, 2H), 3.90 - 3.82 (m, 2H), 2.08 - 2.00 (m, 2H), 1.69 - 1.61 (m, 2H), 1.46 - 1.32 (m, 2H), 1.29 - 1.16 (m, 20H).

[0197] (4) tert-butyl (S)-2-((benzyloxy)carbonyl)amino)-18-(1,3-dioxolan-2-yl)octadec-4-enoate (5-d)

[0198] According to a similar procedure to that of Preparation Example 2(5), compound 2-d (128.9 mg, 0.422 mmol), toluene (3.5 mL), compound 5-c (569.6 mg, 2.02 mmol), and second-generation HOVEYDA-GRUBBS catalyst (30.4 mg, 0.0485 mmol) were added to a recovery flask and stirred at 50°C for 1 hour. The reaction solution was returned to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-25%) as an eluent to obtain the title compound 5-d (137.8 mg, 58%) as a colorless oil.

[0199] HRMS m / z calculation for C 33 H 53 NO6Na + [M + Na] + 582.3765, found 582.3771.

[0200] 1 H NMR (500 MHz, CDCl3) δ 7.38 - 7.28 (m, 5H), 5.59 - 5.46 (m, 1H), 5.31 - 5.22 (m, 2H), 5.15 - 5.08 (m, 2H), 4.84 (t, J = 4.8 Hz, 1H), 4.31 - 4.24 (m, 1H), 4.00 - 3.91 (m, 2H), 3.90 - 3.80 (m, 2H), 2.53 - 2.39 (m, 2H), 2.01 - 1.89 (m, 2H), 1.69 - 1.61 (m, 2H), 1.49 - 1.37 (m, 13H), 1.37 - 1.19 (m, 18H).

[0201] (5) tert-butyl (S)-2-amino-18-(1,3-dioxolan-2-yl) octadecanoate (5-e)

[0202] According to a similar procedure to that of Preparation Example 2(6), compound 5-d (26.7 mg, 0.0477 mmol), tert-butyl alcohol (2.5 mL), and palladium on carbon (Pd 10%, 4.2 mg, 0.00395 mmol) were added to a recovery flask and stirred under a hydrogen gas atmosphere at room temperature for 5 hours. The reaction solution was filtered through Celite to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 1%-5%) as an eluent to give the title compound 5-e (15.9 mg, 78%) as a colorless oil.

[0203] HRMS m / z calculation for C 25 H 50 No. 4 + [M + H] + 428.3734, found 428.3734.

[0204] 1 H NMR (500 MHz, CDCl3) δ 4.84 (t, J= 4.8 Hz, 1H), 4.00 - 3.91 (m, 2H), 3.90 - 3.80 (m, 2H), 3.31 (dd, J = 7.2, 5.5 Hz, 1H), 1.72 - 1.58 (m, 6H), 1.58 - 1.48 (m, 1H), 1.46 (s, 9H), 1.44 - 1.20 (m, 25H).

[0205] (6) di-tert-butyl (((S)-1-(tert-butoxy)-18-(1,3-dioxolan-2-yl)-1-oxooctadecan-2-yl)carbamoyl)-L-glutamate (5-f)

[0206] According to a similar procedure to that of Production Example 1 (6), di-tert-butyl L-glutamate hydrochloride (47.3 mg, 0.160 mmol) and dichloromethane (1.5 mL) were added to a two-necked eggplant-shaped flask and cooled to -78°C. After that, triphosgene (16.1 mg, 0.0543 mmol) and diisopropylethylamine (115 μL, 0.660 mmol) were added and stirred at -78°C for 1 hour. The reaction solution was warmed to room temperature and stirred for 15 minutes. The reaction solution was cooled again to -78°C, and then a dichloromethane solution (1.5 mL) of compound 5-e (56.2 mg, 0.131 mmol) and diisopropylethylamine (115 μL, 0.660 mmol) were added. The mixture was stirred at -78°C for 2 hours, then warmed to room temperature and stirred for 1 hour. Water was added to the reaction solution to terminate the reaction, and the mixture was extracted with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to give the title compound 5-f (72.7 mg, 78%) as a colorless oil.

[0207] HRMS m / z calculation for C 39 H 72 N2O9Na + [M + Na] + 735.5130, found 735.5134.

[0208] 1 H NMR (500 MHz, CDCl3) δ 4.93 (d, J= 7.8 Hz, 1H), 4.87 (d, J = 8.0 Hz, 1H), 4.84 (t, J = 4.8 Hz, 1H), 4.38 - 4.28 (m, 2H), 3.98 - 3.95 (m, 2H), 2.39 - 2.23 (m, 2H), 2.10 - 2.03 (m, 2H), 1.89 - 1.82 (m, 1H), 1.79 - 1.36 (m, 46H), 1.36 - 1. 19 (m, 14H),

[0209] (7) di-tert-butyl (((S)-1-(tert-butoxy)-1,19-dioxononadecan-2-yl)carbamoyl)-L-glutamate (5-g)

[0210] According to a similar procedure to that of Production Example 2(8), compound 5-f (36.3 mg, 0.0509 mmol), 2 M hydrochloric acid (1 mL), and tert-butyl alcohol (1 mL) were added to a recovery flask and stirred at room temperature for 3.5 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution to terminate the reaction, and then water was added and the mixture was extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 16%-50%) as an eluent to obtain the title compound 5-g (27.7 mg, 81%) as a colorless oil.

[0211] HRMS m / z calculation for C 37 H 69 N2O8 + [M + H] + 669.5048, found 669.5056.

[0212] 1 H NMR (500 MHz, CDCl3) δ 9.77 (t, J= 1.9 Hz, 1H), 5.10 (d, J = 8.0 Hz, 1H), 5.05 (d, J = 8.0 Hz, 1H), 4.39 - 4.28 (m, 2H), 2.46 - 2.39 (m, 2H), 2.12 - 2.02 (m, 2H), 1.91 - 1.69 (m, 2H), 1.69 - 1.54 (m, 4H), 1.53 - 1.09 (m, 53H).

[0213] (8) (S)-19-(tert-butoxy)-18-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-19-oxononadecanoic acid (5-h)

[0214] According to a similar procedure to that of Production Example 1(8), compound 5-g (26.4 mg, 0.0395 mmol), tert-butyl alcohol (1.5 mL), water (0.3 mL), 2-methyl-2-butene (22 μL, 0.207 mmol), sodium hypochlorite (16.6 mg, 0.145 mmol), and sodium dihydrogen phosphate dihydrate (9.2 mg, 0.0590 mmol) were added to a recovery flask and stirred at room temperature for 2 hours. The reaction solution was then concentrated under reduced pressure. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to obtain the title compound 5-h (20.4 mg, 75%) as a white powder.

[0215] HRMS m / z calculation for C 37 H 68 N2O9Na + [M + Na] + 707.4817, found 707.4816.

[0216] 1 H NMR (500 MHz, CDCl3) δ 5.36 - 5.28 (m, 2H), 4.40 - 4.24 (m, 2H), 2.39 - 2.24 (m, 4H), 2.12 - 2.00 (m, 1H), 1.94 - 1.78 (m, 1H), 1.78 - 1.69 (m, 1H), 1.69 - 1.55 (m, 3H), 1.55 - 1.39 (m, 27H), 1.39 - 1.12 (m, 26H).

[0217] (9) di-tert-butyl (((S)-1-(tert-butoxy)-19-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidop entan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-1,19-dioxononadecan-2-yl)carbamoyl)-L-glutamate (5-i)

[0218] Following a procedure similar to that of Preparation Example 1(9), compound 5-h (39.7 mg, 0.0580 mmol), dimethylformamide (2 mL), diisopropylethylamine (50 μL, 0.287 mmol), and HATU (27.6 mg, 0.0726 mmol) were added to a recovery flask and stirred at room temperature. After 30 minutes, Val-Cit-PAB-OH (32.2 mg, 0.0849 mmol) was added, and the mixture was stirred at room temperature for 20 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution. Water and methanol were added, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 5%-12%) as an eluent to obtain the title compound 5-i (45.1 mg, 74%) as a white solid.

[0219] HRMS m / z calculation for C 55 H 95 N7O 12 Na + [M + Na] + 1068.6931, found 1068.6925.

[0220] 1H NMR (500 MHz, MeOD) δ 8.03 - 7.96 (m, 1H), 7.59 - 7.52 (m, 2H), 7.35 - 7.27 (m, 2H), 6.40 - 6.30 (m, 1H), 4.55 (s, 2H), 4.53 - 4.48 (m, 1H), 4.23 - 4.09 (m, 3H), 3.27 - 3.16 (m, 1H), 3.15 - 3.04 (m, 1H), 2.37 - 2.23 (m, 4H), 2.13 - 1.98 (m, 2H), 1.95 - 1.85 (m, 1H), 1.85 - 1.68 (m, 3H), 1.68 - 1.52 (m, 6H), 1.52 - 1.19 (m, 52H), 1.02 - 0.92 (m, 6H).

[0221] (10) di-tert-butyl (((S)-1-(tert-butoxy)-19-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)- 1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-1,19-dioxononadecan-2-yl)carbamoyl)-L-glutamate (5-j)

[0222] According to a similar procedure to that of Preparation Example 1 (10), compound 5-i (45.1 mg, 0.0431 mmol), dimethylformamide (2 mL), N,N-diisopropylethylamine (30 μL, 0.172 mmol), and bis-4-nitrophenyl carbonate (40.2 mg, 0.132 mmol) were added to a recovery flask and stirred at room temperature for 17 hours. Water was added to the reaction solution to quench the reaction, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 1%-3%) as an eluent to give the title compound 5-j (34.7 mg, 66%) as a pale yellow solid.

[0223] HRMS m / z calcd. for C 62 H 98 N8O 16 Na + [M + Na] + 1233.6993, found 1233.6985.

[0224] 1 H NMR (500 MHz, MeOD) δ 8.34 - 8.27 (m, 2H), 7.68 - 7.61 (m, 2H), 7.49 - 7.43 (m, 2H), 7.43 - 7.38 (m, 2H), 6.39 - 6.32 (m, 1H), 5.26 (s, 2H), 4.65 - 4.56 (m, 1H), 4.56 - 4.47 (m, 1H), 4.24 - 4.04 (m, 4H), 3.26 - 3.15 (m, 1H), 3.15 - 3.06 (m, 1H), 2.39 - 2.22 (m, 4H), 2.14 - 1.97 (m, 2H), 1.94 - 1.68 (m, 2H), 1.67- 1.53 (m, 6H), 1.51- 1.40 (m, 27H), 1.40- 1.20 (m, 25H), 1.03- 0.93 (m, 6H).

[0225] (11)(((S)-1-carboxy-18-(((S)-1-(((S)-1-((4-(((((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)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-18-oxooctadecyl)carbamoyl)-L-glutamic acid(5)

[0226] Following a procedure similar to that of Preparation Example 1 (11), compound 5-j (34.7 mg, 0.0286 mmol), dimethylformamide (3.5 mL), exatecan mesylate (23.3 mg, 0.0438 mmol), pyridine (80 μL, 0.994 mmol), and hydroxybenzotriazole (9.4 mg, 0.0696 mmol) were added to a recovery flask and stirred at room temperature for 60 hours. Water and methanol were added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and ethyl acetate (concentration gradient: 0%-15%) as the eluent. The resulting residue (31.9 mg), trifluoroacetic acid (1 mL), and dichloromethane (1 mL) were added to a recovery flask and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase high performance liquid chromatography to give the title compound 5 (7.1 mg, 18%) as a gray powder.

[0227] HRMS m / z calculation for C 68 H 92 FN 10 O 17 + [M + H] + 1339.6621, found 1339.6630.

[0228] 11H NMR (500 MHz, DMSO) δ 9.90 (s, 1H), 8.05 (d, J = 7.4 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.3 Hz, 2H), 6.31 (d, J = 8.3 Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 5.99 - 5.95 (m, 1H), 5.41 (s, 2H), 5.12 - 5.06 (m, 1H), 4.40 - 4.35 (m, 2H), 4.22 - 4.15 (m, 2H), 4.13 - 4.00 (m, 2H), 3.25 - 2.84 (m, 2H), 2.50 - 2.31 (m, 2H), 2.28 - 2.09 (m, 4H), 2.02 - 1.80 (m, 1H), 1.74 - 1.11 (m, 49H), 0.91 - 0.76 (m, 9H).

[0229] Production Example 6 Production of Drug Conjugate (Compound 6) (1) tert-butyl ((2-(((2R)-1-((2-(((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-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (6-a)

[0230] Boc-Gly-Gly-Phe-Gly-OH (21.5 mg, 0.04926 mmol), N-hydroxysuccinimide (8.5 mg, 0.07386 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (15.0 mg, 0.07825 mmol), and dichloromethane (3 mL) were added to a recovery flask and stirred at room temperature. After 3 hours, exatecan mesylate (18.0 mg, 0.03386 mmol), dimethylformamide (1.0 mL), and triethylamine (12 μL, 0.08586 mmol) were added, and the mixture was stirred at room temperature for 26 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 0%-10%) as an eluent to give the title compound 6-a (31.1 mg, 74%) as a white solid.

[0231] HRMS m / z calculation for C 44 H 48 FN7O 10 Na + [M + Na] + 876.3339, found 876.3340

[0232] 1H-NMR (500 MHz, DMSO-d6) δ 8.44 (d, J = 8.8 Hz, 1H), 8.37 - 8.33 (m, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.92 - 7.86 (m, 1H), 7.82 (d, J = 10.9 Hz, 1H), 7.32 (s, 1H), 7.27 - 7.15 (m, 5H), 7.02 - 6.99 (m, 1H), 6.55 (s, 1H), 5.61 - 5.57 (m, 1H), 5.45 - 5.40 (m, 2H), 5.29 - 5.24 (m, 2H), 4.46 - 4.43 (m, 1H), 3.76 - 3.68 (m, 2H), 3.60 - 3.50 (m, 2H), 3.21 - 3.17 (m, 2H), 3.02 - 2.95 (m, 1H), 2.80 - 2.74 (m, 1H), 2.42 (s, 3H), 2.22 - 2.18 (m, 2H), 2.17 - 2.11 (m, 1H), 1.90 - 1.82 (m, 2H), 1.41 - 1.36 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H)

[0233] (2)(2R)-2-(2-(2-aminoacetamide)acetamide)-N-(2-(((9S)-9-et hyl-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-oxoethyl)-3-phenylpropanamide (6-b)

[0234] Compound 6-a (31.1 mg, 0.03642 mmol), trifluoroacetic acid (3 mL), and dichloromethane (3 mL) were added to a recovery flask and stirred in an ice bath. After 3 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was washed with dichloromethane to give the title compound 6-b (34.3 mg, containing a small amount of impurity) as a yellow powder.

[0235] HRMS m / z calcd. for C 39 H 40 FN7O8Na + [M + Na] + 776.2814, found 776.2819

[0236] 1 H-NMR (500 MHz, DMSO-d6) δ 8.54 (d, J = 8.6 Hz, 1H), 8.48 (t, J = 5.6 Hz, 1H), 8.43 (t, J = 5.6 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.01 - 7.89(m, 2H), 7.83 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 7.29 - 7.14 (m, 5H), 6.57 (s, 1H), 5.59 (dt, J = 8.8, 4.5 Hz, 1H), 5.49 - 5.37 (m, 2H), 5.26 (d, J = 6.3 Hz, 2H), 4.59 - 4.51 (m, 1H), 3.92 - 3.84 (m, 2H), 3.80 - 3.68 (m, 3H), 3.60 - 3.55 (m, 2H), 3.20 (s, 2H), 3.00 (dd, J = 13.8, 4.7 Hz, 1H), 2.75 (dd, J = 13.8, 9.5 Hz, 1H), 2.21 - 2.18 (m, 1H), 2.17 - 2.11 (m, 1H), 1.93 - 1.80 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H)

[0237] (3)(((1S)-1-carboxy-16-((2-((2-(((2R)-1-((2-(((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]in dolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl) amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-16-oxohexadecyl)carbamoyl)-L-glutamic acid(6)

[0238] Compound 6-b (11.6 mg, 0.01539 mmol), dimethylformamide (2.5 mL), diisopropylethylamine (50 μL, 0.2871 mmol), (S)-17-(tert-butoxy)-16-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-17-oxoheptadecanoic acid (compound 4-h) (19.1 mg, 0.02908 mmol), and HATU (20.6 mg, 0.05418 mmol) were placed in a recovery flask and stirred at 0°C for 3 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 0%-12%) as an eluent. The resulting residue (20.3 mg), trifluoroacetic acid (2 mL), and dichloromethane (2 mL) were added to a 10 mL recovery flask and stirred at room temperature. After 1 hour, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by reverse-phase high-performance liquid chromatography to give the title compound 6 (3.0 mg, 17%) as a pale yellow powder.

[0239] HRMS m / z calculation for C 62 H 78 FN9O 16 Na + [M + Na] +1246.5443, found 1246.5455

[0240] 1 H-NMR (500 MHz, DMSO-d6) δ 12.66 - 12.40 (m, 2H), 12.30 - 11.96 (m, 1H), 8.42 (d, J = 8.8 Hz, 1H), 8.33 (s, 1H), 8.11 - 8.06 (m, 1H), 8.03 - 7.99 (m, 1H), 7.82 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 7.27 - 7.13 (m, 5H), 6.54 (s, 1H), 6.32 - 6.28 (m, 2H), 5.61 - 5.56 (m, 1H), 5.47 - 5.37 (m, 2H), 5.31 - 5.21 (m, 2H), 4.45 - 4.40 (m, 1H), 4.13 - 4.01 (m, 2H), 3.80 - 3.67 (m, 3H), 3.67 - 3.61 (m, 3H), 3.54 (dd, J = 16.7, 5.7 Hz, 1H), 3.19 (s, 2H), 2.99 (dd, J = 13.7, 5.0 Hz, 1H), 2.82 (s, 1H), 2.67 (t, J = 7.2 Hz, 1H), 2.26 - 2.18 (m, 3H), 2.16 - 2.09 (m, 2H), 1.91 - 1.83 (m, 2H), 1.77 - 1.69 (m, 1H), 1.66 - 1.57 (m, 1H), 1.46(s, 3H), 1.40 (s, 2H), 1.29 - 1.19 (m, 24H), 0.88 (t, J = 7.3 Hz, 3H)

[0241] Production Example 7 Production of Drug Conjugate (Compound 7) (1) 18-bromooctadecan-1-ol (7-a)

[0242] 1,18-Octanediol (1.24 g, 4.33 mmol), 47% hydrobromic acid (0.85 mL, 6.92 mmol), and cyclohexane (30 mL) were added to a recovery flask and stirred at 110°C for 14.5 hours. Water was added to the reaction solution to terminate the reaction, followed by extraction with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, and the insoluble material was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 30%-100%) as the eluent to obtain the title compound 7-a (943 mg, 63%) as a white powder.

[0243] 1H NMR (500 MHz, CDCl3) δ3.64 (t, J = 6.9 Hz, 2H), 3.41 (t, J = 6.9 Hz, 2H), 1.90 - 1.81 (m, 2H), 1.61 - 1.56 (m, 2H), 1.45 - 1.37 (m, 2H), 1.38 - 1.17 (m, 28H)

[0244] (2) 2-(17-bromoheptadecyl)-1,3-dioxolane (7-b)

[0245] According to the same procedures as in Production Example 2(1) and Production Example 2(2), compound 7-a (943 mg, 2.70 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl (42.0 mg, 0.269 mmol), iodobenzene diacetate (948 mg, 2.94 mmol), and anhydrous dichloromethane (30 mL) were added to a recovery flask and stirred at room temperature for 3 hours. A mixture of saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate was added to the reaction solution to terminate the reaction, followed by extraction with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, and insoluble matter was filtered off. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using dichloromethane and hexane (concentration gradient: 1%-100%) as the eluent. The residue (1.32 g), ethylene glycol (1.5 mL, 26.9 mmol), paratoluenesulfonic acid monohydrate (51.2 mg, 0.269 mmol), and cyclohexane (30 mL) were added to a 50 mL eggplant-shaped flask and stirred at 100°C. After 17 hours, the reaction solution was returned to room temperature, and water and saturated aqueous sodium bicarbonate were added to terminate the reaction, followed by extraction with ethyl acetate. The combined organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 1%-50%) as an eluent to give the title compound 7-b (789 mg, 75%) as a white powder.

[0246] 1 H NMR (500 MHz, CDCl3) δ 4.84 (t, J = 4.9 Hz, 1H), 4.03 - 3.91 (m, 2H), 3.90 - 3.79 (m, 2H), 3.41 (t, J = 6.9 Hz, 2H), 1.91 - 1.80 (m, 2H), 1.69 - 1.61 (m, 2H), 1.47 - 1.37 (m, 4H), 1.36 - 1.17 (m, 24H).

[0247] (3) 2-(heptadec-16-en-1-yl)-1,3-dioxolane (7-c)

[0248] In a similar manner to that of Production Example 2(3), compound 7-b (789 mg, 2.02 mmol), sodium iodide (1.06 g, 7.05 mmol), and acetone (25 mL) were added to a recovery flask and stirred at 70°C for 15 hours. The reaction solution was returned to room temperature, saturated saline was added to terminate the reaction, and the mixture was extracted with diethyl ether. The collected organic layer was dried over anhydrous sodium sulfate, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure. The residue (817 mg), potassium tert-butoxide (520 mg, 4.65 mmol), and THF (25 mL) were added to a 50 mL recovery flask and stirred at room temperature for 19 hours. Water was added to the reaction solution to terminate the reaction, and the mixture was extracted with ethyl acetate. The collected organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using dichloromethane and hexane (concentration gradient: 10%-100%) as an eluent to give the title compound 7-c (537 mg, 93%) as a colorless oil.

[0249] HRMS m / z calculation for C 20 H 39 BrO + [M + H] + 311.2945, found 311.2943.

[0250] 1 H NMR (500 MHz, CDCl3) δ 5.88 - 5.74 (m, 1H), 5.02 - 4.96 (m, 1H), 4.96 - 4.90 (m, 1H), 4.84 (t, J = 4.9 Hz, 1H), 4.03 - 3.91 (m, 2H), 3.89 - 3.76 (m, 2H), 2.10 - 1.99 (m, 2H), 1.69 - 1.62 (m, 2H), 1.48 - 1.37 (m, 2H), 1.35 - 1.18 (m, 24H).

[0251] (4) tert-butyl (S,E)-2-((benzyloxy)carbonyl)amino)-20-(1,3-dioxolan-2-yl)icos-4-enoate (7-d)

[0252] In a similar manner to that of Preparation Example 2(5), compound 2-d (391.0 mg, 1.28 mmol), toluene (10 mL), compound 7-c (516.0 mg, 1.66 mmol), and second-generation HOVEYDA-GRUBBS catalyst (85.4 mg, 0.136 mmol) were added to a recovery flask and stirred at 50°C for 15 hours. The reaction solution was returned to room temperature and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 10%-20%) as an eluent to obtain the title compound 7-d (295.4 mg, 39%) as a colorless oil.

[0253] HRMS m / z calculation for C 35 H 57 NO6Na + [M + Na] + 610.4078, found 610.4077.

[0254] 1 H NMR (500 MHz, CDCl3) δ 7.41 - 7.29 (m, 6H), 5.56 - 5.46 (m, 1H), 5.32 - 5.22 (m, 1H), 5.17 - 5.08 (m, 2H), 4.84 (t, J = 4.9 Hz, 1H), 4.32 - 4.23 (m, 1H), 4.00 - 3.92 (m, 2H), 3.90 - 3.80 (m, 2H), 2.53 - 2.37 (m, 2H), 2.08 - 1.91 (m, 2H), 1.69 - 1.61 (m, 2H), 1.46 (s, 9H), 1.43 - 1.37 (m, 6H), 1.33 - 1.15 (m, 20H).

[0255] (5) tert-butyl(S)-2-amino-20-(1,3-dioxolan-2-yl)icosanoate (7-e)

[0256] According to a similar procedure to that of Preparation Example 2(6), compound 7-d (605 mg, 1.03 mmol), tert-butyl alcohol (10 mL), and palladium on carbon (Pd 10%, 235 mg, 0.209 mmol) were added to a recovery flask and stirred under a hydrogen gas atmosphere at 30°C for 1 hour. The reaction solution was filtered through Celite to remove insoluble matter, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 1%-5%) as an eluent to give the title compound 7-e (344 mg, 73%) as a colorless oil.

[0257] HRMS m / z calculation for C 37 H 54 No. 4 + [M + H] + 456.4048, found 456.4043.

[0258] 1 H NMR (500 MHz, CDCl3) δ 4.84 (t, J = 4.8 Hz, 1H), 4.01 - 3.91 (m, 2H), 3.90 - 3.81 (m, 2H), 3.33 - 3.26 (m, 1H), 1.68 - 1.63 (m, 2H), 1.58 - 1.48 (m,2H), 1.46 (s, 9H), 1.43 - 1.19 (m, 32H).

[0259] (6) di-tert-butyl (((S)-1-(tert-butoxy)-20-(1,3-dioxolan-2-yl)-1-oxoicosan-2-yl)carbamoyl)-L-glutamate (7-f)

[0260] According to a similar procedure to that of Production Example 1 (6), di-tert-butyl L-glutamate hydrochloride (246 mg, 0.830 mmol) and dichloromethane (18 mL) were added to a two-necked eggplant-shaped flask and cooled to -78°C. After that, triphosgene (112 mg, 0.378 mmol) and diisopropylethylamine (0.65 mL, 3.73 mmol) were added and stirred at -78°C for 1 hour. The reaction solution was warmed to room temperature and stirred for 15 minutes. The reaction solution was again cooled to -78°C, and then a dichloromethane solution (3 mL) of 7-e (344 mg, 0.755 mmol) and diisopropylethylamine (0.65 mL, 3.73 mmol) were added and stirred at -78°C for 2 hours, after which the temperature was warmed to room temperature and stirred for 1 hour. Water was added to the reaction solution to terminate the reaction, and the solution was extracted with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove insoluble material, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to give the title compound 7-f (411 mg, 74%) as a yellow oil.

[0261] HRMS m / z calculation for C 41 H 77 N2O9 + [M + H] + 741.5625, found 741.5623.

[0262] 1 H NMR (500 MHz, CDCl3) δ 5.08 (d, J = 8.0 Hz, 1H), 5.03 (d, J = 8.0 Hz, 1H), 4.84 (t, J = 4.8 Hz, 1H), 4.41 - 4.27 (m, 2H), 4.01 - 3.91 (m, 2H), 3.91 - 3.80 (m, 2H), 2.44 - 2.22 (m, 2H), 2.13 - 2.00 (m, 2H), 1.92 - 1.80 (m, 1H), 1.81 - 1.68 (m, 1H), 1.70 - 1.56 (m, 4H), 1.55 - 1.37 (m, 27H), 1.36 - 1.16 (m, 30H).

[0263] (7) di-tert-butyl (((S)-1-(tert-butoxy)-1,21-dioxohenicosan-2-yl)carbamoyl)-L-glutamate (7-g)

[0264] According to a similar procedure to that of Production Example 2(8), compound 7-f (370 mg, 0.499 mmol), 2 M hydrochloric acid (5 mL), tert-butyl alcohol (4.5 mL), and THF (0.5 mL) were added to a recovery flask and stirred at room temperature for 24 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution to terminate the reaction, and then water was added and the mixture was extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate, and the insoluble matter was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 16%-50%) as an eluent to obtain the title compound 7-g (333 mg, 96%) as a colorless oil.

[0265] HRMS m / z calculation for C 39 H 73 N2O8 + [M + H] + 697.5362, found 697.5361.

[0266] 1 H NMR (500 MHz, CDCl3) δ 9.77 (t, J = 1.9 Hz, 1H), 5.01 (d, J = 7.9 Hz, 1H), 4.96 (d, J = 8.0 Hz, 1H), 4.40 - 4.28 (m, 2H), 2.46 - 2.39 (m, 2H), 2.39 - 2.24 (m, 2H), 2.12 - 1.99 (m, 1H), 1.93 - 1.79 (m, 1H), 1.81 - 1.71 (m, 2H), 1.69 - 1.56 (m, 4H), 1.54 - 1.38 (m, 27H), 1.38 - 1.18 (m, 28H).

[0267] (8) (S)-21-(tert-butoxy)-20-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-21-oxohenicosanoic acid (7-h)

[0268] According to a similar procedure to that of Production Example 1 (8), compound 7-g (326 mg, 0.468 mmol), tert-butyl alcohol (5.5 mL), water (1.1 mL), 2-methyl-2-butene (250 μL, 2.34 mmol), sodium hypochlorite (185 mg, 1.64 mmol), and sodium dihydrogen phosphate dihydrate (109 mg, 0.703 mmol) were added to a recovery flask and stirred at room temperature for 17.5 hours. The reaction solution was then concentrated under reduced pressure. Water was added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using ethyl acetate and hexane (concentration gradient: 25%-50%) as an eluent to obtain the title compound 7-h (245 mg, 74%) as a white powder.

[0269] HRMS m / z calculation for C 39 H 73 N2O9 + [M + H] + 713.511, found 713.5312.

[0270] 1H NMR (500 MHz, CDCl3) δ 5.35 - 5.19 (m, 2H), 4.39 - 4.22 (m, 2H), 2.45 - 2.19 (m, 4H), 2.17 - 2.00 (m, 1H), 1.95 - 1.81 (m, 1H), 1.81 - 1.68 (m, 1H), 1.70 - 1.56 (m, 3H), 1.52 - 1.38 (m, 27H), 1.39 - 1.18 (m, 30H).

[0271] (9) di-tert-butyl (((S)-1-(tert-butoxy)-21-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan- L-glutamate (7-i)

[0272] Following a procedure similar to that of Preparation Example 1(9), compound 7-h (65.6 mg, 0.0897 mmol), dimethylformamide (2.5 mL), diisopropylethylamine (75 μL, 0.449 mmol), and HATU (42.6 mg, 0.112 mmol) were added to a recovery flask and stirred at room temperature. After 30 minutes, Val-Cit-PAB-OH (49.4 mg, 0.130 mmol) was added, and the mixture was stirred at room temperature for 17 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution. Water and methanol were added, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 5%-12%) as the eluent to yield the title compound 7-i (72.1 mg, 75%) as a white solid.

[0273] 1H NMR (500 MHz, MeOD) δ 7.48 - 7.42 (m, 2H), 7.24 - 7.15 (m, 2H), 6.41 - 6.15 (m, 1H), 4.45 (s, 2H), 4.43 - 4.38 (m, 1H), 4.13 - 4.06 (m, 2H), 4.05 - 3.98 (m, 1H), 3.15 - 3.05 (m, 1H), 3.04 - 2.93 (m, 1H), 2.27 - 2.12 (m, 4H), 2.03 - 1.88 (m, 2H), 1.83 - 1.75 (m, 1H), 1.75 - 1.57 (m, 2H), 1.59 - 1.41 (m, 4H), 1.41 - 1.32 (m, 27H), 1.30 - 1.12 (m, 34H), 0.91 - 0.82 (m, 6H).

[0274] (10) di-tert-butyl (((S)-1-(tert-butoxy)-21-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)- 1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-1,21-dioxohenicosan-2-yl)carbamoyl)-L-glutamate (7-j)

[0275] According to a similar procedure to that of Preparation Example 1 (10), compound 7-i (50.4 mg, 0.0469 mmol), dimethylformamide (2 mL), N,N-diisopropylethylamine (33 μL, 0.187 mmol), and bis-4-nitrophenyl carbonate (39.9 mg, 0.131 mmol) were added to a recovery flask and stirred at room temperature for 16 hours. Water was added to the reaction solution to quench the reaction, followed by extraction with ethyl acetate. The combined organic layer was washed with water and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and dichloromethane (concentration gradient: 1%-3%) as an eluent to give the title compound 7-j (53.3 mg, 92%) as a pale yellow solid.

[0276] 1 H NMR (500 MHz, MeOD) δ 8.31 - 8.23 (m, 2H), 7.65 - 7.59 (m, 2H), 7.46 - 7.41 (m, 2H), 7.40 - 7.35 (m, 2H), 6.35 - 6.28 (m, 1H), 5.22 (s, 2H), 4.51 - 4.45 (m, 1H), 4.18 - 4.11 (m, 2H), 4.12 - 4.07 (m, 1H), 3.62 - 3.59 (m, 1H), 3.25 - 3.21 (m, 1H), 3.10 - 2.99 (m, 1H), 2.35 - 2.21 (m, 4H), 2.08 - 1.93 (m, 2H), 1.92 - 1.82 (m, 1H), 1.82 - 1.64 (m, 2H), 1.63 - 1.51 (m, 5H), 1.46 - 1.37 (m, 27H), 1.37 - 1.21 (m, 34H), 0.98 - 0.90 (m, 6H).

[0277] (11)(((S)-1-carboxy-20-(((S)-1-(((S)-1-((4-(((((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)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-20-oxoicosyl)carbamoyl)-L-glutamic acid(7)

[0278] Following a procedure similar to that of Preparation Example 1 (11), compound 7-j (48.4 mg, 0.0391 mmol), dimethylformamide (3 mL), exatecan mesylate (26.1 mg, 0.0491 mmol), pyridine (110 μL, 1.37 mmol), and hydroxybenzotriazole (12.6 mg, 0.0932 mmol) were added to a recovery flask and stirred at room temperature for 68.5 hours. Water and methanol were added to the reaction solution, followed by extraction with ethyl acetate. The combined organic layer was washed with water and saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methanol and ethyl acetate (concentration gradient: 0%-15%) as the eluent. The resulting residue (40.9 mg), trifluoroacetic acid (1 mL), and dichloromethane (2 mL) were added to a 10 mL recovery flask and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by reversed-phase high performance liquid chromatography to give the title compound 7 (12.0 mg, 33%) as a brown powder.

[0279] 1 H NMR (500 MHz, DMSO) δ 10.01 (s, 1H), 8.16 - 8.02 (m, 2H), 7.85 - 7.75 (m, 2H), 7.65 - 7.58 (m, 2H), 7.44 - 7.34 (m, 2H), 7.31 (s, 1H), 6.56 - 6.49 (m, 2H), 6.01 - 5.96 (m, 1H), 5.51 - 5.34 (m, 4H), 5.32 - 5.21 (m, 2H), 5.07 (s, 1H), 4.47 - 4.30 (m, 1H), 4.24 - 4.12 (m, 2H), 4.12 - 3.96 (m, 2H), 3.19 - 2.83 (m, 6H), 2.40 - 2.33 (m, 4H), 2.28 - 2.09 (m, 6H), 2.01 - 1.79 (m, 3H), 1.78 - 1.31 (m, 9H), 1.28 - 1.31 (m, 25H), 0.91 - 0.80 (m, 12H).

[0280] Test Example 1 PSMA inhibitory activity test and cytotoxicity test against prostate cancer cell lines 1. Methods (1) PSMA inhibitory activity test (IC 50 Value) Cultured LNCaP cells 1.0 x 10 7 The cells were washed three times with 1x PBS. Then, the cells were homogenized using 100 μL of lysis buffer (RIPA buffer, 1x Proteinase Inhibition Cocktail) and placed on ice for 20 minutes. The cells were centrifuged at 12,000 rpm at 4°C for 20 minutes to precipitate cell debris. The supernatant was collected and diluted with 1.0x10 LNCaP cell extract. 5 This was further diluted with deionized distilled water to give a 1.0 x 10 4 / μL of LNCaP cell extract was prepared and used in the test.

[0281] 25 μL of LNCaP cell extract, 12.5 μL of 8 μmol / L N-acetyl-aspartyl-glutamate (NAAG), and 25 μL of compounds 1, 2, 4, 5, and 6 (0.2-1000 nmol / L) prepared in Preparation Examples 1, 2, 4, 5, and 6 were mixed and incubated for 2 hours at 37°C. After incubation, 50 μL of working solution from the Amplex Red Glutamic Acid / Glutamate Oxidase Assay Kit (Thermo Fisher) was added, and the mixture was further incubated in the dark at 37°C for 60 minutes, after which the fluorescence intensity was measured.

[0282] The fluorescence intensity was measured using a multifunctional microplate reader (Varioskan Flash (Thermo Fisher), excitation wavelength 555 nm, measurement fluorescence wavelength 580 nm). Based on the obtained fluorescence intensity values ​​at each concentration, IC was calculated using GraphPad Prism 10 (GraphPad Software). 50 The values ​​were calculated. The results are shown in Table 1. Compound 4 was evaluated by repeating the same test twice.

[0283] (2) Cytotoxicity Test on Prostate Cancer Cell Lines 7500 LNCaP (PSMA-positive cell line) cells and 2500 PC3 (PSMA-negative cell line) cells were dissolved in 135 μL of RPMI medium and dispersed into each well of a 96-well plate. Then, the cells were cultured for 16 hours in a 5% CO2 incubator. 15 μL of Compounds 1, 2, 4, 5, and 6 (0.1-100,000 nmol / L) prepared in Preparation Examples 1, 2, 4, 5, and 6 was added to each well of the cultured cell line to make a total of 150 μL (final DMSO concentration in the medium was 1% or less), and the cells were cultured for 72 hours. After discarding the compound and medium from each well, 10 μL of WST-1 reagent (Takara Bio) + 100 μL of RPMI medium was added to each well and cultured at 37°C for 2 hours. The absorbance of each well was measured using a microplate reader (iMark microplate reader (BioRad), absorbance wavelength: 580 nm). Based on the obtained absorbance values ​​at each concentration, IC was calculated using GraphPad Prism 10 (GraphPad Software). 50 The values ​​were calculated. The results are also shown in Table 1. Compound 4 was evaluated by conducting the test on LNCaP cells twice.

[0284] 2. Results (1) As a result of the PSMA inhibitory activity test, it was found that the PSMA binding ability was affected by the length of the hydrocarbon chain (n = 7, 9, 11, 13). For example, Compound 1 (n = 7: IC 50 On the other hand, compound 2 (n = 9: IC 50 = 179.6 nM), Compound 4 (n = 11: IC 50 = 163.9 nM, 21.9 nM), compound 5 (n = 13: IC 50 The PSMA inhibitory activity of Compound 2 (n = 9) and Compound 4 (n = 11) was stronger than that of Compound 5 (n = 13), suggesting that the hydrocarbon chain length was appropriate. Furthermore, Compound 2 (n = 9) and Compound 4 (n = 11) had stronger PSMA inhibitory activity than Compound 5 (n = 13). Therefore, among PSMA-targeted small molecule drug conjugates with long hydrocarbon chains, the hydrocarbon chain lengths of Compound 2 (n = 9) and Compound 4 (n = 11) were found to be optimal for binding to PSMA.

[0285] (2) Cytotoxicity tests showed that compounds 2, 4, and 5 exhibited toxicity equivalent to or greater than that of exatecan against PSMA-expressing prostate cancer cells (LNCaP). The tendency for cytotoxicity against PSMA-expressing prostate cancer cells (LNCaP) was proportional to the PSMA binding ability, and it was found that the hydrocarbon chain length of compounds 2 and 4 (n = 9, 11) was also optimal for cytotoxicity against PSMA-expressing prostate cancer cells (LNCaP). On the other hand, against PSMA-non-expressing prostate cells (PC3), compounds 2, 4, and 5 exhibited cytotoxicity several tens of times weaker than exatecan. This suggests that these compounds bind to PSMA, are transported into cells by endocytosis, and then do not exhibit toxicity unless the peptide linker is cleaved by the intracellular enzyme cathepsin B. It is expected that their toxicity against normal cells will be lower than that of anticancer drugs such as exatecan administered alone. Compounds 2, 4, and 5 all have excellent selective toxicity against PSMA-expressing prostate cancer cells (LNCaP) over PSMA-non-expressing prostate cells (PC3), but compound 4 in particular is thought to be useful as a PSMA-targeted small molecule drug conjugate that selectively kills PSMA-expressing prostate cancer cells.

[0286] (3) Compound 6 exhibited the highest cytotoxicity against LNCaP, but its PSMA inhibitory activity was low. The reason for this is thought to be that while the Gly-Gly-Phe-Gly linker of compound 6 is not optimal for PSMA binding, it is susceptible to cleavage by intracellular enzymes, resulting in gradual intracellular translocation over the 72-hour exposure period. After translocation, compound 4 released the anticancer drug into the cells more quickly than the Val-Cit-PABC linker, resulting in high cytotoxicity. To support this hypothesis, a cathepsin B cleavage test was performed on compound 4 as shown below, and its half-life was determined to be 79 hours, as described above, demonstrating sustained release (Figure 1). Previously reported PSMA ligand small molecule drug conjugates (SMDCs) with cathepsin B cleavable linkers (e.g., Patent Document 1) have reported half-lives of less than 2 hours in cathepsin B cleavage tests, so a sustained release of 79 hours can be considered. The reason for this is thought to be that the combination of the Val-Cit-PABC linker with the long hydrocarbon chain, a characteristic of the product of the present invention, results in sustained cathepsin B release. It is generally known that PSMA is expressed in tissues other than prostate cancer, such as the kidneys and salivary glands, and it has been reported that PSMA ligands also localize to these excretory organs but are rapidly eliminated within a few hours (Yang, X., et al., Journal of Medicinal Chemistry 2016, 59, 206-218). Meanwhile, the rate of elimination of PSMA ligands from prostate cancer is much slower than that from these excretory organs. Therefore, even if Compound 4, which has sustained cathepsin B release properties, localizes to excretory organs where PSMA is expressed, such as the kidneys and salivary glands, the release of the antitumor drug is very slow, and the rate of elimination from these excretory organs is faster, which is expected to reduce toxicity to the kidneys, salivary glands, etc. To confirm the sustained release of cathepsin B from Compound 4, a cytotoxicity test was performed on PSMA-expressing prostate cancer cells (LNCaP) by extending the exposure time from 72 hours to 144 hours. As a result, the cytotoxicity was improved (IC 50 = 51.8 nM), suggesting that Compound 4 is localized in prostate cancer cells for a long period of time and gradually releases the antitumor drug.

[0287]

[0288] <Cathepsin B Cleavage Test> The cathepsin B cleavage test method of Patent Document 1 was partially modified to evaluate the cleavage ability of Compound 4 by cathepsin B. 6.6 μL of Cathepsin B, Human Recombinant (R&D Systems, Inc.) (containing 3.48 mg of Cathepsin B, final concentration: 60 nM) was added to 1.84 mL of buffer solution (25 mM acetate, 1 mM EDTA, pH 5), and the mixture was preheated at 37°C for 5 minutes. 160 μL of a 1 mM solution of Compound 4 was added (final concentration of Compound 4: 80 mM). After 0, 0.5, 1, 2, 4, 6, 24, 48, 120, and 168 hours, 200 mL aliquots of the solution were taken and added to a container containing 20 μL of thioprotease inhibitor E-64 solution (approximately 30 nM: final concentration). The peak areas of each aliquot were then analyzed by HPLC, and the half-life was calculated. The half-life of compound 4 due to cathepsin B was found to be approximately 79 hours (Figure 1).

[0289] Test Example 2 Antitumor activity test using prostate cancer model mice 1. Method LNCap 5.0 x 10 6 The LNCap solution was subcutaneously transplanted into the flank of a BALB / c nu / nu mouse. Six weeks after transplantation, the estimated size of the transplanted tumor was 50 mm. 3 from 150 mm 3 Drug administration was initiated when the tumor size reached 100 mm. The estimated tumor size was measured using a digital caliper (longest diameter 100 mm). 2× minor axis) / 2. A solution of Compound 4 in 10% Tween-80 in PBS (pH 7.4) and a solution of Exatecan in 10% Tween-80 in PBS (pH 7.4) were intraperitoneally administered at 100 μL / dose at concentrations of 4 mg / kg and 1.6 mg / kg, respectively. As a control, a solution of 10% Tween-80 in PBS (pH 7.4) was intraperitoneally administered at 100 μL / dose. Administration was performed twice a week (Monday and Thursday), and the size of the transplanted tumor was measured according to the administration day. After 4 weeks of administration (8 times in total), tumors were collected on the 30th day. The weight of the tumor and the weight of the mouse were measured (n=1 for each).

[0290] 2. Results The results are shown in Table 2, Figures 2, 3, 4, and 5. In control-treated mice, the tumor size increased 8.2 times (53.4 mm) after 28 days. 3 →440mm 3 ), whereas the tumor size of the mice treated with compound 4 decreased by 19% (90.8 mm) after 28 days. 3 →17.4mm 3 In the mice treated with Exatecan, the tumor size decreased by 68% (121 mm) after 28 days. 3 →81.7mm 3 ), but significant weight loss was observed. No weight loss was observed in mice administered Compound 4. These results suggest that Compound 4 increases the antitumor activity of Exatecan by accumulating it in prostate cancer cells, thereby reducing systemic side effects.

[0291]

Claims

1. The following formula (1): [Wherein X is —(CH2) n -CO- or -(CH2) n -NRCO- (wherein R represents a hydrogen atom, a lower alkyl group, a cyclo(lower alkyl) group, an optionally substituted aryl group or an optionally substituted aralkyl group, and n represents an integer of 9 to 17), L represents a peptide linker, and D represents an antitumor drug moiety, or a salt thereof.

2. In formula (1), X is -(CH2) n The drug conjugate or salt thereof according to claim 1, wherein the compound is —CO—.

3. The drug conjugate or salt thereof according to claim 1, wherein n is 9 to 15.

4. L is - [AA] m -or- [AA] m 2. The drug conjugate or salt thereof according to claim 1, which is represented by -B-, wherein AA is a residue of an amino acid selected from Gly, Ala, Val, Phe, Asn, Lys, Glu, and Cit, B is p-aminobenzyloxycarbonyl, and m is 2 to 4.

5. The drug conjugate or salt thereof according to claim 1, wherein L is -Val-Cit-, -Val-Cit-Phe-, or -Glu-Val-Cit-p-aminobenzyloxycarbonyl.

6. The drug conjugate or salt thereof according to claim 1, wherein the antitumor drug is exatecan, monomethyl auristatin F, or monomethyl auristatin E.

7. An anticancer agent containing the drug conjugate or its salt according to claim 1 as an active ingredient.

8. The anticancer agent according to claim 7, wherein the cancer is prostate cancer.

9. Use of the drug conjugate or salt thereof according to claim 1 for the production of an anticancer agent.

10. The drug conjugate or salt thereof according to claim 1 for use in preventing or treating cancer.

11. A method for preventing or treating cancer, which comprises administering to a patient an effective amount of the drug conjugate or salt thereof according to claim 1.

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