Novel manufacturing method of antibody-drug conjugate

Abstract

A method for manufacturing a compound represented by formula (F), wherein R1 represents an amino group protected by a protecting group, and R2 represents an amino group protected by a protecting group, the method comprising the following steps: a step of converting a compound represented by formula (C) into a compound represented by formula (F), wherein R1 represents the same meaning as described above, wherein the step comprises the following sub-steps: (i) reacting the compound represented by formula (C) with a nitrite ester in the presence of a base to introduce a nitroso group at the α-position of the carbonyl group of the compound represented by formula (C); (ii) introducing a protecting group to a nitrogen atom derived from the nitroso group; and (iii) reducing the compound manufactured in sub-step (ii) with hydrogen in the presence of a platinum carbon catalyst.

Description

Technical Field

[0001] The present invention relates to a novel manufacturing method of exatecan, which is a component of an antibody-drug conjugate, and a novel manufacturing method of an antibody-drug conjugate using the same. Prior Art

[0002] An antibody-drug conjugate (ADC) is a conjugate of an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into cells and a cytotoxic drug. By selectively delivering the drug to cancer cells, it is expected to accumulate the drug in cancer cells and cause cancer cell death (Non-Patent Documents 1 to 5).

[0003] As one of the antibody-drug conjugates, an antibody-drug conjugate composed of an antibody and exatecan, which is a topoisomerase I inhibitor, is known (Patent Documents 1 to 8, Non-Patent Documents 6 and 7). These antibody-drug conjugates are currently undergoing clinical trials because of their excellent antitumor effects and safety.

[0004] As a method for manufacturing exatecan, the methods described in Patent Documents 9 to 11 are known. [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2014 / 057687 [Patent Document 2] International Publication No. 2014 / 061277 [Patent Document 3] International Publication No. 2015 / 098099 [Patent Document 4] International Publication No. 2015 / 115091 [Patent Document 5] International Publication No. 2015 / 146132 [Patent Document 6] International Publication No. 2015 / 155976 [Patent Document 7] International Publication No. 2015 / 155998 [Patent Document 8] International Publication No. 2018 / 135501 [Patent Document 9] Japanese Patent Application Laid-Open No. 5-59061 [Patent Document 10] Japanese Patent Application Laid-Open No. 8-337584 [Patent Document 11] WO 96 / 26181

[0006] [Non-Patent Document] [Non-Patent Document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-Patent Document 2] Alley, S. C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [Non-Patent Document 3] Damle N. K. Expert Opin. Biol. Ther. (2004) 4, 1445-1452. [Non-Patent Document 4] Senter P. D., 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

[0007] [Problems to be Solved by the Invention] Irinotecan is a compound represented by the formula (2), which is a compound that is a constituent element of the antibody-drug conjugate of the present invention.

[0008] As a method for producing irinotecan, the methods described in Patent Documents 9 to 11 are known. This production method can be represented as follows. That is, the compound represented by formula (19) is reacted with succinic anhydride to be converted into the compound represented by formula (20), reduced to be converted into the compound represented by formula (21), converted into the compound represented by formula (22) by an intramolecular cyclization reaction, oximated to be converted into the compound represented by formula (23), converted into the compound represented by formula (24) by a Beckmann rearrangement, converted into the compound represented by formula (25) by a ring-opening reaction, the amino group is protected to be converted into the compound represented by formula (26), hydrolyzed to be converted into the compound represented by formula (27), converted into the compound represented by formula (28) by an intramolecular cyclization reaction, reduced to be converted into the compound represented by formula (29), oxidized to be converted into the compound represented by formula (9), and then, a nitrogen atom is introduced to be converted into the compound represented by formula (10), selectively deprotected to be converted into the compound represented by formula (11), converted into the compound represented by formula (12) by a Friedlander reaction with the compound represented by formula (1), and finally, the acetyl group is deprotected to produce the compound represented by formula (2), that is, a method for producing irinotecan. However, since this production method requires repeated ring-opening / ring-closing reactions and oxidation / reduction reactions, it is considered that the number of steps is long and complicated operations are required. Therefore, the development of an industrially excellent production method is eagerly desired.

[0009] One object of the present invention is to find a novel production method for irinotecan with a short number of steps and excellent industrial properties. Another object of the present invention is to construct a novel production method for an antibody-drug conjugate using the same. [Means for Solving the Problem]

[0010] As a result of intensive studies on the production method of irinotecan by the present inventors, a novel production method for irinotecan with a short number of steps and excellent industrial properties was found. Furthermore, a novel production method for an antibody-drug conjugate using irinotecan produced by this production method was constructed.

[0011] That is, the present invention relates to: [1] A method for producing a compound represented by formula (C), which comprises the following steps: By intramolecular cyclization of the compound represented by formula (B) (wherein, R1 represents an amino group protected by a protecting group), and the step of converting to the compound represented by formula (C), (wherein, R1 represents the same meaning as described above). [2] The production method according to [1], wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl. [3] The production method according to [1], wherein R1 is an amino group protected by acetyl or trifluoroacetyl. [4] The production method according to [1], wherein R1 is an amino group protected by acetyl. [5] The production method according to any one of [1] to [4], wherein the intramolecular cyclization is carried out by a method comprising reacting the compound represented by formula (B) with trifluoroacetic anhydride. [6] The production method according to [5], wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid. [7] The production method according to any one of [1] to [4], wherein the intramolecular cyclization is carried out by a method comprising reacting the compound represented by formula (B) with thionyl chloride. [8] The production method according to [7], wherein the intramolecular cyclization is carried out in the presence of aluminum chloride. [9] A production method of a compound represented by formula (C), which comprises the following steps: By carrying out intramolecular cyclization of the compound represented by formula (J) (wherein, Y represents a leaving group, and R1 represents an amino group protected by a protecting group), and the step of converting to the compound represented by formula (C), (wherein, R1 represents the same meaning as described above).

[10] The production method according to [9], wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[11] The production method according to [9], wherein R1 is an amino group protected by acetyl or trifluoroacetyl.

[12] The production method according to [9], wherein R1 is an amino group protected by acetyl.

[13] The production method according to any one of [9] to

[12] , wherein Y is a chloro group.

[14] The production method according to any one of [9] to

[12] , wherein Y is a trifluoroacetoxy group.

[15] The production method according to

[13] , wherein the intramolecular cyclization is carried out in the presence of aluminum chloride.

[16] The production method according to

[14] , wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid.

[17] A production method of a compound represented by formula (C), which comprises the following steps: Coupling a compound represented by formula (D) with 3-butenoic acid (wherein, X represents a leaving group, and R1 represents an amino group protected by a protecting group), And a step of converting it into a compound represented by formula (E) (wherein, R1 represents the same meaning as described above); Next, reducing the compound represented by formula (E) And a step of converting it into a compound represented by formula (B) (wherein, R1 represents the same meaning as described above); Next, carrying out intramolecular cyclization on the compound represented by formula (B) And a step of converting it into a compound represented by formula (C) (wherein, R1 represents the same meaning as described above).

[18] The production method according to

[17] , wherein X is a bromo group, an iodo group, a trifluoromethanesulfonyloxy group, or an arylsulfonyloxy group.

[19] The production method according to

[17] , wherein X is a bromo group.

[20] The production method according to

[17] , wherein X is an iodo group.

[21] The production method according to any one of

[17] to

[20] , wherein R1 is an amino group protected by an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a trimethylacetyl group, a formyl group, or a benzoyl group.

[22] The production method according to any one of [7] to

[10] , wherein R1 is an amino group protected by an acetyl group or a trifluoroacetyl group.

[23] The production method according to any one of

[17] to

[20] , wherein R1 is an amino group protected by an acetyl group.

[24] The production method according to any one of

[17] to

[23] , wherein the step of coupling the compound represented by formula (D) with 3-butenoic acid to convert it into the compound represented by formula (E) is carried out in the presence of a palladium complex prepared from palladium(II) acetate and tris(o-tolyl)phosphine.

[25] The production method according to any one of

[17] to

[24] , which includes: a step of dissolving the compound represented by formula (E) in an alkaline aqueous solution and performing liquid separation washing with a first organic solvent; secondly, adding an acid to the alkaline aqueous solution and performing liquid separation extraction of the compound represented by formula (E) with a second organic solvent.

[26] The production method according to

[25] , wherein the first organic solvent is 2-methyltetrahydrofuran.

[27] The production method according to

[25] or

[26] , wherein the second organic solvent is 2-methyltetrahydrofuran.

[28] The production method according to any one of

[25] to

[27] , wherein the alkaline aqueous solution is an aqueous sodium hydroxide solution.

[29] The production method according to any one of

[17] to

[28] , wherein the step of reducing the compound represented by formula (E) to convert it into the compound represented by formula (B) is carried out by reacting the compound represented by formula (E) with hydrogen in a solvent in the presence of a palladium-carbon catalyst.

[30] The production method according to any one of

[17] to

[29] , wherein the step of subjecting the compound represented by formula (B) to intramolecular cyclization to convert it into the compound represented by formula (C) is carried out by a method including reacting the compound represented by formula (B) with trifluoroacetic anhydride.

[31] The production method according to

[30] , wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid.

[32] The production method according to any one of

[17] to

[29] , wherein the step of subjecting the compound represented by formula (B) to intramolecular cyclization to convert it into the compound represented by formula (C) is carried out by a method including reacting the compound represented by formula (B) with thionyl chloride.

[33] The production method according to

[32] , wherein the intramolecular cyclization is carried out in the presence of aluminum chloride.

[34] A production method of a compound represented by formula (2), characterized by using the compound represented by formula (C) produced by the method according to any one of [1] to

[33] as a starting material, It includes the following steps: The step of converting the compound represented by formula (C) into the compound represented by formula (F) (wherein, R1 represents the same meaning as R1 described in any one of claims 1 to 33, and R2 represents an amino group protected by a protecting group); Next, the step of converting the compound represented by formula (F) into the compound represented by formula (G) (wherein, R2 represents the same meaning as described above); Next, condense the compound represented by formula (G) with the compound represented by formula (1), and convert it into the compound represented by formula (H) (wherein, R2 represents the same meaning as described above); Next, the step of converting the compound represented by formula (H) into the compound represented by formula (2) .

[35] The production method according to

[34] , wherein R2 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[36] The production method according to

[34] , wherein R2 is an amino group protected by acetyl or trifluoroacetyl.

[37] The production method according to

[34] , wherein R2 is an amino group protected by acetyl.

[38] The production method according to any one of

[34] to

[37] , wherein the step of converting the compound represented by formula (C) into the compound represented by formula (F) includes: (i) a stage of reacting with a nitrite in the presence of a base and introducing a nitroso group; next, (ii) a stage of introducing a protecting group to the nitrogen atom derived from the nitroso group; and (iii) a stage of reducing with hydrogen in the presence of a platinum-carbon catalyst.

[39] The production method according to any one of

[34] to

[38] , wherein the step of converting the compound represented by formula (F) into the compound represented by formula (G) is carried out in a solvent containing hydrochloric acid / ethanol.

[40] The production method according to any one of

[34] to

[39] , wherein the step of condensing the compound represented by formula (G) with the compound represented by formula (1) to convert it into the compound represented by formula (H) is carried out in a solvent containing o-cresol.

[41] The production method according to any one of

[34] to

[40] , wherein the step of converting the compound represented by formula (H) into the compound represented by formula (2) is carried out in a solvent containing methanesulfonic acid.

[42] The production method according to any one of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate.

[43] The production method according to any one of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate·m hydrate (where m ranges from 0 to 3).

[44] The production method according to any one of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate anhydride.

[45] The production method according to any one of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate monohydrate.

[46] The production method according to any one of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate dihydrate.

[47] The production method according to any one of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate trihydrate.

[48] A production method of a compound represented by formula (2), which comprises the following steps: Converting the compound represented by formula (3), Into the step of converting it into the compound represented by formula (4), Next, the step of converting the compound represented by formula (4) into the compound represented by formula (5); Next, the step of converting the compound represented by formula (5) into the compound represented by formula (6); Next, the step of coupling the compound represented by formula (6) with 3-butenoic acid to convert it into the compound represented by formula (7); Next, the step of converting the compound represented by formula (7) into the compound represented by formula (8); Next, the step of subjecting the compound represented by formula (8) to intramolecular cyclization to convert it into the compound represented by formula (9); Next, the step of converting the compound represented by formula (9) into the compound represented by formula (10); Next, the step of converting the compound represented by formula (10) into the compound represented by formula (11); Next, condensing the compound represented by formula (11) with the compound represented by formula (1); to convert it into the compound represented by formula (12); Next, the step of converting the compound represented by formula (12) into the compound represented by formula (2); 。

[49] The production method as described in

[48] , wherein the step of coupling the compound represented by formula (6) with 3-butenoic acid to convert it into the compound represented by formula (7) is carried out in the presence of a palladium complex prepared from palladium(II) acetate and tris(o-tolyl)phosphine.

[50] The production method as described in

[48] or

[49] , which includes: the step of dissolving the compound represented by formula (7) in an alkaline aqueous solution and performing liquid separation washing with a first organic solvent; next, adding an acid to the alkaline aqueous solution and performing liquid separation extraction of the compound represented by formula (7) with a second organic solvent.

[51] The production method as described in

[50] , wherein the first organic solvent is 2-methyltetrahydrofuran.

[52] The production method as described in

[50] or

[51] , wherein the second organic solvent is 2-methyltetrahydrofuran.

[53] The production method as described in any one of

[50] to

[52] , wherein the alkaline aqueous solution is a sodium hydroxide aqueous solution.

[54] The production method according to any one of

[50] to

[53] , wherein the step of subjecting the compound represented by formula (8) to intramolecular cyclization to convert it into the compound represented by formula (9) is carried out by a method including reacting the compound represented by formula (8) with trifluoroacetic anhydride.

[55] The production method according to

[54] , wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid.

[56] The production method according to any one of

[48] to

[55] , wherein the step of converting the compound represented by formula (9) into the compound represented by formula (10) includes: (i) A stage of reacting with a nitrite in the presence of a base to introduce a nitro group; secondly, (ii) a stage of introducing a protecting group into the nitrogen atom derived from the nitro group; and (iii) a stage of reducing with hydrogen in the presence of a platinum-carbon catalyst.

[57] The production method according to any one of

[48] to

[56] , wherein the step of converting the compound represented by formula (10) into the compound represented by formula (11) is carried out in a solvent containing hydrochloric acid / ethanol.

[58] The production method according to any one of

[48] to

[57] , wherein the step of condensing the compound represented by formula (11) with the compound represented by formula (1) to convert it into the compound represented by formula (12) is carried out in a solvent containing o-cresol.

[59] The production method according to any one of

[48] to

[58] , wherein the step of converting the compound represented by formula (12) into the compound represented by formula (2) is carried out in a solvent containing methanesulfonic acid.

[60] The production method according to any one of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate.

[61] The production method according to any one of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate·m hydrate (where m is in the range of 0 to 3).

[62] The production method according to any one of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate·anhydride.

[63] The production method according to any one of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate·1 hydrate.

[64] The production method according to any one of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate·2 hydrates.

[65] The production method according to any one of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate·3 hydrates.

[66] A method for producing a compound represented by formula (E), which comprises the following steps: Coupling the compound represented by formula (D) with 3-butenoic acid, (wherein X represents a leaving group, and R1 represents an amino group protected by a protecting group), and a step of converting to a compound represented by formula (E) (wherein R1 represents the same meaning as described above).

[67] The production method according to

[66] , wherein X is bromo, iodo, trifluoromethanesulfonyloxy, or arylsulfonyloxy.

[68] The production method according to

[66] , wherein X is bromo.

[69] The production method according to

[66] , wherein X is iodo.

[70] The production method according to any one of

[66] to

[69] , wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[71] The production method according to any one of

[66] to

[69] , wherein R1 is an amino group protected by acetyl or trifluoroacetyl.

[72] The production method according to any one of

[66] to

[69] , wherein R1 is an amino group protected by acetyl.

[73] The production method according to any one of

[66] to

[72] , which is carried out in the presence of a palladium complex prepared from palladium(II) acetate and tris(o-tolyl)phosphine.

[74] The production method according to any one of

[66] to

[73] , which comprises: dissolving the compound represented by formula (E) in an alkaline aqueous solution, and performing liquid separation and washing with a first organic solvent; secondly, adding an acid to the alkaline aqueous solution, and performing liquid separation and extraction of the compound represented by formula (E) with a second organic solvent.

[75] The production method according to

[74] , wherein the first organic solvent is 2-methyltetrahydrofuran.

[76] The production method according to

[74] or

[75] , wherein the second organic solvent is 2-methyltetrahydrofuran.

[77] The production method according to any one of

[74] to

[76] , wherein the alkaline aqueous solution is an aqueous sodium hydroxide solution.

[78] A method for producing a compound represented by formula (B), which comprises the following steps: Reduce the compound represented by formula (E) (wherein, R1 represents an amino group protected by a protecting group), And the step of converting to the compound represented by formula (B) (wherein, R1 represents the same meaning as described above).

[79] The production method described in

[78] , wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[80] The production method described in

[78] , wherein R1 is an amino group protected by acetyl or trifluoroacetyl.

[81] The production method described in

[78] , wherein R1 is an amino group protected by acetyl.

[82] The production method described in any one of

[78] to

[81] , which is carried out by reacting the compound represented by formula (E) with hydrogen in a solvent in the presence of a palladium-carbon catalyst.

[83] A method for producing a compound represented by formula (F), which comprises the compound represented by formula (C) (wherein, R1 represents an amino group protected by a protecting group), By including the following stages to convert to the compound represented by formula (F) (wherein, R1 represents the same meaning as above, and R2 represents an amino group protected by a protecting group) steps: (i) In the presence of a base, reacting with a nitrite and introducing a nitroso group; secondly, (ii) introducing a protecting group to the nitrogen atom derived from the nitroso group; and (iii) in the presence of a platinum-carbon catalyst, reducing with hydrogen.

[84] The production method described in

[83] , wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[85] The production method described in

[83] , wherein R1 is an amino group protected by acetyl or trifluoroacetyl.

[86] The production method described in

[83] , wherein R1 is an amino group protected by acetyl.

[87] The production method according to any one of

[83] to

[86] , wherein R2 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[88] The production method according to any one of

[83] to

[86] , wherein R2 is an amino group protected by acetyl or trifluoroacetyl.

[89] The production method according to any one of

[83] to

[86] , wherein R2 is an amino group protected by acetyl.

[90] A production method of a compound represented by formula (G), which comprises the following steps: reacting a compound represented by formula (F) (wherein, R1 and R2 represent amino groups protected by protecting groups), in a solvent containing hydrochloric acid / ethanol, to obtain a compound represented by formula (G) (wherein, R2 has the same meaning as described above).

[91] The production method according to

[90] , wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[92] The production method according to

[90] , wherein R1 is an amino group protected by acetyl or trifluoroacetyl.

[93] The production method according to

[90] , wherein R1 is an amino group protected by acetyl.

[94] The production method according to any one of

[90] to

[93] , wherein R2 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[95] The production method according to any one of

[90] to

[93] , wherein R2 is an amino group protected by acetyl or trifluoroacetyl.

[96] The production method according to any one of

[90] to

[93] , wherein R2 is an amino group protected by acetyl.

[97] A production method of a compound represented by formula (H), which comprises the following steps: reacting a compound represented by formula (G) (wherein, R2 represents an amino group protected by a protecting group), The compound represented by formula (1) is condensed in a solvent containing o -cresol, and is converted into the compound represented by formula (H) (wherein, R2 represents the same meaning as described above).

[98] The production method according to

[97] , wherein R2 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, formyl, or benzoyl.

[99] The production method according to

[97] , wherein R2 is an amino group protected by acetyl or trifluoroacetyl.

[100] The production method according to

[97] , wherein R2 is an amino group protected by acetyl.

[101] The production method according to any one of [1] to

[100] , which does not use chromatography.

[102] A compound represented by formula (6) .

[103] A compound represented by formula (34) .

[104] A compound represented by formula (7) .

[105] A compound represented by formula (8) .

[106] A method for producing a compound represented by formula (14), characterized by using the compound represented by formula (2) produced by the method described in any one of

[34] to

[65] as a starting material, which includes: by condensing the compound represented by formula (2) with the compound represented by formula (13), and converting it into the compound represented by formula (14) .

[107] A method for producing an antibody - drug conjugate in which a drug linker (linker) represented by formula (15) is bonded to an antibody through a thioether bond (wherein, A represents the binding position to the antibody), It is characterized in that a compound represented by formula (14) produced by the method described in

[106] is used as a raw material, which comprises the following steps: (i) a step of reducing the antibody; secondly, (ii) a step of reacting the compound represented by formula (14) produced by the above method with the reduced antibody.

[108] The production method as described in

[107] , wherein the antibody is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, or an anti-GPR20 antibody.

[109] The production method as described in

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

[110] The production method as described in

[109] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain composed of the amino acid sequence described in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain composed of the amino acid sequence described in amino acid numbers 1 to 214 of SEQ ID NO: 2; or an antibody comprising a heavy chain composed of the amino acid sequence described in SEQ ID NO: 1 and a light chain composed of the amino acid sequence described in SEQ ID NO: 2.

[111] The production method as described in

[109] or

[110] , wherein the average number of drug linkers per antibody in the antibody-drug conjugate ranges from 7 to 8.

[112] The production method as described in

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

[113] The production method as described in

[112] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain composed of the amino acid sequence described in SEQ ID NO: 3 and a light chain composed of the amino acid sequence described in SEQ ID NO: 4; or an antibody with a lysine residue deletion at the carboxyl terminus of the heavy chain of the antibody.

[114] The production method as described in

[112] or

[113] , wherein the average number of drug linkers per antibody in the antibody-drug conjugate ranges from 7 to 8.

[115] The production method as described in

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

[116] The manufacturing method as described in

[115] , wherein the anti-TROP2 antibody is an antibody comprising a heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain composed of the amino acid sequence described in amino acid numbers 21 to 234 of SEQ ID NO: 6; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[117] The manufacturing method as described in

[115] or

[116] , wherein the average number of drug linkers per antibody in the antibody-drug conjugate ranges from 3 to 5.

[118] The manufacturing method as described in

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

[119] The manufacturing method as described in

[118] , wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain composed of the amino acid sequence described in amino acid numbers 21 to 233 of SEQ ID NO: 8; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[120] The manufacturing method as described in

[118] or

[119] , wherein the average number of drug linkers per antibody in the antibody-drug conjugate ranges from 3 to 5.

[121] The manufacturing method as described in

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

[122] The manufacturing method as described in

[121] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 472 of SEQ ID NO: 9 and a light chain composed of the amino acid sequence described in amino acid numbers 21 to 234 of SEQ ID NO: 10; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[123] The manufacturing method as described in

[121] or

[122] , wherein the average number of drug linkers per antibody in the antibody-drug conjugate ranges from 7 to 8. [Effects of the Invention]

[0012] According to the present invention, a novel manufacturing method of irinotecan with a short number of steps and excellent industrially can be provided. Moreover, a novel manufacturing method of an antibody-drug conjugate using the same can be provided. Brief Description of the Drawings

[0013] FIG. 1 shows the amino acid sequence of the heavy chain of the anti-HER2 antibody (SEQ ID NO: 1). FIG. 2 shows the amino acid sequence of the light chain of the anti-HER2 antibody (SEQ ID NO: 2). Figure 3 shows the amino acid sequence of the heavy chain of the anti-HER3 antibody (Sequence ID No. 3). Figure 4 shows the amino acid sequence of the light chain of the anti-HER3 antibody (Sequence ID No. 4). Figure 5 shows the amino acid sequence of the heavy chain of the anti-TROP2 antibody (Sequence ID No. 5). Figure 6 shows the amino acid sequence of the light chain of the anti-TROP2 antibody (Sequence ID No. 6). Figure 7 shows the amino acid sequence of the heavy chain of the anti-B7-H3 antibody (Sequence ID No. 7). Figure 8 shows the amino acid sequence of the light chain of the anti-B7-H3 antibody (Sequence ID No. 8). Figure 9 shows the amino acid sequence of the heavy chain of the anti-GPR20 antibody (Sequence ID No. 9). Figure 10 shows the amino acid sequence of the light chain of the anti-GPR20 antibody (Sequence ID No. 10). Embodiments

[0014] [Mode for Carrying Out the Invention] Hereinafter, suitable modes for carrying out the present invention will be described. In addition, the embodiments described below show an example of representative embodiments of the present invention, and do not narrowly interpret the scope of the present invention thereby.

[0015] [Antibody-Drug Conjugate]

[0016] The antibody-drug conjugate produced according to the present invention is preferably an antibody-drug conjugate in which the drug linker represented by the formula (15) is bound to the antibody via a thioether bond (wherein, A represents the binding position to the antibody).

[0017] In the present invention, the partial structure composed of the linker and the drug in the antibody-drug conjugate is referred to as the "drug linker". This drug linker binds to the thiol groups (in other words, the sulfur atoms of cysteine residues) generated at the interchain disulfide bond sites of the antibody (at two heavy chain-heavy chain sites and at two heavy chain-light chain sites).

[0018] The drug linker of the present invention includes irinotecan, which is a topoisomerase I inhibitor, as a constituent element. Irinotecan is a compound represented by the formula (2), It is a camptothecin derivative with antitumor effects.

[0019] The antibody-drug conjugate used in the present invention can also be represented by formula (16). Among them, the drug linker is bound to the antibody via a thioether bond. Also, n has the same meaning as the so-called average drug binding number (DAR; Drug-to-Antibody Ratio), representing the average number of drug linkers bound per antibody.

[0020] The antibody-drug conjugate used in the present invention exerts antitumor effects by releasing the compound represented by formula (18) after migrating into cancer cells.

[0021] The compound represented by formula (18) is considered to be the entity of the antitumor activity of the antibody-drug conjugate manufactured according to the present invention and has been confirmed to have topoisomerase I inhibitory activity (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0022] The compound represented by formula (18) is considered to be generated by the decomposition of the aminal structure of the compound represented by formula (17), and the compound represented by formula (17) is considered to be generated by the cleavage of the linker part of the antibody-drug conjugate manufactured according to the present invention.

[0023] It is also known that the antibody-drug conjugate manufactured according to the present invention has a bystander effect (Ogitani Y. et al., Cancer Science(2016)107, 1039-1046).

[0024] The antibody-drug conjugate manufactured according to the present invention, after internalization in cancer cells expressing the target, the released compound represented by formula (18) also exerts antitumor effects on cancer cells near those not expressing the target, thereby exerting this bystander effect.

[0025] [Manufacture of Irinotecan] The manufacture of irinotecan of the present invention can be carried out according to the following method. [Wherein, X represents a leaving group, preferably a bromo group, an iodo group, a trifluoromethanesulfonyloxy group, or an arylsulfonyloxy group, more preferably a bromo group or an iodo group, even more preferably a bromo group; R1 represents an amino group protected by a protecting group, preferably an amino group protected by an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a trimethylacetyl group, a formyl group, or a benzoyl group, more preferably an amino group protected by an acetyl group or a trifluoroacetyl group, even more preferably an amino group protected by an acetyl group; R2 represents a protected amino group, preferably an amino group protected by an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a trimethylacetyl group, a formyl group, or a benzoyl group, more preferably an amino group protected by an acetyl group or a trifluoroacetyl group, even more preferably an amino group protected by an acetyl group. ]

[0026] Step 1: In this step, the compound represented by formula (D) is coupled with 3-butenoic acid to be converted into the compound represented by formula (E). The compound represented by formula (D) can be prepared by referring to well-known methods. The amount of 3-butenoic acid used in this step is not limited as long as the reaction proceeds, but preferably is 1 to 1.5 equivalents relative to the compound represented by formula (D).

[0027] The coupling reaction can be carried out in the presence of a transition metal catalyst, preferably in the presence of a palladium catalyst. The palladium catalyst used in this step can be any as long as the reaction proceeds and is not particularly limited. For example, divalent palladium salts and their complexes such as palladium(II) acetate, palladium(II) trifluoroacetate, palladium(II) chloride, palladium(II) bromide, palladium(II) iodide, dichloro-bis(triphenylphosphine)palladium(II), etc., palladium black, palladium on carbon, tetrakis(triphenylphosphine)palladium(0), bis(dibenzylideneacetone)palladium(0) and other zero-valent palladium metals and their complexes, etc. are preferably used. Palladium(II) acetate is preferably used. The amount of the palladium catalyst used in this step is not limited as long as the reaction proceeds, but preferably is 0.003 to 0.03 equivalents relative to the compound represented by formula (D).

[0028] Furthermore, this step preferably uses, in addition to the above-mentioned palladium catalyst, a ligand for forming a palladium complex in the reaction system. As for the ligand that can be used in this step, for example, triphenylphosphine, tris(o-tolyl)phosphine, tris(3-methoxyphenyl)phosphine, tris(4-chlorophenyl)phosphine, tris(2-furyl)phosphine, tris(2-thienyl)phosphine, 1,2-bis(diphenylphosphino)ethane, and Buchwald ligands (2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos), etc.) can be used. Preferably, tris(o-tolyl)phosphine can be used. The amount of the ligand used in this step is not limited as long as the reaction proceeds, but preferably, it is 0.006 to 0.06 equivalents relative to the compound represented by formula (D).

[0029] This step is preferably carried out in the presence of a base. As for the base used in this step, it is not particularly limited as long as the reaction proceeds, but examples include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methyl quinoline, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, and 1,5-diazabicyclo[4.3.0]undec-7-ene, and inorganic bases such as potassium carbonate, potassium hydroxide, potassium hydrogen carbonate, sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide. Preferably, triethylamine, tributylamine, diisopropylethylamine, potassium carbonate, potassium hydroxide, potassium hydrogen carbonate, sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, and potassium acetate can be mentioned. More preferably, diisopropylethylamine can be mentioned. The amount of the base used in this step is not limited as long as the reaction proceeds, but preferably, it is 2 to 3 equivalents relative to the compound represented by formula (D).

[0030] As for the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction, but for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-d oxane, 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, and water, as well as mixed solvents thereof, can be used. Preferably, tetrahydrofuran can be mentioned.

[0031] The reaction temperature of this step is not restricted as long as the reaction proceeds, but is preferably 45 to 85 °C, more preferably the temperature under heating reflux of tetrahydrofuran. The reaction time of this step is not restricted as long as the reaction proceeds, but is preferably 2.5 to 10 hours.

[0032] The compound represented by formula (E) can be appropriately purified by performing the following steps: dissolving the compound represented by formula (E) in an alkaline aqueous solution and performing a liquid separation washing step using a first organic solvent; then, adding an acid to the alkaline aqueous solution and performing a liquid separation extraction step of the compound represented by formula (E) using a second organic solvent. The first organic solvent is preferably 2-methyltetrahydrofuran. The second organic solvent is preferably 2-methyltetrahydrofuran. The alkaline aqueous solution is preferably an aqueous sodium hydroxide solution.

[0033] Also, in the compound represented by formula (E), there exist an E-form and a Z-form as geometric isomers, but either one is included in the compound represented by formula (E) and is included within the scope of the present invention. The compound represented by formula (E) of the present invention can be a mixture of the E-form and the Z-form, and the mixture can be directly used in the next step.

[0034] Also, in this step, 3-butenoic acid ester can be used instead of 3-butenoic acid. In this case, the compound represented by formula (E) can be obtained by hydrolyzing the product obtained by coupling the compound represented by formula (D) with 3-butenoic acid ester.

[0035] Step 2: This step is a step of converting the compound represented by formula (E) into the compound represented by formula (B) by reduction.

[0036] The reduction method of this step is not restricted as long as the reaction proceeds, but is preferably carried out in a hydrogen atmosphere (preferably under a hydrogen flow of 0.05 to 0.6 MPa) using a palladium catalyst, a platinum catalyst, a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst, more preferably carried out using a palladium catalyst, further preferably carried out using palladium on carbon, and further preferably carried out using 5% palladium on carbon. The amount of 5% palladium on carbon used in this step is not restricted as long as the reaction proceeds, but is preferably 5 to 80% by weight relative to the compound represented by formula (D) used in step 1.

[0037] Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction. Examples include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 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, and water, as well as mixed solvents thereof. Preferably, 2-methyltetrahydrofuran can be mentioned. Alkanes, 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, and water, as well as mixed solvents thereof. Preferably, 2-methyltetrahydrofuran can be mentioned.

[0038] The reaction temperature of this step is not restricted as long as the reaction proceeds, but it is preferably 20 to 60 °C. The reaction time of this step is not restricted as long as the reaction proceeds, but it is preferably 0.5 to 2 hours.

[0039] Step 3: This step is to convert the compound represented by formula (B) into the compound represented by formula (C) through intramolecular cyclization. Intramolecular cyclization is preferably carried out by an intramolecular Friedel-Crafts acylation reaction. Regarding the intramolecular Friedel-Crafts acylation reaction in this step, the method is not restricted as long as the reaction proceeds, but preferably, methods using trifluoroacetic anhydride can be mentioned; or methods using thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphoryl chloride, phosphorus trichloride, or phosphorus pentachloride. More preferably, methods using trifluoroacetic anhydride or thionyl chloride can be mentioned, and even more preferably, the method using trifluoroacetic anhydride can be mentioned. The amount of trifluoroacetic anhydride used in this step is not restricted as long as the reaction proceeds, but it is preferably 1 to 3 equivalents relative to the compound represented by formula (B). The amount of thionyl chloride used in this step is not restricted as long as the reaction proceeds, but it is preferably 1 to 3 equivalents relative to the compound represented by formula (B).

[0040] In the case of the method using trifluoroacetic anhydride, this step is preferably carried out in the presence of an acid, and more preferably in the presence of trifluoroacetic acid. In the case of the method using thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphoryl chloride, phosphorus trichloride, or phosphorus pentachloride, this step is preferably carried out in the presence of aluminum chloride. The amount of aluminum chloride used in this step is not restricted as long as the reaction proceeds, but it is preferably 1 to 5 equivalents relative to the compound represented by formula (B).

[0041] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. Examples thereof include dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, and chlorobenzene, as well as mixed solvents thereof. Dichloromethane is preferably exemplified. Further, when trifluoroacetic acid is used, it is preferred to contain trifluoroacetic acid as the solvent.

[0042] The reaction temperature in this step is not limited as long as the reaction proceeds. However, when trifluoroacetic anhydride is used, it is preferably -10°C to 20°C, and when the method using thionyl chloride is employed, it is preferably 10°C to 40°C. The reaction time in this step is not limited as long as the reaction proceeds. However, when the method using trifluoroacetic anhydride is used, it is preferably 2 hours to 8 hours, and when the method using thionyl chloride is used, it is preferably 1 hour to 4 hours.

[0043] Further, this step can also be carried out in two stages as follows. [In the formula, Y represents a leaving group, preferably a chlorine group, a bromine group, an iodine group, a fluorine group, or a trifluoroacetoxy group, more preferably a chlorine group or a trifluoroacetoxy group; R1 represents an amino group protected by a protecting group, preferably an amino group protected by an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a trimethylacetyl group, a formyl group, or a benzoyl group, more preferably an amino group protected by an acetyl group or a trifluoroacetyl group, and even more preferably an amino group protected by an acetyl group.]

[0044] Step 3A is a step of converting the compound represented by formula (B) into the compound represented by formula (J).

[0045] When Y is a chlorine group, this step is preferably carried out by using thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphoryl chloride, phosphorus trichloride or phosphorus pentachloride, and more preferably by using thionyl chloride. The amount of thionyl chloride used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 3 equivalents relative to the compound represented by formula (B). When Y is trifluoroacetoxy, this step is preferably carried out by using trifluoroacetic anhydride. The amount of trifluoroacetic anhydride used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 3 equivalents relative to the compound represented by formula (B). Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction, and examples thereof include dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, and trifluoroacetic acid, and mixed solvents thereof. In the case of using thionyl chloride, dichloromethane is preferably listed, and in the case of using trifluoroacetic anhydride, trifluoroacetic acid is preferably listed.

[0046] Step 3B is a step of converting the compound represented by formula (J) into the compound represented by formula (C). In the case of using thionyl chloride, this step is preferably carried out in the presence of aluminum chloride. The amount of aluminum chloride used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 5 equivalents relative to the compound represented by formula (B). In the case of using trifluoroacetic anhydride, this step is preferably carried out in the presence of an acid, and more preferably in the presence of trifluoroacetic acid.

[0047] The reaction temperatures of Step 3A and Step 3B are not limited as long as the reaction proceeds, but in the case of using thionyl chloride, it is preferably 10°C to 40°C, and in the case of using trifluoroacetic anhydride, it is preferably -10°C to 20°C. The total reaction time of Step 3A and Step 3B is not limited as long as the reaction proceeds, but in the case of using thionyl chloride, it is preferably 1 hour to 4 hours, and in the case of using trifluoroacetic anhydride, it is preferably 2 hours to 8 hours.

[0048] Step 4: This step is a step of converting the compound represented by formula (C) into the compound represented by formula (F). This step is preferably carried out through the following stages: (i) the stage of nitrosating (or oximating) the α-position of the carbonyl group; (ii) the stage of introducing a protecting group to the nitrogen atom derived from the nitroso (or oxime) group; (iii) the reduction stage. The order of (ii) and (iii) can be exchanged or they can be carried out simultaneously.

[0049] (i) The nitrosating agent (or oximating agent) used in this step is not particularly limited as long as it can nitrosate (or oximize) the α-position of the carbonyl group of the compound represented by formula (C). However, nitrite esters are preferably used, and amyl nitrite, n-butyl nitrite, or tert-butyl nitrite is more preferably used, and amyl nitrite is even more preferably used. The amount of amyl nitrite used in step (i) is not limited as long as the reaction proceeds, but it is preferably 1 to 1.6 equivalents relative to the compound represented by formula (C).

[0050] (i) A base is preferably used in this step. Regarding the base used in step (i), as long as it is applicable to the nitrosation (or oximation) of the α-position of the carbonyl group of the compound represented by formula (C), it is not particularly limited, and potassium tert-butoxide is preferably used. The amount of potassium tert-butoxide used in step (i) is not limited as long as the reaction proceeds, but it is preferably 1 to 1.5 equivalents relative to the compound represented by formula (C).

[0051] Regarding the solvent used in step (i), as long as it does not inhibit the reaction, it is not particularly limited. Examples include diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4- dioxane, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, and dimethyl sulfoxide, as well as mixed solvents thereof. Tetrahydrofuran is preferably listed.

[0052] (i) The reaction temperature in this step is not limited as long as the reaction proceeds, but it is preferably -10 to 20°C. The reaction time for this step is not limited as long as the reaction proceeds, but it is preferably 1.5 to 30 hours.

[0053] (ii) The appropriate reaction conditions can be set according to the type of protecting group of the amino group in R2. In the case where R2 is an amino group protected by an acetyl group, in step (ii), acetic anhydride is preferably used in acetic acid.

[0054] (iii) This step can be carried out under a hydrogen atmosphere (preferably under a hydrogen flow of 0.15 to 1.2 MPa). For example, it can be carried out using a platinum-carbon catalyst or using zinc powder, but it is preferably carried out using a platinum-carbon catalyst, and more preferably carried out using a 2% or 5% platinum-carbon catalyst. The amount of the 2% or 5% platinum-carbon catalyst is not limited as long as the reaction proceeds, but it is preferably 5 to 60% by weight relative to the compound represented by formula (C). The solvent used in step (ii) can be used in step (iii).

[0055] (ii) The reaction temperature in the stage of (ii) and the stage of (iii) is not limited as long as the reaction proceeds, but is preferably 0 to 40 °C. The total reaction time in the stage of (ii) and the stage of (iii) is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours.

[0056] Step 5: This step is a step of selectively deprotecting the aromatic amino protecting group of the compound represented by formula (F) to convert it into the compound represented by formula (G). Appropriate reaction conditions can be set according to the type of the amino protecting group in R1 and R2. In the case where R1 and R2 are amino groups protected by acetyl groups, this step is preferably carried out using hydrochloric acid, and more preferably carried out using 2N hydrochloric acid / ethanol.

[0057] The reaction temperature of this step is not limited as long as the reaction proceeds, but is preferably 40 to 60 °C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 2 to 14 hours.

[0058] Step 6: This step is a step of condensing the compound represented by formula (G) with the compound represented by formula (1) to convert it into the compound represented by formula (H). The compound represented by formula (1) can be produced with reference to the descriptions in US Patent No. 4778891 etc., or commercially available products can also be used. The amount of the compound represented by formula (1) used in this step is not limited as long as the reaction proceeds, but is preferably 0.8 to 1.2 equivalents relative to the compound represented by formula (G).

[0059] This step is carried out in the presence of an acid catalyst. Regarding the acid catalyst used in this step, pyridinium p-toluenesulfonate is preferably cited. The acid catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.03 to 0.3 equivalents relative to the compound represented by formula (G).

[0060] This step is preferably carried out in a solvent containing cresol or phenol, and more preferably in toluene containing o-cresol. This is because the precipitation phase of the compound represented by formula (H) is improved due to the presence of o-cresol or phenol, and effects such as increased yield and shortened reaction time can be seen.

[0061] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 90 to 130 °C, more preferably the temperature under heating reflux of toluene. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 16 to 64 hours.

[0062] Furthermore, this step is considered to proceed via the compound represented by formula (K) and the compound represented by formula (L) which are reaction intermediates.

[0063] Step 7: This step is a step of converting the compound represented by formula (H) into the compound represented by formula (2). The compound represented by formula (2) may be a salt or may further be a hydrate, and either is included in the scope of the "compound represented by formula (2)" in the present invention. This step is preferably carried out in the presence of an acid, more preferably in the presence of methanesulfonic acid and water. Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction, but a solvent containing 2-methoxyethanol and ethylcyclohexane is preferably used. In the case where the above acid is included as the solvent, a mixed solvent of methanesulfonic acid, water, 2-methoxyethanol, and ethylcyclohexane is more preferably used. This step is not limited as long as the reaction proceeds, but is preferably carried out at 80 to 160 °C, more preferably at the temperature under heating reflux of a mixed solvent of methanesulfonic acid, water, 2-methoxyethanol, and ethylcyclohexane. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 4 to 16 hours. The compound represented by formula (2) is preferably obtained as a methanesulfonate, more preferably as a methanesulfonate·m hydrate (where m is 0 to 3), even more preferably as a methanesulfonate·anhydrate, methanesulfonate·1 hydrate, methanesulfonate·2 hydrates, or methanesulfonate·3 hydrates, and even more preferably as a methanesulfonate·2 hydrates, and any of them can be used in the production method of the present invention. The number of hydrates described above can be controlled by adjusting the humidity during crystallization acquisition and drying. The compound represented by formula (2) is more preferably produced according to the following method.

[0064] Step 8: This step is a step of brominating the compound represented by formula (3) to convert it into the compound represented by formula (4). The compound represented by formula (3) can be a compound produced by a well-known method or a commercially available product. Regarding the brominating agent used in this step, there is no limitation as long as the reaction proceeds, but examples thereof include bromine or N-bromosuccinimide, and preferably N-bromosuccinimide can be mentioned. The amount of N-bromosuccinimide used in this step is not limited as long as the reaction proceeds, but preferably it is 1 to 1.5 equivalents relative to the compound represented by formula (3). This step is preferably carried out in a mixed solvent of sulfuric acid and other solvents. Other solvents are not particularly limited as long as they do not inhibit the reaction, but examples thereof include dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, or a mixed solvent thereof, and preferably heptane can be mentioned. The reaction temperature of this step is not limited as long as the reaction proceeds, but preferably it is 50 to 70 °C. The reaction time of this step is not limited as long as the reaction proceeds, but preferably it is 0.5 to 2 hours.

[0065] Step 9: This step is a step of reducing the nitro group of the compound represented by formula (4) to an amino group and converting it into the compound represented by formula (5). The reducing agent used in this step may be one that can selectively reduce the nitro group without performing debromination, and preferably a platinum-carbon catalyst can be used in the presence of hydrogen (preferably under a hydrogen stream of 0.05 to 0.2 MPa), and more preferably a 1% platinum-carbon catalyst can be used. The amount of the platinum-carbon catalyst used in this step is not limited as long as the reaction proceeds, but preferably it is 5 to 40% by weight relative to the compound represented by formula (3) used in step 8. Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction, but examples thereof include methanol, ethanol, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, and water, and a mixed solvent thereof, and preferably ethyl acetate can be mentioned. The reaction temperature of this step is not limited as long as the reaction proceeds, but preferably it is 50 to 70 °C. The reaction time of this step is not limited as long as the reaction proceeds, but preferably it is 2 to 8 hours.

[0066] Step 10: This step is a step of acetylating the amino group of the compound represented by formula (5) to convert it into the compound represented by formula (6). Examples of the acetylating agent used in this step include acetic anhydride or acetyl chloride, and acetic anhydride is preferably cited. The amount of acetic anhydride used in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 1 equivalent relative to the compound represented by formula (3) used in step 8. This step preferably uses a base. Regarding this base, it is not limited as long as the reaction proceeds, but triethylamine is preferably used. The amount of this base is not limited as long as the reaction proceeds, but is preferably 0.75 to 1.5 equivalents relative to the compound represented by formula (3) used in step 8. Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction, and examples include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-di alkane, 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, and water, and mixed solvents thereof, and ethyl acetate is preferably cited. The reaction temperature of 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 3 to 12 hours.

[0067] Step 11: This step is a step of converting the compound represented by formula (6) into the compound represented by formula (7) by coupling with 3-butenoic acid. This step can be carried out in the same manner as the method described in step 1. Furthermore, in the compound represented by formula (7), there exist an E-isomer and a Z-isomer as geometric isomers, but either one is included in the compound represented by formula (7) and is included in the scope of the present invention. The compound represented by formula (7) of the present invention can be a mixture of the E-isomer and the Z-isomer, and the mixture can be directly used in the next step.

[0068] Step 12: This step is a step of converting the compound represented by formula (7) into the compound represented by formula (8) by reduction. For the reduction in this step, the method is not limited as long as the reaction proceeds. However, it is preferably carried out in a hydrogen atmosphere (preferably under a hydrogen flow of 0.05 to 0.2 MPa), and a palladium catalyst, a platinum catalyst, a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst can be used. More preferably, it is carried out by using a palladium catalyst, and further preferably, palladium on carbon can be used. Even more preferably, 5% palladium on carbon can be used. The amount of 5% palladium on carbon used in this step is not limited as long as the reaction proceeds, but it is preferably 5 to 40% by weight relative to the compound represented by formula (7) used in step 11. Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction. Examples include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 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, and water, as well as mixed solvents thereof. Preferably, 2-methyltetrahydrofuran can be mentioned. The reaction temperature in this step is not limited as long as the reaction proceeds, but it is preferably 20 to 60 °C. The reaction time in this step is not limited as long as the reaction proceeds, but it is preferably 0.5 to 2 hours.

[0069] Step 13: This step is a step of converting the compound represented by formula (8) into the compound represented by formula (9) by intramolecular cyclization. This step can be carried out in the same manner as the method described in step 3.

[0070] Step 14: This step is a step of converting the compound represented by formula (9) into the compound represented by formula (10). This step can be carried out in the same manner as the method described in step 4.

[0071] Step 15: This step is a step of selectively deprotecting the protecting group of the aromatic amino group of the compound represented by formula (10) to convert it into the compound represented by formula (11). This step can be carried out in the same manner as the method described in step 5.

[0072] Step 16: This step is a step of condensing the compound represented by formula (11) and the compound represented by formula (1) to convert it into the compound represented by formula (12). This step can be carried out in the same manner as the method described in step 6.

[0073] Also, this step is considered to proceed via the compound represented by formula (30) and / or the compound represented by formula (31) which are reaction intermediates.

[0074] Step 17: This step is a step of converting the compound represented by formula (12) into the compound represented by formula (2). This step can be carried out in the same manner as the method described in Step 7. The compound represented by formula (2) can also be produced according to the following method.

[0075] Step 18: This step is a step of iodinating the compound represented by formula (3) to convert it into the compound represented by formula (32). The compound represented by formula (3) can be a compound produced by a well-known method or a commercially available product. Regarding the iodinating agent used in this step, there is no limitation as long as the reaction proceeds, but examples include iodine or N-iodosuccinimide, and preferably N-iodosuccinimide. The amount of N-iodosuccinimide used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 2 equivalents relative to the compound represented by formula (3). This step is preferably carried out in a mixed solvent of sulfuric acid and other solvents. Other solvents are not particularly limited as long as they do not inhibit the reaction, but examples include dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, or a mixed solvent thereof, and preferably heptane. The reaction temperature of this step is not limited as long as the reaction proceeds, but is preferably -10 to 10 °C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1 to 4 hours.

[0076] Step 19: This step is to reduce the nitro group of the compound represented by formula (32) to an amino group, thereby converting it into the compound represented by formula (33). The reducing agent used in this step can be any one that can selectively reduce the nitro group without deiodination. Preferably, a platinum-carbon catalyst can be used in the presence of hydrogen (preferably under a hydrogen stream of 0.05 - 0.2 MPa). The amount of the platinum-carbon catalyst used in this step is not limited as long as the reaction proceeds, but preferably it is 5 - 40% by weight relative to the compound represented by formula (3) used in step 18. Regarding the solvent used in this step, as long as it does not inhibit the reaction, it is not particularly limited. Examples include methanol, ethanol, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-di alkane, ethyl acetate, and water, as well as mixed solvents thereof. Preferably, ethyl acetate can be cited. The reaction temperature of this step is not limited as long as the reaction proceeds, but preferably it is 50 - 70 °C. The reaction time of this step is not limited as long as the reaction proceeds, but preferably it is 2 - 8 hours.

[0077] Step 20: This step is to acetylate the amino group of the compound represented by formula (33), thereby converting it into the compound represented by formula (34). Examples of the acetylating agent used in this step include acetic anhydride or acetyl chloride. Preferably, acetic anhydride can be cited. The amount of acetic anhydride used in this step is not limited as long as the reaction proceeds, but preferably it is 0.5 - 1 equivalent relative to the compound represented by formula (3) used in step 18. This step preferably uses a base. Regarding this base, as long as the reaction proceeds, it is not limited, but preferably it is triethylamine. The amount of this base is not limited as long as the reaction proceeds, but relative to the compound represented by formula (3) used in step 18, preferably it is 0.75 - 1.5 equivalents. Regarding the solvent used in this step, as long as it does not inhibit the reaction, it is not particularly limited. Examples include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-di alkane, 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, and water, as well as mixed solvents thereof. Preferably, ethyl acetate can be cited. The reaction temperature of this step is not limited as long as the reaction proceeds, but preferably it is 10 - 40 °C. The reaction time of this step is not limited as long as the reaction proceeds, but preferably it is 3 - 12 hours.

[0078] Step 21: This step is a step of converting the compound represented by formula (34) into the compound represented by formula (7) by coupling with 3-butenoic acid. This step can be carried out in the same manner as the method described in Step 1.

[0079] Step 22: This step is a step of converting the compound represented by formula (7) into the compound represented by formula (8) by reduction. This step can be carried out in the same manner as Step 12, but due to the residual iodide ions, there is a possibility of a decrease in catalytic activity. Preferably, it is carried out under a higher catalyst amount and hydrogen pressure than in Step 12. For the reduction in this step, the method is not limited as long as the reaction proceeds, but preferably, it can be carried out in a hydrogen atmosphere (preferably under a hydrogen flow of 0.15 to 0.6 MPa), using a palladium catalyst, a platinum catalyst, a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst. More preferably, it can be carried out using a palladium catalyst, further preferably using palladium on carbon, and even more preferably using 5% palladium on carbon. The amount of 5% palladium on carbon used in this step is not limited as long as the reaction proceeds, but preferably, it is 20 to 160% by weight relative to the compound represented by formula (34) used in Step 21. Regarding the solvent used in this step, it is not particularly limited as long as it does not inhibit the reaction, but examples thereof include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-di alkane, 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, and water, and a mixed solvent thereof. Preferably, 2-methyltetrahydrofuran can be mentioned. The reaction temperature in this step is not limited as long as the reaction proceeds, but preferably, it is 20 to 60 °C. The reaction time in this step is not limited as long as the reaction proceeds, but preferably, it is 4 to 16 hours.

[0080] Step 23: This step is a step of converting the molecule of the compound represented by formula (8) into the compound represented by formula (9) by intramolecular cyclization. This step can be carried out in the same manner as the method described in Step 3.

[0081] Step 24: This step is a step of converting the compound represented by formula (9) into the compound represented by formula (10). This step can be carried out in the same manner as the method described in Step 4.

[0082] Step 25: This step is a step of selectively deprotecting the protecting group of the aromatic amino group of the compound represented by formula (10) and converting it into the compound represented by formula (11). This step can be carried out in the same manner as the method described in step 5.

[0083] Step 26: This step is a step of condensing the compound represented by formula (11) with the compound represented by formula (1) and converting it into the compound represented by formula (12). This step can be carried out in the same manner as the method described in step 16.

[0084] Step 27: This step is a step of converting the compound represented by formula (12) into the compound represented by formula (2). This step can be carried out in the same manner as the method described in step 7.

[0085] For the reactions of the above steps, after the reaction is completed, the target compound of each step can be isolated from the reaction mixture according to the methods well-known in the field of organic chemistry. The target compound can be obtained, for example, by (i) filtering off insolubles such as catalysts as necessary, (ii) adding water and a water-immiscible solvent (such as dichloromethane, diethyl ether, ethyl acetate, or 2-methyltetrahydrofuran, etc.) to the reaction mixture to extract the target compound, (iii) washing the organic layer with water and drying it using a drying agent such as anhydrous magnesium sulfate, and (iv) distilling off the solvent. The obtained target compound can be further purified by methods well-known in the field of organic chemistry (such as recrystallization, reprecipitation, silica gel column chromatography, or high-performance liquid chromatography, etc.) as necessary, but the production method of the present invention preferably does not use chromatography for carrying out.

[0086] The compound represented by formula (2) obtained by the production method of the present invention is preferably used for the production of an antibody-drug conjugate in which a drug linker represented by formula (15) is bound to an antibody via a thioether bond, but is not limited thereto, and can also be used for the production of antibody-drug conjugates having other chemical structures or for other uses.

[0087] [Manufacture of drug linker intermediate] The drug linker intermediate preferably used for the production of the antibody-drug conjugate of the present invention is the compound represented by formula (14).

[0088] The compound represented by formula (14) can be produced in the following manner.

[0089] The compound represented by formula (2) can be produced by the production method of the present invention. The compound represented by formula (13) can be produced with reference to the descriptions in International Publication No. 2014 / 057687, International Publication No. 2015 / 098099, International Publication No. 2015 / 115091, International Publication No. 2015 / 155998, etc.

[0090] The conversion to the compound represented by formula (14) can be carried out by inducing the compound represented by formula (13) into an active ester, mixed anhydride, acid halide, etc., and preferably reacting with the compound represented by formula (2) in the presence of a base.

[0091] The active ester can be produced, for example, by using a condensing agent such as N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD·HCl) to react the compound represented by formula (13) with an additive such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, or p-nitrophenol. Also, the active ester can be produced by reacting the compound represented by formula (13) with a condensing agent such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate pentafluorophenyl ester (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), cyanophosphonic acid diethyl ester, or 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine (DMTMM). -2-yl)-4-methyl morpholine (DMTMM).

[0092] The mixed anhydride can be produced, for example, by reacting the compound represented by formula (13) with isobutyl chloroformate in the presence of a base as necessary.

[0093] The acid halide can be produced by treating with an acid halide such as thionyl chloride or oxalyl chloride in the presence of a base as necessary.

[0094] Regarding the base used in this step, there is no particular limitation as long as the reaction proceeds. Examples include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methyl quinoline, N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]undec-7-ene, etc., or inorganic bases such as potassium carbonate, potassium hydroxide, potassium hydrogen carbonate, sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, etc. Preferred examples include triethylamine, tributylamine, diisopropylethylamine, potassium carbonate, potassium hydroxide, potassium hydrogen carbonate, sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium acetate, and more preferred are triethylamine, diisopropylethylamine, or N-methyl quinoline.

[0095] Regarding the solvent used in this step, there is no particular limitation as long as it does not inhibit the reaction. Examples include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-di oxane, 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, and water, as well as mixed solvents thereof. Preferred examples include acetonitrile, dichloromethane, methanol, tetrahydrofuran, 1,4-di oxane, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents thereof.

[0096] [Manufacture of Antibodies] The antibody used in the manufacture of the antibody-drug conjugate of the present invention can be derived from any species, but is preferably an antibody derived from humans, rats, mice, and rabbits. When the antibody is derived from a species other than humans, it is preferably chimerized or humanized using well-known techniques. The antibody of the present invention can be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

[0097] The antibody used in the production of the antibody-drug conjugate of the present invention preferably has the property of targeting cancer cells, preferably having the characteristics of recognizing cancer cells, binding to cancer cells, being internalized by being taken into cancer cells, and / or having cytotoxic activity against cancer cells, etc.

[0098] The binding property of the antibody to cancer cells can be confirmed using flow cytometry. The uptake of the antibody into cancer cells can be confirmed using the following methods: (1) an analysis method (Cell Death and Differentiation (2008) 15, 751-761) in which a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody is used to visualize the antibody taken into the cells with a fluorescence microscope; (2) an analysis method (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004) in which a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody is used to measure the amount of fluorescence taken into the cells; or (3) the so-called Mab-ZAP analysis method (Bio Techniques 28:162-165, January 2000), which uses an immunotoxin that binds to the therapeutic antibody, and if it is taken into the cells, the toxin is released and cell proliferation is inhibited. For the immunotoxin, a recombinant complex protein of the catalytic region of diphtheria toxin and protein G can also be used.

[0099] The antitumor activity of the antibody can be confirmed in vitro by measuring the inhibitory activity of cell proliferation. For example, a cancer cell line overexpressing the target protein of the antibody can be cultured, the antibody is added to the culture system at various concentrations, and the inhibitory activities against focus formation, colony formation, and spheroid proliferation are measured. In vivo, for example, by administering the antibody to nude mice transplanted with a cancer cell line highly expressing the target protein, the changes in cancer cells are measured, and the antitumor activity can be confirmed.

[0100] Although it is preferable that the antibody itself has an antitumor effect, since the antibody-drug conjugate has a compound that exerts an antitumor effect bound to it, the antitumor effect of the antibody itself is not essential. From the purpose of specifically and selectively exerting the cytotoxicity of the antitumor compound in cancer cells, it is important and preferable that the antibody has the property of being internalized and migrating into cancer cells.

[0101] The antibody used in the production of the antibody-drug conjugate of the present invention can be obtained by well-known means. For example, by using methods commonly practiced in this field, a polypeptide serving as an antigen is immunized to an animal, and the antibody produced in vivo is collected and purified to obtain it. The source of the antigen is not limited to humans, and an antigen derived from an animal other than humans, such as a mouse or a rat, can also be used to immunize the animal. In this case, by testing the cross-reactivity of the obtained antibody with the heterologous antigen and the human antigen, an antibody applicable to human diseases can be selected.

[0102] Furthermore, according to well-known methods (e.g., Kohler and Milstein, Nature (1975) 256, p.495-497; Kennet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, N.Y. (1980)), a hybridoma can be established and a monoclonal antibody can be obtained by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells.

[0103] Moreover, the antigen can be obtained by using genetic manipulation to cause a gene encoding an antigen protein to be produced in a host cell. Specifically, a vector capable of expressing the antigen gene is prepared, introduced into the host cell to express the gene, and the expressed antigen is purified. An antibody can also be obtained by using a method of immunizing an animal with the antigen-expressing cells or cell lines expressing the antigen resulting from the above genetic manipulation.

[0104] The antibody used in the production of the antibody-drug conjugate of the present invention is preferably a genetically recombinant antibody artificially modified for the purpose of reducing its heterologous antigenicity to humans, such as a chimeric antibody, a humanized antibody, or preferably an antibody having only a gene sequence of an antibody derived from humans, i.e., a human antibody. These antibodies can be produced using known methods.

[0105] Examples of chimeric antibodies include antibodies in which the variable region and the constant region of the antibody are heterologous to each other, such as a chimeric antibody in which the variable region of an antibody derived from a mouse or a rat is conjugated with the constant region derived from a human (Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)).

[0106] Examples of humanized antibodies include: antibodies that incorporate only the complementarity determining regions (CDRs) of a heterologous antibody into an antibody derived from humans (Nature (1986) 321, p. 522-525); antibodies that, by the DR transplantation method, transplant not only the sequences of the CDRs of a heterologous antibody but also amino acid residues of a part of the framework of the heterologous antibody into a human antibody (International Publication No. WO 90 / 07861); antibodies humanized using a gene conversion mutagenesis strategy (U.S. Patent No. 5,821,337).

[0107] Examples of human antibodies include antibodies produced using a human antibody-producing mouse having a human chromosome fragment containing genes of the heavy and light chains of a human antibody (see Tomizuka, K. et al., Nature Genetics (1997) 16, 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 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et. al., Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727, etc.). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be mentioned (see Wormstone, I. M. et. al, Investigative Ophthalmology & Visual Science. (2002) 43(7), p. 2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2), p. 189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109(3), p. 427-431, etc.).

[0108] The antibodies used in the production of the antibody-drug conjugates of the present invention also include modified forms of the antibodies. Such modified forms refer to those obtained by chemically or biologically modifying the antibodies of the present invention. Chemical modifications include chemical modified forms having a bond to a chemical moiety of the amino acid backbone, a bond to a chemical moiety of an N-linked or O-linked carbohydrate chain, etc. Biological modified forms include those that have been post-translationally modified (e.g., addition of N-linked or O-linked sugar chains, processing of the N-terminus or C-terminus, deamidation, isomerization of aspartic acid, oxidation of methionine, etc.); those that are expressed using prokaryotic host cells and have a methionine residue added to the N-terminus, etc. Also, those that are labeled for the purpose of detecting or isolating the antibody or antigen of the present invention, such as enzyme-labeled forms, fluorescent-labeled forms, and affinity-labeled forms, are also included in the meaning of such modified forms. Such modified forms of the antibodies of the present invention are useful for improving the stability and blood retention of the antibodies, reducing antigenicity, detecting or isolating the antibody or antigen, etc.

[0109] Furthermore, by regulating the sugar chain modification (glycosylation, defucosylation, etc.) that binds to the antibody of the present invention, the antibody-dependent cell cytotoxic activity can be enhanced. Regarding the techniques for regulating the sugar chain modification of antibodies, International Publication No. 99 / 54342, International Publication No. 00 / 61739, International Publication No. 02 / 31140, etc. are known, but are not limited thereto. The antibodies of the present invention also include antibodies in which such sugar chain modification has been regulated.

[0110] Furthermore, for antibodies produced by mammalian cultured cells, it is known that lysine residues at the carboxyl terminus of the heavy chain are deleted (Journal of Chromatography A, 705: 129-134 (1995)). Also, it is known that two amino acid residues, glycine and lysine, at the carboxyl terminus of the same heavy chain are deleted, and the proline residue at the new carboxyl terminus position is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequences do not affect the antigen-binding ability and effector functions (such as complement activation or antibody-dependent cell cytotoxicity) of the antibody. Therefore, the antibodies of the present invention also include antibodies subjected to such modifications and functional fragments of the antibodies, and also include deletion mutants having 1 or 2 amino acid deletions at the carboxyl terminus of the heavy chain, and amidated deletion mutants thereof (for example, a heavy chain in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector functions are retained, the deletion mutants at the carboxyl terminus of the heavy chain of the antibodies of the present invention are not limited to the above types. The two heavy chains constituting the antibodies of the present invention can be any of the heavy chains selected from the group consisting of the full length and the above deletion mutants, or can be a combination of any two of them. Although the quantitative ratio of each deletion mutant is affected by the type and culture conditions of the mammalian cultured cells producing the antibodies of the present invention, the antibodies of the present invention preferably include those in which one amino acid residue at the carboxyl terminus is deleted in both of the two heavy chains.

[0111] Regarding the isotype of the antibodies of the present invention, examples include IgG (IgG1, IgG2, IgG3, IgG4), etc., and preferably IgG1 or IgG2 can be cited.

[0112] The antibodies that can be used for the production of the antibody-drug conjugates of the present invention are not particularly limited, and examples include anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 antibody, anti-FOLR1 antibody, anti-VEGF antibody, and anti-GPR20 antibody. Preferably, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, and anti-GPR20 antibody can be cited.

[0113] In the present invention, "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (Human Epidermal Growth Factor Receptor Type 2; ErbB-2), and preferably has the activity of internalizing in HER2-expressing cells by binding to HER2.

[0114] Examples of anti-HER2 antibodies include, for example, trastuzumab (U.S. Patent No. 5,821,337) and pertuzumab (International Publication No. 01 / 00245), and trastuzumab is preferably cited.

[0115] In the present invention, "trastuzumab" is a humanized anti-HER2 monoclonal antibody that comprises a heavy chain composed of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 (Figure 1) and a light chain composed of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2 (Figure 2).

[0116] In the present invention, "anti-HER3 antibody" refers to an antibody that specifically binds to HER3 (Human Epidermal Growth Factor Receptor Type 3; ErbB-3), and preferably has the activity of internalizing in HER3-expressing cells by binding to HER3 on the surface of HER3-expressing cells.

[0117] Examples of anti-HER3 antibodies include, for example, patritumab (U3-1287), U1-59 (International Publication No. 2007 / 077028), MM-121 (seribantumab), the anti-ERBB3 antibody described in International Publication No. 2008 / 100624, RG-7116 (lumretuzumab), and LJM-716 (elgemtumab), and patritumab and U1-59 are preferably cited.

[0118] In the present invention, the "anti-TROP2 antibody" refers to an antibody having specific binding to TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2; EGP-1), and preferably having the activity of internalizing in TROP2-expressing cells by binding to TROP2.

[0119] Examples of the anti-TROP2 antibody include, for example, hTINA1-H1L1 (International Publication No. WO2015 / 098099).

[0120] In the present invention, the "anti-B7-H3 antibody" refers to an antibody having specific binding to B7-H3 (B cell antigen #7 homolog 3; PD-L3; CD276), and preferably having the activity of internalizing in B7-H3-expressing cells by binding to B7-H3.

[0121] Examples of the anti-B7-H3 antibody include, for example, M30-H1-L4 (International Publication No. WO2014 / 057687).

[0122] In the present invention, the "anti-GPR20 antibody" refers to an antibody specifically binding to GPR20 (G Protein-coupled receptor 20), and preferably having the activity of internalizing in GPR20-expressing cells by binding to GPR20.

[0123] Examples of the anti-GPR20 antibody include, for example, h046-H4e / L7 (International Publication No. WO2018 / 135501).

[0124] [Conjugate of Antibody and Drug Linker Intermediate] The antibody-drug conjugate of the present invention can be produced by reacting a drug linker intermediate (preferably the compound represented by formula (14)) with an antibody having a thiol group (also referred to as a mercapto group).

[0125] Antibodies having a thiol group can be obtained by methods well-known to those of ordinary skill in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, for each inter-chain disulfide bond in the antibody, 0.3 to 3 molar equivalents of a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is used, and the reaction is carried out with the antibody in a buffer solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), whereby an antibody having a thiol group in which the inter-chain disulfide bond in the antibody is partially or completely reduced can be obtained.

[0126] Furthermore, for each antibody having a thiol group, 2 to 20 molar equivalents of a drug linker intermediate (preferably the compound represented by formula (14)) are used to produce an antibody-drug conjugate in which each antibody has 2 to 8 drug conjugations.

[0127] The calculation of the average number of drug conjugations per molecule of the antibody of the produced antibody-drug conjugate can be carried out by the following methods. For example, a method (UV method) in which the UV absorbance of the antibody-drug conjugate and its conjugate precursor at two wavelengths of 280 nm and 370 nm is measured and calculated; or a method (HPLC method) in which the antibody-drug conjugate is treated with a reducing agent, and each obtained fragment is quantified and calculated by HPLC measurement.

[0128] The calculation of the conjugate of the antibody and the drug linker intermediate and the average number of drug conjugations per molecule of the antibody of the antibody-drug conjugate can be carried out with reference to the descriptions in International Publication No. 2014 / 057687, International Publication No. 2015 / 098099, International Publication No. 2015 / 115091, International Publication No. 2015 / 155998, and International Publication No. 2018 / 135501, etc.

[0129] In the present invention, the "anti-HER2 antibody-drug conjugate" means an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0130] The anti-HER2 antibody preferably comprises a heavy chain composed of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain composed of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2; or an antibody comprising a heavy chain composed of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain composed of the amino acid sequence set forth in SEQ ID NO: 2.

[0131] The average number of drug linkers per antibody of the anti-HER2 antibody-drug conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, still more preferably 7.5 to 8, and still more preferably about 8.

[0132] The anti-HER2 antibody-drug conjugate can be produced by referring to the description in International Publication No. 2015 / 115091 etc. using the drug linker intermediate (preferably the compound represented by formula (14)) produced by the production method of the present invention.

[0133] In the present invention, "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-HER3 antibody.

[0134] The anti-HER3 antibody preferably comprises an antibody composed of a heavy chain composed of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain composed of the amino acid sequence set forth in SEQ ID NO: 4; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0135] The average number of drug linkers per antibody of the anti-HER3 antibody-drug conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, still more preferably 7.5 to 8, and still more preferably about 8.

[0136] The anti-HER3 antibody-drug conjugate can be produced by referring to the description in International Publication No. 2015 / 155998 etc. using the drug linker intermediate (preferably the compound represented by formula (14)) produced by the production method of the present invention.

[0137] In the present invention, "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-TROP2 antibody.

[0138] The anti-TROP2 antibody preferably comprises a heavy chain composed of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain composed of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0139] The average number of drug linkers per antibody of the anti-TROP2 antibody-drug conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 5, still more preferably 3.5 to 4.5, and still more preferably about 4.

[0140] The anti-TROP2 antibody-drug conjugate can be produced by referring to the descriptions in International Publication No. 2015 / 098099 etc. using the drug linker intermediate (preferably the compound represented by formula (14)) produced by the production method of the present invention.

[0141] In the present invention, the "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[0142] The anti-B7-H3 antibody preferably comprises a heavy chain composed of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain composed of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0143] The average number of drug linkers per antibody of the anti-B7-H3 antibody-drug conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 5, still more preferably 3.5 to 4.5, and still more preferably about 4.

[0144] The anti-B7-H3 antibody-drug conjugate can be produced by referring to the descriptions in International Publication No. 2014 / 057687 etc. using the drug linker intermediate (preferably the compound represented by formula (14)) produced by the production method of the present invention.

[0145] In the present invention, the "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[0146] The anti-GPR20 antibody preferably comprises a heavy chain composed of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 9 and a light chain composed of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 10; or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0147] The average number of drug linkers per antibody of the anti-GPR20 antibody-drug conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0148] The anti-GPR20 antibody-drug conjugate can be produced by referring to the descriptions in International Publication No. 2018 / 135501, etc., using the drug linker intermediate (preferably the compound represented by formula (14)) produced by the production method of the present invention.

[0149] [Pharmaceutical Composition] The antibody-drug conjugate produced according to the present invention can be administered containing one or more pharmaceutically suitable components. The pharmaceutically suitable components can be appropriately selected from the formulation additives and others commonly used in this field according to the dosage or concentration of the antibody-drug conjugate produced according to the present invention. For example, the antibody-drug conjugate produced according to the present invention can be administered as a pharmaceutical composition containing a buffer such as a histidine buffer, an excipient such as sucrose or trehalose, and a surfactant such as polysorbate 80 or polysorbate 20.

[0150] The pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention, in addition to being administered to a patient as a systemic therapy, can be locally administered to cancer tissue to expect a therapeutic effect.

[0151] The pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention is preferably used for mammals, but more preferably used for humans.

[0152] The pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention is preferably used as an injection, more preferably used as an aqueous injection or a freeze-dried injection, and even more preferably used as a freeze-dried injection.

[0153] When the pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention is an aqueous injection, it is preferably administered intravenously by drip after being diluted with an appropriate diluent. Examples of the diluent include glucose solutions (preferably 5% glucose solution), physiological saline, and the like.

[0154] When the pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention is a freeze-dried injection, it is preferably dissolved in water for injection, and after diluting the necessary amount with an appropriate diluent, it is administered intravenously by drip. Examples of the diluent include glucose solutions (preferably 5% glucose solution), physiological saline, and the like.

[0155] Examples of the introduction routes that can be used for administering the pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention include, for example, intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, and the intravenous route is preferably listed.

[0156] The antibody-drug conjugate produced according to the present invention can be administered to humans at intervals of once every 1 to 180 days, preferably at intervals of once a week, once every two weeks, once every three weeks, or once every four weeks, and more preferably at intervals of once every three weeks. Also, the antibody-drug conjugate produced according to the present invention can be administered at a dosage of about 0.001 to 100 mg / kg per administration, preferably at a dosage of 0.8 to 12.4 mg / kg per administration. When the antibody-drug conjugate produced according to the present invention is an anti-HER2 antibody-drug conjugate, it is preferably administered at a dosage of 5.4, 6.4, or 7.4 mg / kg per administration, and more preferably at a dosage of 5.4 mg / kg or 6.4 mg / kg per administration.

[0157] A pharmaceutical composition comprising an antibody-drug conjugate manufactured according to the present invention can be used for the treatment of cancer, preferably for the treatment of at least one selected from the group consisting of: breast cancer, gastric cancer (also known as gastric adenocarcinoma), colorectal cancer (also known as 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, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, digestive tract stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, 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, neuroepithelial tissue tumor, schwannoma, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma, and sarcoma. For example, when the antibody-drug conjugate manufactured according to the present invention is an anti-HER2 antibody-drug conjugate, it is more preferably used for the treatment of at least one selected from the group consisting of: breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, and uterine carcinosarcoma. Even more preferably, it is used for the treatment of at least one selected from the group consisting of: breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, and Paget's disease. Even further preferably, it is used for the treatment of breast cancer, gastric cancer, colorectal cancer, or non-small cell lung cancer.

[0158] A pharmaceutical composition comprising an antibody-drug conjugate manufactured according to the present invention can be optionally used as an agent for drug therapy, which is the main treatment method for cancer treatment. As a result, the growth of cancer cells can be delayed, proliferation can be inhibited, and cancer cells can be destroyed. Through these effects, in cancer patients, liberation from cancer-induced symptoms, improvement of QOL, and maintenance of the life of cancer patients can be achieved to obtain a therapeutic effect. Even in the case where cancer cells are not destroyed, by inhibiting or controlling the proliferation of cancer cells, a higher QOL can be achieved in cancer patients, and long-term survival can be achieved.

[0159] In addition to the use of the drug alone in such drug therapy, a pharmaceutical composition comprising an antibody-drug conjugate manufactured according to the present invention can also be used in combination with other therapies in adjuvant therapy, and can be combined with surgery, radiotherapy, hormonal therapy, etc. Moreover, it can also be used as a drug therapy in neoadjuvant therapy.

[0160] In addition to the use of the above treatment, a pharmaceutical composition containing an antibody-drug conjugate produced according to the present invention is also expected to inhibit the proliferation of fine metastatic cancer cells and further achieve preventive effects such as destruction. For example, it is expected to inhibit and destroy cancer cells in body fluids during the metastasis process, and to inhibit and destroy fine cancer cells immediately after they have implanted in any tissue. Therefore, it is particularly expected to have an inhibitory and preventive effect on cancer metastasis after surgical removal of cancer.

[0161] The pharmaceutical composition containing the antibody-drug conjugate produced according to the present invention can also be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect. Examples of other cancer therapeutic agents used for this purpose include 5-fluorouracil (5-FU), Pertuzumab, Trastuzumab, Paclitaxel, Carboplatin, Cisplatin, Gemcitabine, Capecitabine, Irinotecan (CPT-11), Docetaxel, Pemetrexed, Sorafenib, Vinblastin, Vinorelbine, Everolims, Tanespimycin, Bevacizumab, Oxaliplatin, Lapatinib, Trastuzumab emtansine (T-DM1) and the agents described in WO 2003 / 038043, as well as LH-RH analogs (Leuprorelin, Goserelin, etc.), Estramustine Phosphate, estrogen antagonists (Tamoxifen, Raloxifene, etc.), aromatase inhibitors (Anastrozole, Letrozole, Exemestane, etc.), etc. However, it is not limited as long as it is an agent having antitumor activity. [Examples]

[0162] The present invention will be further described in detail by way of the following examples, but the present invention is not limited to these examples.

[0163] In the examples, "1H-NMR" and "13C-NMR" mean "nuclear magnetic resonance spectroscopy". CDCl3 in parentheses means deuterated chloroform as the solvent for measurement, DMSO-d6 means deuterated dimethyl sulfoxide as the solvent for measurement, and D2O means heavy water as the solvent for measurement. TMS (tetramethylsilane) is used as an internal standard substance. The multiplicity in 1H-NMR means s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, and brs = broad singlet.

[0164] (Example 1) N-(3-Bromo-5-fluoro-4-methylphenyl)acetamide A solution of 2-fluoro-1-methyl-4-nitrobenzene (10.0 g, 64.5 mmol) in concentrated sulfuric acid (more than 90%, 50 mL) and heptane (50 mL) was heated to about 60 °C, and then N-bromosuccinimide (13.8 g, 77.4 mmol) was added in 6 portions. After stirring at about 60 °C for 1 hour, it was cooled to room temperature. The obtained reaction solution was added to cold water (50 mL). After adding toluene (50 mL) and separating the layers, the aqueous layer was removed. Next, the organic layer was washed with water (50 mL), 6.5 wt% aqueous sodium hydrogen carbonate solution (50 mL), and 5 wt% aqueous sodium sulfite solution (50 mL). Water (50 mL) and activated carbon (1.0 g) were added to the obtained aqueous layer, and after stirring at room temperature for 1 hour, the insoluble matter was filtered out and the insoluble matter was washed with toluene (20 mL). After removing the aqueous layer from the filtrate, the organic layer was concentrated under reduced pressure. Ethyl acetate (100 mL) was added to the concentrated residue (about 30 mL), and it was concentrated again under reduced pressure to obtain an ethyl acetate solution (about 30 mL) of 1-bromo-3-fluoro-2-methyl-5-nitrobenzene. A suspension of 1% platinum-carbon catalyst (2.0 g) and ethyl acetate (120 mL) was added to a solution of ethyl acetate (about 30 mL) of 1-bromo-3-fluoro-2-methyl-5-nitrobenzene, and the mixture was purged with nitrogen and then with hydrogen. Under a hydrogen stream (0.1 MPa), the mixture was stirred at about 60 °C for 4 hours and then cooled to room temperature. The insoluble matter was filtered out from the obtained suspension, and the insoluble matter was washed with ethyl acetate (30 mL). The filtrate was washed twice with 0.5 N hydrochloric acid solution (100 mL) to obtain an organic layer. The aqueous layer at this time was extracted with ethyl acetate (50 mL) to obtain an organic layer, and the organic layers were combined. Thereafter, the combined organic layer was washed with 6.5 wt% aqueous sodium hydrogen carbonate solution (50 mL) and 5 wt% brine (50 mL), and then concentrated under reduced pressure. Ethyl acetate (50 mL) was added to the concentrated residue (about 30 mL), and the mixture was concentrated again under reduced pressure to obtain a solution of 3-bromo-5-fluoro-4-methylaniline in ethyl acetate (about 30 mL). Triethylamine (7.2 mL, 51.8 mmol) and acetic anhydride (3.3 mL, 34.4 mmol) were added to a solution of ethyl acetate (30 mL) in a solution of ethyl acetate (about 29 mL) of 3-bromo-5-fluoro-4-methylaniline, and the mixture was stirred at room temperature for 6 hours. 10 wt% brine (50 mL) was added to the obtained reaction solution for liquid separation, and the aqueous layer was removed. After concentrating the obtained organic layer under reduced pressure, ethyl acetate (50 mL) was added and the mixture was concentrated again under reduced pressure. Ethyl acetate (80 mL) was added to the concentrated residue (about 30 mL), 4N hydrochloric acid / ethyl acetate solution (10.9 mL, 43.7 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The insoluble matter was filtered out, and the insoluble matter was washed with ethyl acetate (40 mL). 10 wt% brine (40 mL) was added to the filtrate, and 25 wt% aqueous sodium hydroxide solution (5.6 g) was added to adjust the pH to about 7. After removing the aqueous layer, the organic layer was concentrated under reduced pressure. Toluene (100 mL) was added to the concentrated residue (about 30 mL), and the concentrated residue (about 30 mL) concentrated under reduced pressure was stirred at 50 °C for 5 hours and then cooled to room temperature. After stirring at room temperature for 12 hours, the mixture was cooled to 3 °C and stirred for 2 hours. The precipitated crystals were collected by filtration and washed with cold toluene (20 mL) and cold 75% aqueous acetonitrile (20 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain N-(3-bromo-5-fluoro-4-methylphenyl)acetamide (5.7 g, yield 37%) as white crystals. 1H-NMR (500 MHz, CDCl3) δ 7.41 (1H, s), 7.39 (1H, d, J = 9.2 Hz), 7.20 (1H, brs), 2.27 (3H, d, J = 2.0 Hz), 2.17 (3H, s)

[0165] (Example 2) 4-[5-(Acetylamino)-3-fluoro-2-methylphenyl]butyric acid A solution of N-(3-bromo-5-fluoro-4-methylphenyl)acetamide (30.0 g, 121.9 mmol), 3-butenoic acid (12.4 mL, 146.3 mmol), and diisopropylethylamine (46.0 mL, 268.2 mmol) in tetrahydrofuran (120 mL) and water (30 mL) was degassed under reduced pressure and purged with nitrogen. Then, tris(o-tolyl)phosphine (1.1 g, 3.7 mmol) was added. After degassing and purging with nitrogen again under reduced pressure, palladium(II) acetate (0.4 g, 1.8 mmol) was added. After degassing under reduced pressure and purging with nitrogen, the mixture was heated under reflux for 5 hours. Activated carbon (3.0 g) was added to the reaction solution cooled to room temperature, and the mixture was stirred at room temperature for 1 hour. The insoluble material was filtered off and washed with 20% aqueous tetrahydrofuran (60 mL). 2-Methyltetrahydrofuran (300 mL) and water (300 mL) were added to the filtrate, and 25 wt% aqueous sodium hydroxide solution (23.4 g, 146.3 mmol) was added. The organic layer was removed. 2-Methyltetrahydrofuran (300 mL) and concentrated hydrochloric acid (36%, 22.2 g, 219.4 mmol) were added to the aqueous layer, and then sodium chloride (30 g) was added. After liquid separation, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (90 mL). The obtained organic layer was concentrated under reduced pressure to obtain geometric isomer-containing 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid as a 2-methyltetrahydrofuran solution (about 150 mL). 4-[5-(Acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid containing geometric isomers was added with 2-methyltetrahydrofuran (308 mL) and a suspension of 5% palladium on carbon (5.6 g) in a 2-methyltetrahydrofuran solution (about 140 mL). After purging with nitrogen, it was purged with hydrogen. Under a hydrogen stream (0.1 MPa), it was stirred at about 40 °C for 1 hour and cooled to room temperature. The insoluble matter was filtered out from the obtained suspension, and the insoluble matter was washed with 2-methyltetrahydrofuran (112 mL). Water (140 mL) was added to the filtrate, and the pH was adjusted to about 2 with 1N hydrochloric acid. After liquid separation, the aqueous layer was removed, and the obtained organic layer was concentrated under reduced pressure. Ethyl acetate (420 mL) was added to the concentrated residue, and it was concentrated under reduced pressure. Ethyl acetate (420 mL) was added again, and it was concentrated under reduced pressure to obtain a concentrated residue (about 170 mL). After stirring at 50 °C for 5 hours, heptane (140 mL) was added and cooled to room temperature. The precipitated crystals were collected by filtration and washed with ethyl acetate / heptane (3 / 7) (84 mL). The obtained crystals were dried under reduced pressure to obtain 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyric acid as white crystals (26.1 g, yield 91%). 1H-NMR(500MHz,DMSO-d 6)δ12.08(1H,brs),9.97(1H,s),7.42(1H,dd,J=12.5,2.0Hz),7.05(1H,d,J=1.5Hz),2.59-2.54(2H,m),2.28(2H,t,J=7.3Hz),2.10(3H,d,J=2.0Hz),2.02(3H,s),1.71(2H,quint,J=7.5Hz)

[0166] (Example 3-1) N-(3-Fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide A solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyric acid (12.0 g, 47.4 mmol) and trifluoroacetic acid (24 mL) was cooled to 4 °C, and then trifluoroacetic anhydride (13.4 mL, 94.8 mmol) was added dropwise. The mixture was stirred at about 4 °C for 4 hours. The resulting reaction solution was added dropwise to 50% aqueous acetonitrile (120 mL) cooled to 5 °C. After adjusting the pH to about 7 with 25 wt% aqueous sodium hydroxide solution (77.3 g), water (59 mL) was added. Then, the mixture was returned to room temperature and the precipitated crystals were collected by filtration and washed with water (60 mL) and 75% aqueous acetonitrile (60 mL). The obtained crystals were dried under reduced pressure to obtain N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (10.2 g, yield 92%) as pale yellowish-white crystals. 1H-NMR(400MHz,CDCl 3)δ12.31(1H,brs),8.43(1H,d,J=12.8Hz),2.88(2H,t,J=12.0Hz),2.66(2H,dd,J=7.2,6.0Hz),2.22(3H,s),2.17(3H,d,J=2.0Hz),2.09(3H,quint,J=6.4Hz)

[0167] (Example 3-2) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide A solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyric acid (5.0 g, 19.7 mmol) and trifluoroacetic acid (10 mL) was cooled to 2 °C, and then trifluoroacetic anhydride (5.6 mL, 39.5 mmol) was added dropwise. The mixture was stirred at about 5 °C for 3 hours. 50% aqueous acetonitrile (50 mL) was added dropwise to the reaction solution. After adjusting the pH to about 7 with 25 w / v% aqueous sodium hydroxide solution (33 mL), water (17 mL) was added. Then, the mixture was returned to room temperature and the precipitated crystals were collected by filtration and washed with water (25 mL) and 75% aqueous acetonitrile (25 mL). The obtained crystals were dried under reduced pressure to obtain N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4.3 g, yield 92%) as pale yellowish-white crystals.

[0168] (Example 3-3) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide A solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyric acid (5.0 g, 19.7 mmol) and trifluoroacetic acid (10 mL) was cooled to 2 °C, and then trifluoroacetic anhydride (5.6 mL, 39.5 mmol) was added dropwise. The mixture was stirred at about 5 °C for 4 hours. After 17% aqueous acetonitrile (30 mL) was added dropwise to the reaction solution, water (20 mL) was added dropwise. The pH was adjusted to about 7 with 25 w / v% aqueous sodium hydroxide solution (33 mL), and then water (17 mL) was added. Then, the mixture was returned to room temperature and the precipitated crystals were collected by filtration. The crystals were washed with water (25 mL) and 75% aqueous acetonitrile (25 mL). The obtained crystals were dried under reduced pressure to obtain N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4.3 g, yield 93%) as pale yellowish white crystals.

[0169] (Example 4-1) N,N’-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide A solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (5.0 g, 21.3 mmol) in tetrahydrofuran (75 mL) was cooled to 6 °C, and amyl nitrite (3.7 mL, 27.6 mmol) and potassium tert-butoxide (2.9 g, 25.5 mmol) were added. After stirring at 3 °C for 17 hours, acetic acid (25 mL) and acetic anhydride (25 mL) were added and the temperature was raised. 2% platinum on carbon catalyst (1.5 g) was added at about 20 °C, the atmosphere was replaced with nitrogen and then with hydrogen. Under a hydrogen stream (0.3 MPa), the mixture was stirred at room temperature for 4 hours. Insoluble matters were filtered from the obtained suspension, and the insoluble matters were washed with ethyl acetate (25 mL). Activated carbon (0.7 g) was added to the filtrate, and the mixture was stirred at room temperature for 1 hour, then the insoluble matters were filtered out and the insoluble matters were washed with ethyl acetate (25 mL). The filtrate was cooled to 1 °C, and 5N aqueous sodium hydroxide solution (50 mL) was added dropwise. The aqueous layer was removed, 5N aqueous sodium hydroxide solution (50 mL) was added again, and the aqueous layer was removed. Tetrahydrofuran (35 mL) and water (25 mL) were added to the obtained organic layer, and 5N aqueous sodium hydroxide solution (25 mL) was added to adjust the pH to about 7. After raising the temperature to room temperature, the aqueous layer was removed and the organic layer was washed with 10 wt% brine (25 mL). The obtained organic layer was concentrated under reduced pressure to obtain a concentrated residue. Ethyl acetate (50 mL) was added to the concentrated residue, and the mixture was concentrated under reduced pressure. This operation was repeated three times in total. The concentrated residue (about 25 mL) was stirred at 40 °C for 5 hours, cooled to room temperature, and stirred at 2 °C for 3 hours. The precipitated crystals were collected by filtration and washed with cold ethyl acetate (25 mL) and water (25 mL). The obtained crystals were dried under reduced pressure to obtain N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide as white crystals (3.7 g, yield 60%). 1H-NMR(500MHz,CDCl 3)δ11.76(1H,s),8.43(1H,d,J=13.0Hz),6.53(1H,d,J=4.5Hz),4.62(1H,dt,J=14.0,5.4Hz),3.08-2.96(2H,m),2.78-2.72(1H,m),2.23(3H,s),2.15(3H,d,J=1.5Hz),2.11(3H,s),1.88-1.77(1H,m)

[0170] (Example 4-2) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide Cool a solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (35.0 g, 148.8 mmol) in tetrahydrofuran (525 mL) to 4 °C, and add amyl nitrite (25.7 mL, 193.4 mmol) and potassium tert-butoxide (20.0 g, 178.6 mmol). After stirring at 1 °C for 17 hours, add acetic acid (175 mL) and acetic anhydride (175 mL), and then raise the temperature. Add 2% platinum-carbon catalyst (11.8 g) at about 20 °C, replace with nitrogen, and then replace with hydrogen. Stir at room temperature for 3 hours under a hydrogen stream (0.3 MPa). Filter the insoluble matter from the obtained suspension, and wash the insoluble matter with ethyl acetate (175 mL). Add activated carbon (5.3 g) to the filtrate, stir at room temperature for 2 hours, filter the insoluble matter, and wash the insoluble matter with ethyl acetate (175 mL). Cool the filtrate to 1 °C, and dropwise add 5N aqueous sodium hydroxide solution (350 mL). Remove the aqueous layer, add 5N aqueous sodium hydroxide solution (350 mL) again, and remove the aqueous layer. Add tetrahydrofuran (245 mL) and water (175 mL) to the obtained organic layer, and then add 5N aqueous sodium hydroxide solution (150 mL) to adjust the pH to about 7. After raising the temperature to room temperature, remove the aqueous layer and wash the organic layer with 10 wt% brine (175 mL). Concentrate the obtained organic layer under reduced pressure to obtain a concentrated residue. Add ethyl acetate (350 mL) to the concentrated residue and concentrate under reduced pressure. This operation is repeated a total of 3 times. The concentrated residue (about 175 mL) is stirred at 40 °C for 5 hours, cooled to room temperature, and stirred at 2 °C for 3 hours. Filter the precipitated crystals, and wash them with cold ethyl acetate (175 mL) and water (175 mL). Dry the obtained crystals under reduced pressure to obtain white crystals (25.1 g). Heat a suspension of the obtained crystals (24.0 g, 82.1 mmol) in 20% aqueous ethanol (300 mL) to 65 °C. Add activated carbon (4.8 g), stir at 70 °C for 30 minutes, filter the insoluble matter, and wash the insoluble matter with 20% aqueous ethanol (72 mL). Dropwise add water (300 mL) to the filtrate at 60 °C, and then slowly cool to 2 °C and stir for 2 hours. Filter the precipitated crystals, and wash them with cold 60% aqueous ethanol (120 mL). Dry the obtained crystals under reduced pressure to obtain N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide as white crystals (21.6 g, yield 52%). 1H-NMR (500 MHz, CDCl3) δ 11.76 (1H, s), 8.43 (1H, d, J = 13.0 Hz), 6.53 (1H, d, J = 4.5 Hz), 4.62 (1H, dt, J = 14.0, 5.4 Hz), 3.08 - 2.96 (2H, m), 2.78 - 2.72 (1H, m), 2.23 (3H, s), 2.15 (3H, d, J = 1.5 Hz), 2.11 (3H, s), 1.88 - 1.77 (1H, m)

[0171] (Example 4-3) N,N'-(3-Fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide Cool a solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (3.0 g, 12.8 mmol) in tetrahydrofuran (45 mL) to 0 °C, and add amyl nitrite (2.2 mL, 16.6 mmol) and potassium tert-butoxide (1.7 g, 15.3 mmol). After stirring at 3 °C for 3 hours, add acetic acid (15 mL) and acetic anhydride (15 mL), warm to 20 °C, and stir for 1.5 hours. Add 5% platinum on carbon catalyst (0.4 g) at about 3 °C, replace with nitrogen, and then replace with hydrogen. Stir under a hydrogen stream (0.6 MPa) at about 5 °C for 3 hours. Then, stir at about 30 °C for 1 hour, filter out the insoluble matter, and wash the insoluble matter with ethyl acetate (15 mL). Add activated carbon (0.5 g) to the filtrate, stir at room temperature for 2 hours, filter out the insoluble matter, and wash the insoluble matter with ethyl acetate (15 mL). Cool the filtrate to about 5 °C, and dropwise add 5N aqueous sodium hydroxide solution (30 mL). Remove the aqueous layer, add tetrahydrofuran (30 mL) and 5N aqueous sodium hydroxide solution (30 mL), and remove the aqueous layer. Add water (15 mL) to the obtained organic layer, add 5N aqueous sodium hydroxide solution (15 mL), and adjust the pH to about 7. After warming to room temperature, remove the aqueous layer and wash the organic layer with 10 wt% brine (15 mL). Concentrate the obtained organic layer under reduced pressure to obtain a concentrated residue. Add ethyl acetate (30 mL) to the concentrated residue and concentrate under reduced pressure. Stir the concentrated residue (about 15 mL) obtained by repeating this operation three times in total at 40 °C for 5 hours, then cool to room temperature, and stir at 5 °C for more than 2 hours. Filter the precipitated crystals, and wash with cold ethyl acetate (15 mL) and water (15 mL). Dry the obtained crystals under reduced pressure to obtain N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide as white crystals (2.3 g, yield 62%).

[0172] (Example 5-1) N-(8-Amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide A suspension of N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (3.0 g, 10.3 mmol) and 2N hydrochloric acid / ethanol (30 mL) was stirred at 50 °C for 7 hours. Water (45 mL) was added to the resulting reaction solution, and it was cooled to 1 °C. Triethylamine (8.6 mL, 61.6 mmol) was added dropwise at 1 °C, and then sodium sulfite (26 mg, 0.2 mmol) was added. After stirring at 1 °C for 4 hours, the precipitated crystals were collected by filtration and washed with cold 60% aqueous ethanol (30 mL) and water (15 mL). The obtained crystals were dried under reduced pressure to obtain pale green crystals (2.4 g). A suspension of the obtained pale green crystals (1.8 g) in acetone (18 mL) was stirred at 50 °C for 5 hours and then cooled to room temperature. The precipitated crystals were collected by filtration and washed with acetone (9 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide as pale green crystals (1.6 g, yield 82%). 1H-NMR(400MHz,DMSO-d 6)δ8.07(1H,d,J=8.0Hz),7.40(2H,brs),6.38(1H,d,J=13.2Hz),4.52-4.43(1H,m),2.98-2.88(1H,m),2.87-2.76(1H,m),2.18-2.10(1H,m),1.98(3H,d,J=1.2Hz),1.90(3H,s),1.88-1.78(1H,m)

[0173] (Example 5-2) N-(8-Amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide At room temperature, N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (5.0 g, 17.1 mmol) was added in 5 portions to 2N hydrochloric acid / ethanol (75 mL), and the mixture was stirred at 50 °C for 5 hours. Water (113 mL) was added to the obtained reaction solution, and the mixture was cooled to 2 °C. Triethylamine (22.5 mL, 161.4 mmol) was added dropwise at 2 °C, and then sodium sulfite (43 mg, 0.3 mmol) was added. After stirring at 2 °C for 3 hours, the precipitated crystals were collected by filtration and washed with cold 60% aqueous ethanol (50 mL) and water (25 mL). The obtained crystals were dried under reduced pressure to obtain pale blue crystals (3.9 g). A suspension of the obtained crystals (1.8 g) in acetone (18 mL) was stirred at 50 °C for 5 hours and then cooled to room temperature. The precipitated crystals were collected by filtration and washed with acetone (9 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide as pale green crystals (1.6 g, yield 80%). 1H-NMR(400MHz,DMSO-d 6)δ8.07(1H,d,J=8.0Hz),7.40(2H,brs),6.38(1H,d,J=13.2Hz),4.52-4.43(1H,m),2.98-2.88(1H,m),2.87-2.76(1H,m),2.18-2.10(1H,m),1.98(3H,d,J=1.2Hz),1.90(3H,s),1.88-1.78(1H,m)

[0174] (Example 6-1) N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]ind o[1,2-b]quinolin-1-yl]acetamide To N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (170.0 g, 679 mmol) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]ind A suspension of 3,6,10(4H)-trione (196.7 g, 747 mmol) in toluene (8.5 L) was added with o-cresol (510 mL) and pyridinium p-toluenesulfonate (25.6 g, 102 mmol), and refluxed for 32 hours. Toluene (500 mL) was added and cooled to room temperature, and further stirred for 2 hours. The precipitated crystals were filtered, and the filtered crystals were washed with acetone (850 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]ind eno[1,2-b]quinolin-1-yl]acetamide (312.5 g, yield 96%). 1H-NMR (400 MHz, DMSO-d6) δ 0.87 (3H, t, J = 7.3 Hz), 1.79 - 1.88 (2H, m), 1.91 (3H, s), 2.13 - 2.15 (2H, m), 2.39 (3H, s), 3.13 - 3.22 (2H, m), 5.20 (2H, dd, J = 25.6, 18.9 Hz), 5.42 (2H, s), 5.53 - 5.57 (1H, m), 6.52 (1H, s), 6.65 - 6.69 (0.4H, m), 6.75 (0.4H, d, J = 7.9 Hz), 6.95 - 6.99 (0.4H, m), 7.03 (0.4H, d, J = 7.3 Hz), 7.13 - 7.27 (0.4H, m).7.30 (1H, s), 7.79 (1H, d, J = 11.0 Hz), 8.46 (1H, d, J = 9.2 Hz), 9.19 (0.4H, s). 13C-NMR (100 MHz, DMSO-d6) δ 7.7, 10.9, 10.9, 15.9, 22.6, 23.1, 27.7, 30.3, 44.0, 49.5, 65.2, 72.3, 96.6, 109.7, 109.9, 114.5, 118.7, 119.1, 121.4, 123.6, 123.7, 123.7, 125.3, 125.5, 126.6, 128.2, 128.9, 130.5, 136.2, 136.3, 140.4, 145.2, 147.8, 147.9, 149.9, 152.3, 155.3, 156.6, 160.3, 162.8, 169.1, 172.4. MS(ESI) (m / z): 478 ([M + H]+).

[0175] (Example 6-2) N-[(9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]ind o[1,2-b]quinolin-1-yl]acetamide In a suspension of toluene (125 mL) of N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (2.5 g, 9.99 mmol) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]ind -3,6,10(4H)-trione (3.42 g, 12.99 mmol), o-cresol (7.5 mL) and pyridinium p-toluenesulfonate (0.75 g, 3.00 mmol) were added, and the mixture was refluxed for 19 hours (confirmed via the compound represented by formula (30) and the compound represented by formula (31) as intermediates). The compound represented by formula (30): N-[(2S)-8-{[(4S)-4-Ethyl-4-hydroxy-3,10-dioxo-3,4,8,10-tetrahydro-1H-pyrano[3,4-f]ind -6-yl]amino}-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl]acetamide 1H-NMR (500 MHz, CDCl3) δ 1.04 (3H, t, J = 7.5 Hz), 1.80 - 1.93 (2H, m), 2.02 - 2.18 (8H, m), 3.80 - 3.85 (1H, m), 4.62 - 4.68 (1H, m), 4.75 - 4.85 (m, 2H), 5.20 - 5.33 (m, 2H), 5.70 (1H, d, J = 16.0 Hz), 6.35 (1H, s), 6.67 (1H, d, J = 5.5 Hz), 6.88 (1H, s), 6.99 (1H, d, J = 12.0 Hz), 11.14 (1H, s). MS (ESI) (m / z): 496.5 ([M + H]+). The compound represented by formula (31): N-[(2R)-8-{[(4S)-4-ethyl-4-hydroxy-3,10-dioxo-3,4,8,10-tetrahydro-1H-pyrano[3,4-f]ind -6-yl]amino}-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl]acetamide 1H-NMR (500 MHz, CDCl3) δ 1.03 (3H, t, J = 7.5 Hz), 1.80 - 1.92 (2H, m), 2.02 - 2.18 (8H, m), 3.79 (1H, s), 4.60 - 4.68 (1H, m), 4.72 - 4.87 (m, 2H), 5.28 (1H, d, J = 16.0 Hz), 5.70 (1H, d, J = 16.0 Hz), 6.35 (1H, s), 6.68 (1H, d, J = 4.5 Hz), 6.88 (1H, s), 7.00 (1H, d, J = 12.0 Hz), 11.10 (1H, s). MS (ESI) (m / z): 496.6 ([M + H]+). After cooling, the liquid volume was adjusted to 135 mL with toluene and further stirred for 2 hours. The precipitated crystals were filtered, and the filtered crystals were washed with acetone (12.5 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain yellow crystals of N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]ind and [1,2-b]quinolin-1-yl]acetamide (4.58 g, yield 96%). The machine data is the same as that of the compound described in Example 6-1.

[0176] (Example 7-1) 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]ind and [1,2-b]quinolin-1-ammonium 2-hydrate To a suspension of N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]ind and [1,2-b]quinolin-1-yl]acetamide (300.0 g, 628 mmol) in 2-methoxyethanol (1.5 L), water (4.5 L), and ethylcyclohexane (1.5 L), methanesulfonic acid (1.5 L) was added and the mixture was refluxed for 8 hours. After cooling to room temperature, the organic layer was removed by liquid separation and the solution was concentrated under reduced pressure to 3 L. The concentrated solution was heated to 40 °C and methanol (6 L) was added dropwise over 30 minutes. After stirring for 2 hours, the precipitated crystals were filtered and the filtered crystals were washed with methanol (1.5 L). The obtained crystals were dissolved in water (1.2 L), methanol (600 mL), and methanesulfonic acid (1.2 L), activated carbon (15 g) was added and the mixture was stirred for 30 minutes. Cellulose powder (150 g) was added and the mixture was stirred for 30 minutes, then the insoluble matter was filtered off and the insoluble matter was washed with 50% methanesulfonic acid aqueous solution (600 mL) and methanol (600 mL). The filtrate was heated to 40 °C and methanol (4.8 L) was added dropwise over 55 minutes. After stirring for 2 hours, the precipitated crystals were filtered and the filtered crystals were washed with methanol (1.5 L). The obtained crystals were suspended in ethanol (6 L) and water (600 mL), and refluxed for 1.5 hours. After cooling to room temperature, the mixture was stirred for 30 minutes and the precipitated crystals were filtered. The crystals were washed with ethanol (1.5 L). The obtained crystals were dried under reduced pressure at 40 °C, and then conditioned in air at 40% RH for 4 days to obtain colorless crystals of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]ind o[1,2-b]quinolin-1-ammonium 2-hydrate (152.3 g, yield 43%). 1H-NMR (400 MHz, DMSO-d6, D2O) δ 0.89 (3H, t, J = 7.3 Hz), 1.90 (2H, q, J = 7.3 Hz), 2.35 (3H, s), 2.38 - 2.47 (1H, m), 2.64 (3H, s), 3.04 - 3.11 (1H, m), 3.30 - 3.34 (1H, m), 5.08 (1H, s), 5.34 (2H, dd, J = 17.7, 15.9 Hz), 5.50 (2H, dd, J = 17.7, 10.4 Hz), 7.41 (1H, s), 7.59 (1H, d, J = 11.0 Hz). 13C-NMR (125 MHz, DMSO-d6) δ 7.7, 10.9, 11.0, 18.5, 20.8, 24.7, 30.2, 39.5, 44.5, 49.4, 55.9, 65.2, 72.2, 95.3, 96.9, 110.1, 100.3, 119.4, 120.5, 124.6, 124.7, 127.5, 134.2, 135.2, 135.2, 144.8, 147.8, 147.9, 149.9, 152.3, 156.6, 160.6, 162.6, 172.3. MS (ESI) (m / z): 436 ([M + H]+).

[0177] (Example 7-2) (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]ind o[1,2-b]quinolin-1-ammonium In a suspension of N-[(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]indol -[1,2-b]quinolin-1-yl]acetamide (3.5 g, 7.3 mmol) in pure water (53 mL), 2-methoxyethanol (18 mL), and ethylcyclohexane (18 mL), methanesulfonic acid (18 mL) was added, and nitrogen displacement under reduced pressure was repeated 3 times (the operation of stirring under reduced pressure at 50 mbar and then replacing with nitrogen at normal pressure was repeated 3 times). After heating the suspension to 85 °C, it was stirred for 11 hours. After confirming the completion of the reaction, it was cooled to 25 °C. The mixture was concentrated under reduced pressure to 38.5 mL. The concentrated solution was heated to 40 °C, and methanol (18 mL) was added dropwise over 15 minutes. After stirring for 6 hours, methanol (53 mL) was added dropwise over 2 hours. After stirring for another 2 hours, the precipitated crystals were filtered, and the filtered crystals were washed with methanol (35 mL). The obtained crystals were dissolved in a mixture of pure water (14 mL) and methanesulfonic acid (14 mL). After heating to 37 °C, methanol (7 mL), activated carbon (0.35 g), and filter aid (0.70 g, Celpure C1000 diatomaceous earth) were added, and nitrogen displacement under reduced pressure was repeated 3 times (50 mbar, nitrogen displacement at normal pressure 3 times). After stirring the suspension for 20 minutes, the insoluble matter was filtered out, and the insoluble matter was washed with a mixed solution of methanesulfonic acid - pure water - methanol (7 mL, 7 mL, 3.5 mL) and methanol (7 mL). The filtrate was heated to 37 °C, and methanol (10.5 mL) was added dropwise over 15 minutes. After stirring for 6 hours, methanol (42 mL) was added dropwise over 1 hour. After stirring for another 2 hours, the precipitated crystals were filtered, and the filtered crystals were washed with methanol (35 mL). The obtained crystals were suspended in ethanol (70 mL) and water (7 mL), and stirred at 73 °C for 2 hours. After cooling to 25 °C, it was stirred for 2 hours and the precipitated crystals were filtered, and the crystals were washed with ethanol (18 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain 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]indol -[1,2-b]quinolin-1-ammonium (1.72 g, yield 44%). The machine data is the same as that of the compound described in Example 7-1.

[0178] (Example 8) N-(3-Iodo-5-fluoro-4-methylphenyl)acetamide A solution of 2-fluoro-1-methyl-4-nitrobenzene (5.0 g, 32.3 mmol) in concentrated sulfuric acid (more than 90%, 25 mL) and heptane (25 mL) was cooled to about 1 °C, and N-iodosuccinimide (10.2 g, 45.1 mmol) was added in 6 portions. The mixture was stirred at about 2 °C for 2 hours. The resulting reaction solution was added to cold water (25 mL). After adding toluene (25 mL) and separating the layers, the aqueous layer was removed. Next, the organic layer was washed with water (25 mL), 6.5 wt% aqueous sodium hydrogen carbonate solution (25 mL), 5 wt% aqueous sodium sulfite solution (25 mL, 3 times), and finally with water (25 mL). After removing the aqueous layer from the filtrate, the organic layer was concentrated under reduced pressure. Ethyl acetate (50 mL) was added to the concentrated residue (about 30 mL), and it was concentrated again under reduced pressure to obtain an ethyl acetate solution (about 15 mL) of 1-iodo-3-fluoro-2-methyl-5-nitrobenzene. A suspension of 1% platinum-carbon catalyst (1.1 g) and ethyl acetate (45 mL) was added to the ethyl acetate solution (about 15 mL) of 1-iodo-3-fluoro-2-methyl-5-nitrobenzene, and the mixture was purged with nitrogen and then with hydrogen. The mixture was stirred at about 60 °C for 5 hours under a hydrogen stream (0.1 MPa) and cooled to room temperature. The insoluble matter was filtered out from the resulting suspension, and the insoluble matter was washed with ethyl acetate (15 mL). The filtrate was washed twice with 0.5 N hydrochloric acid solution (50 mL, 25 mL) to obtain an organic layer. The aqueous layer at this time was extracted with ethyl acetate (25 mL) to obtain an organic layer, and the organic layers were combined. Thereafter, the combined organic layer was washed with 6.5 wt% aqueous sodium hydrogen carbonate solution (25 mL) and 5 wt% brine (25 mL), and the obtained organic layer was concentrated under reduced pressure to obtain an ethyl acetate solution of 3-iodo-5-fluoro-4-methylaniline. To a solution of 3-bromo-5-fluoro-4-methylaniline in ethyl acetate (25 mL), a solution of ethyl acetate (25 mL) was added, and triethylamine (3.7 mL, 26.8 mmol) and acetic anhydride (1.7 mL, 17.7 mmol) were added, followed by stirring at room temperature for 4 hours. To the resulting reaction solution, 10 wt% brine (25 mL) was added, and after liquid separation, the aqueous layer was removed. The obtained organic layer was concentrated under reduced pressure. To the concentrated residue, ethyl acetate (50 mL) was added, and 4N hydrochloric acid / ethyl acetate solution (5.6 mL, 22.6 mmol) was added, followed by stirring at room temperature for 15 minutes. The insoluble matter was filtered out and washed with ethyl acetate (20 mL). To the filtrate, 10 wt% brine (20 mL) was added, and 25 w / v% aqueous sodium hydroxide solution (2.5 mL) was added to adjust the pH to about 7. After removing the aqueous layer, the organic layer was concentrated under reduced pressure. To the concentrated residue, acetonitrile (38 mL) and water (38 mL) were added, and the mixture was stirred at 25 °C. The precipitated crystals were collected by filtration and washed with 50% aqueous acetonitrile (15 mL). The obtained crystals were dried under reduced pressure at 40 °C to obtain N-(3-iodo-5-fluoro-4-methylphenyl)acetamide (2.8 g, yield 29%) as white crystals. 1H-NMR(500MHz,CDCl 3)δ7.61(1H,s),7.47(1H,d,J=10.8Hz),7.10(1H,brs),2.30(3H,d,J=2.3Hz),2.16(3H,s)

[0179] (Example 9) 4-[5-(Acetylamino)-3-fluoro-2-methylphenyl]butanoic acid A solution of N-(3-iodo-5-fluoro-4-methylphenyl)acetamide (2.0 g, 6.8 mmol), 3-butenoic acid (0.7 mL, 8.2 mmol), and diisopropylethylamine (2.6 mL, 15.0 mmol) in tetrahydrofuran (8 mL) and water (2 mL) was degassed under reduced pressure and purged with nitrogen. Then, tris(o-tolyl)phosphine (62.3 mg, 0.2 mmol) was added. After degassing and purging with nitrogen again under reduced pressure, palladium(II) acetate (23.0 mg, 0.1 mmol) was added. After degassing and purging with nitrogen under reduced pressure, the mixture was heated under reflux for 2 hours. After cooling to room temperature, activated carbon (0.2 g), 2-methyltetrahydrofuran (10 mL), and water (10 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. The insoluble matter was filtered out and washed with 20% aqueous tetrahydrofuran (4 mL). 2-Methyltetrahydrofuran (10 mL) and water (10 mL) were added to the filtrate, and 25 w / v% aqueous sodium hydroxide solution (1.3 mL, 8.2 mmol) was added. The organic layer was removed. 2-Methyltetrahydrofuran (20 mL) and concentrated hydrochloric acid (36%, 1.2 g, 12.3 mmol) were added to the aqueous layer, and then sodium chloride (2 g) was added. After liquid separation, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (6 mL). The obtained organic layer was concentrated under reduced pressure to obtain a residue of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid containing geometric isomers (1.9 g). A suspension of 5% palladium on carbon (1.7 g) in 2-methyltetrahydrofuran (30 mL) was added to the residue of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid containing geometric isomers (1.9 g), and the mixture was purged with nitrogen and then with hydrogen. The mixture was stirred at about 40 °C for 8 hours under a hydrogen stream (0.3 MPa) and then cooled to room temperature. The insoluble matter was filtered out from the obtained suspension and washed with 2-methyltetrahydrofuran (8 mL). Water (10 mL) was added to the filtrate, and the pH was adjusted to about 2 with 1 N hydrochloric acid solution. After liquid separation, the aqueous layer was removed, and the obtained organic layer was concentrated under reduced pressure. Ethyl acetate (10 mL) was added to the concentrated residue, and the mixture was heated to about 50 °C. Then, heptane (10 mL) was added, and the mixture was cooled to room temperature. The precipitated crystals were collected by filtration and washed with ethyl acetate / heptane (3 / 7) (6 mL). The obtained crystals were dried under reduced pressure to obtain 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyric acid as white crystals (1.4 g, yield 81%).

[0180] (Example 10) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide In a solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyric acid (200 mg, 0.79 mmol), thionyl chloride (86 μL, 1.18 mmol), and dichloromethane (4 mL), aluminum chloride (263 mg, 1.97 mmol) was added at room temperature under a nitrogen stream, and the mixture was stirred at room temperature for 2 hours. 1N aqueous hydrochloric acid solution (10 ml) and ethyl acetate (50 ml) were added to the obtained reaction solution. The aqueous layer was removed, and the organic layer was washed with water (10 ml), 6.5 wt% aqueous sodium bicarbonate solution (10 ml), and water (10 ml). The obtained organic layer was dried over sodium sulfate. After filtering off the insoluble matter, the residue obtained by distilling off the solvent under reduced pressure was purified by preparative thin-layer chromatography (hexane:ethyl acetate = 2:1) to obtain N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (125 mg, yield 67%) as pale yellowish-white crystals. The machine data is the same as the compound described in Example 3-1. [Non-keyword text of the sequence listing] Sequence ID No. 1: Amino acid sequence of the heavy chain of the anti-HER2 antibody Sequence ID No. 2: Amino acid sequence of the light chain of the anti-HER2 antibody Sequence ID No. 3: Amino acid sequence of the heavy chain of the anti-HER3 antibody Sequence ID No. 4: Amino acid sequence of the light chain of the anti-HER3 antibody Sequence ID No. 5: Amino acid sequence of the heavy chain of the anti-TROP2 antibody Sequence ID No. 6: Amino acid sequence of the light chain of the anti-TROP2 antibody Sequence ID No. 7: Amino acid sequence of the heavy chain of the anti-B7-H3 antibody Sequence ID No. 8: Amino acid sequence of the light chain of the anti-B7-H3 antibody Sequence ID No. 9: Amino acid sequence of the heavy chain of the anti-GPR20 antibody Sequence ID No. 10: Amino acid sequence of the light chain of the anti-GPR20 antibody

Claims

1. A method for manufacturing a compound represented by formula (F), wherein R1 represents an amino group protected by a protecting group, and R2 represents an amino group protected by a protecting group, the method comprising the following steps: a step of converting the compound represented by formula (C) into the compound represented by formula (F), wherein, R1 represents the same meaning as described above, wherein the step comprises the following sub-steps: (i) reacting the compound represented by formula (C) with a nitrite ester in the presence of a base to introduce a nitroso group at the α-position of the carbonyl group of the compound represented by formula (C); (ii) introducing a protecting group to the nitrogen atom derived from the nitroso group; and (iii) reducing the compound produced in sub-step (ii) with hydrogen in the presence of a platinum-carbon catalyst.

2. The manufacturing method of claim 1, wherein R1 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, methyl, or benzyl.

3. The manufacturing method of claim 1, wherein R1 is an amine group protected by acetyl or trifluoroacetyl.

4. The manufacturing method of claim 1, wherein R1 is an acetyl-protected amino group.

5. The manufacturing method of any one of claims 1 to 4, wherein R2 is an amino group protected by acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, trimethylacetyl, methyl, or benzyl.

6. The manufacturing method of any one of claims 1 to 4, wherein R2 is an amine group protected by acetyl or trifluoroacetyl.

7. The manufacturing method of any one of claims 1 to 4, wherein R2 is an acetyl-protected amino group.

8. A method for manufacturing a compound represented by formula (2), the method comprising the following steps: manufacturing the compound represented by formula (F) by any one of claims 1 to 7; transforming the compound represented by formula (F) into the compound represented by formula (G): wherein R2 has the same meaning as above; then, fused the compound represented by formula (G) with the compound represented by formula (1): and transforming the compound represented by formula (G) into the compound represented by formula (H): wherein R2 has the same meaning as above; and then, transforming the compound represented by formula (H) into the compound represented by formula (2).

9. A method for manufacturing a compound represented by formula (14), the method comprising the steps of: manufacturing the compound represented by formula (2) by the manufacturing method of claim 8; fused the compound represented by formula (2) with the compound represented by formula (13); and transforming the compound represented by formula (2) into the compound represented by formula (14).

10. A method for manufacturing an antibody-drug conjugate, wherein a drug-linker represented by formula (15) is bound to an antibody via a thioether bond, wherein A represents the binding site with the antibody, the method comprising the following steps: (i) manufacturing the compound represented by formula (14) by the manufacturing method of claim 9; (ii) reducing the antibody; and then (iii) reacting the compound represented by formula (14) with the reduced antibody.

11. The manufacturing method of claim 10, wherein the antibody is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, or an anti-GPR20 antibody.

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