Novel method for producing antibody-drug conjugates

Abstract

To provide a novel, industrially superior method for producing exatecan, which is a component of an antibody-drug conjugate, with a small number of steps, and a novel method for producing an antibody-drug conjugate using the same.SOLUTION: A method for producing an intermediate compound represented by formula (C), where R1 represents an amino group protected with a protecting group, comprises a step of subjecting a compound represented by formula (B), where R1 represents the same meaning as above, to intramolecular cyclization to convert the compound into the compound represented by formula (C).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for producing exatecan, a component of antibody-drug conjugates, and a novel method for producing antibody-drug conjugates using the same. [Background technology]

[0002] Antibody-drug conjugates (ADCs), which combine a cytotoxic drug with an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into the cells, are expected to be able to selectively deliver drugs to cancer cells, thereby accumulating the drug within the cancer cells and killing them (Non-Patent Documents 1 to 5).

[0003] One known antibody-drug conjugate is one that contains an antibody and the topoisomerase I inhibitor exatecan as components (Patent Documents 1 to 8, Non-Patent Documents 6 and 7). These antibody-drug conjugates have excellent antitumor effects and are safe, and clinical trials are currently underway.

[0004] As a method for producing 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] Special Publication No. 5-59061 [Patent Document 10] Special Publication No. 8-337584 [Patent Document 11] International Publication No. 96 / 26181

Non-licensed literature

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

Non-patent document 7

[0007] Exatecan is a compound of formula (2)

[0008] [ka]

[0009] and is a compound that serves as a constituent element of the antibody-drug conjugate of the present invention.

[0010] Known methods for producing exatecan include those described in Patent Documents 9 to 11. These production methods can be expressed as follows: That is, a compound represented by formula (19) is reacted with succinic anhydride to convert it to a compound represented by formula (20), which is then reduced to convert it to a compound represented by formula (21), which is then converted to a compound represented by formula (22) by an intramolecular cyclization reaction, which is then oximed to convert it to a compound represented by formula (23), which is then converted to a compound represented by formula (24) by a Beckmann rearrangement, which is then converted to a compound represented by formula (25) by a ring-opening reaction, which is then converted to a compound represented by formula (26) by protecting the amino group, which is then hydrolyzed to convert it to a compound represented by formula (27), which is then converted to a compound represented by formula (28), which is then subjected to an intramolecular cyclization reaction. This method involves converting the compound represented by formula (28) by a cleavage reaction, converting it to a compound represented by formula (29) by reduction, converting it to a compound represented by formula (9) by oxidation, then introducing a nitrogen atom to convert it to a compound represented by formula (10), converting it to a compound represented by formula (11) by selective deprotection, converting it to a compound represented by formula (12) by a Friedlander reaction with the compound represented by formula (1), and finally deprotecting the acetyl group to produce the compound represented by formula (2), i.e., exatecan. However, this production method requires repeated ring-opening and ring-closing reactions and oxidation and reduction reactions, resulting in a long and cumbersome process. Therefore, the development of an industrially superior production method is desirable.

[0011] [ka]

[0012] One objective of the present invention is to discover a novel method for producing exatecan that requires fewer steps and is industrially superior. Another objective of the present invention is to develop a novel method for producing antibody-drug conjugates using the same. [Means for solving the problem]

[0013] As a result of extensive investigation into methods for producing exatecan, the present inventors have discovered a novel method for producing exatecan that requires fewer steps and is industrially superior. Furthermore, they have developed a novel method for producing antibody-drug conjugates using the exatecan produced by this production method. That is, the present invention is [1] Formula (B)

[0014] [ka]

[0015] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group) is intramolecularly cyclized to Formula (C)

[0016] [ka]

[0017] A compound represented by the formula (wherein R 1 has the same meaning as defined above). [2] R 1 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group. [3] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group. [4] R 1 is an amino group protected with an acetyl group. [5] The 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 method according to [5], wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid. [7] The 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 method according to [7], wherein the intramolecular cyclization is carried out in the presence of aluminum chloride. [9] Formula (J)

[0018] [ka]

[0019] (wherein Y represents a leaving group, and R 1 represents an amino group protected by a protecting group) is intramolecularly cyclized to Formula (C)

[0020] [ka]

[0021] A compound represented by the formula (wherein R 1 has the same meaning as defined above).

[10] R 1is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[11] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[12] R 1 is an amino group protected with an acetyl group.

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

[12] , wherein Y is a chloro group.

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

[12] , wherein Y is a trifluoroacetoxy group.

[15] The method according to

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

[16] The method according to

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

[17] Formula (D)

[0022] [ka]

[0023] (wherein X represents a leaving group, and R 1 represents an amino group protected by a protecting group) is coupled with 3-butenoic acid, Formula (E)

[0024] [ka]

[0025] A compound represented by the formula (wherein R 1 has the same meaning as defined above), and then reducing the compound represented by formula (E), Formula (B)

[0026] [ka]

[0027] A compound represented by the formula (wherein R 1 has the same meaning as defined above), and then intramolecularly cyclizing the compound represented by formula (B), Formula (C)

[0028] [ka]

[0029] A compound represented by the formula (wherein R 1 has the same meaning as defined above), A method for producing a compound represented by formula (C), comprising:

[18] The method according to

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

[19] The method according to

[17] , wherein X is a bromo group.

[20] The method according to

[17] , wherein X is an iodo group. [twenty one] R 1 is an amino group protected by an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group. [twenty two] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group. [twenty three] R 1 is an amino group protected with an acetyl group. [twenty four] 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 tri(o-tolyl)phosphine. [twenty five] The production method according to any one of

[17] to

[24] , comprising the steps of dissolving the compound represented by formula (E) in a basic aqueous solution and separating and washing with a first organic solvent, and then adding an acid to the basic aqueous solution and separating and extracting the compound represented by formula (E) with a second organic solvent.

[26] The 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]

[25] The method according to any one of

[25] to

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

[29] The method according to any one of

[17] to

[28] , wherein the step of reducing the compound represented by formula (E) and converting it to 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 method according to any one of

[17] to

[29] , wherein the step of intramolecularly cyclizing the compound represented by formula (B) and converting it to the compound represented by formula (C) is carried out by a method comprising reacting the compound represented by formula (B) with trifluoroacetic anhydride.

[31] The method according to

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

[32] The method according to any one of

[17] to

[29] , wherein the step of intramolecularly cyclizing the compound represented by formula (B) and converting it to the compound represented by formula (C) is carried out by a method comprising reacting the compound represented by formula (B) with thionyl chloride.

[33] The method according to

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

[34] Produced by the method described in any one of [1] to

[33] Formula (C)

[0030] [ka]

[0031] The method is characterized in that a compound represented by the formula (I) is used as a starting material, The compound represented by formula (C) Formula (F)

[0032] [ka]

[0033] A compound represented by the formula (wherein R 1 is R according to any one of claims 1 to 33 1 and R 2 represents an amino group protected by a protecting group), and then converting the compound represented by formula (F) into Formula (G)

[0034] [ka]

[0035] A compound represented by the formula (wherein R 2 has the same meaning as defined above), and then reacting a compound represented by formula (G) with Formula (1)

[0036] [ka]

[0037] Condensing a compound represented by the formula: Formula (H)

[0038] [ka]

[0039] A compound represented by the formula (wherein R 2 has the same meaning as defined above), and then converting the compound represented by formula (H) into Formula (2)

[0040] [ka]

[0041] A method for producing a compound represented by formula (2), comprising a step of converting a compound represented by formula (2) into a compound represented by formula (2).

[35] R 2 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[36] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[37] R 2 is an amino group protected with an acetyl group.

[38] The method according to any one of

[34] to

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

[39] The 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 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 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 method according to any one of

[34] to

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

[43] The method according to any one of

[34] to

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

[44] The method according to any one of

[34] to

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

[45] The method according to any one of

[34] to

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

[46] The method according to any one of

[34] to

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

[47] The method according to any one of

[34] to

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

[48] Formula (3)

[0042] [ka]

[0043] A compound represented by Formula (4)

[0044] [ka]

[0045] and then converting the compound represented by formula (4) into a compound represented by formula (5): Formula (5)

[0046] [ka]

[0047] and then converting the compound represented by formula (5) into a compound represented by formula (6): Formula (6)

[0048] [ka]

[0049] and then coupling the compound represented by formula (6) with 3-butenoic acid, Formula (7)

[0050] [ka]

[0051] and then converting the compound represented by formula (7) into a compound represented by formula (8). Formula (8)

[0052] [ka]

[0053] and then intramolecularly cyclizing the compound represented by formula (8), Formula (9)

[0054] [ka]

[0055] and then converting the compound represented by formula (9) into a compound represented by formula (9): Formula (10)

[0056] [ka]

[0057] and then converting the compound represented by formula (10) into a compound represented by formula (11): Formula (11)

[0058] [ka]

[0059] and then a compound represented by formula (11), Formula (1)

[0060] [ka]

[0061] Condensing a compound represented by the formula: Formula (12)

[0062] [ka]

[0063] and then converting the compound represented by formula (12) into a compound represented by formula (13): Formula (2)

[0064] [ka]

[0065] A method for producing a compound represented by formula (2), comprising a step of converting a compound represented by formula (2) into a compound represented by formula (2).

[49] The production method according to

[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 tri(o-tolyl)phosphine.

[50] The production method according to

[48] or

[49] , comprising the steps of dissolving the compound represented by formula (7) in a basic aqueous solution and separating and washing with a first organic solvent, and then adding an acid to the basic aqueous solution and separating and extracting the compound represented by formula (7) with a second organic solvent.

[51] The method according to

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

[52] The method according to

[50] or

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

[53]

[50] The method according to any one of

[52] to

[53] , wherein the basic aqueous solution is an aqueous solution of sodium hydroxide.

[54] The method according to any one of

[50] to

[53] , wherein the step of intramolecularly cyclizing the compound represented by formula (8) to convert it to the compound represented by formula (9) is carried out by a method comprising reacting the compound represented by formula (8) with trifluoroacetic anhydride.

[55] The method according to

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

[56] The method according to any one of

[48] to

[55] , wherein the step of converting the compound represented by formula (9) to the compound represented by formula (10) comprises (i) a step of reacting with a nitrite ester in the presence of a base to introduce a nitroso group, followed by (ii) a step of introducing a protecting group to the nitrogen atom derived from the nitroso group, and (iii) a step of reducing with hydrogen in the presence of a platinum-carbon catalyst.

[57] The 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 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 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 method according to any one of

[48] to

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

[61] The method according to any one of

[48] to

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

[62] The method according to any one of

[48] to

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

[63] The method according to any one of

[48] to

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

[64] The method according to any one of

[48] to

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

[65] The method according to any one of

[48] to

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

[66] Formula (D)

[0066] [ka]

[0067] (wherein X represents a leaving group, and R 1 represents an amino group protected by a protecting group) is coupled with 3-butenoic acid, Formula (E)

[0068] [ka]

[0069] A compound represented by the formula (wherein R 1 has the same meaning as defined above).

[67] The method according to

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

[68] The method according to

[66] , wherein X is a bromo group.

[69] The method according to

[66] , wherein X is an iodo group.

[70] R 1 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[71] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[72] R 1 is an amino group protected with an acetyl group.

[73] The 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 tri(o-tolyl)phosphine.

[74] The production method according to any one of

[66] to

[73] , comprising the steps of dissolving the compound represented by formula (E) in a basic aqueous solution and separating and washing with a first organic solvent, and then adding an acid to the basic aqueous solution and separating and extracting the compound represented by formula (E) with a second organic solvent.

[75] The 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]

[74] The method according to any one of

[76] to

[77] , wherein the basic aqueous solution is an aqueous solution of sodium hydroxide.

[78] Formula (E)

[0070] [ka]

[0071] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group), Formula (B)

[0072] [ka]

[0073] A compound represented by the formula (wherein R 1 has the same meaning as defined above).

[79] R 1 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[80] R 1is an amino group protected with an acetyl group or a trifluoroacetyl group.

[81] R 1 is an amino group protected with an acetyl group.

[82] The production method according to any one of

[78] to

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

[83] Formula (C)

[0074] [ka]

[0075] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group), (i) a step of introducing a nitroso group by reacting with a nitrite ester in the presence of a base, followed by (ii) a step of introducing a protecting group to the nitrogen atom derived from the nitroso group, and (iii) a step of reducing with hydrogen in the presence of a platinum-carbon catalyst, Formula (F)

[0076] [ka]

[0077] A compound represented by the formula (wherein R 1 has the same meaning as above, and R 2 represents an amino group protected by a protecting group) to form a compound represented by formula (F).

[84] R 1 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[85] R 1is an amino group protected with an acetyl group or a trifluoroacetyl group.

[86] R 1 is an amino group protected with an acetyl group.

[87] R 2 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[88] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[89] R 2 is an amino group protected with an acetyl group.

[90] Formula (F)

[0078] [ka]

[0079] A compound represented by the formula (wherein R 1 and R 2 represents an amino group protected by a protecting group) in a solvent containing hydrochloric acid / ethanol, Formula (G)

[0080] [ka]

[0081] A compound represented by the formula (wherein R 2 has the same meaning as defined above).

[91] R 1is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[92] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[93] R 1 is an amino group protected with an acetyl group.

[94] R 2 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[95] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[96] R 2 is an amino group protected with an acetyl group.

[97] Formula (G)

[0082] [ka]

[0083] A compound represented by the formula (wherein R 2 represents an amino group protected by a protecting group), and Formula (1)

[0084] [ka]

[0085] in a solvent containing o-cresol, Formula (H)

[0086] [ka]

[0087] A compound represented by the formula (wherein R 2 has the same meaning as defined above).

[98] R 2 is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[99] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[0100] R 2 is an amino group protected with an acetyl group.

[0101] A method for producing a compound according to any one of [1] to

[0100] , characterized in that no chromatography is used.

[0102] Formula (6)

[0088] [ka]

[0089] A compound represented by the formula:

[0103] Formula (34)

[0090] [ka]

[0091] A compound represented by the formula:

[0104] Formula (7)

[0092] [ka]

[0093] A compound represented by the formula:

[0105] Formula (8)

[0094] [ka]

[0095] A compound represented by the formula:

[0106] Produced by the method described in any one of

[34] to

[65] Formula (2)

[0096] [ka]

[0097] The method is characterized in that a compound represented by the formula (I) is used as a starting material, A compound represented by formula (2), Formula (13)

[0098] [ka]

[0099] By condensing a compound represented by Formula (14)

[0100] [ka]

[0101] A method for producing a compound represented by formula (14), comprising:

[0107]

[0106] Formula (14)

[0102] [ka]

[0103] The method is characterized in that a compound represented by the formula: (i) reducing the antibody, and then (ii) reacting the compound represented by formula (14) prepared by the above method with a reduced antibody; Including, Formula (15)

[0104] [ka]

[0105] (wherein A represents the binding site to the antibody) A method for producing an antibody-drug conjugate in which a drug linker represented by the formula:

[0108] A manufacturing method described in

[0107] , 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.

[0109] A manufacturing method described in

[0108] , wherein the antibody is an anti-HER2 antibody.

[0110] A manufacturing method described in

[0109] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0111] A manufacturing method described in

[0109] or

[0110] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0112] A manufacturing method described in

[0108] , wherein the antibody is an anti-HER3 antibody.

[0113] A manufacturing method described in

[0112] , in which the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting 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.

[0114] A manufacturing method described in

[0112] or

[0113] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0115] A manufacturing method described in

[0108] , in which the antibody is an anti-TROP2 antibody.

[0116] The method for producing an anti-TROP2 antibody described in

[0115] , wherein the anti-TROP2 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody.

[0117] A manufacturing method described in

[0115] or

[0116] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3 to 5.

[0118] A manufacturing method described in

[0108] , wherein the antibody is an anti-B7-H3 antibody.

[0119] The method for producing an anti-B7-H3 antibody described in

[0118] , wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody.

[0120] A manufacturing method described in

[0118] or

[0119] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3 to 5.

[0121] A manufacturing method described in

[0108] , wherein the antibody is an anti-GPR20 antibody.

[0122] A method for producing an anti-GPR20 antibody described in

[0121] , wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in 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.

[0123] A manufacturing method described in

[0121] or

[0122] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8. Regarding. [Effects of the Invention]

[0106] The present invention provides a novel method for producing exatecan with a short number of steps and excellent industrial viability, and further provides a novel method for producing antibody-drug conjugates using the same. [Brief explanation of the drawings]

[0107] [Figure 1] 1 shows the amino acid sequence of the anti-HER2 antibody heavy chain (SEQ ID NO: 1). [Figure 2] 1 shows the amino acid sequence of the anti-HER2 antibody light chain (SEQ ID NO: 2). [Figure 3]1 shows the amino acid sequence of the anti-HER3 antibody heavy chain (SEQ ID NO: 3). [Figure 4] 1 shows the amino acid sequence of the anti-HER3 antibody light chain (SEQ ID NO: 4). [Figure 5] The amino acid sequence of the anti-TROP2 antibody heavy chain (SEQ ID NO: 5) is shown. [Figure 6] The amino acid sequence of the anti-TROP2 antibody light chain (SEQ ID NO: 6) is shown. [Figure 7] The amino acid sequence of the anti-B7-H3 antibody heavy chain (SEQ ID NO: 7) is shown. [Figure 8] The amino acid sequence of the anti-B7-H3 antibody light chain (SEQ ID NO: 8) is shown. [Figure 9] The amino acid sequence of the anti-GPR20 antibody heavy chain (SEQ ID NO: 9) is shown. [Figure 10] The amino acid sequence of the anti-GPR20 antibody light chain (SEQ ID NO: 10) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0108] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiment described below shows one example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0109] [Antibody-drug conjugates]

[0110] The antibody-drug conjugate produced by the present invention is preferably Formula (15)

[0111] [ka]

[0112] (wherein A represents the binding site to the antibody) and an antibody via a thioether bond.

[0113] In the present invention, the partial structure of an antibody-drug conjugate consisting of a linker and a drug is referred to as a "drug linker." This drug linker is bound to a thiol group (in other words, the sulfur atom of a cysteine ​​residue) generated at the interchain disulfide bond sites of the antibody (two heavy-heavy chain and two heavy-light chain disulfide bond sites).

[0114] The drug linker of the present invention is composed of exatecan, a topoisomerase I inhibitor. Exatecan is represented by the formula (2):

[0115] [ka]

[0116] and is a camptothecin derivative having antitumor effects.

[0117] The antibody-drug conjugate used in the present invention is Formula (16)

[0118] [ka]

[0119] It can also be expressed as

[0120] Here, the drug linker is bound to the antibody via a thioether bond, and n is synonymous with the so-called average number of drug linkers bound (DAR; Drug-to-Antibody Ratio), which indicates the average number of drug linkers bound per antibody. The antibody-drug conjugate used in the present invention, after being transported into cancer cells, Formula (18)

[0121] [ka]

[0122] The compound represented by the formula (I) is released, thereby exerting an antitumor effect.

[0123] The compound represented by formula (18) is believed to be the main component of the antitumor activity of the antibody-drug conjugate produced by 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).

[0124] The compound represented by formula (18) is believed to be generated by cleavage of the linker moiety of the antibody-drug conjugate produced by the present invention. Formula (17)

[0125] [ka]

[0126] It is believed that this occurs due to the decomposition of the aminal structure of the compound represented by the formula: The antibody-drug conjugates produced according to the present invention are also known to have a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). This bystander effect is exerted when the antibody-drug conjugate produced by the present invention is internalized into target-expressing cancer cells, and the released compound represented by formula (18) exerts an antitumor effect even on nearby cancer cells that do not express the target.

[0127] [Exatecan manufacturing] The exatecan of the present invention can be produced according to the following method.

[0128] [ka]

[0129] [In the formula, 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, and even more preferably a bromo group; R 1 represents an amino group protected with a protecting group, preferably represents an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group, more preferably represents an amino group protected with an acetyl group or a trifluoroacetyl group, and even more preferably represents an amino group protected with an acetyl group; R 2 represents a protected amino group, preferably represents an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group, more preferably represents an amino group protected with an acetyl group or a trifluoroacetyl group, and even more preferably represents an amino group protected with an acetyl group.

[0130] Step 1: This step involves coupling a compound represented by formula (D) with 3-butenoic acid to convert it into a compound represented by formula (E). The compound represented by formula (D) can be produced by referring to known methods. The amount of 3-butenoic acid used in this step is not limited as long as the reaction proceeds, but is preferably 1 to 1.5 equivalents relative to the compound represented by formula (D).

[0131] 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 is not particularly limited as long as it allows the reaction to proceed. For example, divalent palladium salts and complexes thereof, such as palladium(II) acetate, palladium(II) trifluoroacetate, palladium(II) chloride, palladium(II) bromide, palladium(II) iodide, and bis(triphenylphosphine)palladium(II) chloride, and zerovalent palladium metals and complexes thereof, such as palladium black, palladium carbon, tetrakis(triphenylphosphine)palladium(0), and bis(dibenzylideneacetone)palladium(0), can be used. Palladium(II) acetate is preferably used. The amount of palladium catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 0.003 to 0.03 equivalents relative to the compound represented by formula (D).

[0132] Furthermore, in addition to the palladium catalyst, this step preferably uses a ligand for forming a palladium complex in the reaction system. Examples of ligands that can be used in this step include triphenylphosphine, tri(o-tolyl)phosphine, tri(3-methoxyphenyl)phosphine, tri(4-chlorophenyl)phosphine, tri(2-furyl)phosphine, tri(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.). Tri(o-tolyl)phosphine is preferably used. The amount of the ligand used in this step is not limited as long as the reaction proceeds, but is preferably 0.006 to 0.06 equivalents relative to the compound represented by formula (D).

[0133] This step is preferably carried out in the presence of a base. The base to be used in this step is not particularly limited as long as the reaction proceeds, but examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, 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; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium Examples of suitable inorganic bases include triethylamine, tributylamine, diisopropylethylamine, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, and potassium acetate, and more preferably diisopropylethylamine. The amount of base used in this step is not limited as long as the reaction proceeds, but is preferably 2 to 3 equivalents relative to the compound represented by formula (D).

[0134] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. Examples of the solvent that can be used 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, and preferred is tetrahydrofuran.

[0135] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 45 to 85°C, more preferably a temperature at which tetrahydrofuran is heated under reflux. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2.5 to 10 hours.

[0136] The compound represented by formula (E) can be suitably purified by dissolving the compound represented by formula (E) in a basic aqueous solution and washing with a first organic solvent, followed by adding an acid to the basic aqueous solution and extracting the compound represented by formula (E) with a second organic solvent. The first organic solvent is preferably 2-methyltetrahydrofuran. The second organic solvent is preferably 2-methyltetrahydrofuran. The basic aqueous solution is preferably an aqueous sodium hydroxide solution.

[0137] The compound represented by formula (E) has geometric isomers, E-isomer and Z-isomer, both of which are included in the compound represented by formula (E) and are within the scope of the present invention. The compound represented by formula (E) of the present invention may be a mixture of E-isomer and Z-isomer, and the mixture can be used as is in the next step.

[0138] In this step, a 3-butenoic acid ester can also be used instead of 3-butenoic acid. In this case, the product obtained by coupling the compound represented by formula (D) with the 3-butenoic acid ester can be converted to the compound represented by formula (E) by hydrolysis.

[0139] Step 2: In this step, the compound represented by formula (E) is converted into the compound represented by formula (B) by reduction.

[0140] The reduction in this step is not limited as long as the reaction proceeds, but is preferably carried out under a hydrogen atmosphere (preferably under a hydrogen stream of 0.05 to 0.6 MPa) using a palladium catalyst, platinum catalyst, nickel catalyst, ruthenium catalyst, or rhodium catalyst, more preferably using a palladium catalyst, even more 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 is preferably 5 to 80% by weight based on the compound of formula (D) used in step 1.

[0141] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, and examples thereof 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, and preferably 2-methyltetrahydrofuran.

[0142] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 20 to 60° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 hours.

[0143] Step 3: This step involves intramolecularly cyclizing a compound represented by formula (B) to convert it to a compound represented by formula (C). The intramolecular cyclization is preferably carried out by intramolecular Friedel-Crafts acylation. The intramolecular Friedel-Crafts acylation reaction in this step is not limited as long as the reaction proceeds, but preferred examples include a method using trifluoroacetic anhydride, or a method using thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride, or phosphorus pentachloride. More preferred examples include a method using trifluoroacetic anhydride or thionyl chloride, and even more preferred examples include a method 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). 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).

[0144] In the case of a method using trifluoroacetic anhydride, this step is preferably carried out in the presence of an acid, more preferably in the presence of trifluoroacetic acid. In the case of a method using thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphorus oxychloride, 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 limited as long as the reaction proceeds, but is preferably 1 to 5 equivalents relative to the compound represented by formula (B).

[0145] The solvent used in this step 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, and chlorobenzene, as well as mixed solvents thereof, and preferably methylene chloride. When trifluoroacetic acid is used, trifluoroacetic acid can preferably be contained as a solvent.

[0146] The reaction temperature in this step is not limited as long as the reaction proceeds, but in the case of a method using trifluoroacetic anhydride, it is preferably −10° C. to 20° C., and in the case of a method using thionyl chloride, it is preferably 10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 hours to 8 hours in the case of a method using trifluoroacetic anhydride, and preferably 1 hour to 4 hours in the case of a method using thionyl chloride.

[0147] This process can also be carried out in the following two stages.

[0148] [ka]

[0149] [In the formula, Y represents a leaving group, preferably a chloro group, a bromo group, an iodo group, a fluoro group, or a trifluoroacetoxy group, more preferably a chloro group or a trifluoroacetoxy group; R 1 represents an amino group protected with a protecting group, preferably represents an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group, more preferably represents an amino group protected with an acetyl group or a trifluoroacetyl group, and even more preferably represents an amino group protected with an acetyl group.

[0150] Step 3A is a step of converting a compound represented by formula (B) into a compound represented by formula (J).

[0151] When Y is a chloro group, this step can be preferably carried out by a method using thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride, or phosphorus pentachloride, and more preferably by a method 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 a trifluoroacetoxy group, this step can preferably be carried out by a method 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). The solvent used in this step 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, as well as mixed solvents thereof. In the case of a method using thionyl chloride, methylene chloride is preferred, and in the case of a method using trifluoroacetic anhydride, trifluoroacetic acid is preferred.

[0152] Step 3B is a step of converting a compound represented by formula (J) into a compound represented by formula (C). In the case of a method using thionyl chloride, this step can be 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 a method using trifluoroacetic anhydride, this step can preferably be carried out in the presence of an acid, more preferably in the presence of trifluoroacetic acid.

[0153] The reaction temperature for Step 3A and Step 3B is not limited as long as the reaction proceeds, but in the case of the method using thionyl chloride, it is preferably 10° C. to 40° C., and in the case of the method using trifluoroacetic anhydride, it is preferably −10° C. to 20° C. The total reaction time for Step 3A and Step 3B is not limited as long as the reaction proceeds, but in the case of the method using thionyl chloride, it is preferably 1 hour to 4 hours, and in the case of the method using trifluoroacetic anhydride, it is preferably 2 hours to 8 hours.

[0154] Step 4: This step is a step of converting a compound represented by formula (C) to a compound represented by formula (F). This step can be preferably carried out by (i) a step of nitrosating (or oximating) the α-position of the carbonyl group, (ii) a step of introducing a protecting group to the nitrogen atom derived from the nitroso group (or oxime group), and (iii) a step of reducing. The order of (ii) and (iii) may be reversed, or they may be carried out simultaneously.

[0155] The nitrosating agent (or oximating agent) used in step (i) is not particularly limited as long as it can nitrosate (or oximate) the α-position of the carbonyl group of the compound represented by formula (C), but preferably a nitrite ester can be used, more preferably amyl nitrite, n-butyl nitrite, or tert-butyl nitrite can be used, and even more preferably amyl nitrite can be used. The amount of amyl nitrite used in step (i) is not limited as long as the reaction proceeds, but is preferably 1 to 1.6 equivalents relative to the compound represented by formula (C).

[0156] A base is preferably used in step (i). The base used in step (i) is not particularly limited as long as it is applicable to nitrosation (or oximation) of the α-position of the carbonyl group of the compound represented by formula (C), but 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 is preferably 1 to 1.5 equivalents relative to the compound represented by formula (C).

[0157] The solvent used in step (i) is not particularly limited as long as it does not inhibit the reaction, and examples thereof 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, and preferably tetrahydrofuran.

[0158] The reaction temperature in step (i) is not limited as long as the reaction proceeds, but is preferably −10 to 20° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1.5 to 30 hours. Step (ii) is R 2 The reaction conditions can be appropriately set depending on the type of protecting group for the amino group in R 2 When is an amino group protected with an acetyl group, step (ii) can be preferably carried out using acetic anhydride in acetic acid.

[0159] Step (iii) can be carried out under a hydrogen atmosphere (preferably under a hydrogen stream of 0.15 to 1.2 MPa) using, for example, a platinum-carbon catalyst or zinc powder, but is preferably carried out using a platinum-carbon catalyst, more preferably 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 is preferably 5 to 60% by weight based on the compound represented by formula (C). Step (iii) can use the solvent used in step (ii). The reaction temperature for steps (ii) and (iii) is not limited as long as the reaction proceeds, but is preferably 0 to 40° C. The total reaction time for steps (ii) and (iii) is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours.

[0160] Step 5: This step is a step of selectively deprotecting the protecting group of the aromatic amino group of the compound represented by formula (F) to convert it into a compound represented by formula (G). 1 and R 2 The reaction conditions can be appropriately set depending on the type of protecting group for the amino group in R 1 and R 2 When is an amino group protected with an acetyl group, this step can be preferably carried out using hydrochloric acid, more preferably using 2N hydrochloric acid / ethanol. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 40 to 60° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 2 to 14 hours.

[0161] Step 6: This step involves condensing a compound represented by formula (G) with a compound represented by formula (1) to convert it into a compound represented by formula (H). The compound represented by formula (1) can be produced by referring to the description in U.S. Pat. No. 4,778,891, etc., or a commercially available compound 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).

[0162] This step is carried out in the presence of an acid catalyst. A suitable example of the acid catalyst used in this step is pyridinium p-toluenesulfonate. The acid catalyst used in this step is not limited as long as the reaction proceeds, but is preferably used in an amount of 0.03 to 0.3 equivalents relative to the compound represented by formula (G).

[0163] This step is preferably carried out in a solvent containing cresol or phenol, more preferably in toluene containing o-cresol, because the presence of o-cresol or phenol improves the precipitation state of the compound represented by formula (H), resulting in improved yields and shorter reaction times.

[0164] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 90 to 130° C., more preferably a temperature at which toluene is heated to reflux. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 16 to 64 hours.

[0165] It is believed that this step proceeds via the compound represented by formula (K) and the compound represented by formula (L) as reaction intermediates.

[0166] [ka]

[0167] Step 7: This step is a step of converting a compound represented by formula (H) into a compound represented by formula (2). The compound represented by formula (2) may be a salt or may be a hydrate, both of which are included in the scope of the "compound represented by formula (2)" in the present invention. This step can be preferably carried out in the presence of an acid, more preferably in the presence of methanesulfonic acid and water.

[0168] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, but a solvent containing 2-methoxyethanol and ethylcyclohexane can be preferably used. When the above-mentioned acid is further included as a solvent, a mixed solvent of methanesulfonic acid, water, 2-methoxyethanol, and ethylcyclohexane can be more preferably used.

[0169] This step is not limited as long as the reaction proceeds, but is preferably carried out at 80 to 160°C, more preferably at a temperature at which a mixed solvent of methanesulfonic acid, water, 2-methoxyethanol, and ethylcyclohexane is heated to reflux. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 4 to 16 hours.

[0170] The compound represented by formula (2) can be obtained preferably as a methanesulfonate salt, more preferably as a methanesulfonate m-hydrate (where m is 0 to 3), even more preferably as anhydrous methanesulfonate, methanesulfonate monohydrate, methanesulfonate dihydrate, or methanesulfonate trihydrate, and even more preferably as a methanesulfonate dihydrate, all of which can be used in the production method of the present invention. The number of hydrates can be controlled by adjusting the humidity during crystal collection and drying. The compound represented by formula (2) can be more preferably produced according to the following method.

[0171] [ka]

[0172] Step 8: In this step, a compound represented by formula (3) is brominated to convert it into a compound represented by formula (4). The compound represented by formula (3) can be produced by a known method or commercially available.

[0173] The brominating agent used in this step is not limited as long as the reaction proceeds, but examples thereof include bromine and N-bromosuccinimide, and N-bromosuccinimide is preferred. The amount of N-bromosuccinimide used in this step is not limited as long as the reaction proceeds, but is preferably 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 another solvent.

[0174] The other solvent 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 mixed solvents thereof, and preferably heptane.

[0175] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 50 to 70° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 hours.

[0176] Step 9: This step involves reducing the nitro group of a compound represented by formula (4) to an amino group, thereby converting it into a compound represented by formula (5). The reducing agent used in this step may be any agent capable of selectively reducing only the nitro group without causing debromination. A platinum-carbon catalyst in the presence of hydrogen (preferably under a hydrogen stream at 0.05 to 0.2 MPa) is preferred, and a 1% platinum-carbon catalyst is more preferred. The amount of platinum-carbon catalyst used in this step is not particularly limited as long as the reaction proceeds, but is preferably 5 to 40 wt % relative to the compound represented by formula (3) used in step 8. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, but examples include methanol, ethanol, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, water, and mixed solvents thereof, with ethyl acetate being preferred. The reaction temperature in this step is not particularly limited as long as the reaction proceeds, but is preferably 50 to 70°C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours.

[0177] Step 10: This step is a step of acetylating the amino group of a compound represented by formula (5) to convert it to a compound represented by formula (6). Examples of the acetylating agent used in this step include acetic anhydride and acetyl chloride, and preferably acetic anhydride. 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. A base can be preferably used in this step. The base is not limited as long as the reaction proceeds, but is preferably triethylamine. The amount of the 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. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, but 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, with ethyl acetate being preferred. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 3 to 12 hours.

[0178] Step 11: In this step, a compound represented by formula (6) is converted into a compound represented by formula (7) by coupling with 3-butenoic acid. This step can be carried out in the same manner as in step 1. The compound represented by formula (7) has geometric isomers, E-isomer and Z-isomer, both of which are included in the compound represented by formula (7) and are within the scope of the present invention. The compound represented by formula (7) of the present invention may be a mixture of E-isomer and Z-isomer, and the mixture can be used as is in the next step.

[0179] Step 12: In this step, the compound represented by formula (7) is converted to a compound represented by formula (8) by reduction.

[0180] The reduction in this step is not limited as long as the reaction proceeds, but is preferably carried out under a hydrogen atmosphere (preferably under a hydrogen stream at 0.05 to 0.2 MPa) using a palladium catalyst, platinum catalyst, nickel catalyst, ruthenium catalyst, or rhodium catalyst, more preferably using a palladium catalyst, even more 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 is preferably 5 to 40% by weight based on the compound of formula (7) used in step 11.

[0181] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, and examples thereof 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, and preferably 2-methyltetrahydrofuran.

[0182] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 20 to 60° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 hours.

[0183] Step 13: This step is a step of converting a compound represented by formula (8) into a compound represented by formula (9) by intramolecular cyclization. This step can be carried out in the same manner as in step 3.

[0184] Step 14: This step is a step of converting a compound represented by formula (9) into a compound represented by formula (10). This step can be carried out in the same manner as in step 4.

[0185] 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 a compound represented by formula (11). This step can be carried out in the same manner as in step 5.

[0186] Step 16: This step is a step of condensing a compound represented by formula (11) with a compound represented by formula (1) to convert it into a compound represented by formula (12). This step can be carried out in the same manner as in step 6. It is believed that this step proceeds via a compound represented by formula (30) and / or a compound represented by formula (31) as a reaction intermediate.

[0187] [ka]

[0188] Step 17: This step is a step of converting a compound represented by formula (12) into a compound represented by formula (2). This step can be carried out in the same manner as in step 7. The compound represented by formula (2) can also be produced according to the following method.

[0189] [ka]

[0190] Step 18: In this step, a compound represented by formula (3) is iodized to convert it into a compound represented by formula (32). The compound represented by formula (3) can be produced by a known method or commercially available. The iodinating agent used in this step is not limited as long as the reaction proceeds, but examples thereof include iodine and N-iodosuccinimide, and N-iodosuccinimide is preferred. 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 another solvent.

[0191] The other solvent 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 mixed solvents thereof, and preferably heptane. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably −10 to 10° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 1 to 4 hours.

[0192] Step 19: This step involves reducing the nitro group of a compound represented by formula (32) to an amino group, thereby converting the compound to a compound represented by formula (33). The reducing agent used in this step may be any agent capable of selectively reducing only the nitro group without causing deiodination. A preferred reducing agent is a platinum-carbon catalyst in the presence of hydrogen (preferably under a hydrogen stream at 0.05 to 0.2 MPa). The amount of platinum-carbon catalyst used in this step is not limited as long as the reaction proceeds, but is preferably 5 to 40 wt % relative to the compound represented by formula (3) used in step 18. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, but examples include methanol, ethanol, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, water, and mixed solvents thereof, with ethyl acetate being preferred. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 50 to 70°C. The reaction time for this step is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours.

[0193] Step 20: This step is a step of acetylating the amino group of a compound represented by formula (33) to convert it to a compound represented by formula (34). Examples of the acetylating agent used in this step include acetic anhydride and acetyl chloride, and acetic anhydride is preferred. 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 18. A base can be used in this step. The base is not limited as long as the reaction proceeds, but is preferably triethylamine. The amount of the 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 18. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, but 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, with ethyl acetate being preferred. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 3 to 12 hours.

[0194] Step 21: In this step, a compound represented by formula (34) is coupled with 3-butenoic acid to convert it into a compound represented by formula (7). This step can be carried out in the same manner as in step 1.

[0195] Step 22: In this step, the compound represented by formula (7) is converted to a compound represented by formula (8) by reduction.

[0196] This step can be carried out in the same manner as step 12, but since residual iodide ions may reduce catalytic activity, it is preferable to carry out the step using a higher amount of catalyst and hydrogen pressure than in step 12.

[0197] The reduction in this step is not limited as long as the reaction proceeds, but is preferably carried out under a hydrogen atmosphere (preferably under a hydrogen stream at 0.15 to 0.6 MPa) using a palladium catalyst, platinum catalyst, nickel catalyst, ruthenium catalyst, or rhodium catalyst, more preferably using a palladium catalyst, even more 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 is preferably 20 to 160% by weight based on the compound of formula (34) used in step 21.

[0198] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction, and examples thereof 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, and preferably 2-methyltetrahydrofuran.

[0199] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 20 to 60° C. The reaction time in this step is not limited as long as the reaction proceeds, but is preferably 4 to 16 hours.

[0200] Step 23: This step is a step of converting a compound represented by formula (8) into a compound represented by formula (9) by intramolecular cyclization. This step can be carried out in the same manner as in step 3.

[0201] Step 24: This step is a step of converting a compound represented by formula (9) into a compound represented by formula (10). This step can be carried out in the same manner as in step 4.

[0202] 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) to convert it into a compound represented by formula (11). This step can be carried out in the same manner as in step 5.

[0203] Step 26: This step is a step of condensing a compound represented by formula (11) with a compound represented by formula (1) to convert it into a compound represented by formula (12). This step can be carried out in the same manner as in step 16.

[0204] Step 27: This step is a step of converting a compound represented by formula (12) into a compound represented by formula (2). This step can be carried out in the same manner as in step 7. After completion of the reaction in each of the above steps, the target compound of each step can be isolated from the reaction mixture according to methods well known in the field of organic chemistry. The target compound can be obtained, for example, by (i) filtering off insoluble materials such as the catalyst, if necessary; (ii) adding water and a water-immiscible solvent (e.g., methylene chloride, diethyl ether, ethyl acetate, or 2-methyltetrahydrofuran) to the reaction mixture to extract the target compound; (iii) washing the organic layer with water and drying it with a drying agent such as anhydrous magnesium sulfate; and (iv) distilling off the solvent. The resulting target compound can be further purified, if necessary, by methods well known in the field of organic chemistry (e.g., recrystallization, reprecipitation, silica gel column chromatography, or high-performance liquid chromatography). However, the production method of the present invention can be preferably performed without using chromatography.

[0205] The compound represented by formula (2) obtained by the production method of the present invention is preferably a compound represented by formula (15): and an antibody via a thioether bond, but the present invention is not limited thereto and can also be used to produce antibody-drug conjugates having other chemical structures or for other applications.

[0206] [Preparation of Drug Linker Intermediates]

[0207] A drug linker intermediate preferably used in the production of the antibody-drug conjugate of the present invention is a compound represented by formula (14).

[0208] [ka]

[0209] The compound represented by formula (14) can be prepared as follows.

[0210] [ka]

[0211] 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 by referring to the descriptions in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, etc.

[0212] The conversion to the compound represented by formula (14) can be carried out by converting the compound represented by formula (13) into an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacting the resulting product with the compound represented by formula (2), preferably in the presence of a base.

[0213] The activated ester can be prepared, for example, by reacting the compound of formula (13) with an additive such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, or p-nitrophenol using a condensing agent such as N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD HCl). Alternatively, the activated ester can be prepared by reacting the compound of formula (13) with a condensing agent such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate pentafluorophenyl trifluoroacetate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), diethyl cyanophosphonate, or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).

[0214] The mixed acid anhydride can be produced, for example, by reacting the compound represented by formula (13) with isobutyl chlorocarbonate, if necessary in the presence of a base.

[0215] The acid halide can be prepared by treating with an acid halide such as thionyl chloride or oxalyl chloride, if necessary in the presence of a base.

[0216] The base used in this step is not particularly limited as long as the reaction proceeds, and examples thereof include organic bases such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, 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; potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, and potassium Examples of the base include inorganic bases such as tert-butoxide, and preferred examples include triethylamine, tributylamine, diisopropylethylamine, potassium carbonate, potassium hydroxide, potassium hydrogencarbonate, sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, and potassium acetate, and more preferred examples include triethylamine, diisopropylethylamine, and N-methylmorpholine.

[0217] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. Examples of the solvent 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, water, and mixed solvents thereof. Preferred examples include acetonitrile, dichloromethane, methanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, water, and mixed solvents thereof.

[0218] [Antibody production]

[0219] The antibody used to produce the antibody-drug conjugate of the present invention may be derived from any species, but is preferably derived from human, rat, mouse, or rabbit. When the antibody is derived from a species other than human, it is preferably chimerized or humanized using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

[0220] The antibody used in the production of the antibody-drug conjugate of the present invention preferably has the property of being able to target cancer cells, and preferably has the ability to recognize cancer cells, the ability to bind to cancer cells, the ability to be taken up and internalized within cancer cells, and / or cytocidal activity against cancer cells.

[0221] Antibody binding to cancer cells can be confirmed using flow cytometry. Antibody uptake into cancer cells can be confirmed using (1) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to visualize the antibody uptake by the cell using a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which a fluorescently labeled secondary antibody that binds to the therapeutic antibody is used to measure the amount of fluorescence uptake by the cell (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released upon intracellular uptake, inhibiting cell proliferation (BioTechniques 28:162-165, January 2000). A recombinant complex protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin.

[0222] The antitumor activity of an antibody can be confirmed in vitro by measuring its inhibitory activity against cell proliferation. For example, a cancer cell line overexpressing the antibody's target protein is cultured, and the antibody is added to the culture system at various concentrations to measure its inhibitory activity against focus formation, colony formation, and spheroid growth. In vivo, antitumor activity can be confirmed by administering the antibody to nude mice transplanted with a cancer cell line overexpressing the target protein and measuring changes in the cancer cells.

[0223] Although it is preferable for the antibody itself to have antitumor activity, the antibody-drug conjugate is not necessarily antitumor because it is bound to a compound that exerts antitumor activity. However, for the purpose of specifically and selectively exerting the cytotoxicity of the antitumor compound on cancer cells, it is important and preferable for the antibody to have the property of being internalized and transported into cancer cells.

[0224] The antibodies used in producing the antibody-drug conjugates of the present invention can be obtained by known means. For example, they can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0225] Alternatively, a monoclonal antibody can be obtained by fusing antibody-producing cells that produce an antibody against an antigen with myeloma cells to establish a hybridoma according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0226] Antigens can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce it. Specifically, a vector capable of expressing the antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above-mentioned genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[0227] The antibody used to produce the antibody-drug conjugate of the present invention is preferably a genetically engineered antibody that has been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as a chimeric antibody or a humanized antibody, or is preferably an antibody having only the genetic sequence of a human-derived antibody, i.e., a human antibody. These antibodies can be produced using known methods.

[0228] Chimeric antibodies include antibodies in which the variable and constant regions are of different species, such as chimeric antibodies in which the variable regions of a mouse- or rat-derived antibody are joined to the constant regions of a human-derived antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0229] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody has been incorporated into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies in which not only the CDR sequence of a heterologous antibody but also some framework amino acid residues of the heterologous antibody have been grafted onto a human antibody using a CDR grafting method (WO 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0230] Examples of human antibodies include antibodies produced using human antibody-producing mice carrying human chromosomal fragments containing the heavy and light chain genes of human antibodies (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 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, pp. 722-727). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be used (see, for example, Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427-431).

[0231] The antibodies used to produce the antibody-drug conjugates of the present invention also include modified antibodies. The term "modified antibody" refers to an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include those in which a chemical moiety is attached to the amino acid backbone or an N- or O-linked carbohydrate chain. Biological modifications include those that have undergone post-translational modification (e.g., addition of an N- or O-linked sugar chain, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, etc.), and those in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Also included within the meaning of such modified antibodies are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens.

[0232] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the antibody of the present invention. Techniques for modulating antibody sugar chain modification are known, including, but not limited to, those disclosed in International Publication Nos. 99 / 54342, 00 / 61739, and 02 / 31140. Antibodies of the present invention also include antibodies with modified sugar chain modification.

[0233] It is known that antibodies produced in cultured mammalian cells lose the lysine residue at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (e.g., complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the antibodies of the present invention also include antibodies that have undergone such modifications and functional fragments of such antibodies, including deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and amidated deletions (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody of the present invention are not limited to the above types. The two heavy chains constituting the antibody of the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above-mentioned deletions, or a combination of any two types. The quantitative ratio of each deletion may be affected by the type and culture conditions of the cultured mammalian cells producing the antibody of the present invention, but preferred antibodies of the present invention include those in which one amino acid residue is deleted at the carboxyl terminus of each of the two heavy chains.

[0234] The isotype of the antibody according to the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG2.

[0235] The antibodies that can be used to produce the antibody-drug conjugates of the present invention are not particularly limited, and examples thereof include anti-HER2 antibodies, anti-HER3 antibodies, anti-TROP2 antibodies, anti-B7-H3 antibodies, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD56 antibodies, anti-CD98 antibodies, anti-DR5 antibodies, anti-EGFR antibodies, anti-EPHA2 antibodies, anti-FGFR2 antibodies, anti-FGFR4 antibodies, anti-FOLR1 antibodies, anti-VEGF antibodies, and anti-GPR20 antibodies, and preferred examples thereof include anti-HER2 antibodies, anti-HER3 antibodies, anti-TROP2 antibodies, anti-B7-H3 antibodies, and anti-GPR20 antibodies.

[0236] In the present invention, the term "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 being internalized into HER2-expressing cells upon binding to HER2.

[0237] Examples of anti-HER2 antibodies include trastuzumab (US Pat. No. 5,821,337) and pertuzumab (WO 01 / 00245), with trastuzumab being preferred.

[0238] In the present invention, "trastuzumab" is a humanized anti-HER2 monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 (Figure 1) and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2 (Figure 2).

[0239] In the present invention, the term "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 binding to HER3 on the surface of HER3-expressing cells and being internalized into the HER3-expressing cells.

[0240] Examples of anti-HER3 antibodies include patritumab (U3-1287), U1-59 (WO 2007 / 077028), MM-121 (seribantumab), the anti-ERBB3 antibody described in WO 2008 / 100624, RG-7116 (lumretuzumab), and LJM-716 (elgemtumab), and preferred examples include patritumab and U1-59.

[0241] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (Tumor-associated calcium signal transducer 2 (TACSTD2); EGP-1), and preferably has the activity of being internalized into TROP2-expressing cells upon binding to TROP2.

[0242] An example of an anti-TROP2 antibody is hTINA1-H1L1 (WO 2015 / 098099).

[0243] In the present invention, the term "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B cell antigen #7 homolog 3; PD-L3; CD276), and preferably has the activity of being internalized into B7-H3-expressing cells upon binding to B7-H3.

[0244] An example of an anti-B7-H3 antibody is M30-H1-L4 (WO 2014 / 057687).

[0245] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that specifically binds to GPR20 (G protein-coupled receptor 20), and preferably has the activity of being internalized into GPR20-expressing cells upon binding to GPR20. An example of an anti-GPR20 antibody is h046-H4e / L7 (WO 2018 / 135501).

[0246] [Conjugation of antibodies and drug-linker intermediates]

[0247] The antibody-drug conjugate of the present invention can be produced by reacting a drug linker intermediate (preferably a compound represented by formula (14)) with an antibody having a thiol group (also referred to as a sulfhydryl group).

[0248] Antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, G. T., Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, antibodies having sulfhydryl groups in which intra-chain disulfides have been partially or completely reduced can be obtained by reacting an antibody with a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in an amount of 0.3 to 3 molar equivalents per intra-chain disulfide in the antibody in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

[0249] Furthermore, antibody-drug conjugates having 2 to 8 drugs bound to each antibody can be prepared by using 2 to 20 molar equivalents of a drug linker intermediate (preferably a compound represented by formula (14)) per antibody having a sulfhydryl group.

[0250] The average number of drugs bound per antibody molecule in the produced antibody-drug conjugate can be calculated, for example, by a method in which the UV absorbance of the antibody-drug conjugate and its conjugation precursor is measured at two wavelengths, 280 nm and 370 nm (UV method), or by a method in which the antibody-drug conjugate is treated with a reducing agent, and each of the resulting fragments is quantified by HPLC measurement and then calculated (HPLC method).

[0251] Conjugation of an antibody and a drug linker intermediate, and calculation of the average number of drugs bound per antibody molecule in an antibody-drug conjugate can be carried out with reference to the descriptions in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, WO 2018 / 135501, etc.

[0252] In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-HER2 antibody.

[0253] The anti-HER2 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting 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 consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0254] The average number of drug linkers bound per antibody in the anti-HER2 antibody-drug conjugates produced by 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.

[0255] The anti-HER2 antibody-drug conjugate can be produced using a drug linker intermediate (preferably a compound represented by formula (14)) produced by the production method of the present invention, with reference to the descriptions in WO 2015 / 115091 and the like.

[0256] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-HER3 antibody.

[0257] The anti-HER3 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody.

[0258] The average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugates produced by 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.

[0259] The anti-HER3 antibody-drug conjugate can be produced using a drug linker intermediate (preferably a compound represented by formula (14)) produced by the production method of the present invention, with reference to the descriptions in WO 2015 / 155998 and the like.

[0260] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-TROP2 antibody.

[0261] The anti-TROP2 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody.

[0262] The average number of drug linkers bound per antibody in the anti-TROP2 antibody-drug conjugates produced according to the present invention is preferably 2 to 8, more preferably 3 to 5, even more preferably 3.5 to 4.5, and even more preferably about 4.

[0263] The anti-TROP2 antibody-drug conjugate can be produced using a drug linker intermediate (preferably a compound represented by formula (14)) produced by the production method of the present invention, with reference to the descriptions in WO 2015 / 098099 and the like.

[0264] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-B7-H3 antibody.

[0265] The anti-B7-H3 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody.

[0266] The average number of drug linkers bound per antibody in the anti-B7-H3 antibody-drug conjugates produced by the present invention is preferably 2 to 8, more preferably 3 to 5, even more preferably 3.5 to 4.5, and even more preferably about 4.

[0267] The anti-B7-H3 antibody-drug conjugate can be produced using a drug linker intermediate (preferably a compound represented by formula (14)) produced by the production method of the present invention, with reference to the descriptions in WO 2014 / 057687 and the like.

[0268] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-GPR20 antibody.

[0269] The anti-GPR20 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 10, or an antibody lacking a lysine residue at the carboxyl terminus of the heavy chain of the antibody.

[0270] The average number of drug linkers bound per antibody in the anti-GPR20 antibody-drug conjugate produced by 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.

[0271] The anti-GPR20 antibody-drug conjugate can be produced using a drug linker intermediate (preferably a compound represented by formula (14)) produced by the production method of the present invention, with reference to the descriptions in WO 2018 / 135501 and the like.

[0272] [Pharmaceutical composition] The antibody-drug conjugates produced according to the present invention can be administered containing one or more pharmaceutically compatible ingredients. The pharmaceutically compatible ingredients can be appropriately selected from formulation additives and other ingredients commonly used in this field depending on the dose, administration concentration, etc. of the antibody-drug conjugates produced according to the present invention. For example, the antibody-drug conjugates 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.

[0273] Pharmaceutical compositions containing the antibody-drug conjugates produced by the present invention can be applied to patients as systemic therapy, and can also be applied locally to cancer tissues to achieve therapeutic effects.

[0274] Pharmaceutical compositions containing antibody-drug conjugates produced according to the present invention can be preferably used in mammals, and more preferably in humans.

[0275] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be preferably used as an injection, more preferably as an aqueous injection or a lyophilized injection, and even more preferably as a lyophilized injection.

[0276] When the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention is in the form of an aqueous injection, it can be administered intravenously by infusion after being diluted with an appropriate diluent, such as a glucose solution (preferably a 5% glucose solution) or physiological saline.

[0277] When the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention is in the form of a lyophilized injection, it is preferably dissolved in water for injection, and then diluted with an appropriate diluent at the required amount, and then administered intravenously by infusion. The diluent may include a glucose solution (preferably a 5% glucose solution) or physiological saline.

[0278] Examples of introduction routes that can be used to administer a pharmaceutical composition containing an antibody-drug conjugate produced according to the present invention include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, and preferably include the intravenous route.

[0279] The antibody-drug conjugates produced by the present invention can be administered to humans at intervals of once every 1 to 180 days, preferably once every 1, 2, 3, or 4 weeks, and even more preferably once every 3 weeks. Furthermore, the antibody-drug conjugates produced by the present invention can be administered at a single dose of approximately 0.001 to 100 mg / kg, preferably at a single dose of 0.8 to 12.4 mg / kg. When the antibody-drug conjugates produced by the present invention are anti-HER2 antibody-drug conjugates, they can be administered at a single dose of preferably 5.4, 6.4, or 7.4 mg / kg, and even more preferably at a single dose of 5.4 mg / kg or 6.4 mg / kg.

[0280] Pharmaceutical compositions comprising antibody-drug conjugates produced by the present invention can be used for the treatment of cancer, and are preferably used to treat breast cancer, gastric cancer (sometimes referred to as gastric adenocarcinoma), colon cancer (sometimes referred to 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, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, gastrointestinal 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, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, and sarcoma, and for example, when the antibody-drug conjugate produced by the present invention is an anti-HER2 antibody-drug conjugate, it can be more preferably used to treat at least one cancer 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, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, and uterine carcinosarcoma, more preferably used to treat at least one cancer 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, bile duct cancer, and Paget's disease, and even more preferably used to treat breast cancer, gastric cancer, colorectal cancer, or non-small cell lung cancer.

[0281] Pharmaceutical compositions containing antibody-drug conjugates produced by the present invention can be selected and used as drugs for drug therapy, a major treatment for cancer, and as a result, can slow the growth of cancer cells, suppress their proliferation, and even destroy them. These effects can relieve cancer patients from cancer-related symptoms, improve their quality of life, and achieve therapeutic effects while preserving the lives of cancer patients. Even if cancer cells are not destroyed, the inhibition and control of cancer cell proliferation can enable cancer patients to achieve a higher quality of life and longer survival.

[0282] In addition to using the drug alone in such drug therapy, pharmaceutical compositions containing the antibody-drug conjugates produced by the present invention can also be used in adjuvant therapy in combination with other therapies, such as surgery, radiation therapy, and hormone therapy, and can also be used as drug therapy in neoadjuvant therapy.

[0283] In addition to the therapeutic uses described above, pharmaceutical compositions containing antibody-drug conjugates produced by the present invention can also be expected to have preventive effects, such as suppressing the growth of and even destroying micrometastatic cancer cells. For example, they can be expected to have effects such as suppressing and destroying cancer cells present in body fluids during the metastasis process, and suppressing and destroying microscopic cancer cells immediately after implantation in any tissue. Therefore, they can be expected to have an inhibitory and preventive effect on cancer metastasis, particularly after surgical removal of cancer.

[0284] Pharmaceutical compositions containing the antibody-drug conjugates produced by the present invention can be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect. Other cancer therapeutic agents that can be used for this purpose include 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), docetaxel ( Docetaxel, Pemetrexed, Sorafenib, Vinblastine, Vinorelbine, Everolimus, Tanespimycin, Bevacizumab, Oxaliplatin, Lapatinib, Trastuzumab Emtansine Examples of such antitumor agents include T-DM1 (T-DM1), drugs described in WO 2003 / 038043, as well as LH-RH analogs (e.g., leuprorelin, goserelin), estramustine phosphate, estrogen antagonists (e.g., tamoxifen, raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, and exemestane), but are not limited thereto as long as they have antitumor activity. [Example]

[0285] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the example, 1 H-NMR" and " 13"C-NMR" means "nuclear magnetic resonance spectrum," and in parentheses, CDCl3 means the measurement solvent, deuterated chloroform, DMSO-d6 means the measurement solvent, deuterated dimethyl sulfoxide, and DO means the measurement solvent, deuterium oxide. TMS (tetramethylsilane) was used as the internal standard. 1 Multiplicities in H-NMR mean s=singlet, d=doublet, t=triplet, q=quartet, quint=quintet, m=multiplet, and brs=broad singlet.

[0286] Example 1 N-(3-bromo-5-fluoro-4-methylphenyl)acetamide

[0287] [ka]

[0288] A solution of 2-fluoro-1-methyl-4-nitrobenzene (10.0 g, 64.5 mmol) in concentrated sulfuric acid (≥90%, 50 mL) and heptane (50 mL) was heated to approximately 60 °C, and then N-bromosuccinimide (13.8 g, 77.4 mmol) was added in six portions. The mixture was stirred at approximately 60 °C for 1 hour and then cooled to room temperature. The resulting reaction solution was added to cold water (50 mL). Toluene (50 mL) was added, and the mixture was separated. The aqueous layer was then removed. The organic layer was then washed with water (50 mL), 6.5 wt% aqueous sodium bicarbonate 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 resulting aqueous layer, and the mixture was stirred at room temperature for 1 hour. The insoluble matter was then filtered off and washed with toluene (20 mL). The aqueous layer was removed from the filtrate, and the organic layer was concentrated under reduced pressure. Ethyl acetate (100 mL) was added to the concentrated residue (about 30 mL), and the mixture was concentrated again under reduced pressure to obtain a solution of 1-bromo-3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (about 30 mL).

[0289] A suspension of 1-bromo-3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (approximately 30 mL) containing 1% platinum-on-carbon catalyst (2.0 g) and ethyl acetate (120 mL) was purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen atmosphere (0.1 MPa) at approximately 60°C for 4 hours and then cooled to room temperature. Insoluble matter was filtered off from the resulting suspension and washed with ethyl acetate (30 mL). The filtrate was washed twice with 0.5 N aqueous hydrochloric acid (100 mL) to obtain an organic layer. The aqueous layer was extracted with ethyl acetate (50 mL) to obtain the organic layer, and the organic layer was combined. The mixture was then washed with 6.5 wt% aqueous sodium bicarbonate (50 mL) and 5 wt% brine (50 mL), and the resulting organic layer was 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). A solution of 3-bromo-5-fluoro-4-methylaniline in ethyl acetate (approximately 29 mL) was added with ethyl acetate (30 mL), and triethylamine (7.2 mL, 51.8 mmol) and acetic anhydride (3.3 mL, 34.4 mmol) were added and stirred at room temperature for 6 hours. 10 wt% brine (50 mL) was added to the resulting reaction mixture, and the layers were separated. The aqueous layer was removed. The resulting organic layer was concentrated under reduced pressure, followed by the addition of ethyl acetate (50 mL) and further concentration under reduced pressure. Ethyl acetate (80 mL) was added to the concentrated residue (approximately 30 mL), and a 4N hydrochloric acid / ethyl acetate solution (10.9 mL, 43.7 mmol) was added. The mixture was stirred at room temperature for 1 hour. The insoluble matter was filtered off and washed with ethyl acetate (40 mL). 10 wt% brine (40 mL) was added to the filtrate, and then 25 wt% aqueous sodium hydroxide solution (5.6 g) was added to adjust the pH to approximately 7. After removing the aqueous layer, the organic layer was concentrated under reduced pressure. Toluene (100 mL) was added to the concentrated residue (approximately 30 mL), and the concentrated residue (approximately 30 mL) was concentrated under reduced pressure. The resulting mixture 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 as white crystals (5.7 g, yield 37%).

[0290] 1 H-NMR (500MHz,CDCl3)δ7.41(1H,s),7.39(1H,d,J=9.2Hz),7.20(1H,brs),2.27(3H,d,J=2.0Hz),2.17(3H,s)

[0291] Example 2 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid

[0292] [ka]

[0293] 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, followed by the addition of tri(o-tolyl)phosphine (1.1 g, 3.7 mmol). The mixture was again degassed under reduced pressure and purged with nitrogen, followed by the addition of palladium(II) acetate (0.4 g, 1.8 mmol). The mixture was then degassed under reduced pressure and purged with nitrogen, followed by heating to reflux for 5 hours. Activated carbon (3.0 g) was added to the reaction mixture, which was then cooled to room temperature and stirred at room temperature for 1 hour. The insoluble matter was filtered off and washed with 20% aqueous tetrahydrofuran (60 mL). To the filtrate, 2-methyltetrahydrofuran (300 mL) and water (300 mL) were added, followed by the addition of 25 wt% aqueous sodium hydroxide (23.4 g, 146.3 mmol). The organic layer was removed, and to the aqueous layer, 2-methyltetrahydrofuran (300 mL) and concentrated hydrochloric acid (36%, 22.2 g, 219.4 mmol) were added, followed by the addition of sodium chloride (30 g). After separation, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (90 mL). The resulting organic layer was concentrated under reduced pressure to give 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid containing geometric isomers as a 2-methyltetrahydrofuran solution (approximately 150 mL).

[0294] A suspension of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid (containing a geometric isomer) in 2-methyltetrahydrofuran (approximately 140 mL) was prepared by adding 2-methyltetrahydrofuran (308 mL) and 5% palladium on carbon (5.6 g). The atmosphere was then purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen atmosphere (0.1 MPa) at approximately 40°C for 1 hour and then cooled to room temperature. Insoluble matter was filtered off from the resulting suspension and washed with 2-methyltetrahydrofuran (112 mL). Water (140 mL) was added to the filtrate, and the pH was adjusted to approximately 2 with 1N aqueous hydrochloric acid. After separation, the aqueous layer was removed, and the resulting organic layer was concentrated under reduced pressure. Ethyl acetate (420 mL) was added to the concentrated residue, and the mixture was concentrated under reduced pressure. Ethyl acetate (420 mL) was added again, and the mixture was concentrated under reduced pressure to obtain a concentrated residue (approximately 170 mL). After stirring at 50°C for 5 hours, heptane (140 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) (84 mL). The obtained crystals were dried under reduced pressure to obtain 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid as white crystals (26.1 g, yield 91%).

[0295] 1 H-NMR (500MHz,DMSO-d6)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)

[0296] Example 3-1 N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0297] [ka]

[0298] A solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic 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 approximately 4°C for 4 hours. The resulting reaction solution was added dropwise to 50% aqueous acetonitrile (120 mL) cooled to 5°C. The pH was adjusted to approximately 7 with 25 wt% aqueous sodium hydroxide (77.3 g), and water (59 mL) was added. The mixture was then 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 as pale yellowish white crystals (10.2 g, yield 92%).

[0299] 1 H-NMR (400MHz,CDCl3)δ12.31(1H,brs),8.43(1H,d,J=12.8Hz),2.88(2H,t,J=12.0Hz),2.6 6(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)

[0300] (Example 3-2) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0301] [ka]

[0302] A solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid (5.0 g, 19.7 mmol) and trifluoroacetic acid (10 mL) was cooled to 2 °C, and trifluoroacetic anhydride (5.6 mL, 39.5 mmol) was added dropwise. The mixture was stirred at approximately 5 °C for 3 hours. 50% aqueous acetonitrile (50 mL) was added dropwise. The pH was adjusted to approximately 7 with 25 w / v% aqueous sodium hydroxide (33 mL), and water (17 mL) was added. The mixture was then 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 resulting crystals were dried under reduced pressure to give N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide as pale yellow-white crystals (4.3 g, 92% yield).

[0303] (Example 3-3) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0304] [ka]

[0305] A solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic 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 approximately 5°C for 4 hours. 17% aqueous acetonitrile (30 mL) was added dropwise, followed by water (20 mL). The pH was adjusted to approximately 7 with 25 w / v% aqueous sodium hydroxide (33 mL), and then water (17 mL) was added. The mixture was then 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 as pale yellowish white crystals (4.3 g, yield 93%).

[0306] (Example 4-1) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide

[0307] [ka]

[0308] 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 mixture was heated. At approximately 20 °C, 2% platinum-on-carbon catalyst (1.5 g) was added, and the atmosphere was purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen stream (0.3 MPa) at room temperature for 4 hours. Insoluble matter was filtered from the resulting suspension, and the insoluble matter was washed with ethyl acetate (25 mL). Activated carbon (0.7 g) was added to the filtrate and stirred at room temperature for 1 hour. The insoluble matter was then filtered off and 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, and 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 resulting organic layer, followed by the addition of 5N aqueous sodium hydroxide solution (25 mL) to adjust the pH to approximately 7. After warming to room temperature, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (25 mL). The resulting organic layer was concentrated under reduced pressure to give a concentrated residue. Ethyl acetate (50 mL) was added to the concentrated residue and concentrated under reduced pressure. This procedure was repeated three times. The concentrated residue (approximately 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, washed with cold ethyl acetate (25 mL) and water (25 mL), and dried under reduced pressure to give 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%).

[0309] 1 H-NMR (500MHz,CDCl3)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)

[0310] (Example 4-2) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide

[0311] [ka]

[0312] 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) was cooled to 4 °C, and amyl nitrite (25.7 mL, 193.4 mmol) and potassium tert-butoxide (20.0 g, 178.6 mmol) were added. After stirring at 1 °C for 17 hours, acetic acid (175 mL) and acetic anhydride (175 mL) were added, and the temperature was raised. At approximately 20 °C, 2% platinum-on-carbon catalyst (11.8 g) was added, and the atmosphere was purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen stream (0.3 MPa) at room temperature for 3 hours. Insoluble matter was filtered from the resulting suspension and washed with ethyl acetate (175 mL). Activated carbon (5.3 g) was added to the filtrate and stirred at room temperature for 2 hours. The insoluble matter was then filtered off and washed with ethyl acetate (175 mL). The filtrate was cooled to 1°C, and 5N aqueous sodium hydroxide solution (350 mL) was added dropwise. The aqueous layer was removed, and 5N aqueous sodium hydroxide solution (350 mL) was added again, and the aqueous layer was removed. Tetrahydrofuran (245 mL) and water (175 mL) were added to the resulting organic layer, followed by the addition of 5N aqueous sodium hydroxide solution (150 mL) to adjust the pH to approximately 7. After warming to room temperature, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (175 mL). The resulting organic layer was concentrated under reduced pressure to give a concentrated residue. Ethyl acetate (350 mL) was added to the concentrated residue and concentrated under reduced pressure. This procedure was repeated three times. The concentrated residue (approximately 175 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, washed with cold ethyl acetate (175 mL) and water (175 mL), and dried under reduced pressure to obtain white crystals (25.1 g). A suspension of the obtained crystals (24.0 g, 82.1 mmol) in 20% aqueous ethanol (300 mL) was heated to 65 °C. Activated carbon (4.8 g) was added and the mixture was stirred at 70 °C for 30 minutes. The insoluble matter was filtered off and washed with 20% aqueous ethanol (72 mL). Water (300 mL) was added dropwise to the filtrate at 60 °C, followed by gradual cooling to 2 °C and stirring for 2 hours. The precipitated crystals were collected by filtration and washed with cold 60% aqueous ethanol (120 mL). The obtained crystals were dried under reduced pressure to give N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide as white crystals (21.6 g, 52% yield).

[0313] 1 H-NMR (500MHz,CDCl3)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)

[0314] (Example 4-3) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide

[0315] [ka]

[0316] 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) was cooled to 0 °C, and amyl nitrite (2.2 mL, 16.6 mmol) and potassium tert-butoxide (1.7 g, 15.3 mmol) were added. After stirring at 3 °C for 3 hours, acetic acid (15 mL) and acetic anhydride (15 mL) were added, the mixture was heated to 20 °C, and stirred for 1.5 hours. At approximately 3 °C, 5% platinum-on-carbon catalyst (0.4 g) was added, and the atmosphere was purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen stream (0.6 MPa) at approximately 5 °C for 3 hours. After stirring at approximately 30 °C for 1 hour, the insoluble material was filtered off and washed with ethyl acetate (15 mL). Activated carbon (0.5 g) was added to the filtrate, and the mixture was stirred at room temperature for 2 hours. The insoluble matter was then filtered off and washed with ethyl acetate (15 mL). The filtrate was cooled to approximately 5°C, and 5N aqueous sodium hydroxide solution (30 mL) was added dropwise. The aqueous layer was removed, and tetrahydrofuran (30 mL) and 5N aqueous sodium hydroxide solution (30 mL) were added, and the aqueous layer was removed. Water (15 mL) was added to the resulting organic layer, followed by the addition of 5N aqueous sodium hydroxide solution (15 mL) to adjust the pH to approximately 7. After warming to room temperature, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (15 mL). The resulting organic layer was concentrated under reduced pressure to give a concentrated residue. Ethyl acetate (30 mL) was added to the concentrated residue, and the mixture was concentrated under reduced pressure. This procedure was repeated three times. The concentrated residue (approximately 15 mL) was stirred at 40°C for 5 hours, cooled to room temperature, and stirred at 5°C for at least 2 hours. The precipitated crystals were collected by filtration, washed with cold ethyl acetate (15 mL) and water (15 mL), and dried under reduced pressure to give 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%).

[0317] (Example 5-1) N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide

[0318] [ka]

[0319] 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) in 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 the mixture was cooled to 1°C. Triethylamine (8.6 mL, 61.6 mmol) was added dropwise at 1°C, followed by the addition of sodium sulfite (26 mg, 0.2 mmol). After stirring at 1°C for 4 hours, the precipitated crystals were filtered and washed with cold 60% aqueous ethanol (30 mL) and water (15 mL). The resulting 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, washed with acetone (9 mL), and dried at 40° C. under reduced pressure 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%).

[0320] 1 H-NMR (400MHz,DMSO-d6)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)

[0321] (Example 5-2) N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide

[0322] [ka]

[0323] 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 five portions to 2N hydrochloric acid / ethanol (75 mL) at room temperature and stirred at 50 °C for 5 hours. Water (113 mL) was added to the resulting reaction solution and cooled to 2 °C. Triethylamine (22.5 mL, 161.4 mmol) was added dropwise at 2 °C, followed by the addition of sodium sulfite (43 mg, 0.3 mmol). After stirring at 2 °C for 3 hours, the precipitated crystals were filtered and washed with cold 60% aqueous ethanol (50 mL) and water (25 mL). The resulting 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, washed with acetone (9 mL), and dried at 40° C. under reduced pressure 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%).

[0324] 1 H-NMR (400MHz,DMSO-d6)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)

[0325] (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]indolizino[1,2-b]quinolin-1-yl]acetamide

[0326] [ka]

[0327] To a suspension of 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]indolizine-3,6,10(4H)-trione (196.7 g, 747 mmol) in toluene (8.5 L), o-cresol (510 mL) and pyridinium p-toluenesulfonate (25.6 g, 102 mmol) were added and refluxed for 32 hours. Toluene (500 mL) was added, cooled to room temperature, and stirred for an additional 2 hours. The precipitated crystals were filtered and washed with acetone (850 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]indolizino[1,2-b]quinolin-1-yl]acetamide (312.5 g, yield 96%).

[0328] 1 H-NMR(400MHz,DMSO-d6)δ0.87(3H,t,J=7.3Hz),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, 18.9Hz),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=7.9Hz),6.95-6.99(0.4H,m) ,7.03(0.4H,d,J=7.3Hz),7.13-7.27(0.4H,m).7.30(1H,s),7.79(1H,d,J=11.0Hz),8.46(1H,d,J=9.2Hz),9.19(0.4H,s). 13C-NMR (100MHz,DMSO-d6)δ7.7, 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] + ).

[0329] (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]indolizino[1,2-b]quinolin-1-yl]acetamide

[0330] [ka]

[0331] To a suspension 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]indolizine-3,6,10(4H)-trione (3.42 g, 12.99 mmol) in toluene (125 mL), 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 (it was confirmed that the compound represented by formula (30) and the compound represented by formula (31) were produced via intermediates).

[0332] Compounds 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]indolizin-6-yl]amino}-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl]acetamide

[0333] [ka]

[0334] 1 H-NMR(500MHz,CDCl3)δ1.04(3H,t,J=7.5Hz),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=16.0Hz),6.35(1H,s),6.67(1H,d,J=5.5Hz),6.88(1H,s),6.99(1H,d,J=12.0Hz),11.14(1H,s). MS(ESI)(m / z):496.5([M+H] + ).

[0335] Compounds 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]indolizin-6-yl]amino}-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl]acetamide

[0336] [ka]

[0337] 1H-NMR(500MHz,CDCl3)δ1.03(3H,t,J=7.5Hz),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=16.0Hz),5.70(1H,d,J=16.0Hz),6.35(1H,s),6.68(1H,d,J=4.5Hz),6.88(1H,s),7.00(1H,d,J=12.0Hz),11.10(1H,s). MS(ESI)(m / z):496.6([M+H] + ).

[0338] After cooling, the volume was adjusted to 135 mL with toluene and stirred for an additional 2 hours. The precipitated crystals were filtered and washed with acetone (12.5 mL). The resulting crystals were dried under reduced pressure at 40 °C to give 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]indolizino[1,2-b]quinolin-1-yl]acetamide (4.58 g, 96% yield).

[0339] The instrumental data was similar to that of the compound described in Example 6-1.

[0340] Example 7-1 (1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-aminium methanesulfonate dihydrate

[0341] [ka]

[0342] 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]indolizino[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) was added with methanesulfonic acid (1.5 L) and refluxed for 8 hours. After cooling to room temperature, the organic layer was removed and the mixture was concentrated under reduced pressure to 3 L. The concentrate 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 washed with methanol (1.5 L).

[0343] The resulting crystals were dissolved in water (1.2 L), methanol (600 mL), and methanesulfonic acid (1.2 L), and activated carbon (15 g) was added and stirred for 30 minutes. Cellulose powder (150 g) was added and stirred for 30 minutes, after which the insoluble matter was filtered off and washed with 50% methanesulfonic acid water (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 washed with methanol (1.5 L).

[0344] The resulting crystals were suspended in ethanol (6 L) and water (600 mL) and refluxed for 1.5 hours. After cooling to room temperature and stirring for 30 minutes, the precipitated crystals were filtered and washed with ethanol (1.5 L). The resulting crystals were dried under reduced pressure at 40 °C and then conditioned under 40% RH air for 4 days to yield 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]indolizino[1,2-b]quinoline-1-aminium methanesulfonate dihydrate (152.3 g, 43% yield).

[0345] 1H-NMR (400MHz, DMSO-d6, D2O) δ0.89(3H,t,J=7.3Hz),1.90(2H,q,J=7.3Hz),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, 15.9Hz),5.50(2H,dd,J=17.7, 10.4Hz),7.41(1H,s),7.59(1H,d,J=11.0Hz). 13 C-NMR (125MHz,DMSO-d6)δ7.7, 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] + ).

[0346] (Example 7-2) Methanesulfonic acid (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-aminium

[0347] [ka]

[0348] 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]indolizino[1,2-b]quinolin-1-yl]acetamide (3.5 g, 7.3 mmol) in purified water (53 mL), 2-methoxyethanol (18 mL), and ethylcyclohexane (18 mL) was added with methanesulfonic acid (18 mL), and the mixture was subjected to three cycles of vacuum nitrogen purge (stirring under a 50 mbar vacuum followed by atmospheric pressure nitrogen purge three times). The suspension was heated to 85°C, stirred for 11 hours, and cooled to 25°C after confirming the completion of the reaction. The mixture was concentrated under reduced pressure to 38.5 mL, the concentrate 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 washed with methanol (35 mL).

[0349] The resulting crystals were dissolved in a mixture of purified water (14 mL) and methanesulfonic acid (14 mL). The mixture was heated to 37°C, and then methanol (7 mL), activated carbon (0.35 g), and filter aid (0.70 g, diatomaceous earth: Celpure C1000) were added. The mixture was then subjected to nitrogen substitution under reduced pressure three times (50 mbar, atmospheric nitrogen substitution three times). After stirring the suspension for 20 minutes, the insoluble material was filtered off and washed with a methanesulfonic acid-purified water-methanol mixture (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 an additional 2 hours, the precipitated crystals were filtered and washed with methanol (35 mL).

[0350] The resulting crystals were suspended in ethanol (70 mL) and water (7 mL) and stirred at 73 °C for 2 hours. After cooling to 25 °C and stirring for 2 hours, the precipitated crystals were filtered and washed with ethanol (18 mL). The resulting crystals were dried under reduced pressure at 40 °C to give (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-aminium methanesulfonate (1.72 g, 44% yield).

[0351] The instrumental data was similar to that of the compound described in Example 7-1.

[0352] Example 8 N-(3-iodo-5-fluoro-4-methylphenyl)acetamide

[0353] [ka]

[0354] A solution of 2-fluoro-1-methyl-4-nitrobenzene (5.0 g, 32.3 mmol) in concentrated sulfuric acid (≥90%, 25 mL) and heptane (25 mL) was cooled to approximately 1 °C, and then N-iodosuccinimide (10.2 g, 45.1 mmol) was added in six portions. The mixture was stirred at approximately 2 °C for 2 hours. The resulting reaction solution was added to cold water (25 mL). Toluene (25 mL) was added, and the layers were separated. The aqueous layer was then removed. The organic layer was then washed with water (25 mL), 6.5 wt% aqueous sodium bicarbonate solution (25 mL), 5 wt% aqueous sodium sulfite solution (25 mL, three 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 the mixture was concentrated again under reduced pressure to obtain an ethyl acetate solution of 1-iodo-3-fluoro-2-methyl-5-nitrobenzene (about 15 mL).

[0355] A suspension of 1-iodo-3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (approximately 15 mL) containing 1% platinum-on-carbon catalyst (1.1 g) and ethyl acetate (45 mL) was purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen atmosphere (0.1 MPa) at approximately 60°C for 5 hours and then cooled to room temperature. The insoluble material was filtered off from the resulting suspension and washed with ethyl acetate (15 mL). The filtrate was washed twice with 0.5 N aqueous hydrochloric acid (50 mL, 25 mL) to obtain an organic layer. The aqueous layer was extracted with ethyl acetate (25 mL) to obtain the organic layer, and the organic layer was combined. The mixture was then washed with 6.5 wt% aqueous sodium bicarbonate (25 mL) and 5 wt% brine (25 mL). The resulting organic layer was concentrated under reduced pressure to obtain a solution of 3-iodo-5-fluoro-4-methylaniline in ethyl acetate.

[0356] Ethyl acetate (25 mL) was added to a solution of 3-bromo-5-fluoro-4-methylaniline in ethyl acetate, and triethylamine (3.7 mL, 26.8 mmol) and acetic anhydride (1.7 mL, 17.7 mmol) were added to the resulting reaction mixture and stirred at room temperature for 4 hours. 10 wt% brine (25 mL) was added to the resulting reaction mixture, and the layers were separated. The aqueous layer was removed. The resulting organic layer was concentrated under reduced pressure. Ethyl acetate (50 mL) was added to the concentrated residue, and a 4N hydrochloric acid / ethyl acetate solution (5.6 mL, 22.6 mmol) was added. The mixture was stirred at room temperature for 15 minutes. The insoluble material was filtered off and washed with ethyl acetate (20 mL). 10 wt% brine (20 mL) was added to the filtrate, and then 25 w / v% aqueous sodium hydroxide solution (2.5 mL) was added to adjust the pH to approximately 7. The aqueous layer was removed, and the organic layer was concentrated under reduced pressure. Acetonitrile (38 mL) and water (38 mL) were added to the concentrated residue and 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 as white crystals (2.8 g, yield 29%).

[0357] 1H-NMR (500MHz,CDCl3)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)

[0358] Example 9 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid

[0359] [ka]

[0360] 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, followed by the addition of tri(o-tolyl)phosphine (62.3 mg, 0.2 mmol). After degassing and purging with nitrogen again under reduced pressure, palladium(II) acetate (23.0 mg, 0.1 mmol) was added. The mixture was degassed under reduced pressure and purged with nitrogen, and then heated to reflux for 2 hours. Activated carbon (0.2 g), 2-methyltetrahydrofuran (10 mL), and water (10 mL) were added to the reaction mixture, which was then stirred at room temperature for 1 hour. The insoluble matter was filtered off and washed with 20% aqueous tetrahydrofuran (4 mL). To the filtrate, 2-methyltetrahydrofuran (10 mL) and water (10 mL) were added, followed by the addition of 25 w / v% aqueous sodium hydroxide solution (1.3 mL, 8.2 mmol). The organic layer was removed, and to the aqueous layer, 2-methyltetrahydrofuran (20 mL) and concentrated hydrochloric acid (36%, 1.2 g, 12.3 mmol) were added, followed by the addition of sodium chloride (2 g). After separation, the aqueous layer was removed, and the organic layer was washed with 10 wt% brine (6 mL). The resulting organic layer was concentrated under reduced pressure to give 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid residue (1.9 g) containing geometric isomers.

[0361] A suspension of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid residue (1.9 g) containing a geometric isomer was added to 2-methyltetrahydrofuran (30 mL) and 5% palladium on carbon (1.7 g). The atmosphere was then purged with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen atmosphere (0.3 MPa) at approximately 40°C for 8 hours and then cooled to room temperature. Insoluble matter was filtered off from the resulting suspension and washed with 2-methyltetrahydrofuran (8 mL). Water (10 mL) was added to the filtrate, and the pH was adjusted to approximately 2 with 1N aqueous hydrochloric acid. After separation, the aqueous layer was removed, and the resulting organic layer was concentrated under reduced pressure. Ethyl acetate (10 mL) was added to the concentrated residue, and the mixture was heated to approximately 50°C. 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]butanoic acid as white crystals (1.4 g, yield 81%).

[0362] Example 10 N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0363] [ka]

[0364] To a solution of 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid (200 mg, 0.79 mmol), thionyl chloride (86 μL, 1.18 mmol), and methylene chloride (4 mL) was added aluminum chloride (263 mg, 1.97 mmol) at room temperature under a nitrogen stream and stirred at room temperature for 2 hours. To the resulting reaction solution, 1N aqueous hydrochloric acid (10 mL) and ethyl acetate (50 mL) were added. The aqueous layer was removed, and the air layer was washed with water (10 mL), 6.5 wt% aqueous sodium bicarbonate (10 mL), and water (10 mL). The resulting organic layer was dried over sodium sulfate. After filtering off the insoluble matter, the solvent was distilled off under reduced pressure and the resulting residue 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 as pale yellow-white crystals (125 mg, yield 67%).

[0365] The instrumental data was similar to that of the compound described in Example 3-1. [Sequence List Free Text]

[0366] SEQ ID NO: 1: Amino acid sequence of the anti-HER2 antibody heavy chain SEQ ID NO: 2: Amino acid sequence of the anti-HER2 antibody light chain SEQ ID NO: 3: Amino acid sequence of the heavy chain of the anti-HER3 antibody SEQ ID NO: 4: Amino acid sequence of the light chain of the anti-HER3 antibody SEQ ID NO: 5: Amino acid sequence of the anti-TROP2 antibody heavy chain SEQ ID NO: 6: Amino acid sequence of the anti-TROP2 antibody light chain SEQ ID NO: 7: Amino acid sequence of the anti-B7-H3 antibody heavy chain SEQ ID NO: 8: Amino acid sequence of the anti-B7-H3 antibody light chain SEQ ID NO: 9: Amino acid sequence of the anti-GPR20 antibody heavy chain SEQ ID NO: 10: Amino acid sequence of the anti-GPR20 antibody light chain SEQUENCE LISTING <110> DAIICHI SANKYO COMPANY, LIMITED <120> NOVEL METHOD FOR PRODUCING ANTIBODY-DRUG CONJUGATE <130> PD20A-0090D <150> JP2017-167690 <151> 2017-08-31 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-HER2 antibody <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-HER2 antibody <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-HER3 antibody <400> 3 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Lys Trp Thr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 4 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-HER3 antibody <400> 4 Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 5 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-TROP2 antibody <400> 5 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Ala Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala 65 70 75 80 Glu Asp Phe Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp 115 120 125 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 6 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-TROP2 antibody <400> 6 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Ile Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 7 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-B7-H3 antibody <400> 7 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 8 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-B7-H3 antibody <400> 8 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 9 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-GPR20 antibody <400> 9 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Lys Tyr Met Gly Phe Ile Asn Pro Gly Ser Gly His Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Thr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Ala Gly Gly Phe Leu Arg Ile Ile Thr Lys 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 10 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-GPR20 antibody <400> 10 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser 35 40 45 Val Ser Thr Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Gln Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Gly Asn Leu Glu Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Asn Tyr Tyr Cys Gln Gln Ile Asn 100 105 110 Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230

Claims

1. Formula (E) 【Chemical 1】 A compound represented by the formula (wherein R １ represents an amino group protected by a protecting group), Formula (B) 【Chemistry 2】 A compound represented by the formula (wherein R １ has the same meaning as defined above).

2. R １ 2. The method according to claim 1, wherein is an amino group protected with an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a trichloroacetyl group, a pivaloyl group, a formyl group, or a benzoyl group.

3. R １ The method according to claim 1, wherein is an amino group protected with an acetyl group or a trifluoroacetyl group.

4. R １ The method according to claim 1, wherein is an amino group protected with an acetyl group.

5. The method according to any one of claims 1 to 4, which is carried out by a method comprising reacting a compound represented by formula (E) with hydrogen in a solvent in the presence of a palladium-carbon catalyst.

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