Improved production method for antibody-drug conjugates

Abstract

To provide: an improved production method for a drug-linker intermediate that is industrially superior and does not require purification by chromatography; and an improved production method for an antibody-drug conjugate using the improved production method for the drug-linker intermediate.SOLUTION: The invention provides a crystal of a compound represented by formula (1), a method for producing the same, and a method for producing an antibody-drug conjugate using the crystal.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an improved method for preparing a drug linker intermediate for an antibody-drug conjugate, and The present invention relates to an improved method for producing antibody-drug conjugates using the above method. [Background technology]

[0002] Antibodies that bind to antigens that are expressed on the surface of cancer cells and can be internalized into the cells are used to produce cytotoxic antibodies. Antibody-drug conjugates (Antibody-Drug Conjugates) ADCs (anti-cancer drug delivery systems) are effective in treating cancer by selectively delivering drugs to cancer cells. It is expected that drugs will accumulate inside cells and kill cancer cells (Non-patent documents 1-5). .

[0003] One of the antibody-drug conjugates is a combination of an antibody and an exon, a topoisomerase I inhibitor. Antibody-drug conjugates containing satecan as a component are known (Patent Documents 1 to 5, Non-patent documents 6, 7) These antibody-drug conjugates have excellent antitumor effects and safety. Clinical trials are currently underway.

[0004] A method for producing a drug linker intermediate for producing the above antibody-drug conjugate is The methods described in Patent Documents 1 to 4 are known. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 057687 [Patent Document 2] International Publication No. 2015 / 098099 [Patent Document 3] International Publication No. 2015 / 115091 [Patent Document 4] International Publication No. 2015 / 155998 [Patent Document 5] International Publication No. 2018 / 135501 [Non-patent literature]

[0006] [Non-Patent Document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-patent document 2] Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [Non-patent document 3] Damle NK Expert Opin. Biol. Ther. (2004) 4, 1445-1452. [Non-patent document 4] Senter PD, et al., Nature Biotechnology (2012) 30, 631-637. [Non-patent document 5] Howard A. et al., J Clin Oncol 29: 398-405. [Non-patent document 6] Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108. [Non-Patent Document 7] Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046. Summary of the Invention [Problem to be solved by the invention]

[0007] The drug linker intermediate for producing the antibody-drug conjugate of the present invention is represented by the formula (1):

[0008] [ka]

[0009] It is a compound represented by the formula:

[0010] As a method for producing the compound represented by formula (1), the methods described in Patent Documents 1 to 4 are known. However, it is known that the compound represented by formula (1) can be obtained as a crystal. It is necessary to carry out complicated procedures such as purification by chromatography. Therefore, the development of an industrially superior manufacturing method is desired.

[0011] One object of the present invention is to provide an industrially excellent drug that does not require purification by chromatography. Another object of the present invention is to find an improved method for preparing the linker intermediate. Improved manufacturing method for antibody-drug conjugates using an improved manufacturing method for linker intermediates To do this. [Means for solving the problem]

[0012] As a result of extensive investigation into methods for producing drug linker intermediates, the present inventors have surprisingly found that It was found that the compound represented by formula (1) can be obtained as a crystal. As a result of improving the manufacturing method of the compound, it is possible to produce an industrially viable compound without the need for chromatographic purification. Furthermore, the present inventors have discovered an antibody- An improved method for producing drug conjugates was developed, and the present invention was completed.

[0013] That is, the present invention provides: [1] Formula (1)

[0014] [ka]

[0015] A crystal of the compound represented by the formula: [2] In powder X-ray diffraction obtained by irradiating copper Kα rays, the values ​​were 5.6±0.2° and 15.5±0 The main peaks are at diffraction angles (2θ) of 0.2° and 22.0±0.2°, as described in [1]. Crystals of the same species. [3] Formula (1)

[0016] [ka]

[0017] and then, from the solution, a compound represented by formula (1) is dissolved in the solution. and precipitating a crystal of the compound represented by formula (1). method. [4] The crystals of the compound represented by formula (1) are shown in powder X-ray diffraction obtained by irradiating with copper Kα rays. The diffraction angles (2θ) were 5.6±0.2°, 15.5±0.2°, and 22.0±0.2°. ) shows a major peak in the production method described in [3]. [5] The solution in which the compound represented by formula (1) is dissolved is prepared by using a lower ketone and a lower alcohol as solvents. The method according to [3] or [4], comprising: [6] The method according to [5], wherein the lower ketone is acetone. [7] The method according to [5], wherein the lower ketone is methyl ethyl ketone. [8] [5] to [7], wherein the lower alcohol is 1-propanol. Manufacturing method. [9] The process according to any one of [5] to [7], wherein the lower alcohol is 2-butanol. Construction method.

[10] Any of [3] to [9], which includes a step of adding seed crystals of a compound represented by formula (1). 10. The manufacturing method according to any one of claims 1 to 9.

[11] The compound represented by formula (1) is produced by production method (I): [3] to

[10] . (Here, the production method (I) is Formula (B)

[0018] [ka]

[0019] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group, and R 2 is a protecting group Deprotecting the amino and carboxy protecting groups of the compound (representing a protected carboxy group), Formula (8)

[0020] [ka]

[0021] and then a compound represented by formula (8), Formula (C)

[0022] [ka]

[0023] (wherein X represents an active ester group or a carboxy group) By doing so, Formula (10)

[0024] [ka]

[0025] and then converting the compound represented by formula (10) into a compound represented by formula (11):

[0026] [ka]

[0027] By condensing a compound represented by Formula (1)

[0028] [ka]

[0029] and (b) converting the compound represented by formula (1) into a compound represented by formula (1).

[12] The compound represented by formula (1) is produced by production method (II): the manufacturing method according to any one of [3] to

[10] ; (Here, the production method (II) is Formula (B)

[0030] [ka]

[0031] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group, and R 2 is a protecting group By deprotecting the protecting group of the amino group of the compound (representing a protected carboxy group), Formula (D)

[0032] [ka]

[0033] A compound represented by the formula (wherein R 2 has the same meaning as above), and then a step of converting the compound of formula ( D) a compound represented by the formula Formula (C)

[0034] [ka]

[0035] (wherein X represents an active ester group or a carboxy group) By doing so, Formula (E)

[0036] [ka]

[0037] A compound represented by the formula (wherein R 2 has the same meaning as above), and then a step of converting the compound of formula ( E) by deprotecting the protecting group of the carboxy group of the compound represented by Formula (10)

[0038] [ka]

[0039] and then converting the compound represented by formula (10) into a compound represented by formula (11):

[0040] [ka]

[0041] By condensing a compound represented by Formula (1)

[0042] [ka]

[0043] and (b) converting the compound represented by formula (1) into a compound represented by formula (1).

[13] A step of dissolving the compound represented by formula (10) in a solvent containing 1,2-dimethoxyethane. Then, crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) were precipitated.

[11] or

[12] , comprising the step of:

[14] The crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) are In the powder X-ray diffraction obtained by irradiation, the angles were 19.0±0.2° and 25.0±0.2° The manufacturing method described in

[13] shows a main peak at a diffraction angle (2θ) of

[15] By condensing a compound represented by formula (10) with a compound represented by formula (11), The step of converting to the compound represented by formula (1) is carried out by using an aqueous solution of sodium sulfate and tetrahydrofuran. The method according to any one of

[11] to

[14] , wherein the method is carried out in a two-phase system.

[16] The compound represented by formula (1) is produced by production method (III). the manufacturing method according to any one of [3] to

[10] ; (Here, the production method (III) is Formula (B)

[0044] [ka]

[0045] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group, and R 2 is a protecting group By deprotecting the protecting group of the carboxy group of Formula (F)

[0046] [ka]

[0047] A compound represented by the formula (wherein R 1 has the same meaning as above), and then a step of converting the compound of formula ( F) a compound represented by the formula Formula (11)

[0048] [ka]

[0049] By condensing a compound represented by Formula (G)

[0050] [ka]

[0051] A compound represented by the formula (wherein R 1 has the same meaning as above), and then a step of converting the compound of formula ( G) by deprotecting the protecting group of the amino group of the compound represented by the formula: Formula (16)

[0052] [ka]

[0053] and then a compound represented by formula (16), Formula (C)

[0054] [ka]

[0055] (wherein X represents an active ester group or a carboxy group) By doing so, Formula (1)

[0056] [ka]

[0057] and (b) converting the compound represented by formula (1) into a compound represented by formula (1).

[17]

[11] to

[16] , wherein the compound represented by formula (11) is a methanesulfonate salt. 10. The manufacturing method according to any one of claims 1 to 9.

[18] The compound represented by formula (11) is methanesulfonate·m hydrate (where m is 0 to 3 The method according to any one of

[11] to

[16] , wherein the number of the saturates is within a range of 100 to 150.

[19] The method according to any one of

[11] to

[0016] , wherein the compound represented by formula (11) is methanesulfonate anhydride.

[20] The compound represented by formula (11) is methanesulfonate monohydrate,

[11]

[16] The method according to any one of the preceding items. [twenty one] The compound represented by formula (11) is methanesulfonate dihydrate,

[11]

[16] The method according to any one of the preceding items. [twenty two] The compound represented by formula (11) is methanesulfonate trihydrate,

[11]

[16] The method according to any one of the preceding items. [twenty three] The compound represented by formula (B) is produced by production method (IV): the manufacturing method according to any one of

[11] to

[22] ; (Here, the production method (IV) is Formula (H)

[0058] [ka]

[0059] A compound represented by the formula (wherein R 3 indicates a protected amino group) and lead tetraacetate. By reacting, Formula (J)

[0060] [ka]

[0061] A compound represented by the formula (wherein R 3 has the same meaning as above), and then a step of converting the compound of formula ( J) a compound represented by Formula (K)

[0062] [ka]

[0063] A compound represented by the formula (wherein R 2 is R according to any one of claims 11 to 22. 2 Same as (which has the meaning of "a compound of formula (I)") in the presence of an acid or a base, Formula (L)

[0064] [ka]

[0065] A compound represented by the formula (wherein R 2 and R 3 has the same meaning as above), By deprotecting the protecting group of the amino group of the compound represented by formula (L), Formula (M)

[0066] [ka]

[0067] A compound represented by the formula (wherein R 2 has the same meaning as above), and then a step of converting the compound of formula ( M) a compound represented by the formula: Formula (N)

[0068] [ka]

[0069] A compound represented by the formula (wherein R 1 is R according to any one of

[11] to

[22] 1 and By condensing Formula (B)

[0070] [ka]

[0071] A compound represented by the formula (wherein R 1 and R 2 has the same meaning as above), (This is a manufacturing method that involves [twenty four] The compound of formula (H) is reacted with lead tetraacetate to give the compound of formula (J). The method according to

[23] , wherein the step of converting into the compound is carried out in the presence of acetic acid. [twenty five] A compound represented by formula (J) is reacted with a compound represented by formula (K) to obtain a compound represented by formula (L).

[23] or

[24] The manufacturing method described in

[24] .

[26] A compound represented by formula (J) is reacted with a compound represented by formula (K) to obtain a compound represented by formula (L). The step of converting the compound into a compound having the formula The method according to

[23] or

[24] ,

[27] The protecting group of the amino group of the compound represented by formula (L) is removed to give a compound represented by formula (M). After the step of converting the compound represented by formula (M) into the compound represented by formula (M), an acid is added to the compound represented by formula (M). The method according to any one of

[23] to

[26] , further comprising a step of precipitating a salt of method.

[28] The method according to

[27] , wherein the acid is 1-hydroxybenzotriazole.

[29] R 1 is an amino group protected by a benzyloxycarbonyl group,

[11] the method for producing the compound according to any one of

[0028] to

[11] .

[30] R 1 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. The method for producing a semiconductor device according to any one of

[11] to

[28] .

[31] R 2 is a carboxy group protected by a benzyl group, any of

[11] to

[30] 1. The method of manufacturing according to claim 1.

[32] R 3 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. The manufacturing method according to any one of

[23] to

[31] .

[33] X is a (2,5-dioxopyrrolidin-1-yl)oxycarbonyl group.

[11]

[32] The method for producing a compound according to any one of

[32] to

[32] .

[34] The compound represented by formula (1) is produced by production method (V). [3] to

[10] . (Here, the production method (V) is Formula (2)

[0072] [ka]

[0073] By reacting the compound represented by the formula with lead tetraacetate, Formula (3)

[0074] [ka]

[0075] and then converting the compound represented by formula (3) into a compound represented by formula (4) in the presence of an acid or a base. by reacting with benzyl glycolate in the presence of Formula (4)

[0076] [ka]

[0077] and then protecting the amino group of the compound represented by formula (4). By deprotecting the group, Formula (5)

[0078] [ka]

[0079] and then a compound represented by formula (5), Formula (6)

[0080] [ka]

[0081] By condensing a compound represented by Formula (7)

[0082] [ka]

[0083] and then, a step of converting the amino group and carboxyl group of the compound represented by formula (7) into a compound represented by formula (8). The protecting group of the carboxy group is deprotected, Formula (8)

[0084] [ka]

[0085] and then a compound represented by formula (8), Formula (9)

[0086] [ka]

[0087] By condensing a compound represented by Formula (10)

[0088] [ka]

[0089] and then converting the compound represented by formula (10) into a compound represented by formula (11):

[0090] [ka]

[0091] By condensing a compound represented by Formula (1)

[0092] [ka]

[0093] and (b) converting the compound represented by formula (1) into a compound represented by formula (1).

[35] A step of dissolving the compound represented by formula (10) in a solvent containing 1,2-dimethoxyethane. Then, crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) were precipitated.

[34] The manufacturing method according to

[34] , comprising the step of:

[36] The crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) are In the powder X-ray diffraction obtained by irradiation, the angles were 19.0±0.2° and 25.0±0.2° The manufacturing method described in

[35] shows a main peak at a diffraction angle (2θ) of

[37] By condensing a compound represented by formula (10) with a compound represented by formula (11), The step of converting to the compound represented by formula (1) is carried out by using an aqueous solution of sodium sulfate and tetrahydrofuran. The method according to any one of

[34] to

[36] , wherein the method is carried out in a two-phase system.

[38] The compound represented by formula (1) is produced by production method (VI): the manufacturing method according to any one of [3] to

[10] ; (wherein, production method (VI) is Formula (2)

[0094] [ka]

[0095] By reacting the compound represented by the formula with lead tetraacetate, Formula (3)

[0096] [ka]

[0097] and then converting the compound represented by formula (3) into a compound represented by formula (4) in the presence of an acid or a base. by reacting with benzyl glycolate in the presence of Formula (4)

[0098] [ka]

[0099] and then protecting the amino group of the compound represented by formula (4). By deprotecting the group, Formula (5)

[0100] [ka]

[0101] and then a compound represented by formula (5), Formula (12)

[0102] [ka]

[0103] By condensing a compound represented by Formula (13)

[0104] [ka]

[0105] and then, a step of converting the carboxy group of the compound represented by formula (13) into a compound represented by formula (14). By deprotecting the protecting group of Formula (14)

[0106] [ka]

[0107] and then a compound represented by formula (14), Formula (11)

[0108] [ka]

[0109] By condensing a compound represented by Formula (15)

[0110] [ka]

[0111] and then, a step of converting the amino group of the compound represented by formula (15) into a compound represented by formula (16). By removing the protecting group, Formula (16)

[0112] [ka]

[0113] and then a compound represented by formula (16), Formula (9)

[0114] [ka]

[0115] By condensing a compound represented by Formula (1)

[0116] [ka]

[0117] and (b) converting the compound represented by formula (1) into a compound represented by formula (1).

[39] The compound represented by formula (2) is reacted with lead tetraacetate to give the compound represented by formula (3).

[34] to

[38] , wherein the step of converting the compound into the compound is carried out in the presence of acetic acid. The manufacturing method described above.

[40] The step of converting the compound represented by formula (3) into the compound represented by formula (4) comprises the step of: The method according to any one of

[34] to

[39] , wherein the method is carried out in the presence of an aqueous sodium solution. Law.

[41] The step of converting the compound represented by formula (3) into the compound represented by formula (4) is carried out by using a tris(pentaerythritol)- Any one of

[34] to

[39] , which is carried out in the presence of (pentafluorophenyl)borane The manufacturing method described in paragraph .

[42] The protecting group of the amino group of the compound represented by formula (4) is removed to give the compound represented by formula (5). After the step of converting the compound represented by formula (5) into the compound represented by formula (6), an acid is added to the compound represented by formula (5). The method according to any one of

[34] to

[41] , further comprising a step of precipitating a salt of method.

[43] The method according to

[42] , wherein the acid is 1-hydroxybenzotriazole.

[44] The compound represented by formula (6) Formula (23)

[0118] [ka]

[0119] and N-hydroxysuccinimide, Formula (24)

[0120] [ka]

[0121] and then reacting the compound represented by formula (24) with L-phenyl and converting guaranaline into a compound represented by formula (6)

[43] The manufacturing method according to any one of

[34] to

[43] , .

[45] The compound represented by formula (9) Formula (17)

[0122] [ka]

[0123] By reacting a compound represented by the formula: with maleic anhydride, Formula (18)

[0124] [ka]

[0125] and then reacting the compound represented by formula (18) with an N-hydrogen group. Add thionyl chloride to a mixed solution containing succinimide and 2,6-lutidine. and converting the compound into a compound represented by formula (9). The method for producing a compound according to any one of

[34] to

[44] ,

[46]

[34] to

[45] , wherein the compound represented by formula (11) is a methanesulfonate salt. 10. The manufacturing method according to any one of claims 1 to 9.

[47] The compound represented by formula (11) is methanesulfonate·m hydrate (where m is 0 to 3 The method according to any one of

[34] to

[45] , wherein the number of the hydroxyl groups is within a range of 100 to 150.

[48] The method according to any one of

[34] to

[0045] , wherein the compound represented by formula (11) is methanesulfonate anhydride.

[49] From

[34] , where the compound represented by formula (11) is methanesulfonate monohydrate.

[45] The manufacturing method according to any one of the items.

[50] The compound represented by formula (11) is methanesulfonate dihydrate,

[34]

[45] The manufacturing method according to any one of the items.

[51] The compound represented by formula (11) is methanesulfonate trihydrate,

[34]

[45] The manufacturing method according to any one of the items.

[52] Formula (H)

[0126] [ka]

[0127] A compound represented by the formula (wherein R 3 indicates a protected amino group) in the presence of acetic acid by reacting with lead tetraacetate under Formula (J)

[0128] [ka]

[0129] A compound represented by the formula (wherein R 3 is as defined above), ) A method for producing a compound represented by the formula:

[53] R 3 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. The manufacturing method described in

[52] .

[54] Formula (J)

[0130] [ka]

[0131] A compound represented by the formula (wherein R 3 represents an amino group protected by a protecting group), and Formula (K)

[0132] [ka]

[0133] A compound represented by the formula (wherein R 2 indicates a carboxyl group protected by a protecting group) is hydroxylated The reaction is carried out in the presence of aqueous sodium or tris(pentafluorophenyl)borane. Therefore, Formula (L)

[0134] [ka]

[0135] A compound represented by the formula (wherein R 2 and R 3 has the same meaning as above) A method for producing a compound represented by formula (L).

[55] The process according to

[54] , characterized in that the reaction is carried out in the presence of an aqueous sodium hydroxide solution. Construction method.

[56] The production method described in

[0054] , characterized in that the reaction is carried out in the presence of tris(pentafluorophenyl)borane.

[57] R 2 is a carboxy group protected by a benzyl group, any of

[54] to

[56] 1. The method of manufacturing according to claim 1.

[58] R 3 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. The method for producing a semiconductor device according to any one of

[54] to

[57] .

[59] Formula (L)

[0136] [ka]

[0137] A compound represented by the formula (wherein R 2 represents a carboxyl group protected by a protecting group, and R 3 is protected By deprotecting the protecting group of the amino group of Formula (M)

[0138] [ka]

[0139] A compound represented by the formula (wherein R 2 has the same meaning as defined above), and then a step of precipitating a salt of the compound represented by formula (M) with an acid by adding a compound represented by formula (M) A method for producing a salt of a compound represented by (M) with an acid.

[60] The method according to

[59] , wherein the acid is 1-hydroxybenzotriazole.

[61] R 2 is a carboxy group protected by a benzyl group. Construction method.

[62] R 3 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. The manufacturing method according to any one of

[59] to

[61] .

[63] Formula (18)

[0140] [ka]

[0141] and a mixture containing N-hydroxysuccinimide and 2,6-lutidine. By adding thionyl chloride to the solution, Formula (9)

[0142] [ka]

[0143] A method for producing a compound represented by formula (9), comprising a step of converting a compound represented by formula (9) into a compound represented by formula (9).

[64] The compound represented by formula (18) Formula (17)

[0144] [ka]

[0145] and maleic anhydride. The manufacturing method described in

[63] .

[65] In a solvent containing 1,2-dimethoxyethane, Formula (10)

[0146] [ka]

[0147] and then dissolving the compound represented by formula (10) in a 1,2-dichloroisothiazolinone solution. and precipitating crystals of a methoxyethane adduct of the compound represented by formula (10). A method for producing crystals of 1,2-dimethoxyethane adduct.

[66] The crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) are In the powder X-ray diffraction obtained by irradiation, the angles were 19.0±0.2° and 25.0±0.2° The manufacturing method described in

[65] , wherein the diffraction angle (2θ) is 0.05.

[67] Formula (10)

[0148] [ka]

[0149] and a compound represented by Formula (11)

[0150] [ka]

[0151] The compound represented by the formula (I) is condensed in a two-phase system of aqueous sodium sulfate and tetrahydrofuran. By doing so, Formula (1)

[0152] [ka]

[0153] A method for producing a compound represented by formula (1), comprising a step of converting a compound represented by formula (1) into a compound represented by formula (1).

[68] The method according to

[67] , wherein the compound represented by formula (11) is a methanesulfonate salt. Law.

[69] The compound represented by formula (11) is methanesulfonate·m hydrate (where m is 0 to 3 The manufacturing method described in

[67] , wherein the number of the hydroxyl groups is within the range of 100 to 1500.

[70] The compound represented by formula (11) is methanesulfonate anhydride, as described in

[67] . Manufacturing method.

[71] The compound represented by formula (11) is methanesulfonate monohydrate, as described in

[67] . The manufacturing method described above.

[72] The compound represented by formula (11) is the methanesulfonate dihydrate, as described in

[67] . The manufacturing method described above.

[73] The compound represented by formula (11) is the methanesulfonate trihydrate, as described in

[67] . The manufacturing method described above.

[74] Any one of [3] to

[73] , characterized in that no chromatography is used. The manufacturing method described in paragraph .

[75] Formula (10)

[0154] [ka]

[0155] Crystal of the 1,2-dimethoxyethane adduct of the compound represented by the formula:

[76] Powder X-ray diffraction obtained by irradiating with copper Kα rays showed 19.0±0.2° and 25. The crystal described in

[75] exhibits a major peak at a diffraction angle (2θ) of 0±0.2°.

[77] Formula (5)

[0156] [ka]

[0157] A salt of a compound represented by the formula (I) with an acid.

[78] The salt according to

[77] , wherein the acid is 1-hydroxybenzotriazole.

[79] Produced by the method described in any one of [3] to

[51] Formula (1)

[0158] [ka]

[0159] The method is characterized in that a crystal of a compound represented by the formula (I) is used as a starting material, i) reducing the antibody, and then ii) adding a solution in which the crystals of the compound represented by formula (1) prepared by the above method are dissolved; reacting with reduced antibody; Including, Formula (19)

[0160] [ka]

[0161] (wherein A represents the binding site to the antibody) and an antibody-drug conjugate in which the antibody is bound to a drug linker represented by the formula: Method for producing Jugate.

[80] The antibody is anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, or The method for producing the antibody according to

[79] , wherein the antibody is an anti-GPR20 antibody.

[81] The method for production according to

[80] , wherein the antibody is an anti-HER2 antibody.

[82] The anti-HER2 antibody comprises an amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1. a heavy chain consisting of a sequence of amino acids 1 to 214 in SEQ ID NO: 2; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. The manufacturing method described above.

[83] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 7 to The manufacturing method according to

[81] or

[82] , wherein the number of the components is in the range of 8.

[84] The method for production according to

[80] , wherein the antibody is an anti-HER3 antibody.

[85] The anti-HER3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a An antibody comprising a light chain consisting of the amino acid sequence set forth above, or the carboxyl terminal of the heavy chain of said antibody. The method for producing an antibody according to

[84] , wherein the lysine residue is deleted.

[86] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 7 to The manufacturing method according to

[84] or

[85] , wherein the number of the particles is in the range of 8.

[87] The method for production according to

[80] , wherein the antibody is an anti-TROP2 antibody.

[88] The anti-TROP2 antibody comprises the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO:5. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6; an antibody comprising a light chain consisting of a lysine residue at the carboxyl terminus of a heavy chain of said antibody; The method for producing an antibody according to

[87] , wherein the antibody is an antibody having a deletion of a group.

[89] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 3 to The manufacturing method according to

[87] or

[88] , wherein the number of the components is in the range of 5.

[90] The method for production according to

[80] , wherein the antibody is an anti-B7-H3 antibody.

[91] The anti-B7-H3 antibody is selected from the group consisting of amino acids 20 to 471 in SEQ ID NO:7. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8; an antibody comprising a light chain consisting of a lysine residue at the carboxyl terminus of a heavy chain of said antibody; The method for producing an antibody according to

[90] , wherein the antibody is an antibody having a deletion of a group.

[92] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 3 to The manufacturing method according to

[90] or

[91] , wherein the number of the components is in the range of 5.

[93] The method for production according to

[80] , wherein the antibody is an anti-GPR20 antibody.

[94] The anti-GPR20 antibody is a polypeptide comprising the amino acids 20 to 472 of SEQ ID NO: 9. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10; an antibody comprising a light chain consisting of the carboxyl-terminal lysine of said antibody heavy chain, The method for producing an antibody according to

[93] , wherein the antibody has deleted residues.

[95] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 7 to The manufacturing method according to

[93] or

[94] , wherein the number of the components is in the range of 8. Regarding. [Effects of the Invention]

[0162] According to the present invention, it is possible to obtain crystals of the compound represented by formula (1), and crystals of the compound represented by formula ( The compound represented by formula 1) can be obtained. The present invention provides an industrially excellent method for producing the compound represented by formula (1) without using the above-mentioned process. Furthermore, an improved method for producing antibody-drug conjugates using this can be provided. [Brief explanation of the drawings]

[0163] [Figure 1] 1 shows the amino acid sequence of the anti-HER2 antibody heavy chain (SEQ ID NO: 1). [Figure 2] 1 shows the amino acid sequence of the anti-HER2 antibody light chain (SEQ ID NO: 2). [Figure 3] 1 shows powder X-ray diffraction of a crystal of a 1,2-dimethoxyethane adduct of the compound represented by formula (10). [Figure 4] 1 shows the powder X-ray diffraction of the crystals of the compound represented by formula (1). [Figure 5] 1 shows the amino acid sequence of the anti-HER3 antibody heavy chain (SEQ ID NO: 3). [Figure 6] 1 shows the amino acid sequence of the anti-HER3 antibody light chain (SEQ ID NO: 4). [Figure 7] The amino acid sequence of the anti-TROP2 antibody heavy chain (SEQ ID NO: 5) is shown. [Figure 8] The amino acid sequence of the anti-TROP2 antibody light chain (SEQ ID NO: 6) is shown. [Figure 9] The amino acid sequence of the anti-B7-H3 antibody heavy chain (SEQ ID NO: 7) is shown. [Figure 10] The amino acid sequence of the anti-B7-H3 antibody light chain (SEQ ID NO: 8) is shown. [Figure 11] The amino acid sequence of the anti-GPR20 antibody heavy chain (SEQ ID NO: 9) is shown. [Figure 12] The amino acid sequence of the anti-GPR20 antibody light chain (SEQ ID NO: 10) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0164] Preferred embodiments of the present invention will now be described with reference to the drawings. The embodiment described below is an example of a typical embodiment of the present invention. This will not narrow the scope of the present invention.

[0165] [Antibody-drug conjugates] The antibody-drug conjugates produced according to the present invention are Formula (19)

[0166] [ka]

[0167] (wherein A represents the binding site to the antibody) and an antibody-drug conjugate in which the antibody is bound to a drug linker represented by the formula: It's Jugate.

[0168] In the present invention, the portion of the antibody-drug conjugate consisting of a linker and a drug The structure is called a "drug linker." This drug linker binds to the disulfide bond between the antibody chains. Thiol groups (in other words) generated at positions (two heavy chain-heavy chain interchains and two heavy chain-light chain interchains) In other words, it is bound to the sulfur atom of a cysteine ​​residue.

[0169] The drug linker of the present invention comprises exatecan, a topoisomerase I inhibitor, as a component. Exatecan is Formula (11)

[0170] [ka]

[0171] It is a camptothecin derivative having an antitumor effect, represented by the formula:

[0172] The antibody-drug conjugate used in the present invention is Formula (20)

[0173] [ka]

[0174] It can also be expressed as

[0175] Here, the drug linker is bound to the antibody via a thioether bond. The so-called average number of drug-antibody bindings (DAR; Drug-to-Antibody Ratio) It is synonymous with and indicates the average number of drug linkers bound per antibody.

[0176] The antibody-drug conjugate used in the present invention is capable of transporting the drug into cancer cells. Formula (22)

[0177] [ka]

[0178] The compound represented by the formula (I) is released, thereby exerting an antitumor effect. The compound represented by formula (22) is an antibody-drug conjugate produced by the present invention. It is believed to be the main cause of the antitumor activity of α-tocopherol, and has been confirmed to have topoisomerase I inhibitory activity. (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):509 7-5108, Epub 2016 Mar 29).

[0179] The compound represented by formula (22) is an antibody-drug conjugate produced by the present invention. This is thought to be caused by cleavage of the linker portion of Formula (21)

[0180] [ka]

[0181] It is believed that this occurs due to the decomposition of the aminal structure of the compound represented by the formula:

[0182] The antibody-drug conjugates produced by the present invention have a bystander effect It is also known that cancer cells are involved in the regulation of inflammatory bowel disease (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046).

[0183] This bystander effect is due to the fact that the antibody-drug conjugates produced according to the present invention After being internalized into the target-expressing cancer cells, the released compound represented by formula (22) expresses the target. This is achieved by exerting an antitumor effect on nearby cancer cells that have not yet expressed the tumor.

[0184] [Drug-linker intermediates used in the production of antibody-drug conjugates] The drug linker intermediate used in the production of the antibody-drug conjugate of the present invention is Formula (1)

[0185] [ka]

[0186] According to the present invention, the compound represented by formula (1) can be obtained as a crystal. The crystals can be suitably used for producing the antibody-drug conjugates of the present invention. It is possible.

[0187] The quality of the crystals of the compound represented by formula (1) can be determined, for example, by the impurity content, the amount of residual solvent, and The evaluation can be performed using the appearance as an index. 3 months, 6 months, 12 months, 24 months under conditions such as ℃ / 75%RH The storage stability can be evaluated using the indicators of storage stability over 36 months.

[0188] These quality evaluations confirmed the superiority of the compound represented by the amorphous formula (1). It is also possible to do so.

[0189] The production method of the present invention comprises: The crystals of the compound represented by formula (1) are produced by precipitating the crystals of the compound represented by formula (1). This allows the compound represented by formula (1) to be produced with high purity and consistent quality. Crystals of the compound can be produced.

[0190] The crystals of the compound represented by formula (1) are preferably obtained by powder X-ray diffraction obtained by irradiating with copper Kα rays. In the diffraction, the main peaks are at diffraction angles (2θ) of 5.6°, 15.5°, and 22.0°. Generally, the diffraction angle (2θ) in powder X-ray diffraction is ±0. Since there is a margin of error within 2°, the above diffraction angle values ​​are also within ±0.2°. (Regarding the technical common sense of measurement and evaluation by powder X-ray diffraction, For example, see the 16th edition of the Japanese Pharmacopoeia, p. 64-68 (2.58 Powder X-ray Diffraction Measurement Method) or the 17th edition of the Japanese Pharmacopoeia. The Revised Japanese Pharmacopoeia, pp. 71-74 (2.58 Powder X-ray Diffraction Measurement Method) can be referred to.

[0191] Therefore, not only the crystals that perfectly match the diffraction angles mentioned above, but also 5.6±0.2°, 15. Crystals with major peaks at diffraction angles (2θ) of 5±0.2° and 22.0±0.2° are also the same and are included in the present invention. In the present invention, "±0.2°" means that the It indicates a value in the range of -0.2° to +0.2°. For example, "5.6±0.2 "°" indicates a numerical value in the range of 5.4° to 5.8°.

[0192] The solution for precipitating the crystals of the compound represented by formula (1) is preferably a mixture of acetone and low Similarly, a solution containing a lower ketone and a lower alcohol as a solvent is A solution containing the compound represented by formula (1) is also suitable as a solution for precipitating crystals of the compound represented by formula (1). can be used for.

[0193] In the present invention, the term "lower ketone" refers to a ketone having 3 to 6 carbon atoms, for example, Acetone, methyl ethyl ketone, methyl propyl ketone, methyl isopropyl ketone, methyl butyl ketone, methyl isobutyl ketone, methyl tert-butyl ketone, ethyl ether Examples include ethyl ketone, ethyl propyl ketone, and ethyl isopropyl ketone. Preferred examples include acetone and methyl ethyl ketone, and more preferred examples include acetone and methyl ethyl ketone. Seton can be mentioned.

[0194] In the present invention, the term "lower alcohol" refers to an alcohol having 1 to 4 carbon atoms. For example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol 2-methyl-1-propanol, 2-butanol, and tert-butanol. and preferred examples thereof include 1-propanol and 2-butanol. More preferred is 1-propanol.

[0195] Therefore, the solution for precipitating the crystals of the compound represented by formula (1) is preferably acetone. A solution containing acetone and 1-propanol, or a solution containing acetone and 2-butanol. More preferably, it is a solution containing acetone and 1-propanol.

[0196] The crystals of the compound represented by formula (1) can be precipitated by adding the following to a solution containing the compound represented by formula (1): This can also be achieved by adding seed crystals of the compound of formula (1).

[0197] Seed crystals of the compound represented by formula (1) can also be obtained by directly carrying out the above method. However, preferably, a small amount of the compound of formula (1) is purified by chromatography. Afterwards, a solvent containing acetone and 1-propanol or a solvent containing acetone and 2-butanol was added. It can be obtained by dissolving it in a solvent and crystallizing it from the solution.

[0198] The compound represented by formula (1) can be prepared according to the method disclosed in WO 2014 / 057687 and WO 20 15 / 098099, International Publication No. 2015 / 115091, International Publication No. 2015 / 1 Although it can be produced by referring to the description in No. 55998, it is preferable to use the following production method (I ), (II), (III), (V), (VI), and (IX) This allows for high-throughput purification without the use of chromatography in all steps. Crystals of the compound represented by formula (1) can be produced in a high yield.

[0199] [Manufacturing method (I)] The production method (I) is a method for producing a compound represented by formula (1) from a compound represented by formula (B) through steps 1 to 3. Steps 1 to 3 are explained in detail below.

[0200] [ka]

[0201] [In the formula, R 1 represents an amino group protected with a protecting group, and is preferably a benzyloxycarbonyl represents an amino group protected by a methyl group, and R 2 represents a carboxy group protected by a protecting group, and represents a carboxy group protected by a benzyl group, and X represents an active ester group or a carboxyl group. represents a hydroxyl group, and preferably a (2,5-dioxopyrrolidin-1-yl)oxycarbonyl group. ]

[0202] Step 1: This step is carried out to remove the protecting groups of the amino and carboxy groups of the compound represented by formula (B). This is a process for converting the compound represented by formula (8) into a compound represented by formula (8).

[0203] The deprotection of the amino and carboxy protecting groups of the compound represented by formula (B) can be performed by a person skilled in the art. This can be done by well-known methods (see, for example, Peter GM Wuts, Theodora W. Greene, Greene, ne's Protective Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscie nce et al.).

[0204] R 1 is an amino group protected with a benzyloxycarbonyl group, and R 2 is a benzyl group In the case of a protected carboxy group, this step can be preferably carried out by the following method. do.

[0205] The protecting groups of the amino group and the carboxy group of the compound represented by formula (B) are deprotected as the reaction proceeds. The method is not limited as long as it is carried out, but preferably, the reaction is carried out under a hydrogen atmosphere using a palladium catalyst or a platinum catalyst. The reaction can be carried out using a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst, and more preferably This can be carried out using a palladium catalyst, and more preferably, using palladium on carbon. More preferably, 5% palladium on carbon can be used. The amount of 5% palladium carbon used in the process is not limited as long as the reaction proceeds. In particular, the content is 5 to 40% by weight based on the compound represented by formula (B).

[0206] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example of the solvent is a mixed solvent of tetrahydrofuran and water.

[0207] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1 to 5 hours. is.

[0208] Step 2: This step involves condensing a compound represented by formula (8) with a compound represented by formula (C). The compound represented by formula (C) is commercially available. Those commercially available, those produced by known methods, or those produced in accordance with the following production method (VII) The compound prepared by the method described above can be used. In the case where the hydroxyl group is a (1-yl)oxycarbonyl group, this step is preferably carried out by the following method: can be done.

[0209] The amount of the compound represented by formula (C) used in this step is not limited as long as the reaction proceeds. However, it is preferably 1 to 4 equivalents relative to the compound represented by formula (8).

[0210] In this step, a base is preferably used. The base used in this step is particularly There is no limitation, but examples thereof include triethylamine, tributylamine, and diisopropylamine. amine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine. A preferred example is N,N-diisopropylethylamine. The amount of N,N-diisopropylethylamine used in this step is constant as long as the reaction proceeds. Although not limited thereto, it is preferably 0.5 to 2 equivalents relative to the compound represented by formula (8). .

[0211] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example of the solvent is a mixed solvent of acetonitrile and water.

[0212] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 7 to 30 hours. It is between.

[0213] The compound represented by formula (10) is preferably a crystal of a 1,2-dimethoxyethane adduct. can be obtained by doing so.

[0214] The quality of the crystals of the 1,2-dimethoxyethane adduct of the compound of formula (10) is For example, the impurity content, the amount of residual solvent, and the appearance can be used as indicators for evaluation. For example, 3 months under conditions such as 25°C / 60%RH or 40°C / 75%RH. The storage stability was evaluated using the following indicators: 1, 6, 12, 24, and 36 months. These quality evaluations allow us to evaluate the quality of the amorphous compound represented by formula (10). It is also possible to confirm the advantages of

[0215] The crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) are preferably In powder X-ray diffraction obtained by irradiating copper Kα rays, diffraction angles of 19.0° and 25.0° Generally, the diffraction pattern in powder X-ray diffraction is characterized by a major peak at the angle (2θ). The diffraction angle (2θ) may have an error within the range of ±0.2°, so the above values ​​of the diffraction angle are ± It should be understood that this also includes values ​​within a range of 0.2° (measured by powder X-ray diffraction). For the technical common sense of determination and evaluation, see, for example, the 16th Edition of the Japanese Pharmacopoeia, p. 64-68 (2.58 Powder X-ray Diffraction measurement method), or see the 17th Edition of the Japanese Pharmacopoeia, p. 71-74 (2.58 Powder X-ray Diffraction Measurement Method), etc. Therefore, not only crystals that perfectly match the above diffraction angles, but also 19. Crystals with major peaks at diffraction angles (2θ) of 0±0.2° and 25.0±0.2° are also the same and are included in the present invention.

[0216] Step 3: This step involves condensing a compound represented by formula (10) with a compound represented by formula (11). This is a process for converting the compound represented by formula (1) into a compound represented by formula (11). The product can be preferably used as a methanesulfonate salt, more preferably as a methanesulfonate salt. sulfonate·m hydrate (where m is 0 to 3), Preferably, methanesulfonate anhydrous, methanesulfonate monohydrate, methanesulfonate It can be used as the methanesulfonate dihydrate or the methanesulfonate trihydrate, More preferably, it can be used as the methanesulfonate dihydrate. The number of hydrates mentioned above depends on the humidity at the time of crystal acquisition and drying. It can be controlled by adjusting

[0217] The amount of the compound represented by formula (11) used in this step is not limited as long as the reaction proceeds. However, it is preferably 0.5 to 2 equivalents relative to the compound represented by formula (10).

[0218] The compound represented by formula (10) can be preferably converted to an active ester to give a compound represented by formula ( 11) can be condensed with the compound represented by the formula (11). The method for the introduction is not limited as long as the reaction proceeds. For example, 1-ethyl-3-(3-dimethylamino)- N,N'-diethylaminopropyl)carbodiimide hydrochloride (WSCD HCl), or N,N'-di Using a condensing agent such as cyclohexylcarbodiimide (DCC), 1-hydroxybenzoxazole 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, ethyl cyano(hydroxyimino)acetate, or p-nitro This can be carried out by reacting with an additive such as 3-(3-phenylphenol). Dimethylaminopropyl)carbodiimide hydrochloride and cyano(hydroxyimino)acetic acid ester The 3-(3-dimethylaminopropyl) The amount of carbodiimide hydrochloride is not limited as long as the reaction proceeds. The amount of the cyano( The amount of ethyl hydroxyiminoacetate is not limited as long as the reaction proceeds, but is preferably The amount is 0.02 to 0.2 equivalents relative to the compound represented by formula (10).

[0219] In this step, a base is preferably used. The base used in this step is not particularly limited as long as the reaction proceeds. There is no limitation to, for example, triethylamine, tributylamine, diisopropylamine amines, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, etc. Examples of the methyl morpholine used in this step include N-methylmorpholine. The amount of N-methylmorpholine that can be used is not limited as long as the reaction proceeds, but is preferably The amount is 0.5 to 2 equivalents relative to the compound represented by formula (10).

[0220] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example of the solvent is a mixed solvent of tetrahydrofuran and water.

[0221] The methanesulfonate of the compound represented by formula (11) is neutralized with a base to form a free The reaction proceeds after the compound represented by formula (11) is converted into methanesulfonic acid. The acid salt is hydrophilic, whereas the free form of the compound represented by formula (11) is lipophilic. Therefore, in order to efficiently proceed with a series of reactions, this step is preferably carried out in two phases, an aqueous layer and an organic layer. When the organic layer is tetrahydrofuran, it is difficult to mix with it. As the aqueous layer, an aqueous solution with a high ionic strength, for example, an aqueous sodium sulfate solution, is preferably used. This can be done.

[0222] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 It's time.

[0223] [Manufacturing method (II)] The production method (II) is a method for producing a compound represented by formula (1) from a compound represented by formula (B) through steps 4 to 7. Steps 4 to 7 are explained in detail below.

[0224] [ka]

[0225] [In the formula, R 1 represents an amino group protected by a protecting group, and R 2 represents a carboxyl group protected by a protecting group. X represents an active ester group or a carboxy group, and is preferably (2,5-dihydroxybenzoyl) represents a 1-oxopyrrolidin-1-yl)oxycarbonyl group.

[0226] Step 4: In this step, the protecting group of the amino group in the compound represented by formula (B) is removed to obtain a compound represented by formula (D)

[0227] The protecting group of the amino group of the compound represented by formula (B) can be deprotected by a method well known to those skilled in the art. (See, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective (See Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc.).

[0228] Step 5: This step is carried out by condensing a compound represented by formula (D) with a compound represented by formula (C). This is a process for converting the compound represented by formula (E) into a compound represented by formula (E).

[0229] This step can be carried out in the same manner as in step 2 of production method (I).

[0230] Step 6: This step is carried out by deprotecting the protecting group of the carboxy group of the compound represented by formula (E). This is a process of converting the compound represented by formula (10).

[0231] The protecting group of the carboxy group of the compound represented by formula (B) can be deprotected by a method well known to those skilled in the art. (See, e.g., Peter GM Wuts, Theodora W. Greene, Greene's Protect see "Vital Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc." ).

[0232] The compound represented by formula (10) can be preferably produced by the same method as in step 2 of production method (I). It can be obtained as a crystal of the 1,2-dimethoxyethane adduct.

[0233] Step 7: This step involves condensing a compound represented by formula (10) with a compound represented by formula (11). This is a process for converting the compound represented by formula (1) into the compound represented by formula (1).

[0234] This step can be carried out in the same manner as in step 3 of production method (I).

[0235] [Manufacturing method (III)] In the production method (III), a compound represented by formula (B) is converted to a compound represented by formula (1) through steps 8 to 11. The method is a method for converting a compound represented by the formula: Steps 8 to 11 will be explained in detail below.

[0236] [ka]

[0237] [In the formula, R 1 represents an amino group protected by a protecting group, and is preferably (9H-fluorene-9 -(-ylmethoxy)carbonyl-protected amino group, and R 2 is protected by a protecting group represents a carboxy group, preferably a carboxy group protected by a benzyl group, and X is an active It represents an ester group or a carboxy group, and is preferably (2,5-dioxopyrrolidine-1- represents a (hydroxy)carbonyl group.

[0238] Step 8: This step is carried out by deprotecting the protecting group of the carboxy group of the compound represented by formula (B). , a process of converting the compound represented by formula (F) into a compound represented by formula (F).

[0239] The protecting group of the carboxy group of the compound represented by formula (B) can be deprotected by a method well known to those skilled in the art. (See, e.g., Peter GM Wuts, Theodora W. Greene, Greene's Protect see "Vital Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc." ).

[0240] R 2 When is a carboxy group protected with a benzyl group, this step is preferably carried out by the following reaction This can be done in a method.

[0241] The deprotection of the protecting group of the carboxy group of the compound represented by formula (B) can be carried out in any manner as long as the reaction proceeds. The method is not limited, but preferably, the reaction is carried out under a hydrogen atmosphere using a palladium catalyst, a platinum catalyst, or a nickel catalyst. The reaction can be carried out using a catalyst such as a ruthenium catalyst or a rhodium catalyst, and more preferably a palladium catalyst. This can be carried out using a catalyst, and even more preferably using palladium on carbon. More preferably, a palladium carbon-ethylenediamine complex can be used. The amount of palladium carbon-ethylenediamine complex used in this step is adjusted as long as the reaction proceeds. Although not limited thereto, it is preferable that the amount of the compound represented by formula (B) is 34 to 136% by weight. be.

[0242] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example of the solvent is a mixed solvent of tetrahydrofuran and water.

[0243] The reaction temperature in this step is preferably 10 to 40°C, but is not limited to this range as long as the reaction proceeds. The reaction time in this step is preferably 1 to 54 hours, but is not limited thereto as long as the reaction proceeds. This includes, but is not limited to:

[0244] Step 9: This step involves condensing a compound represented by formula (F) with a compound represented by formula (11). This is a process for converting the compound represented by formula (G) into a compound represented by formula (G).

[0245] The compound represented by formula (F) can be preferably converted to an active ester to give a compound represented by formula (1 The compound represented by formula (11) used in this step can be condensed with the compound represented by formula (1). The amount of the compound to be used is not limited as long as the reaction proceeds, but it is preferable to use a compound represented by formula (F) The amount of the compound is 0.7 to 1.3 equivalents. As long as the reaction proceeds, the method is not limited. For example, 1-ethyl-3-(3-dimethylamine) N,N'-dicyclohexane)carbodiimide hydrochloride (WSCD HCl) or N,N'-dicyclohexane Using a condensation agent such as dihydroxycarbodiimide (DCC), 1-hydroxybenzotriazole 1-Hydroxy-7-azabenzotriazole (HOAt), N-Hydroxybenzotriazole (HOBt), ethyl cyano(hydroxyimino)acetate, or p-nitrophenoxy This can be carried out by reacting with an additive such as 3-(3-dimethylpropyl)methylpropional. Aminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole The 3-(3-dimethylaminopropyl)carboxime used in this step The amount of amide hydrochloride is not limited as long as the reaction proceeds, but is preferably The amount of 1-hydroxybenzoate used in this step is 0.7 to 1.3 equivalents. The amount of benzotriazole is not limited as long as the reaction proceeds, but is preferably a compound represented by formula (F). The amount is 0.7 to 1.3 equivalents relative to the compound to be treated.

[0246] In this step, a base is preferably used. The base used in this step is not particularly limited as long as the reaction proceeds. There is no limitation to, for example, triethylamine, tributylamine, diisopropylamine amines, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, etc. Examples of the amines used in this step include triethylamine. The amount of triethylamine used is not limited as long as the reaction proceeds, but is preferably a compound represented by the formula (F): The amount is 0.7 to 1.3 equivalents relative to the compound represented by the formula:

[0247] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. The solvent can be selected from the group consisting of tetrahydrofuran, tetrahydrofuran being preferred.

[0248] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1 to 4 hours. is.

[0249] Step 10: In this step, the protecting group of the amino group in the compound represented by formula (G) is removed to obtain a compound represented by formula This is a process for converting the compound represented by formula (16).

[0250] The protecting group of the amino group of the compound represented by formula (G) can be deprotected by a method well known to those skilled in the art. (See, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective (See Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc.).

[0251] R 1 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. In some cases, this step can be preferably carried out in the following manner.

[0252] The deprotection of the protecting group of the amino group of the compound represented by formula (G) is not particularly restricted as long as the reaction proceeds. Examples include, but are not limited to, 1,8-diazabicyclo[5,4,0]-7-undecene, trime Chirguanidine, 1,5,7-triazabicyclo[4,4,0]dec-5-ene, or 7 -methyl-1,5,7-triazabicyclo[4,4,0]dec-5-ene 1,5-diaza Bicyclo[4,3,0]-5-nonene can be used, preferably 1,8-dia This can be done using zabicyclo[5,4,0]-7-undecene. The amount of 1,8-diazabicyclo[5,4,0]-7-undecene added is determined as long as the reaction proceeds. Although not limited thereto, it is preferable to use 0.5 to 2 equivalents of the compound represented by formula (15). be.

[0253] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. The solvent can be selected from the group consisting of tetrahydrofuran and tetrahydrofuran.

[0254] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1 to 5 hours. is.

[0255] Step 11: This step involves condensing a compound represented by formula (16) with a compound represented by formula (C). The compound represented by formula (C) is Commercially available products, products produced by known methods, or products produced by the following production method (VII) The compound prepared by the method described above can be used. -yl)oxycarbonyl group, this step can be preferably carried out by the following method. can.

[0256] The amount of the compound represented by formula (C) used in this step is not limited as long as the reaction proceeds. However, it is preferably 0.5 to 2 equivalents relative to the compound represented by formula (16).

[0257] In this step, a base is preferably used. The base used in this step is not particularly limited as long as the reaction proceeds. There is no limitation to, for example, triethylamine, tributylamine, diisopropylamine amines, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, etc. Examples of the amines used in this step include triethylamine. The amount of triethylamine used is not limited as long as the reaction proceeds, but it is preferably a triethylamine represented by the formula (16) The amount is 0.75 to 6 equivalents relative to the compound represented by the formula (I).

[0258] This step preferably further comprises using pyridinium p-toluenesulfonate. The amount of pyridinium p-toluenesulfonate used in this step is As long as the reaction proceeds, there is no limitation, but preferably, the reaction is carried out in a ratio of 1 to 4 with respect to the compound represented by formula (16). It is equivalent.

[0259] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Methyl-2-pyrrolidone, dimethyl sulfoxide, pyridine, and water, and mixtures thereof Suitable solvents include pyridine, acetonitrile, and tetrahydrofuran. A mixture of orchid solvents can be mentioned.

[0260] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1.5 to 7 It's time.

[0261] In the production methods (I) to (III), the compound represented by formula (B) is preferably The compound produced by the production method (IV) can be used.

[0262] [Manufacturing method (IV)] In the production method (IV), a compound represented by formula (H) is converted to a compound represented by formula (B) through steps 12 to 15. Steps 12 to 15 are explained in detail below. .

[0263] [ka]

[0264] [In the formula, R 1 represents an amino group protected with a protecting group, and is preferably a benzyloxycarbonyl an amino group protected by a (9H-fluoren-9-ylmethoxy)carbonyl group; indicates R 2 represents a carboxy group protected by a protecting group, preferably a carboxy group protected by a benzyl group. indicates the carboxyl group, and R 3 represents an amino group protected with a protecting group, and preferably represents an amino group protected with a protecting group. -fluoren-9-ylmethoxy) carbonyl-protected amino group, and X is an active It represents an ester group or a carboxy group, and is preferably (2,5-dioxopyrrolidine-1- represents a (hydroxy)carbonyl group. Step 12: In this step, a compound represented by formula (H) is reacted with lead tetraacetate to give a compound represented by formula (J): The compound represented by formula (H) is commercially available or Lead tetraacetate produced by a known method can be used. The amount is not limited as long as the reaction proceeds, but is preferably in the range of 100 to 1500 ppm relative to the compound represented by formula (H). The amount is 1 to 3 equivalents.

[0265] This step can be preferably carried out in the presence of acetic acid or pyridine, more preferably It can be carried out in the presence of acetic acid.

[0266] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyl ethyl tetrahydrofuran, 1,4-dioxane, ethyl acetate, N,N-dimethylformamide amide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, and dimethyl sulfone and mixed solvents thereof, preferably tetrahydrofuran. Examples include:

[0267] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 45 to 85°C. The reaction temperature in this step is preferably a temperature at which tetrahydrofuran is heated to reflux. The reaction time is not limited as long as the reaction proceeds, but is preferably 0.5 to 3 hours.

[0268] Step 13: In this step, a compound represented by formula (J) and a compound represented by formula (K) are reacted in the presence of an acid or a base. This step converts the compound represented by formula (L) into a compound represented by formula (L) by reacting the compound in the presence of The amount of the compound represented by formula (K) used in the step (a) is not limited as long as the reaction proceeds. Preferably, it is 1 to 4 equivalents relative to the compound represented by formula (J).

[0269] This step can be carried out in the presence of a base or an acid. The aqueous sodium hydroxide solution used in this step is preferably an aqueous sodium hydroxide solution. The amount is not limited as long as the reaction proceeds, but is preferably The acid used in this step is preferably tris(pentafluoro) The compound used in this process is tris(pentafluorophenyl)borane. The amount of the amine is not limited as long as the reaction proceeds, but it is preferably In contrast, the amount is 0.01 to 0.1 equivalents.

[0270] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydro Furan and 1,4-dioxane are preferred, and 1,2-dimethoxyethane is preferred. Tan is one example.

[0271] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably −10 to 15° C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 minutes. It's 6 hours.

[0272] Step 14: In this step, the protecting group of the amino group in the compound represented by formula (L) is removed to obtain a compound represented by formula (M)

[0273] The protecting group of the amino group of the compound represented by formula (L) can be deprotected by a method well known to those skilled in the art. (See, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective (See Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc.).

[0274] R 3 is an amino group protected with a (9H-fluoren-9-ylmethoxy)carbonyl group. In some cases, this step can be preferably carried out in the following manner. The deprotection of the amino protecting group of the compound represented by formula (L) is not particularly restricted as long as the reaction proceeds. Examples include, but are not limited to, 1,8-diazabicyclo[5,4,0]-7-undecene, trime Chirguanidine, 1,5,7-triazabicyclo[4,4,0]dec-5-ene, or 7 -methyl-1,5,7-triazabicyclo[4,4,0]dec-5-ene 1,5-diaza Bicyclo[4,3,0]-5-nonene can be used, preferably 1,8-dia This can be done using zabicyclo[5,4,0]-7-undecene. The amount of 1,8-diazabicyclo[5,4,0]-7-undecene added is determined as long as the reaction proceeds. Although not limited thereto, it is preferable to use 0.25 to 1 equivalent amount of the compound represented by formula (L). be.

[0275] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. Suitable solvents include acetonitrile and N,N-dimethylacetamide. It is possible.

[0276] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours. is.

[0277] The compound represented by formula (M) can be precipitated from the reaction solution by forming a salt with an acid, and preferably This allows for the efficient isolation and purification of by-products that may inhibit the reaction in the subsequent steps. The product can be removed. The acid is preferably 1-hydroxybenzotriazole. Hydroxybenzotriazole serves as one of the condensing agents in the next step 15. Similarly, acids other than 1-hydroxybenzotriazole can also be used as condensing agents. Any substance that functions as such can be suitably used in this step.

[0278] Step 15: This step is carried out by condensing a compound represented by formula (M) with a compound represented by formula (N). The compound represented by formula (N) is commercially available. The commercially available product, the product produced by a known method, or the product produced by the following production method (VIII) The compound represented by formula (N) used in this step can be prepared by the method The amount of the compound is not limited as long as the reaction proceeds, but it is preferable to use a compound represented by formula (M) The amount is 0.7 to 1.3 equivalents relative to the substance.

[0279] The compound represented by formula (M) can be preferably converted to an active ester to give a compound represented by formula (N The derivative into an active ester can be, for example, condensed with a compound represented by 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD·HC l), or using a condensing agent such as N,N'-dicyclohexylcarbodiimide (DCC), 1-Hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole Azoles (HOAt), N-hydroxysuccinimide, cyano(hydroxyimino)acetic acid This can be achieved by reacting the compound with an additive such as ethyl acetate or p-nitrophenol. Preferably, 3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzoyl This can be done using 3-(3-dimethylbenzotriazole). The amount of (ethylaminopropyl)carbodiimide hydrochloride is not limited as long as the reaction proceeds. However, it is preferably 0.7 to 1.3 equivalents relative to the compound represented by formula (5). The amount of 1-hydroxybenzotriazole used in the process is not limited as long as the reaction proceeds. However, it is preferably 0.7 to 1.3 equivalents relative to the compound represented by formula (M).

[0280] Furthermore, when the compound represented by formula (M) is a 1-hydroxybenzotriazole salt, Preferably, this step is carried out without adding any additional 1-hydroxybenzotriazole. This can be done.

[0281] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example of the solvent is a mixed solvent of acetonitrile and water.

[0282] In step 14, the compound represented by formula (M) may be continuously subjected to this step without isolating it. When this step is carried out, the solvent used in step 14 can be used as it is in this step. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably −10 to 15° C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1.5 to 1.5 minutes. It's 7 hours.

[0283] In a more specific embodiment, the compound represented by formula (1) is preferably produced by the following production method: The compound prepared by (V) or (VI) can be used.

[0284] [Manufacturing method (V)] The production method (V) is a method for producing a compound represented by formula (1) from a compound represented by formula (2) through steps 16 to 22. Steps 16 to 22 are explained in detail below.

[0285] [ka]

[0286] Step 16: In this step, the compound represented by formula (2) is reacted with lead tetraacetate to give the compound represented by formula (3): The compound represented by formula (2) is commercially available or This step is the same as in the production method (IV). This can be done in the same manner as in step 12.

[0287] Step 17: In this step, the compound represented by formula (3) is reacted with benzoyl glycolate in the presence of an acid or a base. This step converts the compound into a compound represented by formula (4). This can be carried out in the same manner as in step 13 of production method (IV).

[0288] Step 18: In this step, the protecting group of the amino group in the compound represented by formula (4) is removed to obtain a compound represented by formula This step is the same as step 14 of the production method (IV). This can be done in the same manner as above.

[0289] Step 19: This step involves condensing a compound represented by formula (5) with a compound represented by formula (6). The compound represented by formula (6) is converted into a compound represented by formula (7) by the following reaction: It is commercially available, produced by a known method, or produced by the following production method (VIII). This step can be carried out in the same manner as step 15 of production method (IV). This can be done by:

[0290] Step 20: This step is carried out to remove the protecting groups of the amino and carboxy groups of the compound represented by formula (7). This step is a step of converting the compound represented by formula (8) into a compound represented by formula (8). This can be carried out in the same manner as in step 1 of I).

[0291] Step 21: This step involves condensing a compound represented by formula (8) with a compound represented by formula (9). This step is a step of converting the compound represented by formula (10) into a compound represented by formula (10) in the production method (I). This can be done in the same manner as in step 2.

[0292] Step 22: This step involves condensing a compound represented by formula (10) with a compound represented by formula (11). This step is a process for converting the compound represented by formula (1) into a compound represented by formula (1). This can be carried out in the same manner as in step 3 of the above.

[0293] [Manufacturing method (VI)] In the production method (VI), a compound represented by formula (2) is converted to a compound represented by formula (1) through steps 23 to 30. Steps 23 to 30 will be explained in detail below. .

[0294] [ka]

[0295] Step 23: In this step, the compound represented by formula (2) is reacted with lead tetraacetate to give the compound represented by formula (3): The compound represented by formula (2) is commercially available or This step is the same as that of the production method (IV). This can be done in the same manner as in step 12.

[0296] Step 24: In this step, the compound represented by formula (3) is reacted with benzyl glycolate in the presence of an acid or a base. This step converts the compound represented by formula (4) into a compound represented by formula (4) by reacting the compound with This can be carried out in the same manner as in step 13 of production method (IV).

[0297] Step 25: In this step, the protecting group of the amino group in the compound represented by formula (4) is removed to obtain a compound represented by formula This step is the same as step 14 of the production method (IV). This can be done in the same manner as above.

[0298] Step 26: This step involves condensing a compound represented by formula (5) with a compound represented by formula (12). This is a process for converting the compound represented by formula (13) into a compound represented by formula (13). Commercially available products or products produced by known methods can be used. This can be carried out in the same manner as in step 15 of the production method (IV).

[0299] Step 27: This step involves deprotecting the protecting group of the carboxy group of the compound represented by formula (13). This step is a step of converting the compound represented by formula (14) into a compound represented by formula (14). This can be done in the same manner as in step 8 of the previous paragraph.

[0300] Step 28: This step involves condensing a compound represented by formula (14) with a compound represented by formula (11). This step is a step of converting the compound represented by formula (15) into a compound represented by formula (15). This can be carried out in the same manner as in step 9 of II).

[0301] Step 29: This step involves deprotecting the protecting group of the amino group of the compound represented by formula (15), This step is a step of converting the compound represented by formula (16) into a compound represented by formula (16). This can be done in the same manner as in step 10.

[0302] Step 30: This step involves condensing a compound represented by formula (16) with a compound represented by formula (9). This step is a step of converting the compound represented by formula (1) into a compound represented by formula (1) by the following method. This can be carried out in the same manner as in step 11 of the above.

[0303] [Manufacturing method (VII)] The compound represented by formula (9) is preferably produced by the following production method (VII): This will not affect the quality of the compound produced in the subsequent steps. It is possible to suppress impurities that may cause adverse reactions, and obtain a high-quality compound represented by formula (1). This can contribute to profits.

[0304] [ka]

[0305] Step 31: In this step, a compound represented by formula (17) is condensed with maleic anhydride to obtain a compound represented by formula ( 18) The amount of maleic anhydride used in this step is not limited as long as the reaction proceeds, but is preferably The amount is 0.7 to 1.3 equivalents.

[0306] This step is preferably carried out in acetic acid.

[0307] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 80 to 120°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 8 to 32 It's time.

[0308] Step 32: This step involves condensing the compound represented by formula (18) with N-hydroxysuccinimide. This is a process for converting the N-hydroxybenzoate used in this process into a compound represented by formula (9). The amount of hydroxysuccinimide is not limited as long as the reaction proceeds, but it is preferably a compound represented by the formula (1 7) is 0.7 to 1.3 equivalents relative to the compound represented by formula (7).

[0309] The compound represented by formula (18) may be an active ester, a mixed acid anhydride, an acid halide, or the like. can be condensed with N-hydroxysuccinimide by derivatizing , by derivatizing it into an acid halide, and condensing it with N-hydroxysuccinimide. can be done.

[0310] The derivatization into the acid halide can be preferably carried out using thionyl chloride. The amount of thionyl chloride used in this step is not limited as long as the reaction proceeds, but it is preferably a thionyl chloride represented by the formula The amount is 0.5 to 1.5 equivalents relative to the compound represented by (18). The base used in this step is preferably 2,6-lutidine. The amount of 2,6-lutidine to be used is not limited as long as the reaction proceeds, but it is preferably a compound represented by the formula (1 8) is 1 to 3 equivalents relative to the compound represented by formula (8).

[0311] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, diethyl ether, 1, 2-Dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene toluene, chlorobenzene, and mixed solvents thereof. Examples of the solvent include acetonitrile.

[0312] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably −25° C. to 0° C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 10 minutes. It's 2 hours.

[0313] [Manufacturing method (VIII)] The compound represented by formula (6) was prepared by the following production method (VIII). It is possible.

[0314] [ka]

[0315] Step 33: This step involves condensing the compound represented by formula (23) with N-hydroxysuccinimide. This is a process for converting the compound of formula (24) into a compound of formula (24). The amount of the compound represented by formula (23) is not limited as long as the reaction proceeds. The amount is 0.7 to 1.5 equivalents relative to the compound represented by formula (3).

[0316] The compound represented by formula (23) may be an active ester, a mixed acid anhydride, an acid halide, or the like. can be condensed with N-hydroxysuccinimide by derivatizing By deriving the activated ester, it can be condensed with N-hydroxysuccinimide. This can be done.

[0317] The active esterification is preferably carried out using 3-(3-dimethylaminopropyl)carbodiimide. The 3-(3-dimethylaminopropyl) The amount of carbodiimide hydrochloride is not limited as long as the reaction proceeds. The amount is 0.7 to 1.5 equivalents relative to the compound represented by (23).

[0318] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example is acetonitrile.

[0319] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours. is.

[0320] Step 34: This step involves condensing the compound represented by formula (24) with L-phenylalanine to form This is a process for converting the L-phenyl benzoate used in this process into a compound represented by formula (6). The amount of alanine is not limited as long as the reaction proceeds, but is preferably alanine represented by formula (24): The amount is 0.7 to 1.3 equivalents relative to the compound.

[0321] In this step, a base is preferably used. The base used in this step is not particularly limited as long as the reaction proceeds. There is no limitation to, for example, triethylamine, tributylamine, diisopropylamine amines, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, etc. Examples of the amines used in this step include triethylamine. The amount of triethylamine used is not limited as long as the reaction proceeds, but it is preferably a triethylamine represented by the formula (24 The amount is 0.7 to 1.3 equivalents relative to the compound represented by the formula (I).

[0322] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example of the solvent is a mixed solvent of acetonitrile and water.

[0323] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably a temperature The reaction time of this step is not limited as long as the reaction proceeds. is 1 to 4 hours.

[0324] [Manufacturing method (IX)] The compound represented by formula (1) is produced by the following production method (IX). It is also possible.

[0325] [ka]

[0326] [In the formula, R 1 represents an amino group protected by a protecting group, and R 2 represents a carboxyl group protected by a protecting group. represents a silyl group, and R 3 represents an amino group protected by a protecting group, and X represents an active ester group or a carboxyl group. It represents a carboxy group, and preferably represents a (2,5-dioxopyrrolidin-1-yl)oxycarbonyl group. represents a methyl group.]

[0327] Step 35: In this step, a compound represented by formula (H) is reacted with lead tetraacetate to give a compound represented by formula (J): The compound represented by formula (H) is commercially available or a publicly available compound. This step is the same as that of the production method (IV). This can be done in the same manner as in step 12.

[0328] Step 36: In this step, a compound represented by formula (J) is reacted with a compound represented by formula (K) in the presence of an acid or a base. This is a process for converting the compound represented by formula (L) into a compound represented by formula (L). This step can be carried out in the same manner as in step 13 of production method (IV).

[0329] Step 37: This step is carried out by deprotecting the protecting group of the carboxy group of the compound represented by formula (L). , a process of converting the compound represented by formula (O)

[0330] The protecting group of the carboxy group of the compound represented by formula (L) can be deprotected by a method well known to those skilled in the art. (See, e.g., Peter GM Wuts, Theodora W. Greene, Greene's Protect see "Vital Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc." ).

[0331] Step 38: This step involves condensing a compound represented by formula (O) with a compound represented by formula (11). This is a process of converting the compound represented by formula (P) into a compound represented by formula (P).

[0332] The compound represented by formula (O) can be preferably converted to an active ester to give a compound represented by formula (1 1) can be condensed with the compound represented by the formula:

[0333] Step 39: In this step, the protecting group of the amino group in the compound represented by formula (P) is removed to obtain a compound represented by formula This is a process for converting the compound represented by formula (25).

[0334] The protecting group of the carboxy group of the compound represented by formula (P) can be deprotected by a method well known to those skilled in the art. (See, e.g., Peter GM Wuts, Theodora W. Greene, Greene's Protect see "Vital Groups in Organic Synthesis 4th Edition (2007), Wiley-Interscience, etc." ).

[0335] Step 40: This step involves condensing a compound represented by formula (25) with a compound represented by formula (N). This is a process for converting the compound represented by formula (G) into a compound represented by formula (G).

[0336] The compound represented by formula (N) can be preferably converted to an active ester to give a compound represented by formula (2 5) can be condensed with a compound represented by the formula:

[0337] Step 41: In this step, the protecting group of the amino group in the compound represented by formula (G) is removed to obtain a compound represented by formula This step is a step of converting the compound represented by formula (16) into a compound represented by formula (16). This can be done in the same manner as in 10.

[0338] Step 42: This step involves condensing a compound represented by formula (16) with a compound represented by formula (C). This step is a step of converting the compound represented by formula (1) into a compound represented by formula (1) by the following method. This can be carried out in the same manner as in step 11 of the above.

[0339] [Antibodies used in the production of antibody-drug conjugates] The antibodies used to produce the antibody-drug conjugates of the present invention may be derived from any species. The antibodies may be derived from any of various origins, but are preferably derived from humans, rats, mice, and rabbits. If derived from a species other than human, it is preferred to chimerize or humanize it using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Although antibodies may be used, monoclonal antibodies are preferred.

[0340] The antibody used to produce the antibody-drug conjugate of the present invention preferably targets cancer cells. It has the property of being able to recognize cancer cells and bind to them. characteristics, ability to be taken up and internalized by cancer cells, and / or cytocidal activity against cancer cells It is preferable that the material has properties such as

[0341] The binding of antibodies to cancer cells can be confirmed using flow cytometry. Antibody uptake into the cell can be achieved by (1) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody. Cell Death and Differentiation Assay (Cell Death and Differentiation Assay) on (2008) 15, 751-761), (2) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody to detect the cells. Assay to measure the amount of fluorescence taken up into the vesicles (Molecular Biology of the Cell Vol. 1 5, 5268-5282, December 2004), or (3) using immunotoxins that bind to therapeutic antibodies. When taken up into cells, Mab-ZA releases toxins that inhibit cell proliferation. This can be confirmed using the P assay (BioTechniques 28:162-165, January 2000). The toxin is a recombinant complex of the catalytic domain of diphtheria toxin and protein G. Proteins can also be used.

[0342] Anti-tumor activity of antibodies can be assessed in vitro by measuring their ability to inhibit cell proliferation. For example, a cancer cell line that overexpresses the antibody's target protein can be cultured and Antibodies were added at various concentrations to the cells, and the effects on focus formation, colony formation, and spheroid growth were evaluated. In vivo, for example, the inhibitory activity of a target protein can be measured. The antibody is administered to nude mice transplanted with the cancer cell line expressing the antibody, and changes in the cancer cells are measured. By this, antitumor activity can be confirmed.

[0343] It is preferable that the antibody itself has an anti-tumor effect, but the antibody-drug conjugate may also have an anti-tumor effect. Since the antibody is bound to a compound that exerts an antitumor effect, the antibody itself does not necessarily have an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxicity of antitumor compounds on cancer cells, It is important and preferred that the antibody has the property of being internalized and transported into cancer cells.

[0344] The antibody used to produce the antibody-drug conjugate of the present invention can be obtained by known means. For example, antigenic polynucleotides can be prepared by a method commonly used in the art. It is obtained by immunizing an animal with the peptide and collecting and purifying the antibodies produced in the body. The origin of the antigen is not limited to humans, but can be derived from animals other than humans, such as mice and rats. In this case, the antigen that binds to the obtained heterologous antigen can be used to immunize animals. Antibodies applicable to human diseases are selected by testing their cross-reactivity with human antigens. can.

[0345] Also, known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Ke nnet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)). By fusing antibody-producing cells that produce antibodies against antigens with myeloma cells, It is also possible to establish hybridomas and obtain monoclonal antibodies.

[0346] The antigen is produced by genetically manipulating the gene encoding the antigen protein in the host cell. Specifically, a vector capable of expressing an antigen gene is prepared. This can then be introduced into host cells to express the gene, and the expressed antigen can be purified. Immunizing animals with the antigen-expressing cells or cell lines expressing the antigens obtained by the above genetic manipulation. Antibodies can also be obtained by using the method.

[0347] The antibody used to produce the antibody-drug conjugate of the present invention is a heterologous antigen for humans. Recombinant antibodies that have been artificially modified to reduce their sensitivity, for example, Preferably, the antibody is a chimeric antibody or a humanized antibody. or an antibody having only the gene sequence of a human-derived antibody, i.e., a human antibody. These antibodies can be produced using known methods.

[0348] Chimeric antibodies include antibodies in which the variable and constant regions of the antibody are heterologous, such as mouse antibodies. Examples of such antibodies include chimeric antibodies in which the variable regions of a mouse or rat-derived antibody are joined to constant regions of human origin. (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0349] Humanized antibodies are characterized by the complementarity determining regions (CDRs) of heterologous antibodies. The antibody was developed by incorporating only the ity determining region (IL-1) into a human-derived antibody. (Nature (1986) 321, p. 522-525), the CDR sequence of a heterologous antibody is grafted by CDR grafting. In addition, some framework amino acid residues of the heterologous antibody are also transplanted into a human antibody ( WO 90 / 07861, gene conversion mutagenesis Antibodies humanized using a ion mutagenesis strategy (U.S. Patent No. 5,649,496) No. 821337).

[0350] Human antibodies include those derived from humans having human chromosome fragments containing the heavy and light chain genes of human antibodies. Antibodies generated using antibody-producing mice (Tomizuka, K. et al., Nature Genetics (1997) 1 6, p.133-143;Kuroiwa, Y. et. al., Nucl. Acids Res.(1998) 26, p.3447-3448;Yoshida , H. et. al., Animal Cell Technology:Basic and Applied Aspects vol.10, p.69-73(K itagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; (See Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.) Alternatively, a phage display selected from a human antibody library can be used. The antibody obtained by the method (Wormstone, IM et al., Investigative Ophthalmology & Vis (2002)43 (7), p.2301-2308;Carmen, S. et. al., Briefings in Function al Genomics and Proteomics(2002), 1(2), p.189-203;Siriwardena, D. et. al., Ophth See, for example, Almology (2002) 109(3), pp. 427-431.

[0351] The antibodies used to produce the antibody-drug conjugates of the present invention also include modified antibodies. The modified antibody is an antibody according to the present invention that has been chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, N-bonds or O-bonds. - Chemical modifications include those with chemical moieties attached to the carbohydrate chain. The body contains post-translational modifications (e.g., addition of N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, etc. By expressing this in a prokaryotic host cell, a methionine residue is added to the N-terminus. In addition, the present invention also includes those that enable the detection or isolation of the antibody or antigen of the present invention. Such modifications include those labeled with an enzyme label, a fluorescent label, and an affinity label. Such modified antibodies according to the present invention are included in the meaning of the term "antibody." The modified antibodies according to the present invention are intended to improve the stability and circulation of the antibodies. These are useful for improving the affinity, reducing the antigenicity, detecting or isolating antibodies or antigens, etc.

[0352] Furthermore, it is possible to regulate sugar chain modification (glycosylation, decarboxylation) of the antibody of the present invention. Antibody-dependent cellular cytotoxicity can be enhanced by glycosylation of antibodies. Techniques for controlling chain modification include those described in WO 99 / 54342 and WO 00 / 6173 No. 9, International Publication No. 02 / 31140, etc. are known, but are not limited to these. The antibodies according to the present invention also include antibodies in which the glycosylation has been regulated.

[0353] In addition, antibodies produced in mammalian cultured cells have a lysine residue at the carboxyl terminal of the heavy chain. It is known that the hydroxyl group is deleted (Journal of Chromatography A, 705: 129-134 (1995) ), and also, two amino acid residues, glycine and lysine, are deleted at the carboxyl terminal of the heavy chain. It is known that the proline residue at the carboxyl terminus is newly amidated ( Analytical Biochemistry, 360: 75-83(2007)). However, the deletion and Modifications affect the antigen-binding ability and effector functions of antibodies (complement activation and antibody-dependent cellular cytotoxicity). Therefore, the antibody of the present invention does not have any effect on the antibody's function (use, etc.). Also included are antibodies and functional fragments of such antibodies, which have one or two amino acids at the carboxyl terminus of the heavy chain. Deletions in which the acid is deleted and amidated deletions (e.g., carboxyl-terminal However, the antigen-binding ability and the effector - As long as the function is maintained, the carboxyl-terminal deletion of the heavy chain of the antibody according to the present invention can be carried out in the manner described above. The two heavy chains constituting the antibody of the present invention are not limited to the type. The heavy chain may be any one of heavy chains selected from the group consisting of α- and β-blockers, or a combination of any two of heavy chains. The ratio of the amounts of each deletion fragment is determined based on the mammalian culture medium producing the antibody of the present invention. Although the type of cultured cells and culture conditions may affect the antibody of the present invention, it is preferable to use two antibodies. List those in which one amino acid residue is deleted at the carboxyl terminus of both heavy chains. can be done.

[0354] The isotype of the antibody of the present invention may be, for example, IgG (IgG1, IgG2, Ig Examples of the antibody include IgG1 and IgG2. It is possible.

[0355] The antibodies that can be used to produce the antibody-drug conjugates of the present invention are not particularly limited, but examples include: For example, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD 3 antibodies, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD9 8 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FG Examples of antibodies include anti-FR4 antibodies, anti-FOLR1 antibodies, anti-VEGF antibodies, and anti-GPR20 antibodies. and preferably an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, Examples of antibodies include anti-GPR20 antibodies.

[0356] In the present invention, the term "anti-HER2 antibody" refers to an antibody against HER2 (Human Epidermal Growth Factor-2) l Growth Factor Receptor Type 2; ErbB-2) and preferably binds to HER2, thereby being internalized in HER2-expressing cells. The figures show antibodies with localizing activity.

[0357] Examples of anti-HER2 antibodies include trastuzumab (US Patent No. 5821337) and pertuzumab (International Publication No. No. 01 / 00245), and preferably trastuzumab. do.

[0358] In the present invention, "trastuzumab" refers to the amino acid sequence of SEQ ID NO: 1 (FIG. 1). a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 2) to 449; A humanized anti-HER2 monoclonal antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NOs. 1 to 214. It is a clonal antibody.

[0359] In the present invention, the term "anti-HER3 antibody" refers to an antibody against HER3 (Human Epidermal Growth Factor-3) l Growth Factor Receptor Type 3; ErbB-3) and preferably binds to HER3 on the surface of HER3-expressing cells to bind to the HER3. Antibodies with the activity of being internalized into expressing cells are shown.

[0360] Examples of anti-HER3 antibodies include patritumab (U3-1 287), U1-59 (International Publication No. 2007 / 077028), MM-121 (ser ibantumab), the anti-ERBB3 antibody described in WO 2008 / 100624, R G-7116 (lumretuzumab) and LJM-716 (elgemtuma b) and preferably include patritumab and U1-59. do.

[0361] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor r-associated calcium signal transducer 2 ; EGP-1) and preferably binds to TROP2, thereby 1 shows antibodies that have the activity of being internalized into ROP2-expressing cells.

[0362] Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. 2015 / 003366). No. 098099).

[0363] In the present invention, the term "anti-B7-H3 antibody" refers to a B7-H3 (B cell antibody PD-L3; CD276) and specifically binds to Preferably, the antibody has the activity of being internalized into B7-H3-expressing cells by binding to B7-H3. The antibodies shown are:

[0364] Examples of anti-B7-H3 antibodies include M30-H1-L4 (International Publication No. 2014 / 05 No. 7687).

[0365] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that binds to GPR20 (G Protein-coated Specifically binds to GPR20, preferably GPR20. The figures show antibodies that have the activity of being internalized into GPR20-expressing cells when combined with GPR20. Examples of anti-GPR20 antibodies include h046-H4e / L7 (International Publication No. 2018 / 003366). No. 135501).

[0366] [Conjugation of antibodies and drug-linker intermediates] The antibody-drug conjugate of the present invention comprises a compound represented by formula (1) and a thiol group (or It can be produced by reacting an antibody with a hydroxyl group (also called a sulfhydryl group). Cut.

[0367] The crystals of the compound represented by formula (1) of the present invention are preferably dissolved in a solvent and then crystalline The solution containing the compound to be reacted can be used in the reaction. The solvent is not particularly limited as long as it does not inhibit the reaction, but dimethyl sulfoxide is preferred. sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone A solvent containing dimethyl sulfoxide can be used, and more preferably, a solvent containing dimethyl sulfoxide can be used. This can be done.

[0368] Antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press(1996)). For example, a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 0.3 to 3 molar equivalents per intra-chain disulfide of antibody are used, and ethylenediamine By reacting with the antibody in a buffer containing a chelating agent such as EDTA, Antibodies with partially or completely reduced interchain disulfides and sulfhydryl groups can be obtained.

[0369] Furthermore, 2 to 20 molar equivalents of a compound represented by formula (1) per antibody having a sulfhydryl group are used. Using the compounds shown in the above, antibody-drug complexes with 2 to 8 drugs bound to each antibody were prepared. Conjugates can be produced.

[0370] The average number of drugs bound per antibody molecule of the produced antibody-drug conjugate can be calculated using the following formula: For example, antibody-drug conjugates and their conjugates at two wavelengths of 280 nm and 370 nm. The UV method is used to calculate the denaturation rate by measuring the UV absorbance of the denaturation precursor. The compound-drug conjugate was treated with a reducing agent, and each of the resulting fragments was analyzed by HPLC. This can be done by a method of quantitative determination and calculation (HPLC method).

[0371] Conjugation of the antibody with a drug linker intermediate (a compound represented by formula (1)), and The average number of drugs bound per antibody molecule in an antibody-drug conjugate was calculated according to the method described in International Publication No. 2002 / 002444. 014 / 057687, International Publication No. 2015 / 098099, International Publication No. 2015 / 115091, WO 2015 / 155998, and WO 2018 / 13 This can be done by referring to the descriptions in No. 5501 etc.

[0372] In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate. Antibody-drug conjugates in which the antibody in the gate is an anti-HER2 antibody are shown.

[0373] The anti-HER2 antibody preferably comprises the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1. a heavy chain consisting of the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence of SEQ ID NO: 1 to 214; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1; and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:2.

[0374] Drug per antibody in the anti-HER2 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 8, and More preferably, it is 7 to 8, even more preferably, it is 7.5 to 8, and even more preferably, it is It is about 8.

[0375] The anti-HER2 antibody-drug conjugate is a compound represented by the formula (1) produced by the production method of the present invention. Using a crystal of a compound represented by the formula (I), the following is obtained by referring to the description in International Publication No. 2015 / 115091, etc. can be manufactured in

[0376] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate. Antibody-drug conjugates in which the antibody in the gate is an anti-HER3 antibody are shown.

[0377] The anti-HER3 antibody preferably comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a An antibody comprising a light chain consisting of the amino acid sequence set forth in No. 4, or a heavy chain carbohydrate of said antibody. It is an antibody lacking the lysine residue at the xyl terminus.

[0378] Drug per antibody in the anti-HER3 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 8, and More preferably, it is 7 to 8, even more preferably, it is 7.5 to 8, and even more preferably, it is It is about 8.

[0379] The anti-HER3 antibody-drug conjugate is a compound represented by the formula (1) produced by the production method of the present invention. Using a crystal of a compound represented by the formula (I), the following is obtained by referring to the description in International Publication No. 2015 / 155998 etc. can be manufactured in

[0380] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate. 1 shows an antibody-drug conjugate in which the antibody in the conjugate is an anti-TROP2 antibody.

[0381] The anti-TROP2 antibody preferably has a sequence similar to that shown in amino acids 20 to 470 of SEQ ID NO:5. and a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 6 at amino acid numbers 21 to 234. An antibody comprising a light chain consisting of the amino acid sequence set forth above, or the carboxyl terminal of the heavy chain of said antibody. It is an antibody in which the lysine residue is deleted.

[0382] The amount of drug per antibody in the anti-TROP2 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 5, and More preferably, it is 3.5 to 4.5, and even more preferably, it is about 4.

[0383] The anti-TROP2 antibody-drug conjugate is a compound of the formula (1) produced by the production method of the present invention. ) and the like. It can be manufactured in a convenient manner.

[0384] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate. 1 shows an antibody-drug conjugate in which the antibody in the conjugate is an anti-B7-H3 antibody.

[0385] The anti-B7-H3 antibody preferably comprises the amino acid sequence set forth in SEQ ID NO:7 from amino acid number 20 to 471. and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8, which is represented by amino acid numbers 21 to 233. An antibody comprising a light chain consisting of the amino acid sequence set forth above, or the carboxyl terminal of the heavy chain of said antibody. It is an antibody in which the lysine residue is deleted.

[0386] The drug content per antibody of the anti-B7-H3 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 5, and More preferably, it is 3.5 to 4.5, and even more preferably, it is about 4.

[0387] The anti-B7-H3 antibody-drug conjugate is a compound of the formula (1) produced by the production method of the present invention. ) and the like. It can be manufactured in a convenient manner.

[0388] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate. 1 shows an antibody-drug conjugate in which the antibody in the conjugate is an anti-GPR20 antibody.

[0389] The anti-GPR20 antibody preferably comprises a sequence corresponding to amino acid numbers 20 to 472 in SEQ ID NO:9. a heavy chain consisting of the amino acid sequence set forth above and a heavy chain consisting of amino acid sequences 21 to 234 in SEQ ID NO: 10; An antibody comprising a light chain consisting of the amino acid sequence described above, or the heavy chain carboxyl terminal of said antibody. This is an antibody in which the terminal lysine residues are deleted.

[0390] The drug content per antibody of the anti-GPR20 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 8, and More preferably, it is 7 to 8, even more preferably, it is 7.5 to 8, and even more preferably, it is is about 8.

[0391] The anti-GPR20 antibody-drug conjugate is a compound of the formula (1) produced by the production method of the present invention. ) using a crystal of a compound represented by the formula (I) and referring to the description in WO 2018 / 135501 etc. It can be manufactured in a convenient manner.

[0392] [Pharmaceutical composition] The antibody-drug conjugates produced according to the present invention may be prepared by administering to the patient one or more pharmaceutically compatible The pharmaceutically compatible component can be administered in the form of an antibody-drug mixture prepared according to the present invention. Depending on the dosage and administration concentration of the compound conjugate, a formulation commonly used in this field may be used. Additives and other additives can be appropriately selected and applied. For example, The antibody-drug conjugate may be prepared by adding a buffer such as a histidine buffer, sucrose or trehalose. and surfactants such as polysorbate 80 or polysorbate 20. The compound may be administered as a pharmaceutical composition containing the compound.

[0393] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be administered to a patient. In addition to being applied as a systemic therapy, it can also be applied locally to cancer tissues to expect therapeutic effects. can.

[0394] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be administered to a mammal. It can be preferably used for animals, but more preferably for humans. .

[0395] The pharmaceutical composition comprising the antibody-drug conjugate produced by the present invention is preferably It can be used as an injection, more preferably as an aqueous injection or a freeze-dried injection. It can be used as a lyophilized injection, and more preferably, it can be used as a lyophilized injection.

[0396] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention is an aqueous injection. In some cases, it may be preferably administered intravenously after dilution with an appropriate diluent. Examples of diluents include glucose solution (preferably 5% glucose solution) and physiological saline solution. It is possible.

[0397] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention is freeze-dried and can be used for injection. In the case of a pharmaceutical preparation, it is preferably dissolved in water for injection, and then diluted with an appropriate diluent to the required amount. After that, it can be administered intravenously by drip infusion. As a diluent, a glucose solution (preferably 5% Examples of the solution include glucose solution and physiological saline.

[0398] For administering a pharmaceutical composition containing an antibody-drug conjugate produced according to the present invention. Routes of introduction that can be used include, for example, intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal. The route may be mentioned, preferably the intravenous route.

[0399] The antibody-drug conjugates produced according to the present invention can be administered to humans for 1 to 180 days. The dose may be administered once every week, two weeks, three weeks, or four weeks. It can be administered at intervals, and even more preferably, at intervals of once every three weeks. Furthermore, the antibody-drug conjugates produced according to the present invention can be administered in a single dose of approximately 0. It can be administered at a dose of 0.001 to 100 mg / kg, preferably 0.8 mg / kg per dose. The antibody produced by the present invention can be administered at a dose of up to 12.4 mg / kg. When the drug conjugate is an anti-HER2 antibody-drug conjugate, preferably It can be administered at a dose of 5.4, 6.4, or 7.4 mg / kg per dose, and even more preferably More preferably, it is administered at a dose of 5.4 mg / kg or 6.4 mg / kg per dose. can be done.

[0400] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be used in the treatment of cancer. It can be used for the treatment of breast cancer, gastric cancer (sometimes called gastric adenocarcinoma), and the like. , colon cancer (sometimes called colorectal cancer, including colon cancer and rectal cancer), lung cancer ( including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma , bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate Adenocarcinoma, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal Liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, uterine cancer, kidney cancer, Vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neurological Epithelial tissue tumors, nerve sheath tumors, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer It is used for the treatment of at least one cancer selected from the group consisting of carcinoma, mesothelioma, and sarcoma. For example, the antibody-drug conjugate produced by the present invention can be used as an anti-H In the case of an ER2 antibody-drug conjugate, it is more preferably an ER2 antibody-drug conjugate for use in treating breast cancer, gastric cancer, or colon cancer. , non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, bile duct cancer, page at least one selected from the group consisting of pancreatic cancer, ovarian cancer, and uterine carcinosarcoma and more preferably, it can be used for the treatment of breast cancer, stomach cancer, colon cancer, Non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, bile duct cancer, and pegylated The present invention can be used to treat at least one cancer selected from the group consisting of: and even more preferably for the treatment of breast cancer, gastric cancer, colon cancer, or non-small cell lung cancer. can be used for.

[0401] The pharmaceutical composition comprising the antibody-drug conjugate produced by the present invention is useful for the treatment of cancer. It can be used as a drug of choice for drug therapy, which is the main treatment, and as a result, As a result, it can slow the growth of cancer cells, inhibit their proliferation, and even destroy them. These effects can help cancer patients relieve symptoms caused by cancer and improve their quality of life. This can achieve improvement, preserving the life of cancer patients and achieving therapeutic effects. Even if the drug is not effective, it may have beneficial effects on cancer patients by suppressing or controlling the growth of cancer cells. It is possible to achieve a higher quality of life and longer survival.

[0402] In addition to the use of the drug alone in such drug therapy, the antibody produced by the present invention -Pharmaceutical compositions containing drug conjugates may be used in adjuvant therapy in combination with other therapies. It can also be used in combination with surgery, radiation therapy, hormone therapy, etc. Furthermore, it can be used as a drug therapy in neoadjuvant therapy. do.

[0403] In addition to the above-mentioned therapeutic uses, the antibody-drug conjugates produced by the present invention can be used in various therapeutic applications. The pharmaceutical composition containing the compound has a preventive effect of suppressing the growth of minute metastatic cancer cells and even destroying them. For example, it can suppress and destroy cancer cells in body fluids during the metastasis process. and the effects of suppressing or destroying minute cancer cells immediately after implantation in any tissue. Therefore, it is expected to be effective in suppressing and preventing cancer metastasis, especially after surgical removal of cancer. The effect is expected.

[0404] Pharmaceutical compositions containing antibody-drug conjugates produced according to the present invention may be used in other cancer treatments. It can also be administered in combination with other therapeutic agents, which can enhance the antitumor effect. Other cancer treatments used for this purpose include 5-fluorouracil (5-FU). , Pertuzumab, Trastuzumab , Paclitaxel, Carboplatin ), Cisplatin, Gemcitabine, Capecitabine, Irinotecan ( CPT-11, docetaxel, pemetrexed exed), sorafenib, vinblastine in), vinorelbine, everolimus ), Tanespimycin, Bevacizumab mab), oxaliplatin, lapatinib ib), trastuzumab emtansine (T-DM1) and the drugs described in WO 2003 / 038043, and further LH-RH Analogues (Leuprorelin, Goserelin) in), etc.), Estramustine Phos phate), estrogen antagonists (tamoxifen, raloxifene) Raloxifene, etc.), aromatase inhibitors (anastrozole, astrozole, letrozole, and exemestane However, as long as the drug has antitumor activity, There is no limitation. [Example]

[0405] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. It is not something that can be done.

[0406] In the example, 1 H-NMR" and " 13 "C-NMR" stands for "nuclear magnetic resonance spectrum" The CDCl3 in parentheses means the measurement solvent, deuterated chloroform, and DMSO-d6 means the measurement solvent, deuterated dimethyl sulfoxide, and DO means the measurement solvent, deuterated water. MeOH-d4 refers to the measurement solvent, deuterated methanol. S (tetramethylsilane) was used. 1 The multiplicity in H-NMR is s=single t, d=doublet, t=triplet, q=quartet, m=multi plet, and brs = broad singlet.

[0407] Example 1 2,5-Dioxopyrrolidin-1-yl N-[(benzyloxy)carbonyl]glycerin glycinate

[0408] [ka]

[0409] N-[(benzyloxy)carbonyl]glycylglycine (200.00g, 0.75 A mixture of N-hydroxysuccinimide (9 mol) and acetonitrile (2.0 L) was 5.10 g, 0.826 mol), 1-(3-dimethylaminopropyl)-3-ethyl carboxylate Add rubodimide hydrochloride (172.80 g, 0.901 mol) and stir at room temperature for approximately 4 hours. The reaction mixture was cooled to 1°C and stirred for about 3 hours. The precipitate was filtered, and the separated powder was The powder was dried under reduced pressure at 40°C and then washed with acetonitrile (400 mL). -Dioxopyrrolidin-1-yl N-[(benzyloxy)carbonyl]glycylglycerin Cinnate (221.6 g, 0.610 mol, yield 81.2%) was obtained.

[0410] 1 H-NMR (400MHz,DMSO-d6)δ2.81(4H,s),δ3.69 (2H,d,6.7Hz),δ4.28(2H,d,6.1Hz),δ5.04(2H, s),δ7.29-7.39(5H,m),δ7.56(1H,t,6.4Hz),δ8 .55(1H,t,5.8Hz). 13 C-NMR (100MHz,DMSO-d6)δ25.4,38.2,43.3, 65.6, 127.7, 127.8, 128.3, 137.0, 156.5, 166.3 ,170.0,170.0. MS(ESI)(m / z):364([M+H] + ).

[0411] Example 2 N-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine

[0412] [ka]

[0413] L-Phenylalanine (80.0 g, 0.487 mol) in acetonitrile (400 m A mixture of triethylamine (74.7 mL, 0.536 mo l), 2,5-dioxopyrrolidin-1-yl N-[(benzyloxy)carbonyl] Add glycylglycinate (212.4 g, 0.585 mol) and stir at room temperature for approximately 2 hours. To the reaction mixture, water (800 mL) and concentrated hydrochloric acid (40.6 mL) were added, and then N-[(benzyl) Add [(hydroxy)carbonyl]glycylglycyl-L-phenylalanine (80 mg), The mixture was stirred at room temperature for about 6 hours, and the precipitate was filtered off, and the filtered powder was washed with water (160 mL). The obtained powder was dried under reduced pressure at 40°C to obtain N-[(benzyloxy)carbonyl]glycerin. Glycyl-L-phenylalanine (157.2 g, 0.380 mol, yield 78.0%) ) was obtained.

[0414] 1 H-NMR (400MHz,DMSO-d6)δ2.86-2.91(1H,m), δ3.03-3.08(1H,m),δ3.64-3.78(4H,m),δ4.41- 4.47(1H,m),δ5.04(2H,s),δ7.18-7.40(10H,m) ,δ7.50(1H,t,6.1Hz),δ8.05(1H,t,5.8Hz),δ8. 17 (1H, d, 7.9 Hz), δ 12.77 (1H, s). 13 C-NMR (100MHz,DMSO-d6)δ36.8,41.6,43.5, 53.5,65.5,126.5,127.7,127.8,128.2,128.3, 129.1, 137.0, 137.4, 156.5, 168.6, 169.3, 172. 7. MS(ESI)(m / z):412([MH] - ).

[0415] Example 3 ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl ethyl acetate

[0416] [ka]

[0417] N-[(9H-fluoren-ylmethoxy)carbonyl]glycylglycine (650. 0g, 1.834mol) in tetrahydrofuran (9.75L) and acetic acid (1.95L) Add lead tetraacetate (1301.3 g, 2.935 mol) and heat to 40°C to dissolve. The mixture was refluxed for about 1.5 hours, cooled to room temperature, and the insoluble matter was removed by filtration. The resulting solution was washed with 20 (w / v)% citric acid (3.25 L) and combined with the filtrate. Aqueous trisodium dihydrate solution (3.25 L) was added and stirred, and the mixture was separated and the aqueous layer was removed. The obtained organic layer was diluted with 20 (w / v)% aqueous solution of trisodium citrate dihydrate (3.25 L The organic layer was then concentrated under reduced pressure to 6.5 L. Water (1.95 L) was added. Then, ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino Methyl acetate (0.65 g) was added and stirred at room temperature for about 1 hour. Water (6.5 L) The mixture was cooled to 0-5°C and stirred for about 3 hours. The precipitate was filtered, and the separated powder was cooled to 3°C. The powder was washed with 0 (v / v)% aqueous tetrahydrofuran solution (2.6 L). The mixture was dried at 40°C under reduced pressure and then cooled to 100°C. (lysyl)amino)methyl acetate (617.1 g, 1.675 mol, yield 91.3%) ) was obtained.

[0418] 1 H-NMR (400MHz,CDCl3)δ2.06(3H,s),δ3.90(2 H,d,4.9Hz),δ4.23(1H,t,6.7Hz),δ4.45(2H,d, 6.7Hz),δ5.25(2H,d,7.3Hz),δ5.39(1H,brs),δ 7.05(1H,brs),δ7.30-7.34(2H,m),δ7.41(2H,t ,7.3Hz),δ7.59(2H,d,7.3Hz),δ7.77(2H,d,7.3 Hz). 13C-NMR (100MHz,CDCl3)δ20.8,44.4,47.0,63 .9,67.2,120.0,125.0,127.1,127.7,141.3,14 3.6, 156.6, 169.8, 171.7. MS(ESI)(m / z):369([M+H] + ).

[0419] Example 4 Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl} Amino)methoxy]acetate

[0420] [ka]

[0421] ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino) Methyl acetate (610.0 g, 1.656 mol) in 1,2-dimethoxyethane (9 To the 0.15 L mixture, add benzyl glycolate (470 mL, 3.312 mol) The mixture was cooled to 0-5°C. 10 mol / L sodium hydroxide solution (162.6 mL, 1.62 Acetic acid (47.4 mL) was added and stirred at 1°C for about 1 hour. After stirring, water (2.0 L) was added dropwise and benzyl[({N-[(9H-fluorene-9-yl] (trimethoxy)carbonyl]glycyl}amino)methoxy]acetate (0.61 g) was added. Water (4.7 L) was added dropwise and the mixture was stirred at 0 to 5°C for about 2.5 hours. The precipitate was filtered, and the filtered powder was dissolved in cold 50 (v / v)% 1,2-dimethoxyethanol. The resulting wet powder was washed with 1,2-dimethoxyethane (9 After adding water (3.66 L), the mixture was stirred at room temperature for about 30 minutes to dissolve. , benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl To the mixture was added 0.61 g of 1,000 ml of 2,000 ml ... (3.05 L) was added dropwise and stirred at room temperature for about 1 hour. The mixture was cooled to 0-5°C and stirred for about 1 hour. The precipitate was then filtered, and the filtered powder was dissolved in cold 50 (v / v)% 1,2-dimethoxyethane water. The resulting wet powder was washed with 1,2-dimethoxyethane (9.0 L). After adding water (3.6 L), the mixture was stirred at room temperature for about 30 minutes to dissolve. aryl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino ) methoxy] acetate (0.01 g) was added and stirred at room temperature for about 1 hour. The mixture was cooled to 0-5°C and stirred for about 1 hour, after which precipitation The mixture was filtered, and the filtered powder was dissolved in a cold (v / v) 50% aqueous solution of 1,2-dimethoxyethane (2. The resulting wet powder was washed with 1,2-dimethoxyethane (9.0 L). The mixture was stirred at room temperature for about 20 minutes to dissolve the compound. Water (3.6 L) was added, and then benzyl [({N- [(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy] Acetate (0.15 g) was added and stirred at room temperature for about 2 hours. Water (3.0 L) was added dropwise. The mixture was stirred at room temperature for about 1 hour, cooled to 0-5°C, stirred for about 2 hours, and then the precipitate was filtered. The filtered powder was washed with cold 50 (v / v)% 1,2-dimethoxyethane aqueous solution (2.4 L). The obtained powder was dried under reduced pressure at 40°C to obtain crude benzyl [({N-[(9H-fluorene The resulting product was methyl-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate. The obtained crude benzyl [({N-[(9H-fluoren-9-ylmethoxy)carbonyl] Add toluene (12 L) to the glycyl(amino)methoxy)acetate and heat to 70°C. The mixture was cooled to 0-5°C and stirred for about 2 hours, after which the precipitate was filtered off. The powder was washed with cold toluene (2.4 L). The obtained powder was dried at 40°C under reduced pressure and aryl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino ) methoxy] acetate (575.5 g, 1.213 mol, yield 73.2%).

[0422] 1 H-NMR (400MHz, CDCl3)δ3.82(2H,d,4.9Hz),δ 4.19-4.22(3H,m),δ4.45(2H,d,6.7Hz),δ4.83( 2H,d,6.7Hz),δ5.15(2H,s),δ5.34(1H,brs),δ6 .95(1H,brs),δ7.29-7.37(7H,m),δ7.40(2H,t, 7.3Hz), δ7.58(2H,d,7.3Hz),δ7.76(2H,d,7.9H z). 13 C-NMR (100MHz,CDCl3)δ44.5,47.1,66.6,66 .8,67.1,70.6,120.0,124.9,127.1,127.8,128 .4,128.5,128.6,135.2,141.3,143.6,170.2,1 70.2,170.4. MS(ESI)(m / z):475([M+H] + ).

[0423] Example 5 Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycol Cinamide

[0424] [ka]

[0425] Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl }amino)methoxy]acetate (340.0 g, 0.717 mol) in acetonitrile (10.2 L) mixture, 1,8-diazabicyclo[5,4,0]-7-undecene (5 The mixture was stirred at room temperature for approximately 2 hours. 1-Hydroxybenzotriazole monohydrate (132.0 g, 0.862 mol), N- [(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine (311. 0g, 0.752mol) was added, and then 1-(3-dimethylaminopropyl)-3-ethyl Add tricarbodiimide hydrochloride (158.0 g, 0.824 mol) in portions and heat at 0-5°C. After stirring for approximately 1 hour, 10 (w / v)% phosphate buffer (pH 3, 3.4 L) was added and the mixture was cooled to room temperature. The mixture was heated at RT. After separating the layers and removing the aqueous layer, the mixture was concentrated under reduced pressure to 3.7 L. Ethyl acetate (3 10% (w / v) carbonated water was added to the mixture, and the aqueous layer was removed. Aqueous potassium chloride solution (3.4 L) was added and stirred, and the mixture was separated and the aqueous layer was removed. v) 3.4 L of 100% aqueous potassium bicarbonate solution was added and stirred, and the mixture was separated and the aqueous layer was removed. Add water (3.4 L), stir, separate, remove the aqueous layer, and then concentrate under reduced pressure to 1.5 L. 2-Methoxyethanol (3.74 L) was added, and the mixture was concentrated under reduced pressure to 3.06 L. Transfer to a 0 L autoclave and add tetrahydrofuran (1.36 L), water (3.4 L), 5% Palladium carbon (72.6 g, water content 53.2%) was added, and the atmosphere was replaced with hydrogen. After stirring at rt for about 19 hours, the atmosphere was replaced with nitrogen, water (360 mL) was added, and the mixture was stirred at room temperature for about 3 The mixture was stirred for 10 minutes, and the palladium carbon was filtered off and washed with water (1.36 L). The mixture was combined with the filtrate. Ethyl acetate (0.85 L) and n-heptane (2.55 L) were added and stirred. After stirring, the organic layer was removed by separation and concentrated under reduced pressure to 1.6 L. After adding 2-methoxyethanol (126 mL), glycylglycyl-L-phenylalanine was added. Ranyl-N-[(carboxymethoxy)methyl]glycinamide (0.34 g) was added. The mixture was heated to 40°C and stirred for about 19 hours. Ethanol (3.4 L) was added dropwise, and the mixture was stirred at room temperature for about 1 hour. The mixture was stirred for 8 hours, and the precipitate was filtered off. The filtered powder was washed with ethanol (1.02 L). The resulting powder was dried under reduced pressure at 40°C to give glycylglycyl-L-phenylalanyl-N -[(carboxymethoxy)methyl]glycinamide (243.7 g, 0.576 mol The yield was 80.3%.

[0426] 1 H-NMR (400MHz,D2O)δ3.03-3.20(2H,m),δ3.7 9-3.96(6H,m),δ3.97(2H,s),δ4.64(1H,t,7.9H z), δ4.67-4.75(2H,m),δ7.29-7.42(5H,m). 13 C-NMR (100MHz,D2O)δ37.3,41.0,42.7,43.2 ,56.0,67.3,70.0,127.8,129.4,129.8,136.9, 168.4, 171.7, 172.9, 174.4, 178.1. MS(ESI)(m / z):422([MH] - ).

[0427] Example 6 Benzyl [(glycylamino)methoxy]acetate 1H-benzotriazole-1- Oar

[0428] [ka]

[0429] Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl }amino)methoxy]acetate (50.00 g, 105.4 mmol) of acetonitrile A mixture of 1,8-diazabicyclo[5,4,0]-7-undecene (8 0.02g, 52.7mmol) was added and stirred at room temperature for about 4 hours. Benzotriazole monohydrate (35.51 g, 231.9 mmol) was added in portions, and the mixture was stirred for about 3 The mixture was stirred for 10 minutes, cooled to 1°C, and stirred for approximately 11 hours. The precipitate was filtered, and the filtered powder was The powder was washed with acetonitrile (250 mL). The powder was dried under reduced pressure at 40°C and Dimethyl[(glycylamino)methoxy]acetate 1H-benzotriazole-1-ol The resulting product was 38.98 g, 100.6 mol, yield 95.4%.

[0430] 1 H-NMR (500MHz,MeOH-d4)δ3.63-3.68(2H,brs ), δ4.19-4.23(2H,brs),δ4.79(2H,s),δ5.16-5 .20(2H,brs),δ7.25-7.38(7H,m),δ7.64-7.72( 2H,dd,17.3Hz,7.8Hz). 13C-NMR (125MHz,MeOH-d4)δ41.9,66.3,67.6, 71.0, 112.3, 118.7, 125.24, 125.27, 128.8, 129 .25, 129.30, 129.5, 136.9, 144.4, 169.5, 171.8

[0431] Example 7 N-[(benzyloxy)carboxyl]glycylglycyl-L-phenylalanyl-N -{[2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide

[0432] [ka]

[0433] N-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine (1 0.99 g, 26.58 mmol) in acetonitrile (120 mL) and water (20 mL) The mixture contains benzyl [(glycylamino)methoxy]acetate 1H-benzotriazo Add 1-ol (10.00 g, 25.81 mmol), cool to 2°C, and (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.70g, 29 0.73 mmol) was added and the mixture was stirred at 0-5°C for about 3.5 hours. Water (130 mL) and water (150 mL) were added and stirred at room temperature for 14 hours. The mixture was stirred for 2 hours, cooled to 1°C, and stirred for about 1 hour. The precipitate was filtered. The powder was collected by filtration and washed with acetonitrile:water=1:2 (60 mL). Dry under reduced pressure at 40°C and add N-[(benzyloxy)carboxyl]glycylglycyl-L- Phenylanyl-N-{[2-(benzyloxy)-2-oxoethoxy]methyl}glucan Lysinamide (15.34 g, 23.68 mmol, 91.7% yield) was obtained.

[0434] 1 H-NMR (500MHz,DMSO-d6)δ2.79(1H,dd,14Hz, 9.2Hz), δ3.06(1H, dd, 14Hz, 4.5Hz), δ3.55-3.8 0(6H,m),δ4.15(2H,s),δ4.51(1H,ddd,Hz,9.2H z,8.6Hz,4.5Hz),δ4.63(2H,d,6.5Hz),δ5.03(2 H,s),δ5.15(2H,s),δ7.15-7.40(15H,m),δ7.15 -7.40(15H,m),δ7.50(1H,t,6Hz),δ8.02(1H,t, 5.8Hz), δ8.15(1H,d,8.6Hz),δ8.33(1H,t,5.8H z), δ8.60(1H,t,7Hz) 13 C-NMR (125MHz,DMSO-d6)δ37.4,41.9,42.2, 43.6,54.2,64.5,65.6,65.7,69.1,126.3,127. 77,127.85,128.10,128.14,128.2,128.4,128. 5,129.2,135.8,137.0,137.9,156.6,168.9,16 9.5, 169.9, 170.2, 171.5.

[0435] Example 8 Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycol Cinamide

[0436] [ka]

[0437] N-[(benzyloxy)carboxyl]glycylglycyl-L-phenylalanyl- N-{[2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide (15 0.0 g, 23.16 mmol) in tetrahydrofuran (315 mL), water (210 mL) To the mixture was added 5% palladium on carbon (3.31 g, water content 54.7%), and the atmosphere was changed to hydrogen. After stirring at room temperature for about 2.5 hours, the atmosphere was replaced with nitrogen, and palladium carbon was added. The palladium on carbon was filtered off, washed with water (60 mL) and combined with the filtrate. Ethanol (150 mL) was added and the mixture was concentrated under reduced pressure to 180 mL. The mixture was concentrated under reduced pressure to 135 mL. Ethanol (300 mL) was added and the mixture was stirred for 17 hours. The precipitate was filtered, and the filtered powder was washed with ethanol (75 mL). was dried under reduced pressure at 40°C, and glycylglycyl-L-phenylalanyl-N-[(carboxymethyl)-L-phenylalanyl] [(dimethylamino)methyl]glycinamide (8.95 g, 21.14 mmol, yield 91.3 %) was obtained.

[0438] 1 H-NMR (400MHz,D2O)δ3.03-3.20(2H,m),δ3.7 9-3.96(6H,m),δ3.97(2H,s),δ4.64(1H,t,7.9H z), δ4.67-4.75(2H,m),δ7.29-7.42(5H,m). 13 C-NMR (100MHz,D2O)δ37.3,41.0,42.7,43.2 ,56.0,67.3,70.0,127.8,129.4,129.8,136.9, 168.4, 171.7, 172.9, 174.4, 178.1. MS(ESI)(m / z):422([MH] - ).

[0439] Example 9 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid

[0440] [ka]

[0441] A solution of 6-aminohexanoic acid (2.5 kg, 19.1 mol) in acetic acid (10 L) was A solution of maleic acid (1.87 kg, 19.1 mol) in acetic acid (10 L) was heated at 25-30°C. The mixture was added dropwise over 1 hour and stirred at the same temperature for 2 hours. After the temperature was raised to 100°C, the reaction mixture was stirred for 16 hours. After cooling to 100°C, the mixture was concentrated under reduced pressure to 7.0 L. The obtained solution was added to cold water (20 L) at 0 to 5°C under stirring. The concentrated solution (approximately 7.0 L) was added dropwise over 1 hour, and the mixture was stirred at the same temperature for 1 hour. The precipitate was filtered. The powder was collected by filtration, washed with cold water (5.0 L), and dried at 40°C under reduced pressure. , 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (1.46 kg, 6.95 mol, yield 36.4%).

[0442] The resulting 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) Xanthic acid (1.40 kg, 6.66 mol) in acetic acid (2.1 L) and purified water (1.4 L) Dissolve in the mixture at 25-30°C, add purified water (0.7 L), and then cool to 20-25°C. After that, the mixture was stirred for 2 hours. Purified water (7.0 L) was added dropwise to the resulting suspension over 1 hour. After cooling to 5°C, the mixture was stirred for 1 hour. The precipitate was filtered, and the filtered powder was washed with cold water (2.1 L). The resulting powder was dried under reduced pressure at 40°C to give 6-(2,5-dioxo-2,5-dihydroxybenzoate). 1H-pyrrol-1-yl)hexanoic acid (1.27 kg, 6.02 mol, yield 9 0.4%) was obtained.

[0443] 1 H-NMR (400MHz,DMSO-d6)δ1.18-1.24(2H,m), δ1.45-1.52(4H,m),δ2.18(2H,t,7.5Hz),δ3.38 (2H,t,7.5Hz),δ7.01(2H,s),δ11.98(1H,s). Example 10 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl }-1H-pyrrole-2,5-dione

[0444] [ka]

[0445] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (5.0 g, 23.6 mmol), N-hydroxysuccinimide (3.0 g, 26.0 In a mixture of 1-(3-dimethylaminopropyl) )-3-ethylcarbodiimide hydrochloride (5.45 g, 28.4 mmol) was added, and the mixture was stirred at room temperature. The mixture was stirred for about 3.5 hours. Water (100 mL) and toluene (100 mL) were added and stirred. The organic layer was washed twice with water (50 mL), and the organic layer was extracted with 25 mL The mixture was concentrated under reduced pressure to 100°C. The concentrate was then charged onto a silica gel cartridge (KP-sil 10 g) and The eluate was collected by passing toluene:acetone = 9:1 (100 mL) through the column, and the pressure was reduced to 25 mL. After the mixture was concentrated, 1-butanol (50 mL) was added, followed by 1-[6-[(2,5-dioxopyrrolidone) (roridin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-diol After adding 10 mg of ethanol, the mixture was stirred at room temperature for 1 hour. 1-Butanol (50 mL) was added dropwise. The mixture was cooled to -10°C and stirred. The precipitate was filtered, and the filtered powder was dissolved in cold 1-butanol (2 The resulting powder was dried under reduced pressure at 40°C and washed with 1-{6-[(2,5-dichloro-2,6-diphenyl ... oxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2, The 5-dione (6.52 g, 21.1 mmol, 89.4% yield) was obtained.

[0446] 1 H-NMR (400MHz,DMSO-d6)δ1.27-1.35(2H,m), δ1.48-1.56(2H,m),δ1.59-1.67(2H,m),δ2.65( 2H,t,7.3Hz),δ2.81(4H,s),δ3.39(2H,t,7.0Hz ),δ7.00(2H,s). 13 C-NMR (100MHz,DMSO-d6)δ23.7,25.1,25.4, 27.4, 30.0, 36.8, 134.4, 168.9, 170.2, 171.1. MS(ESI)(m / z):309([M+H] + ).

[0447] Example 11 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl }-1H-pyrrole-2,5-dione

[0448] [ka]

[0449] 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (1.1 kg, 5.21 mol), N-hydroxysuccinimide (0.72 kg, 6. A mixture of 2,6-lutidyl 25 mol) and acetonitrile (11 L) was cooled to -15°C and After adding thionyl chloride (0.74 kg, 6 0.25 mol) was added dropwise over 1 hour at -15°C to -10°C. Water (11 L), toluene (11 L) was added and stirred, then the layers were separated and the aqueous layer was removed. The organic layer was washed with cold water ( Wash twice with 11 L of 20% brine (11 L) at 0-5°C, and reduce the organic layer to 5.5 L. After concentration under reduced pressure, toluene (5.5 L) was added and the mixture was again concentrated under reduced pressure to 5.5 L. The funnel was filled with neutral silica gel (Silicagel 60N, 3.3 kg) moistened with water. The concentrated liquid was passed through the column and washed with toluene:acetone=9:1 (29 L) to obtain a filtrate. The filtrate was concentrated under reduced pressure to 5.5 L, and 1-butanol (8.8 L) was added. The mixture was stirred at 5°C for 16 hours, and 1-butanol (13.2 L) was added dropwise, cooled to -15°C, and The mixture was stirred for 1 hour, and the precipitate was filtered off, and the filtered powder was washed with cold 1-butanol (4.4 L). The obtained powder was dried under reduced pressure at 40°C to obtain 1-[6-[(2,5-dioxopyrrolidinyl) 1H-pyrrole-2,5-dione(1H-pyrrole-1-yl)oxy]-6-oxohexyl 0.45 kg, 4.72 mol, yield 90.5%) was obtained.

[0450] 1H-NMR (400MHz,DMSO-d6)δ1.27-1.35(2H,m), δ1.48-1.56(2H,m),δ1.59-1.67(2H,m),δ2.65( 2H,t,7.3Hz),δ2.81(4H,s),δ3.39(2H,t,7.0Hz ),δ7.00(2H,s). 13 C-NMR (100MHz,DMSO-d6)δ23.7,25.1,25.4, 27.4, 30.0, 36.8, 134.4, 168.9, 170.2, 171.1. MS(ESI)(m / z):309([M+H] + ).

[0451] Example 12 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa [(carboxymethoxy)methyl]glycylglycyl-L-phenylalanyl [L]glycinamide

[0452] [ka]

[0453] 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl {1H-pyrrole}-1H-pyrrole-2,5-dione (291.3 g, 0.945 mol) in acetonitrile To a solution of glycylglycyl-L-phenylalanyl-N-[(carbohydrate) [(2-methoxy)methyl]glycinamide (200.0 g, 0.472 mol), water (4. 2 L), N,N-diisopropylethylamine (48.8 g, 0.378 mol) was added. The mixture was stirred at room temperature for about 9 hours. Add 400.0 g of ethanol and 26.0 g of anhydrous disodium hydrogen phosphate, stir, and then The organic layer was separated and removed. Tetrahydrofuran (1.0 L), ethyl acetate (1.0 L) ), anhydrous sodium dihydrogen phosphate (160.0 g) was added and stirred, and the aqueous layer was separated. 10 (w / v)% phosphate buffer (pH 3.4, 0.6 L) was added and stirred. After separating the layers and removing the aqueous layer, the mixture was concentrated under reduced pressure to 1.0 L. 1,2-Dimethoxyethane (4.0 L) was added and concentrated under reduced pressure to 1.0 L. Acetonitrile (0.4 L) was added, and the mixture was concentrated under reduced pressure to 1.0 L. The water content of the solution was measured and found to be 6.1% (equivalent to 18.8 mL of water). (19 mL) was added, and then N-[6-(2,5-dioxo-2,5-dihydro-1H-pi glycylglycyl-L-phenylalanyl-N-[(( Add 1,2-dimethoxymethyl]glycinamide (0.2 g) and Diethane (0.8 L) was added dropwise, and the mixture was stirred at room temperature for approximately 16 hours. (3.2 L) was added dropwise, and the mixture was concentrated under reduced pressure to 4.0 L. The mixture was cooled to 0-5°C and stirred for about 19.5 hours. The precipitate was filtered off. The powder was washed with 1,2-dimethoxyethane (0.8 L). The resulting powder was heated at 40°C under reduced pressure. Dry and N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl] )Hexanoyl]glycylglycyl-L-phenylalanyl-N-[(carboxymethox [(c)methyl]glycinamide (268.3 g, 0.435 mol, 92.1% yield) was obtained. Ta.

[0454] 1 H-NMR (400MHz,DMSO-d6)δ1.15-1.23(2H,m), δ1.43-1.52(4H,m),δ2.11(2H,t,7.3Hz),δ2.78 -2.84(1H,m),δ3.04-3.09(1H,m),δ3.37(2H,t, 7.0Hz)δ3.61-3.79(6H,m),δ3.94(2H,s),δ4.47 -4.52(1H,m),δ4.61(2H,d,6.7Hz),δ6.99(2H,s ), δ7.15-7.27(5H,m),δ8.11-8.15(2H,m),δ8.2 2(1H,d,8.5Hz),δ8.31(1H,t,5.8Hz),δ8.63(1H ,t,6.4Hz). 13 C-NMR (100MHz,DMSO-d6)δ24.6,25.8,27.8, 34.9, 37.0, 37.2, 41.9, 42.1, 42.1, 54.2, 65.1, 69.2, 126.2, 128.1, 129.1, 134.4, 137.9, 168.9 ,169.5,169.8,171.1,171.4,171.9,172.6. MS(ESI)(m / z):615([MH] - ).

[0455] Example 13 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa [(carboxymethoxy)methyl]glycylglycyl-L-phenylalanyl 1,2-Dimethoxyethane adduct of [chloro]glycinamide

[0456] [ka]

[0457] 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl {1H-pyrrole}-1H-pyrrole-2,5-dione (72.8 g, 0.236 mol) acetonite In a solution of glycylglycyl-L-phenylalanyl-N-[(carboxymethyl)-L-phenylalanyl] [(carboxymethoxy)methyl]glycinamide (50.0 g, 0.118 mol), water (1 050.0 mL), N,N-diisopropylethylamine (16.5 mL, 0.095 m The mixture was stirred at room temperature for about 15 hours. Sodium dihydrogen phosphate (100.0 g), anhydrous disodium hydrogen phosphate (6.5 g) After adding and stirring, the mixture was separated and the organic layer was removed. After adding 1,2-dimethoxyethane (250 0.0mL), ethyl acetate (250.0mL), acetonitrile (25.0mL), anhydrous Sodium dihydrogen phosphate (400.0 g) was added and stirred, then the solution was separated and the aqueous layer was removed. Acetonitrile (750.0 mL), water (113.0 mL), sodium chloride (30. 0g), anhydrous sodium dihydrogen phosphate (7.5g), phosphoric acid (85%, 1.5g, 0.0 The mixture was stirred and separated to remove the aqueous layer. Add sodium (30.0 g) and anhydrous sodium dihydrogen phosphate (7.5 g) and stir. The aqueous layer was removed by separation. Add sodium dihydrogen phosphate (7.5 g) and stir. After separating the solution and removing the aqueous layer, The mixture was concentrated under reduced pressure to 0.0 mL. After adding 1,2-dimethoxyethane (750.0 mL), The solution was concentrated under reduced pressure to 500.0 mL. The water content of the solution was measured and found to be 6.9% (water 31.3 g phase). After adding water (9.5 mL) and 1,2-dimethoxyethane (1.0 L), The mixture was stirred at room temperature for approximately 13 hours. 1,2-Dimethoxyethane (250.0 mL) was added dropwise. After stirring at room temperature for about 5 hours, the mixture was concentrated under reduced pressure to 1.0 L. 1,2-Dimethoxyethane (1. After stirring at room temperature for about 1 hour, the mixture was concentrated under reduced pressure to 1.0 L. Add 250 mL of ethoxyethane dropwise, stir at room temperature for approximately 16 hours, and then filter the precipitate. The powder was filtered off and washed with 1,2-dimethoxyethane (250.0 L). Add 1,2-dimethoxyethane (2.0 L) and water (65.0 mL) to the powder and heat to 45°C. After stirring for 30 minutes, the sodium chloride was filtered off and the sodium chloride was dissolved in 1,2-dichloromethane. Wash with methoxyethane / water (97 / 3, 150 mL) and reduce the filtrate to 1.0 L. The mixture was concentrated under reduced pressure. 1,2-Dimethoxyethane (1.0 L) was added and stirred at room temperature for about 3 hours. The mixture was concentrated under reduced pressure to 1.0 L. 1,2-Dimethoxyethane (1.0 L) was added dropwise to the mixture to make a 1.0 L solution. The mixture was concentrated under reduced pressure to a volume of 1 L. 1,2-Dimethoxyethane (250.0 mL) was added dropwise, and the mixture was stirred at room temperature for about After stirring for 16 hours, the precipitate was filtered off, and the filtered powder was dissolved in 1,2-dimethoxyethane (25 The resulting powder was dried under reduced pressure (4 kPa) at 25°C and purified by the method described above. 2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycine Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycine 1,2-Dimethoxyethane adduct of amide (65.7 g, 0.107 mol, yield 90. 3%) was obtained as crystals.

[0458] 1H-NMR (500MHz,DMSO-d6)δ1.16-1.23(2H,m), δ1.44-1.52(4H,m),δ2.11(2H,t,7.5Hz),δ2.79 -2.84(1H,m),δ3.05-3.09(1H,m),δ3.24(6H,s) ,δ3.37(2H,t,7.3Hz),δ3.43(4H,s),δ3.56-3.7 8(6H,m),δ3.99(2H,s),δ4.48-4.52(1H,m),δ4. 61(2H,d,6.5Hz),δ7.00(2H,s),δ7.16-7.27(5H ,m),δ8.02-8.10(2H,m),δ8.15(1H,d,8.0Hz),δ 8.32(1H,t,6.0Hz),δ8.58(1H,t,6.8Hz).δ12.6 1(1H,brs) 13 C-NMR (100MHz,DMSO-d6)δ25.5,26.8,28.7, 35.9,37.9,38.2,42.8,43.0,43.1,55.1,59.0, 65.2,69.8,72.0,127.2,129.0,130.1,135.4,1 38.8, 169.8, 170.4, 170.9, 172.0, 172.3, 172.4 ,173.6. MS(ESI)(m / z):615([MH] - ).

[0459] Powder X-ray diffraction: The crystals of the title compound were subjected to powder X-ray diffraction analysis using copper Kα radiation. The results are shown in Table 1 and Figure 3. The main peaks were observed at diffraction angles (2θ) of 19.0° and 25.0°. was recognized.

[0460] [Table 1]

[0461] Example 14 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa N-[(2-[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox (ethoxy)methyl]glycinamide

[0462] [ka]

[0463] Methanesulfonic acid (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]chino Phosphorus-1-aminium dihydrate (gross weight 154.6g, moisture content 2.95% corrected) To a suspension of 150.0 g (0.282 mol) of 5 (w / v)% aqueous sodium sulfate solution (1.5 L), N-methylmorpholine (28.5 g, 0. 282 mol) was added and stirred at 32°C for about 1 hour. Chill (8.0 g, 56.3 mmol), N-[6-(2,5-dioxo-2,5-dihydro- (1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanine N-[(carboxymethoxy)methyl]glycinamide (gross amount 232.0g, 1 ,2-Dimethoxyethane 2.50% (contents: 226.2g, 0.367mol), 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (108.2g, 0.564 mol) was added, and the mixture was stirred at 29-32°C for about 1 hour, after which the aqueous layer was removed by separation. Ethyl acetate (1.8 L) and 5 (v / v)% acetic acid aqueous solution (0.45 L) were added and stirred. The mixture was separated and the aqueous layer was removed. Activated carbon (15.0 g, Kyokuryoku Shirasagi (Osaka Gas Chemicals Co., Ltd.) After adding the activated carbon, the mixture was stirred at room temperature for about 30 minutes, and the activated carbon was filtered off. The filtrate was combined and concentrated under reduced pressure to 0.75 L. Add propanol (1.5 L), concentrate under reduced pressure to 0.75 L, and mix to obtain a mixture of acetone:1-propanol. The mixture was added with 1:1 ethanol (3.0 L). 1H-Pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl- N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,1 2H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolinol 1-(2-hydroxybenzoyl)amino}-2-oxoethoxymethyl]glycinamide (0.15g) The precipitate was filtered, and the powder was dissolved in acetone:1- The resulting wet powder was washed with propanol = 1:1 (0.6 L). (1.5 L) and water (0.3 L) were added and dissolved, and the mixture was concentrated under reduced pressure to 0.75 L. Add 1.5 L of acetone and concentrate under reduced pressure to 0.75 L. = 1:1 (3.0 L) was added. N-[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N -[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12 H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline -1-yl]amino}-2-oxoethoxy)methyl]glycinamide (0.15 g) The mixture was added and stirred at room temperature for about 24 hours. The precipitate was filtered, and the filtered crystals were dissolved in acetone:1-propanol. The resulting crystals were dried under reduced pressure at 40°C and washed with 1:1 propyl alcohol (0.6 L). -[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexano yl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9 -ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3 ,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxo Ethoxy)methyl]glycinamide (254.6 g, 87.3% yield) was obtained as crystals. .

[0464] 1 H-NMR (400MHz,DMSO-d6)δ0.87(3H,t,7.3Hz) ,δ1.14-1.21(2H,m),δ1.41-1.50(4H,m),δ1.78 -1.93(2H,m),δ2.09(2H,t,7.3Hz),δ2.13-2.23 (2H,m),δ2.36(3H,s),δ2.74-2.80(1H,m),δ3.0 0 - 3.04 (1H, m), δ 3.08 - 3.25 (2H, m), δ 3.32 - 3.37 (2H, m), δ 3.56 - 3.77 (6H, m), δ 4.02 (2H, s), δ 4.4 4 - 4.50 (1H, m), δ 4.64 (2H, d, 6.7 Hz), δ 5.17 (2H, d, 5.5 Hz), δ 5.41 (2H, s), δ 5.57 - 5.62 (1H, m), δ 6 .51 (1H, s), δ 6.99 (2H, s), δ 7.14 - 7.26 (5H, m), δ 7.30 (1H, s), δ 7.75 (1H, d, 11.0 Hz), δ 8.00 (1H, t , 5.8 Hz), δ 8.06 (1H, t, 5.3 Hz), δ 8.12 (1H, d, 7.9 Hz), δ 8.29 (1H, t, 5.8 Hz), δ 8.49 (1H, d, 8.5 Hz), δ 8.62 (1H, t, 6.7 Hz). 13 C - NMR (100 MHz, DMSO - d6) δ 7.6, 10.8, 10.9, 2 3.5, 24.6, 25.7, 27.7, 30.2, 30.6, 34.8, 36.9, 3 7.1, 41.7, 42.0, 44.4, 49.5, 54.1, 65.1, 66.9, 6 9.7, 72.2, 96.6, 109.6, 109.8, 119.0, 121.5, 12 3.4, 123.6, 125.3, 126.2, 128.0, 129.0, 134.3, 136.2, 136.3, 137.7, 140.4, 145.0, 147.7, 147. 8, 149.9, 152.2, 156.6, 160.2, 162.7, 168.8, 16 9.1, 169.3, 170.0, 171.0, 171.3, 172.3, 172.5. MS(ESI)(m / z): 1034 ([M + H] + ).

[0465] Powder X - ray diffraction: The crystals of the title compound were subjected to powder X-ray diffraction analysis using copper Kα radiation. The results are shown in Table 2 and Figure 4. The main diffraction angles (2θ) were 5.6°, 15.5°, and 22.0°. A significant peak was observed.

[0466] [Table 2]

[0467] Example 15 ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl ethyl acetate

[0468] [ka]

[0469] Under a nitrogen atmosphere, N-9-fluorenylmethoxycarbonylglycylglycine (2.85 To a suspension of acetic acid (2 kg, 8.04 mol) in anhydrous tetrahydrofuran (38.0 kg), 0.41 kg, 40.1 mol), lead(IV) tetraacetate (5.35 kg, 12.0 mol) The mixture was refluxed for 1.5 hours, cooled to room temperature, and the precipitated solid was filtered off. The solid was washed with tetrahydrofuran (10.1 kg). The obtained filtrate was diluted under reduced pressure. The concentrate was concentrated to about 16 L, and the resulting concentrate was added with ethyl acetate (26 kg), 10% Aqueous phosphoric acid solution (17.1 L) and 20% saline solution (5.7 L) were added, stirred, and then the mixture was separated. The organic layer was diluted with 10% aqueous citric acid (17.1 L), 9% aqueous sodium bicarbonate (28 The organic layer was washed with 20% sodium chloride solution (14.3 L) and then with 20% sodium chloride solution (14.3 L). Add ethanol 60 (5.7 kg) and ethyl acetate (10.3 kg), stir for 1 hour, and then filter the solid. The solid was separated by filtration, and washed with ethyl acetate (7.7 kg). The liquid was concentrated until the volume was about 5 L, and cyclopentyl methyl ether (24.5 kg) was added. The mixture was concentrated again under reduced pressure until the volume was about 5 L, and cyclopentene was added to the resulting concentrate. C. Dimethyl methyl ether (14.7 kg) was added and the mixture was stirred at about 5.degree. C. for 1 hour. The crystals were filtered and the resulting crystals were added to cyclopentyl methyl ether (4.9 mL) cooled to about 5°C. The resulting crystals were dried under reduced pressure at 40°C to give the title compound (2.01 kg, The compound was obtained as a colorless solid (68% yield).

[0470] Example 16 Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl} Amino)methoxy]acetate

[0471] [ka]

[0472] Under a nitrogen atmosphere, ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine (2.01 kg, 5.46 mol) of anhydrous 1,2-dimethylamino)methyl acetate A suspension of 21 kg of methyltriethoxyethane was added to 1.81 kg of benzyl glycolate (10.9 mo The mixture was cooled to about 0°C. Tris(pentafluorophenyl)borane (142g , 0.27 mol) was added, and the mixture was stirred at the same temperature for 3 hours. % aqueous potassium bicarbonate solution was added, the temperature was raised to room temperature, and the layers were separated. Brine (20.1 L) was added and the mixture was separated and washed. The obtained organic layer was then washed under reduced pressure until the volume was reduced to about 4 L. The mixture was concentrated until the liquid volume reached about 4 L, and methanol (15.7 kg) was added. The mixture was concentrated until the liquid volume was reduced under reduced pressure. The concentrate was concentrated to about 4 L. Methanol (12.5 kg) was added to the resulting concentrate, The mixture was cooled to about 5°C and stirred for 1 hour. The precipitated crystals were filtered and the obtained crystals were cooled to about 5°C. The solid was washed with chilled methanol (4.7 kg), dried under reduced pressure at 40°C, and The title compound was obtained (2.28 kg, 88% yield).

[0473] Example 17 Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl Lysyl-L-phenylalanyl(amino)methoxy]acetate

[0474] [ka]

[0475] Under a nitrogen atmosphere, benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbamate N-[(2-methyl-2-methyl-1,2-diphenyl)glycyl}amino)methoxy]acetate (2.28 kg, 4.81 mol) ,N-dimethylacetamide (15.0 kg) solution of 1,8-diazabicyclo[5.4. 0]Undec-7-ene (0.37 kg, 2.4 mol) was added and stirred at room temperature for 30 minutes. Pyridinium p-toluenesulfonate (0.60 kg, 2.4 mol), 1-hydroxybenzoate Roxybenzotriazole monohydrate (0.74 kg, 4.8 mol), N-[(9H- Fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (2.19 kg, 4.37 mol), 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide hydrochloride (0.84 kg, 4.37 mol) was added and stirred at room temperature for 3 hours. Ethyl acetate (21.0 kg) and 10% brine (34 L) were added, and the mixture was stirred and then separated. The separated organic layer was washed with 10% aqueous citric acid solution (11.4 L). Tetrahydrofuran (20 kg) and 15% potassium bicarbonate aqueous solution (22.8 L) were added to the The resulting organic layer was washed with 10% brine (22.8 L). The organic layer was concentrated under reduced pressure until the liquid volume was about 6.8 L, and 2-propanol (12. The mixture was concentrated again under reduced pressure until the liquid volume was about 6.8 L. 2-propanol (30.2 kg) was added to the solution while heating it to about 50°C. After stirring, the mixture was cooled to about 5°C and stirred for another 2 hours. The precipitated solid was filtered. The crystals were washed with 2-propanol (14.2 kg) cooled to about 5°C. The mixture was suspended in 2-propanol (36 kg) and stirred at about 5°C for 1 hour. The solid was filtered off, and the filtered solid was added to 2-propanol (28.5 kg) cooled to about 5°C. The obtained crystals were dried at 50°C under reduced pressure to give the title compound (3.34 kg, yield 9 4%).

[0476] 1 H-NMR (400MHz,DMSO-d6)δ2.79(1H,dd,J=14. 0, 9.8Hz),3.05(1H,dd,J=14.0, 4.3Hz),3.58 -3.79(6H,m),4.15(2H,s),4.20-4.24(1H,m),4 .28-4.30(2H,m),4.48-4.53(1H,m),4.63(2H,d ,J=6.7Hz),5.14(2H,s),7.15-7.43(13H,m),7. 58(1H,t,J=6.1Hz),7.71(2H,d,J=7.3Hz),7.89 (2H,d,J=7.9Hz),8.01(1H,t,J=5.5Hz),8.15(1 H,d,J=7.9Hz),8.33(1H,t,J=5.8Hz),8.59(1H, t,J=6.4Hz). 13 C-NMR (100MHz,DMSO-d6)δ37.3, 41.8, 42. 1, 43.5, 46.6, 54.1, 64.4, 65.6, 65.7, 6 9.0, 120.1, 125.2, 126.3, 127.1, 127.6, 128.0, 128.1,128.1, 128.4, 129.1, 135.8, 137.8, 140.7, 143.8, 156.5, 168.8, 169. 4, 169.9, 170.1, 171.4. MS(ESI)(m / z):736([M+H] + ).

[0477] Example 18 N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-fluor Phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

[0478] [ka]

[0479] Benzyl[({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl Glycyl-L-phenylalanyl(amino)methoxy]acetate (367g, 0.49 9 mol) in tetrahydrofuran (5.88 kg) and water (1.61 L). Add 28 g of ethylenediamine-carbon complex and heat at room temperature under atmospheric pressure for 1 hour. The mixture was stirred for 3 hours from the start of the reaction. The catalyst was filtered off, and the filtered catalyst was dissolved in tetrahydrofuran (1.63kJ The above reaction and catalyst filtration procedures were repeated nine times. The filtrate and washings were combined and concentrated under reduced pressure until the volume was about 17 L. 2-Propanol (39 kg) was added to the liquid, and the liquid was concentrated under reduced pressure until the volume was about 17 L. This procedure was repeated three times. Ethyl acetate (45 kg) was added to the resulting concentrate, and the mixture was stirred at room temperature. The suspension was stirred at 5°C for 6 hours. The suspension was further stirred at 5°C for 1 hour. The precipitated solid was filtered. The filtered solid was cooled to about 5°C and then soaked in a 1:3 mixture of 2-propanol and ethyl acetate (2 The resulting crystals were dried under reduced pressure at 40°C to obtain the crude title compound (2. The crude product (400 g, 0.62 mol) was added to tetrahydrofuran (18 kg, 75% yield). Suspended in 2.4 L of dolofuran and 5.6 L of ethyl acetate, and added to 1% aqueous potassium hydrogen sulfate. The solution (4 L) was added, and the mixture was heated to about 32°C and stirred to dissolve. The resulting organic layer was separated. The organic layer was separated and washed with water (2 L). The obtained organic layer was concentrated under reduced pressure to a volume of about 2 L. Acetonitrile (6 L) was added to the concentrated solution, and the solution was concentrated under reduced pressure to a volume of approximately 2.8 L. Ethyl acetate (6 L) was added, and the mixture was stirred at room temperature for 18 hours. The mixture was cooled to rt and stirred for 3 hours. The precipitated solid was filtered off, and the filtered crystals were dissolved in acetonitrile. The solid was washed with a 1:2 mixture of ethyl acetate and hexane (7 L). The solid was dried at 40°C under reduced pressure. The title compound (356 g, yield 89%) was obtained.

[0480] 1H-NMR(400MHz,DMSO-d6)δ2.79(1H,dd,J=14.0 , 9.8Hz),3.06(1H,dd,J=13.7, 4.6Hz),3.58- 3.79(6H,m),3.98(2H,s),4.21-4.25(1H,m),4. 28-4.30(2H,m),4.48-4.54(1H,m),4.61(2H,d, J=6.7Hz),7.16-7.20(1H,m),7.22-7.27(4H,m) ,7.33(2H,t,J=7.3Hz),7.42(2H,t,J=7.3Hz),7 .59(1H,t,J=6.1Hz),7.71(2H,d,J=7.3Hz),7.8 9(2H,d,J=7.3Hz),8.03(1H,t,J=5.5Hz),8.16( 1H,d,J=7.9Hz),8.33(1H,t,J=5.8Hz),8.57(1H ,t,J=6.7Hz). 13 C-NMR (100MHz,CDCl3)δ37.4, 41.8, 42.1, 43.5, 46.6, 54.1, 64.2, 65.7, 68.8, 120 .1, 125.2, 126.3, 127.1, 127.6, 128.1, 1 29.1, 137.8, 140.7, 143.8, 156.5, 168.8, 169.4, 170.0, 171.4, 171.4. MS(ESI)(m / z):646([M+H] + ).

[0481] Example 19 N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-fluor Phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxybenzoate Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydroxy Hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycine amino Do

[0482] [ka]

[0483] Under a nitrogen atmosphere, (1S,9S)-9-ethyl-5-fluoro-9-methanesulfonate Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexyl Sahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[ 1,2-b]Quinoline-1-aminium dihydrate (260g, 0.458mol) Methyl sulfoxide (1.8 L), triethyl methyl sulphoxide (1.3 L) suspension in tetrahydrofuran Amine (55.6 g, 0.549 mol), 1-hydroxybenzotriazole monohydrate Solvate (84.2g, 0.549mol), N-[(9H-fluoren-9-ylmethoxy )carbonyl]glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy )methyl]glycinamide (325g, 0.503mol), 1-ethyl-3-(3-di Methylaminopropyl)-carbodiimide hydrochloride (114 g, 0.595 mol) was added. The mixture was stirred at room temperature for 2 hours. Tetrahydrofuran (3.9 L), ethyl acetate (2.6 L), An 11% aqueous solution of potassium bicarbonate (5.2 L) was added, and the mixture was stirred and separated. 9% citric acid solution (3.9 L), 22% potassium bicarbonate solution (2.6 L), 18% The organic layer was washed with brine (0.78 L) and then activated carbon (52 g) was added. After stirring for 10 minutes, tetrahydrofuran (0.78 L), anhydrous magnesium sulfate (0.78 g) was added and stirred for 30 minutes. The solid was filtered off, and the filtered solid was dissolved in tetrahydrofuran (0 The filtrate was concentrated under reduced pressure to a volume of approximately 200 mL. Ethyl acetate (1.3 L) was added to the concentrated solution, and the solution was concentrated under reduced pressure to a volume of approximately 200 mL. Tetrahydrofuran (1.8 L) was added to the obtained concentrated solution. A mixture of ethyl acetate (1.3 L) and cyclopentyl methyl ether (1.3 L) prepared in To this suspension was added cyclopentyl methyl ether (2.6 L). After stirring for 18 hours, the mixture was cooled to about 5°C and stirred for another hour. The filtered solid was dissolved in tetrahydrofuran and cyclopentyl methyl ether in a ratio of 1:3. The resulting solid was dried under reduced pressure at 40°C to give the title compound (4 0.8 g, yield 84%).

[0484] 1 H-NMR(400MHz,DMSO-d6)δ0.86(3H,t,J=7.3), 1.79-1.90(2H,m),2.11-2.22(2H,m),2.37(3H, s),2.77(1H,dd,J=14.0, 9.8Hz),3.02(1H,dd, J=13.7, 4.6Hz),3.07-3.25(2H,m),3.58-3.79 (6H,m),4.02(2H,s),4.18-4.23(1H,m),4.26-4 .30(2H,m),4.45-4.54(1H,m),4.64(2H,d,J=6. 7Hz), 5.17 (2H, dd, J = 23.5, J = 19.2Hz), 5.40 (2H , s), 5.56 - 5.61 (1H, m), 6.52 (1H, s), 7.14 - 7.43 (10H, m), 7.58 (1H, t, J = 6.1Hz), 7.68 (2H, d, J = 7 .3Hz), 7.76 (1H, d, J = 11.0Hz), 7.86 (2H, d, J = 7. 3Hz), 8.02 (1H, t, J = 5.5Hz), 8.15 (1H, d, J = 7.9H z), 8.32 (1H, t, J = 5.8Hz), 8.50 (1H, d, J = 8.5Hz) , 8.63 (1H, t, J = 6.4Hz). 13 C - NMR (100MHz, DMSO - d6) δ7.7, 10.9, 11.0, 23.1, 23.7, 27.8, 30.3, 31.4, 37.3, 41. 8, 42.1, 43.5, 44.6, 46.6, 49.6, 54.2, � *.6, 65.2, 65.8, 67.0, 69.8, 72.3, 82.0, 96.7, 109.7, 109.9, 119.1, 120.0, 121.6 , 123.5, 123.7, 125.2, 125.3, 126.3, 127 .0, 127.6, 128.1, 129.1, 136.3, 136.4, 13 7.8, 140.5, 140.7, 143.8, 143.8, 145.1,<00039​​​​​​​​​​​​ Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethoxy] 5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4': 6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy [Ci)methyl]glycinamide

[0486] [ka]

[0487] Under a nitrogen atmosphere, N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl Lysyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-phenylalanyl] Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]i Indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl ] Glycinamide (400 g, 0.376 mol) suspended in anhydrous tetrahydrofuran (8 L) The suspension was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (51.6 g, 0.3 39 mol) was added in eight portions every 5 minutes, and the mixture was stirred for 2.5 hours. The solid was filtered off and washed with tetrahydrofuran (2.4 L). The solid was dried under reduced pressure at 40°C to give a mixture containing the title compound (363 g, yield 115%). ) was obtained.

[0488] 1 H-NMR(400MHz,DMSO-d6)δ0.87(3H,t,J=7.3), 1.57-1.67(6H,m),1.80-1.92(2H,m),2.06-2.2 5(2H,m),2.35-2.38(3H,m),2.61-2.63(2H,m), 2.73-2.89(1H,m),3.00-3.79(29H,m),3.80(1H ,dd,J=16.2, 7.0Hz),3.99-4.10(2H,m),4.30- 4.51(1H,m),4.58(1H,dd,J=9.8, 6.1Hz),4.63 -4.69(1H,m),5.01(0.5H,br),5.15(1H,t,J=18 .3Hz),5.24(1H,t,J=18.3Hz),5.41(2H,s),5.5 4-5.62(1H,m),6.52(0.6H,br),7.11-7.31(6H, m),7.75-7.79(1H,m),8.12-8.15(0.6H,m),8.2 2(0.2H,d,J=8.5Hz),8.36(0.2H,t,J=5.8Hz),8 .52(0.2H,t,J=5.5Hz),8.66(0.2H,t,J=6.4Hz) ,8.93(0.6H,t,J=5.5Hz),9.10(1H,dd,J=20.1, 9.2Hz), 9.82(0.6H,br). MS(ESI)841:(M+H) +

[0489] Example 21 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa N-[(2-[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox Preparation of seed crystals of [ethoxymethyl]glycinamide.

[0490] [ka]

[0491] Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-en 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-oxoeto [(hydroxy)methyl]glycinamide (200 mg, 0.24 mmol) in pyridine (0.2 m A suspension of pyridine in tetrahydrofuran (2.0 mL) and acetonitrile (0.6 mL) was Dinium p-toluenesulfonate (120 mg, 0.48 mmol), triethylamine amine (100 μL, 0.72 mmol), 6-maleimidohexanoic acid N-succinimide The reaction mixture was added with ethanol (73 mg, 0.24 mmol) and stirred at room temperature for 3 hours. Flash column chromatography (Biotage AB) [tetrahydrofuran The mixture was purified using a solvent-free solvent mixture containing 1,000 ethanol and 1,000 ethanol (v / v): acetone = 3:7 to 7:3 (v / v) to obtain the title compound as an oil. The resulting oil (19.5 mg) was dissolved in acetone (0.4 mL) and 2-butanol (0.2 mL). The mixture was heated to about 60°C, and the precipitated solid was filtered at room temperature. The extract was washed with ethanol (approximately 0.2 mL) to give the title compound (14.3 mg) as a colorless powder. The resulting powder was used as seed crystals in the next reaction.

[0492] Example 22 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa N-[(2-[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox (ethoxy)methyl]glycinamide

[0493] [ka]

[0494] Under a nitrogen atmosphere, pyridine (0.35 L), acetonitrile (1.1 L), tetrahydrofuran Pyridinium p-toluenesulfonate (209g, 0.832L) in furan (3.5L) mol), N-succinimidyl 6-maleimidohexanoate (128g, 0.415m ol), glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox After dissolving [isoethoxymethyl]glycinamide (350 g, 0.416 mol), Triethylamine (63.2 g, 0.625 mol) was added, and the mixture was stirred at room temperature for 3.5 hours. Tetrahydrofuran (3.5 L), 19% aqueous citric acid solution (3.5 L), ethyl acetate ( After stirring, the organic layer was separated. 9% citric acid solution (2.5 L) and 18% saline solution (2.5 L) were added, stirred, and then separated. The resulting organic layer was washed with 22% aqueous potassium bicarbonate (2.1 L) and then with 18% saline (1 L). The resulting organic layer was washed with acetone (0.8 L) and the resulting solution was washed with activated carbon (35 g) in a separate container. The mixture was added dropwise to a suspension of nitrile (35 L) and stirred for 30 minutes, after which the activated carbon was filtered off. The charcoal was washed with acetonitrile (1.8 L). The filtrate was then heated under reduced pressure at an external temperature of about 40°C. The mixture was concentrated until the solvent stopped distilling. The concentrated residue was diluted with acetone (1.8 L), 1-propanol (1.8 L), and 1-propanol (1.8 L). Add propyl alcohol (3.5 L) and heat to 55°C to dissolve, then cool to room temperature. The powder (0.2 g) obtained in Example 21 was added as a seed crystal, and the mixture was stirred for 86 hours. The solid was filtered off and washed with acetone (1.1 L). The crystals were dried at 40°C under reduced pressure to give the title compound (191 g, yield 44%) as crystals.

[0495] Example 23 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa N-[(2-[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox (ethoxy)methyl]glycinamide

[0496] [ka]

[0497] Anhydrous sodium sulfate (1.8 g), ethyl cyano(hydroxyimino)acetate (0.16 g, 1.13 mmol), N-[6-(2,5-dioxo-2,5-dihydro-1H-pi glycylglycyl-L-phenylalanyl-N-[(( carboxymethoxy)methyl]glycinamide (gross amount 5.76g, 1,2-dimethoxy Purified water containing 12.40% tetrahydrofuran (contents 5.05 g, 8.18 mmol) and tetrahydrofuran A mixture of 24 mL and 18 mL of tetrahydrofuran was heated at 20-30°C with methanesulfonate. Acid (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-amini A mixture of purified water and tetrahydrofuran (9 mL and 1 mL) containing ammonium hydroxide (3.0 g, 5.64 mmol) was The mixture was added with tetrahydrofuran (9 mL) and N-methyl ... A solution of methylparaben (0.63 g, 6.23 mmol) in 7.5 mL of tetrahydrofuran After stirring at the same temperature for 15 minutes, 1-(3-dimethylaminopropyl)-3-ethyl Rubodimide hydrochloride (2.16 g, 11.27 mmol) and purified water and tetrahydrofuran The mixture was then heated at 20-30°C for at least 30 minutes. After stirring and confirming the completion of the reaction, the mixture was separated and the aqueous layer was removed. The organic layer was then cooled to 15 to 25°C. The mixture was diluted with ethyl acetate (36 mL), anhydrous sodium sulfate (1.26 g), N-methyl ammonium chloride (1.26 g), and HCl. Purified water (24 mL) containing holin (0.14 g, 1.38 mmol) was added and stirred. The aqueous layer was removed. Purified water (9 mL) was added to the organic layer, and the mixture was stirred and separated. Purified water (9 mL) containing activated carbon (0.45 mL) was added, stirred, and the layers were separated to obtain an organic layer. Add 0.30 g of Strong Shirasagi (Osaka Gas Chemicals Co., Ltd.) and stir at room temperature for approximately 15 minutes. After stirring, the activated carbon was filtered off, washed with tetrahydrofuran (9 mL), and the filtrate was The concentrate was added with tetrahydrofuran (75 mL) and concentrated under reduced pressure to 30 mL. The mixture was concentrated under reduced pressure to 30 mL, and tetrahydrofuran (45 mL) was added. After confirming that the water content in the concentrate was 8.0% (v / v) or less, acetone and 1- A mixture of propanol (30 mL and 71 mL) was added. -2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L -phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9 -hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexyl 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycine The amide (30 mg) was added and stirred at 20-30°C for 12 hours or more. After cooling to 100°C, the mixture was stirred for 24 hours or more, and the precipitate was filtered off. The filtered powder was then acetone-treated at 0-5°C. The resulting crystals were washed with a 1:1 mixture of ethanol and 1-propanol (30 mL). Dry at 35°C and add N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole- 1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[ (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13 -Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d e]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]a Crude crystals of [(amino)-2-oxoethoxy)methyl]glycinamide (5.23 g, yield 89%) .6%).

[0498] N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl N-[(2-[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3 ',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-o Crude crystals of [(2-(ethoxy)methyl)glycinamide] (4.50 g, 4.35 mmol) were added to acetic acid. A mixture of acetone and purified water (10.6 mL and 2.9 mL) containing acid (15 μL) was added. The mixture was stirred at 34-38°C for at least 1 hour, and after dissolution was confirmed, it was cooled to 20-25°C. A mixture of 1-propanol (31.5 mL and 64.8 mL) was added, and N-[6-(2,5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycyl Lysyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-phenylalanyl] Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]i Indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl ] Glycinamide (27 mg) was added, and the mixture was stirred at 20 to 25°C for 24 hours or more. After cooling to 0-5°C, the mixture is stirred for 12 hours or more, and the precipitate is filtered. The resulting crystals were washed with a 1:1 mixture of acetone and 1-propanol (27 mL). Dry under reduced pressure at 35°C and add N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolidine]. glycylglycyl-L-phenylalanyl-N-[(2 -{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[a]pyridine Zo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl Purified crystals of [(2-(2-(2-hydroxyethoxy)amino)methyl]glycinamide (4.37 g, yield The success rate was 93.0%. The instrumental data was similar to that of the compound described in Example 14.

[0499] Example 24 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexa N-[(2-[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox (ethoxy)methyl]glycinamide

[0500] [ka]

[0501] Anhydrous sodium sulfate (1.8 g), ethyl cyano(hydroxyimino)acetate (0.16 g, 1.13 mmol), N-[6-(2,5-dioxo-2,5-dihydro-1H-pi glycylglycyl-L-phenylalanyl-N-[(( carboxymethoxy)methyl]glycinamide (gross amount 5.76g, 1,2-dimethoxy Purified water containing 12.40% tetrahydrofuran (contents 5.05 g, 8.18 mmol) and tetrahydrofuran A mixture of 24 mL and 18 mL of tetrahydrofuran was heated at 20-30°C with methanesulfonate. Acid (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-amini A mixture of purified water and tetrahydrofuran (9 mL and 1 mL) containing ammonium hydroxide (3.0 g, 5.64 mmol) was The mixture was added with tetrahydrofuran (9 mL) and N-methyl ... A solution of methylparaben (0.63 g, 6.23 mmol) in 7.5 mL of tetrahydrofuran After stirring at the same temperature for 15 minutes, 1-(3-dimethylaminopropyl)-3-ethyl Rubodimide hydrochloride (2.16 g, 11.27 mmol) and purified water and tetrahydrofuran The mixture was then heated at 20-30°C for at least 30 minutes. After stirring and confirming the completion of the reaction, the mixture was separated and the aqueous layer was removed. The organic layer was then cooled to 15 to 25°C. The mixture was diluted with ethyl acetate (36 mL), anhydrous sodium sulfate (1.26 g), N-methyl ammonium chloride (1.26 g), and HCl. Purified water (24 mL) containing holin (0.14 g, 1.38 mmol) was added and stirred. The aqueous layer was removed. Purified water (9 mL) was added to the organic layer, and the mixture was stirred and separated. Purified water (9 mL) containing activated carbon (0.45 mL) was added, stirred, and the layers were separated to obtain an organic layer. Add 0.30 g of Strong Shirasagi (Osaka Gas Chemicals Co., Ltd.) and let it sit at room temperature for about 15 minutes. After stirring, the activated carbon was filtered off and washed with tetrahydrofuran (9 mL). The mixture was combined and concentrated under reduced pressure to 30 mL. Tetrahydrofuran (75 mL) was added to the concentrated solution, Concentrate under reduced pressure to 30 mL, add tetrahydrofuran (45 mL), and reduce the pressure to 30 mL After confirming that the water content in the concentrate was 8.0% (v / v) or less, Add a mixture of 1-propanol (30 mL and 71 mL) and stir at 20-30°C for 22 hours. After cooling the suspension to 0-5°C, it was further stirred for 24 hours or more, and the precipitate was filtered and separated. The powder was washed with a 1:1 mixture of acetone and 1-propanol (30 mL) at 0-5°C. The crystals were dried under reduced pressure at 35°C to give N-[6-(2,5-dioxo-2,5-dihydro- -1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl -N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]chino Crystals of [phosphon-1-yl]amino}-2-oxoethoxy)methyl]glycinamide (5. 0.8g, yield 87.0%).

[0502] The instrumental data was similar to that of the compound described in Example 14. [Sequence List Free Text]

[0503] SEQ ID NO: 1: Amino acid sequence of the anti-HER2 antibody heavy chain SEQ ID NO: 2: Amino acid sequence of the anti-HER2 antibody light chain SEQ ID NO: 3: Amino acid sequence of the heavy chain of the anti-HER3 antibody SEQ ID NO: 4: Amino acid sequence of the light chain of the anti-HER3 antibody SEQ ID NO: 5: Amino acid sequence of the anti-TROP2 antibody heavy chain SEQ ID NO: 6: Amino acid sequence of the anti-TROP2 antibody light chain SEQ ID NO: 7: Amino acid sequence of the anti-B7-H3 antibody heavy chain SEQ ID NO: 8: Amino acid sequence of the anti-B7-H3 antibody light chain SEQ ID NO: 9: Amino acid sequence of the anti-GPR20 antibody heavy chain SEQ ID NO: 10: Amino acid sequence of the anti-GPR20 antibody light chain

Claims

[Claim 1] The invention described herein.

Images