crystals, methods for producing crystals and an antibody-drug conjugate, and, salt.

专利摘要:
  A crystal of the compound represented by formula (1), a method for producing the crystal and a method for producing an antibody-drug conjugate using said crystals are provided.
公开号:BR112020003646A2
申请号:R112020003646-3
申请日:2018-08-30
公开日:2020-09-01
发明作者:Tatsuya Yamaguchi；Tsuyoshi Ueda；Shinji Matuura；Kei Kurahashi；Yutaka Kitagawa；Tatsuya Nakamura；Takashi Kouko；Shigeru Noguchi；Yohei Yamane；Fumikatsu Kondo；Takahiro Aoki；Tadahiro Takeda；Kohei Sakanishi；Hitoshi Sato
申请人:Daiichi Sankyo Company, Limited；
IPC主号:

专利说明:

[001] The present invention relates to an improved method of producing a linker-drug intermediate for an antibody-drug conjugate and improved method for producing an antibody-drug conjugate, in which the method mentioned above is used. Fundamentals of the invention
[002] [002] An antibody-drug conjugate (ADC) is expected to consist of a drug with cytotoxicity conjugated to an antibody, the antigen of which is expressed on the surface of cancer cells, and which also binds to an antigen capable of cell internalization, and may, therefore, deliver the drug selectively to cancer cells, which thereby causes the drug to accumulate within cancer cells and eliminate cancer cells (Non-Patent Literature 1 to 5).
[003] [003] As one of such antibody-drug conjugates, an antibody-drug conjugate is known which comprises, as components, an antibody and exactecane, which is an inhibitor of topoisomerase I (Patent Literature 1 to 5 and Non-Patent Literature Patent 6 and 7). Due to their superior antitumor effect and safety, these antibody-drug conjugates are currently in clinical studies.
[004] [004] As methods for producing linker-drug intermediates for the production of the described antibody-drug conjugates, the methods described in Patent Literature 1 to 4 are known. List of Citations Patent Literature
[005] [005] Patent Literature 1: International Publication No WO 2014/057687 Patent Literature 2: International Publication No WO
[006] [006] Non-Patent Literature 1: Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. Non-Patent Literature 2: Alley, S. C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537. Non-Patent Literature 3: Damle N. K. Expert Opin. Biol. The R. (2004) 4, 1445-1452. Non-Patent Literature 4: Senter P. D., et al., Nature Biotechnology (2012) 30, 631-637. Non-Patent Literature 5: Howard A. et al., J Clin Oncol 29: 398-405. Non-Patent Literature 6: Ogitani Y. et al., Clinical Cancer Research (2016) 22 (20), 5097-5108. Non-Patent Literature 7: Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046. Summary of the invention Technical problem
[007] [007] A linker-drug intermediate for the production of an antibody-drug conjugate of the present invention is the compound represented by formula (1): [Chemical formula 1]
[008] [008] As a method for producing the compound represented by formula (1), the methods described in Patent Literature 1 to 4 are known. However, it was not known that the compound represented by formula (1) can be obtained as crystals , and complicated operations such as the necessary purification by chromatography are required. There is, therefore, a demand for the development of an industrially better production method.
[009] [009] An object of the present invention is to find an industrially excellent method for producing a binder-drug intermediate without the need for purification by chromatography. Another object of the present invention is to create an improved method for the production of an antibody-drug conjugate, in which the improved method mentioned above is used to produce a linker-drug intermediate. Solution to the problem
[0010] [0010] The present inventors have conducted diligent studies on a method to produce a drug-binding intermediate and, consequently, have found that, surprisingly, the compound represented by formula (1) can be obtained as crystals. The present inventors also improved a method for producing the compound represented by formula (1) and, consequently, discovered an industrially excellent production method without the need for purification by
[0011] [0011] Specifically, the present invention relates to the following.
[0012] [0012] [1] Crystals of the compound represented by formula (1): [Chemical formula 2] (1)
[0013] [0013] [2] The crystals according to [1], where the crystals show major peaks at the diffraction angles (2θ) of 5.6 ± 0.2 °, 15.5 ± 0.2 ° and 22, 0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[0014] [0014] [3] A method for producing crystals of the compound represented by formula (1): [Chemical formula 3] (1)
[0015] [0015] [4] The production method according to [3], in which the crystals of the compound represented by the formula (1) show main peaks at the diffraction angles (2θ) of 5.6 ± 0.2 °, 15 , 5 ± 0.2 ° and 22.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[0016] [0016] [5] The production method according to [3] or [4], wherein the solution in which the compound represented by formula (1) is dissolved comprises a lower ketone and a lower alcohol as solvents.
[0017] [0017] [6] The production method according to [5], in which the lower ketone is acetone.
[0018] [0018] [7] The production method according to [5], in which the lower ketone is methyl ethyl ketone.
[0019] [0019] [8] The production method according to any of [5] to
[7] [7], where the lower alcohol is 1-propanol.
[0020] [0020] [9] The production method according to any of [5] to
[7] [7], where the lower alcohol is 2-butanol.
[0021] [0021] [10] The production method according to any one of [3] to [9], comprising a step of adding a seed crystal of the crystals of the compound represented by the formula (1).
[0022] [0022] [11] The production method according to any one of [3] to [10], in which the compound represented by formula (1) is produced by a production method (I), in which the production method production (I) is a production method that comprises the steps of: unprotecting protective groups for an amino group and a carboxyl group of a compound represented by formula (B):
[0023] [0023] [12] The production method according to any one of [3] to [10], in which the compound represented by formula (1) is produced by a production method (II), in which the production method production (II) is a production method
[0024] [0024] [13] The production method according to [11] or [12], which comprises the steps of: dissolving the compound represented by formula (10) in a solvent containing 1,2-dimethoxyethane; and then precipitating crystals from a 1,2-dimethoxyethane adduct of the compound represented by formula (10).
[0025] [0025] [14] The production method according to [13], in which the crystals of the adduct 1,2-dimethoxyethane of the compound represented by the formula (10) show main peaks at the diffraction angles (2θ) of 19,0 ± 0.2 ° and 25.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[0026] [0026] [15] The production method according to any of
[11] [11] to [14], in which the condensation step of the compound represented by formula (10) and the compound represented by formula (11), in order to convert it into the compound represented by formula (1) is carried out in a biphasic system of an aqueous solution of sodium sulphate and tetrahydrofuran.
[0027] [0027] [16] The production method according to any one of [3] to [10], in which the compound represented by formula (1) is produced by a production method (III), in which the production method production (III) is a production method that comprises the steps of:
[0028] [0028] [17] The production method according to any of
[11] [11] to [16], in which the compound represented by formula (11) is in the form of a salt of methanesulfonic acid.
[0029] [0029] [18] The production method according to any of
[11] [11] to [16], where the compound represented by formula (11) is in the form of a salt of methanesulfonic acid m-hydrate, where m is in the range 0 to 3.
[0030] [0030] [19] The production method according to any of
[11] [11] to [16], in which the compound represented by formula (11) is in the form of anhydride of a salt of methanesulfonic acid.
[0031] [0031] [20] The production method according to any of
[11] [11] to [16], where the compound represented by formula (11) is in the form of a salt of methanesulfonic acid monohydrate.
[0032] [0032] [21] The production method according to any of
[11] [11] to [16], in which the compound represented by formula (11) is in the form of a dihydrated methanesulfonic acid salt.
[0033] [0033] [22] The production method according to any of
[11] [11] to [16], wherein the compound represented by formula (11) is in the form of a methanesulfonic acid salt trihydrate.
[0034] [0034] [23] The production method according to any of
[11] [11] to [22], in which the compound represented by formula (B) is produced by a production method (IV), in which the production method (IV) is a production method that comprises the steps of: reacting a compound represented by the formula (H): [Chemical formula 24] (H) where R3 represents an amino group, protected with a protective group, with lead tetracetate in order to convert it into a compound represented by the formula (J) : [Chemical formula 25] (J) where R3 represents the same meaning as above; then react the compound represented by formula (J) with a compound represented by formula (K): [Chemical formula 26] (K) where R2 represents the same meaning as R2 according to any one of claims 11 to 22, in presence of an acid or a base, in order to convert it into a compound represented by the formula (L): [Chemical formula 27]
[0035] [0035] [24] The production method according to [23], in which the reaction step of the compound represented by the formula (H) with lead tetracetate in order to convert it into the compound represented by the formula (J) is performed in the presence of acetic acid.
[0036] [0036] [25] The production method according to [23] or [24], in which the reaction step of the compound represented by the formula (J) with the compound represented by the formula (K), in order to convert it in the compound represented by the formula (L) is carried out in the presence of an aqueous solution of sodium hydroxide.
[0037] [0037] [26] The production method according to [23] or [24], in which the reaction step of the compound represented by the formula (J) with the compound represented by the formula (K), in order to convert it in the compound represented by the formula (L) is carried out in the presence of tris (pentafluorophenyl) borane.
[0038] [0038] [27] The production method according to any of
[23] [23] to [26], comprising a step of adding an acid to precipitate a salt of the compound represented by the formula (M) and the acid after the step of deprotection of the protecting group to the amino group of the compound represented by the formula (L ) in order to convert it into the compound represented by the formula (M).
[0039] [0039] [28] The production method according to [27], in which the acid is 1-hydroxybenzotriazole.
[0040] [0040] [29] The production method according to any of
[11] [11] to [28], where R1 is an amino group protected with a benzyloxycarbonyl group.
[0041] [0041] [30] The production method according to any of
[11] [11] to [28], where R1 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
[0042] [0042] [31] The production method according to any of
[11] [11] to [30], where R2 is a carboxyl group protected with a benzyl group.
[0043] [0043] [32] The production method according to any of
[23] [23] to [31], where R3 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
[0044] [0044] [33] The production method according to any of
[11] [11] to [32], where X is an (2,5-dioxopyrrolidin-1-yl) oxycarbonyl group.
[0045] [0045] [34] The production method according to any one of [3] to [10], in which the compound represented by formula (1) is produced by a production method (V), in which the production method production (V) is a production method that comprises the steps of: reacting the compound represented by formula (2): [Chemical formula 31] (2) with lead tetracetate in order to convert it into the compound represented by formula (3 ): [Chemical formula 32]
[0046] [0046] [35] The production method according to [34], which comprises the steps of: dissolving the compound represented by formula (10) in a solvent containing 1,2-dimethoxyethane; and then precipitating crystals from a 1,2-dimethoxyethane adduct of the compound represented by formula (10).
[0047] [0047] [36] The production method according to [35], in which the crystals of the adduct 1,2-dimethoxyethane of the compound represented by the formula (10) show main peaks at the diffraction angles (2θ) of 19,0 ± 0.2 ° and 25.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[0048] [0048] [37] The production method according to any of
[34] [34] to [36], in which the condensation step of the compound represented by formula (10) with the compound represented by formula (11), in order to convert it into the compound represented by formula (1) is carried out in a biphasic system of an aqueous solution of sodium sulphate and tetrahydrofuran.
[0049] [0049] [38] The production method according to any one of [3] to [10], in which the compound represented by formula (1) is produced by a production method (VI), in which the production method production (VI) is a production method that comprises the steps of:
[0050] [0050] [39] The production method according to any of
[34] [34] to [38], where the reaction step of the compound represented by the formula
[0051] [0051] [40] The production method according to any of
[34] [34] to [39], wherein the step of converting the compound represented by formula (3) into the compound represented by formula (4) is carried out in the presence of an aqueous sodium hydroxide solution.
[0052] [0052] [41] The production method according to any of
[34] [34] to [39], in which the step of converting the compound represented by formula (3) into the compound represented by formula (4) is carried out in the presence of tris (pentafluorophenyl) borane.
[0053] [0053] [42] The production method according to any of
[34] [34] to [41], comprising a step of adding an acid to precipitate a salt of the compound represented by the formula (5) and the acid after the step of deprotection of the protecting group to the amino group of the compound represented by the formula (4 ) in order to convert it into the compound represented by formula (5).
[0054] [0054] [43] The production method according to [42], in which the acid is 1-hydroxybenzotriazole.
[0055] [0055] [44] The production method according to any of
[34] [34] to [43], in which the compound represented by formula (6) is produced by a method that comprises the steps of: condensing the compound represented by formula (23): [Chemical formula 54] (23) with N- hydroxysuccinimide in order to convert it into the compound represented by formula (24): [Chemical formula 55]
[0056] [0056] [45] The production method according to any of
[34] [34] to [44], in which the compound represented by formula (9) is produced by a method comprising the steps of: reacting the compound represented by formula (17): [Chemical formula 56] (17) with maleic anhydride in order to convert it into the compound represented by formula (18): [Chemical formula 57] (18); and then add thionyl chloride to the compound represented by formula (18) and a mixed solution containing N-hydroxysuccinimide and 2,6-lutidine in order to convert it to the compound represented by formula (9).
[0057] [0057] [46] The production method according to any of
[34] [34] to [45], where the compound represented by formula (11) is in the form of a salt of methanesulfonic acid.
[0058] [0058] [47] The production method according to any of
[34] [34] to [45], where the compound represented by formula (11) is in the form of a salt of methanesulfonic acid m-hydrate, where m is in the range 0 to 3.
[0059] [0059] [48] The production method according to any of
[34] [34] to [45], wherein the compound represented by formula (11) is in the form of anhydride of a salt of methanesulfonic acid.
[0060] [0060] [49] The production method according to any of
[34] [34] to [45], wherein the compound represented by formula (11) is in the form of a salt of methanesulfonic acid monohydrate.
[0061] [0061] [50] The production method according to any of
[34] [34] to [45], where the compound represented by formula (11) is in the form of a dihydrated methanesulfonic acid salt.
[0062] [0062] [51] The production method according to any of
[34] [34] to [45], wherein the compound represented by formula (11) is in the form of a methanesulfonic acid salt trihydrate.
[0063] [0063] [52] A method for the production of a compound represented by formula (J), which comprises the step of: reacting a compound represented by formula (H): [Chemical formula 58] (H) where R3 represents an amino group, protected with a protective group, with lead tetracetate in the presence of acetic acid in order to convert it into the compound represented by formula (J): [Chemical formula 59] (J)
[0064] [0064] [53] The production method according to [52], wherein R3 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
[0065] [0065] [54] A method for the production of a compound represented by the formula (L), which comprises the step of: reacting a compound represented by the formula (J): [Chemical formula 60] (J) where R3 represents an amino group, protected with a protecting group, with a compound represented by the formula (K): [Chemical formula 61] (K) where R2 represents a carboxyl group, protected with a protecting group, in the presence of an aqueous solution of hydroxide sodium or tris (pentafluorophenyl) borane, in order to convert it into the compound represented by the formula (L): [Chemical formula 62] (L) where R2 and R3 represent the same meaning as above.
[0066] [0066] [55] The production method according to [54], in which the reaction is carried out in the presence of an aqueous sodium hydroxide solution.
[0067] [0067] [56] The production method according to [54], in which the reaction is carried out in the presence of tris (pentafluorophenyl) borane.
[0068] [0068] [57] The production method according to any of
[54] [54] to [56], where R2 is a carboxyl group protected with a benzyl group.
[0069] [0069] [58] The production method according to any of
[54] [54] to [57], where R3 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
[0070] [0070] [59] A method for producing a salt of a compound represented by the formula (M) and an acid, comprising the steps of: deprotecting a protecting group to an amino group of a compound represented by the formula (L) : [Chemical formula 63] (L) where R2 represents a carboxyl group, protected with a protecting group, and R3 represents an amino group, protected with a protecting group, in order to convert it into the compound represented by formula (M) : [Chemical formula 64] (M) where R2 represents the same meaning as above; and then add an acid to precipitate the salt of the compound represented by the formula (M) and the acid.
[0071] [0071] [60] The production method according to [59], in which the acid is 1-hydroxybenzotriazole.
[0072] [0072] [61] The production method according to [59] or [60], wherein R2 is a carboxyl group protected with a benzyl group.
[0073] [0073] [62] The production method according to any of
[59] [59] to [61], where R3 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
[0074] [0074] [63] A method for the production of the compound represented by formula (9), which comprises the step of: adding thionyl chloride to the compound represented by formula (18): [Chemical formula 65] (18) and a mixed solution containing N-hydroxysuccinimide and 2,6-lutidine in order to convert it into the compound represented by formula (9): [Chemical formula 66] (9)
[0075] [0075] [64] The production method according to [63], in which the compound represented by formula (18) is produced by a method comprising the step of: reacting the compound represented by formula (17): [Formula chemistry 67] (17) with maleic anhydride.
[0076] [0076] [65] A method for the production of crystals from a 1,2-dimethoxyethane adduct of the compound represented by formula (10), which comprises the steps of: dissolving the compound represented by formula (10): [Chemical formula 68]
[0077] [0077] [66] The production method according to [65], in which the crystals of the 1,2-dimethoxyethane adduct of the compound represented by the formula (10) show peaks in the diffraction angles (2θ) of 19.0 ± 0.2 ° and 25.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[0078] [0078] [67] A method for the production of the compound represented by formula (1), which comprises the step of: condensing the compound represented by formula (10): [Chemical formula 69] (10) and the compound represented by formula (11): [Chemical formula 70] (11)
[0079] [0079] [68] The production method according to [67], in which the compound represented by formula (11) is in the form of a salt of methanesulfonic acid.
[0080] [0080] [69] The production method according to [67], in which the compound represented by formula (11) is in the form of a salt of methanesulfonic acid m-hydrate, in which m is in the range 0 to 3 .
[0081] [0081] [70] The production method according to [67], in which the compound represented by formula (11) is in the form of anhydride of a salt of methanesulfonic acid.
[0082] [0082] [71] The production method according to [67], wherein the compound represented by formula (11) is in the form of a salt of methanesulfonic acid monohydrate.
[0083] [0083] [72] The production method according to [67], wherein the compound represented by formula (11) is in the form of a salt of methanesulfonic acid dihydrate.
[0084] [0084] [73] The production method according to [67], wherein the compound represented by formula (11) is in the form of an acid salt
[0085] [0085] [74] The production method according to any one of [3] to [73], in which no chromatography is used.
[0086] [0086] [75] Crystals of a 1,2-dimethoxyethane adduct of the compound represented by formula (10): [Chemical formula 72] (10)
[0087] [0087] [76] The crystals according to [75], where the crystals show main peaks at the diffraction angles (2θ) of 19.0 ± 0.2 ° and 25.0 ± 0.2 ° in the diffraction of powder x-rays, obtained by irradiation with copper Kα radiation.
[0088] [0088] [77] A salt of the compound represented by formula (5): [Chemical formula 73] (5) and an acid.
[0089] [0089] [78] The salt according to [77], wherein the acid is 1-hydroxybenzotriazole.
[0090] [0090] [79] A method for the production of an antibody-drug conjugate, in which a linker-drug represented by formula (19): [Chemical formula 75]
[51] [51], are used as starting material, and the method comprises the steps of: i) reducing an antibody; and then ii) adding a solution in which the crystals of the compound represented by formula (1), produced in the method mentioned above, are dissolved to react the solution with the reduced antibody.
[0091] [0091] [80] The production method according to [79], wherein the antibody is an anti-HER2 antibody, an anti-HER3 antibody, an antibody
[0092] [0092] [81] The production method according to [80], wherein the antibody is an anti-HER2 antibody.
[0093] [0093] [82] The production method according to [81], wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.
[0094] [0094] [83] The production method according to [81] or [82], in which the average number of units of the ligand-drug conjugated per antibody molecule in the antibody-drug conjugate is in the range between 7 and 8.
[0095] [0095] [84] The production method according to [80], wherein the antibody is an anti-HER3 antibody.
[0096] [0096] [85] The production method according to [84], wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of amino acid sequence represented by SEQ ID NO: 4, or an antibody variant in which a lysine residue in the terminal carboxyl of the antibody heavy chain is deleted.
[0097] [0097] [86] The production method according to [84] or [85], wherein the average number of units of the conjugated drug-ligand per antibody molecule in the antibody-drug conjugate is in the range between 7 and 8.
[0098] [0098] [87] The production method according to [80], wherein the antibody is an anti-TROP2 antibody.
[0099] [0099] [88] The production method according to [87], in which the
[00100] [00100] [89] The production method according to [87] or [88], in which the average number of units of the conjugated drug-ligand per antibody molecule in the antibody-drug conjugate is in the range between 3 and 5.
[00101] [00101] [90] The production method according to [80], wherein the antibody is an anti-B7-H3 antibody.
[00102] [00102] [91] The production method according to [90], wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 8, or an antibody variant in which a lysine residue in the terminal carboxyl of the antibody heavy chain is deleted.
[00103] [00103] [92] The production method according to [90] or [91], in which the average number of units of the conjugated drug-ligand per antibody molecule in the antibody-drug conjugate is in the range between 3 and 5.
[00104] [00104] [93] The production method according to [80], wherein the antibody is an anti-GPR20 antibody.
[00105] [00105] [94] The production method according to [93], wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 10, or an antibody variant in which a
[00106] [00106] [95] The production method according to [93] or [94], wherein the average number of units of the conjugated drug-ligand per antibody molecule in the antibody-drug conjugate is in the range between 7 and 8. Advantageous effects of the invention
[00107] [00107] The present invention allows to obtain the compound represented by formula (1) in crystals and can supply the compound represented by formula (1) with a given quality. The present invention can also provide an industrially excellent method for producing the compound represented by formula (1) without the need for purification by chromatography. The present invention can further provide an improved method for producing an antibody-drug conjugate in which the method mentioned above is used. Brief description of the drawings
[00108] [00108] Figure 1 shows an amino acid sequence of an anti-HER2 antibody heavy chain (SEQ ID NO: 1).
[00109] [00109] Figure 2 shows an amino acid sequence of an anti-HER2 antibody light chain (SEQ ID NO: 2).
[00110] [00110] Figure 3 shows the X-ray diffraction of crystal powder from a 1,2-dimethoxyethane adduct of the compound represented by formula (10).
[00111] [00111] Figure 4 shows the X-ray diffraction of crystalline powder from the compound represented by formula (1).
[00112] [00112] Figure 5 shows an amino acid sequence of an anti-HER3 antibody heavy chain (SEQ ID NO: 3).
[00113] [00113] Figure 6 shows an amino acid sequence of an anti-HER3 antibody light chain (SEQ ID NO: 4).
[00114] [00114] Figure 7 shows an amino acid sequence of an anti-TROP2 antibody heavy chain (SEQ ID NO: 5).
[00115] [00115] Figure 8 shows an amino acid sequence of a
[00116] [00116] Figure 9 shows an amino acid sequence of an anti-B7-H3 antibody heavy chain (SEQ ID NO: 7).
[00117] [00117] Figure 10 shows an amino acid sequence of an anti-B7-H3 antibody light chain (SEQ ID NO: 8).
[00118] [00118] Figure 11 shows an amino acid sequence of an anti-GPR20 antibody heavy chain (SEQ ID NO: 9).
[00119] [00119] Figure 12 shows an amino acid sequence of an anti-GPR20 antibody light chain (SEQ ID NO: 10). Description of modalities
[00120] [00120] In the following, preferred ways of carrying out the present invention are described with reference to the drawings. The embodiments described below are provided simply to illustrate an example of a typical embodiment of the present invention and are not intended to limit the scope of the present invention. Antibody-drug conjugate
[00121] [00121] The antibody-drug conjugate produced by the present invention is an antibody-drug conjugate in which a linker-drug represented by formula (19): [Chemical formula 76] (19) where A represents the connection position with an antibody ,
[00122] [00122] In the present invention, the partial structure consisting of a linker and a drug in the antibody-drug conjugate is referred to as a "linker-drug". The drug ligand is attached to a thiol group (in other words, the sulfur atom of a cysteine residue) formed at the disulfide binding site between the chains (two sites between heavy chains and two sites between a heavy chain and a chain light) in the antibody.
[00123] The linker-drug of the present invention includes exactecane, which is a topoisomerase I inhibitor, as a component. Exatecan is the compound represented by the formula (11): [Chemical formula 77] (11) and is a derivative of camptothecin with an anti-tumor effect.
[00124] [00124] The antibody-drug conjugate used in the present invention can also be represented by formula (20): [Chemical formula 78] Antibody n (20) in which, the linker-drug is conjugated to an antibody through a thioether bond. The meaning of n is equal to what is called the
[00125] [00125] After migrating to cancer cells, the antibody-drug conjugate used in the present invention releases the compound represented by formula (22): [Chemical formula 79] (22) and thus exerts an anti-tumor effect.
[00126] [00126] It is inferred that the compound represented by formula (22) is the original source of the anti-tumor activity of the antibody-drug conjugate produced by the present invention, and has been confirmed to have a topoisomerase I inhibiting effect (Ogitani Y. et al ., Clinical Cancer Research, 2016, Oct 15; 22 (20): 5097-5108, Epub 2016 Mar 29).
[00127] [00127] It is inferred that the compound represented by formula (22) is formed by the decomposition of an amine structure of the compound represented by formula (21): [Chemical formula 80] (21)
[00128] [00128] The antibody-drug conjugate produced by the present invention is known to have a bystander (spectator) effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046).
[00129] [00129] The bystander effect is exerted through a process in which the antibody-drug conjugate produced according to the present invention is internalized in cancer cells that express a target, and the compound represented by the formula (22) released then exerts an effect antitumor also in cancer cells that are around them and do not express the target. Drug-binding intermediary for use in the production of the antibody-drug conjugate
[00130] [00130] A linker-drug intermediate for use in the production of the antibody-drug conjugate of the present invention is the compound represented by formula (1): [Chemical formula 81] (1)
[00131] [00131] According to the present invention, the compound represented by formula (1) can be obtained as crystals, and the crystals can preferably be used to produce the antibody-drug conjugate of the present invention.
[00132] [00132] The quality of the crystals of the compound represented by formula (1) can be evaluated, for example, based on indices such as impurity content, the amount of a residual solvent and aspect. In addition, it can also be evaluated using, as an index, stability, conservation for 3 months, 6 months, 12 months, 24 months and 36 months in an environment of 25 ºC / 60% RH or 40 ºC / 75% RH , for example.
[00133] [00133] With such an assessment of quality, the superiority over an amorphous compound represented by formula (1) can also be confirmed.
[00134] [00134] The production method of the present invention comprises precipitating crystals of the compound represented by formula (1) from a solution in which the compound represented by formula (1) is dissolved, to produce crystals of the compound represented by formula (1). As a result, highly pure crystals of the compound represented by formula (1) of a given quality can be produced.
[00135] [00135] The crystals of the compound represented by formula (1) preferably show main peaks at the diffraction angles (2θ) of 5.6 °, 15.5 ° and 22.0 ° in the powder x-ray diffraction, obtained by irradiation with copper Kα radiation. As diffraction angles (2θ) in powder X-ray diffraction can generally cause an error within the range of ± 0.2 °, it should be understood that the values described above the diffraction angles include numerical values within the range of ± 0.2 ° (for good technical sense regarding the measurement and evaluation by powder X-ray diffraction, see, for example, the Japanese Pharmacopoeia, 16th edition, p. 64-68 (2.58 X-Ray Diffraction Method Powder) or the Japanese Pharmacopoeia, 17th edition, pp. 71-74 (2.58 Powder X-Ray Diffraction Method)).
[00136] [00136] Thus, crystals having diffraction angles that completely agree with the diffraction angles described above are identical to crystals having main peaks at the diffraction angles (2θ) of 5.6 ± 0.2 °, 15.5
[00137] [00137] The solution to precipitate the crystals of the compound represented by formula (1) is preferably a solution containing acetone and a lower alcohol as a solvent. Likewise, a solution containing a lower ketone and a lower alcohol as a solvent can also preferably be used as the solution to precipitate the crystals of the compound represented by formula (1).
[00138] [00138] In the present invention, the term "lower ketone" refers to a ketone containing 3 to 6 carbon atoms. Its examples may include acetone, methyl ethyl ketone, methyl propyl ketone, methyl isopropyl ketone, methyl butyl ketone, methyl isobutyl ketone, methyl tert-butyl ketone, ethyl ketone, ethyl propyl ketone and ethyl isopropyl ketone, and acetone and methyl ethyl ketone preferably be exemplified, with acetone being more preferably exemplified.
[00139] [00139] In the present invention, the term "lower alcohol" refers to an alcohol containing 1 to 4 carbon atoms. Its examples can include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, 2-butanol and tert-butanol, and 1-propanol and 2-butanol can be preferably exemplified, being that 1-propanol can be more preferably exemplified.
[00140] Thus, the solution for precipitating the crystals of the compound represented by formula (1) is preferably a solution containing acetone and 1-propanol or a solution containing acetone and 2-butanol, and more preferably a solution containing acetone and 1-propanol .
[00141] [00141] The precipitation of the crystals of the compound represented by formula (1) can also be carried out by adding a seed crystal of the
[00142] [00142] The seed crystal of the crystals of the compound represented by formula (1) can be obtained by directly performing the method described above, but it can preferably be obtained by purifying a small amount of the compound represented by formula (1) by chromatography, then dissolving it in a solvent containing acetone and 1-propanol or a solvent containing acetone and 2-butanol and crystallizing the solution.
[00143] [00143] The compound represented by formula (1) can be produced with reference to the descriptions in International Publication No WO 2014/057687, International Publication No WO 2015/098099, International Publication No WO 2015/115091, International Publication No WO 2015/155998 and so on, but compounds produced by the production methods (I), (II), (III), (V), (VI), and (IX) described below can preferably be used. As a result, the crystals of the compound represented by formula (1) can be produced in high yield, without the use of chromatography, at all stages. Production method (I)
[00144] [00144] The production method (I) is a method for converting a compound represented by formula (B) into the compound represented by formula (1) through steps 1 to 3. Next, steps 1 to 3 will be described in detail . [Chemical formula 82]
[00145] [00145] In the scheme, R1 represents an amino group protected with a protecting group and preferably represents an amino group protected with a benzyloxycarbonyl group, R2 represents a carboxyl group protected with a protecting group and preferably represents a carboxyl group protected with a benzyl group and X represents an active ester group or a carboxyl group and preferably represents an (2,5-dioxopyrrolidin-1-yl) oxycarbonyl group. Step 1:
[00146] [00146] This step is a step of deprotection of protecting groups for an amino group and a carboxyl group of a compound represented by formula (B), in order to convert it into the compound represented by formula (8).
[00147] [00147] The deprotection of the protective groups for the amino group and the carboxyl group of the compound represented by formula (B) can be
[00148] [00148] In the case that R1 is an amino group protected with a benzyloxycarbonyl group and R2 is a carboxyl group protected with a benzyl group, this step can be carried out, preferably, by the following method.
[00149] [00149] The deprotection of the protecting groups for the amino group and the carboxyl group of the compound represented by formula (B) is not limited by its method, as long as the reaction proceeds. It can preferably be carried out using a palladium catalyst, a platinum catalyst, a nickel catalyst, a ruthenium catalyst or a rhodium catalyst under a hydrogen atmosphere, it can be more preferably carried out using a palladium catalyst and can be even more preferably made using palladium-carbon, and palladium-carbon 5% can be even more preferably used. The amount of 5% palladium-carbon used in this step is not limited, as long as the reaction proceeds. It is preferably 5 to 40% by weight with respect to the compound represented by formula (B).
[00150] [00150] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may 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, dimethylsulfoxide and water, and mixed solvents thereof, and a mixed solvent of tetrahydrofuran and water can preferably be exemplified.
[00151] [00151] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 1 to 5
[00152] [00152] This step is a step of condensing the compound represented by formula (8) with a compound represented by formula (C), in order to convert it into the compound represented by formula (10). As the compound represented by formula (C), a commercially available product or a compound produced by a known method or a compound produced by a method according to the production method (VII) described below can be used. If X is an oxycarbonyl (2,5-dioxopyrrolidin-1-yl) group, this step can be carried out, preferably, by the method below.
[00153] [00153] The amount of the compound represented by formula (C) used in this step is not limited, as long as the reaction proceeds. It is preferably 1 to 4 equivalents with respect to the compound represented by formula (8).
[00154] [00154] This step preferably employs a base. The base used in this step is not particularly limited, as long as the reaction proceeds. Their examples can include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, and N-methylpiperidine, and N, N-diisopropylethylamine can be preferably exemplified. The amount of N, N-diisopropylethylamine used in this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 equivalents with respect to the compound represented by formula (8).
[00155] [00155] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may 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-
[00156] [00156] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 7 to 30 hours.
[00157] [00157] The compound represented by formula (10) can preferably be obtained as crystals of a 1,2-dimethoxyethane adduct.
[00158] [00158] The quality of the crystals of the 1,2-dimethoxyethane adduct of the compound represented by formula (10) can be evaluated, for example, based on indices such as an impurity content, a quantity of a residual solvent and aspect. In addition, it can be assessed using, as an index, conservation stability for 3 months, 6 months, 12 months, 24 months and 36 months in an environment of 25 ºC / 60% RH or 40 ºC / 75% RH, for example. With such an assessment of quality, the superiority over an amorphous compound represented by formula (10) can also be confirmed.
[00159] [00159] The crystals of the adduct 1,2-dimethoxyethane of the compound represented by the formula (10), preferably, show main peaks in the diffraction angles (2θ) of 19,0 ° and 25,0 ° in the x-ray diffraction dust, obtained by irradiation with copper Kα radiation. As diffraction angles (2θ) in powder x-ray diffraction can generally cause an error within the ± 0.2 ° range, it should be understood that the values described above the diffraction angles include numerical values within the range of ± 0.2 ° (for good technical sense regarding the measurement and evaluation by powder X-ray diffraction, see, for example, the Japanese Pharmacopoeia, 16th edition, p. 64-68 (2.58 X-Ray Diffraction Method Powder) or the Japanese Pharmacopoeia, 17th edition, pp. 71-74 (2.58 Powder X-Ray Diffraction Method)). So crystals having angles
[00160] [00160] This step is a step of condensing the compound represented by formula (10) with the compound represented by formula (11), in order to convert it into the compound represented by formula (1). The compound represented by formula (11) can preferably be used in the form of a salt of methanesulfonic acid, it can be more preferably used in the form of a salt of methanesulfonic acid m-hydrate, where m is 0 to 3, it can be even more preferably used in the form of anhydride of a salt of methanesulfonic acid, a salt of methanesulfonic acid monohydrate, a salt of methanesulfonic acid dihydrate or a salt of methanesulfonic acid trihydrate, and can be even more preferably used in the form of a salt of methanesulfonic acid dihydrate, but all of these can be used in the production method of the present invention. The number of water molecules in the hydrate described above can be controlled by adjusting the moisture when obtaining or drying the crystals.
[00161] [00161] The amount of the compound represented by the formula (11) used in this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 equivalents with respect to the compound represented by formula (10).
[00162] [00162] The compound represented by formula (10) can preferably be derivatized into an active ester and condensed with the compound represented by formula (11). Derivatization in the active ester at this stage is not limited by your method, as long as the reaction proceeds. It can be carried out, for example, using a condensing agent, such as
[00163] [00163] This step preferably employs a base. The base used in this step is not particularly limited, as long as the reaction proceeds. Their examples can include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine and N-methylpiperidine, and N-methylmorpholine can be preferably exemplified. The amount of N-methylmorpholine used in this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 equivalents with respect to the compound represented by formula (10).
[00164] [00164] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and a mixed solvent of tetrahydrofuran and water can preferably be exemplified.
[00165] [00165] The methanesulfonic acid salt of the compound represented by formula (11) is neutralized with a base to prepare a free form, and then the reaction proceeds. Here, the methanesulfonic acid salt of the compound represented by formula (11) is hydrophilic, whereas the free form of the compound represented by formula (11) is lipophilic. Therefore, so that a series of reactions can proceed efficiently, this step can preferably be carried out in a two-phase system of an aqueous layer and an organic layer. If the organic layer contains tetrahydrofuran, an aqueous solution of high ionic strength, for example, an aqueous solution of sodium sulfate, can preferably be used as an aqueous layer less miscible with it.
[00166] [00166] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 hours. Production method (II)
[00167] [00167] The production method (II) is a method for converting a compound represented by formula (B) into the compound represented by formula (1) through steps 4 to 7. Next, steps 4 to 7 will be described in detail . [Chemical formula 83]
[00168] [00168] In the scheme, R1 represents an amino group protected with a protecting group, R2 represents a carboxyl group protected with a protecting group and X represents an active ester group or a carboxyl group and preferably represents a group (2.5 -dioxopyrrolidin-1-yl) oxycarbonyl. Step 4:
[00169] [00169] This step is a step of deprotecting a protecting group to an amino group of a compound represented by formula (B), in order to convert it into a compound represented by formula (D).
[00170] [00170] The deprotection of the protecting group to the amino group of the compound represented by formula (B) can be accomplished by a method
[00171] [00171] This step is a step of condensing the compound represented by formula (D) with a compound represented by formula (C), in order to convert it into a compound represented by formula (E).
[00172] [00172] This step can be performed in the same way as in step 2 of the production method (I). Step 6:
[00173] [00173] This step is a step of deprotection of the protecting group to the carboxyl group of the compound represented by formula (E), in order to convert it into the compound represented by formula (10).
[00174] [00174] The deprotection of the protecting group to the carboxyl group of the compound represented by formula (B) can be accomplished by a method well known in the art (see, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition (2007), Wiley-Interscience).
[00175] [00175] The compound represented by formula (10) can preferably be obtained as crystals of a 1,2-dimethoxyethane adduct in the same way as in step 2 of the production method (I). Step 7:
[00176] [00176] This step is a step of condensing the compound represented by formula (10) with the compound represented by formula (11), in order to convert it into the compound represented by formula (1).
[00177] [00177] This step can be performed in the same way as in step 3 of the production method (I). Production method (III)
[00178] [00178] The production method (III) is a method for converting
[00179] [00179] In the scheme, R1 represents an amino group protected with a protecting group and preferably represents an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group, R2 represents a carboxyl group protected with a protecting group and preferably represents a group
[00180] [00180] This step is a step of deprotection of a protecting group to a carboxyl group of a compound represented by formula (B), in order to convert it into a compound represented by formula (F).
[00181] [00181] The deprotection of the protecting group to the carboxyl group of the compound represented by formula (B) can be accomplished by a method well known in the art (see, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition (2007), Wiley-Interscience).
[00182] [00182] If R2 is a carboxyl group protected with a benzyl group, this step can be carried out, preferably, by the following method.
[00183] [00183] The deprotection of the protecting group to the carboxyl group of the compound represented by formula (B) is not limited by its method, as long as the reaction proceeds. It can preferably be carried out using a palladium catalyst, a platinum catalyst, a nickel catalyst, a ruthenium catalyst or a rhodium catalyst under a hydrogen atmosphere, it can be more preferably carried out using a palladium catalyst and can be even more preferably carried out using palladium-carbon, and a palladium-carbon-ethylenediamine complex can be even more preferably used. The amount of the palladium-carbon-ethylenediamine complex used in this step is not limited, as long as the reaction proceeds. It is preferably 34 to 136% by weight with respect to the compound represented by formula (B).
[00184] [00184] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile,
[00185] [00185] The reaction temperature of this step is preferably 10 to 40 ºC, but is not limited to it, as long as the reaction proceeds. The reaction time for this step is preferably 1 to 54 hours, but is not limited to it, as long as the reaction proceeds. Step 9:
[00186] [00186] This step is a step of condensing the compound represented by formula (F) with the compound represented by formula (11), in order to convert it into a compound represented by formula (G).
[00187] [00187] The compound represented by the formula (F) can preferably be derivatized in an active ester and condensed with the compound represented by the formula (11). The amount of the compound represented by the formula (11) used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (F). Derivatization in the active ester at this stage is not limited by your method, as long as the reaction proceeds. It can be carried out, for example, using a condensing agent, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD-HCl) or N, N'-dicyclohexylcarbodiimide (DCC) , and reacting with an additive such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, cyano (hydroxyimino) ethyl acetate or p-nitrophenol, and can preferably be carried out using 3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole. The amount of
[00188] [00188] This step preferably employs a base. The base used in this step is not particularly limited, as long as the reaction proceeds. Their examples can include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine and N-methylpiperidine, and triethylamine can be preferably exemplified. The amount of triethylamine used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (F).
[00189] [00189] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and tetrahydrofuran can preferably be exemplified.
[00190] [00190] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 1 to 4 hours. Step 10:
[00191] [00191] This stage is a stage of deprotection of the protecting group to
[00192] [00192] The deprotection of the protecting group to the amino group of the compound represented by formula (G) can be accomplished by a method well known in the art (see, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition (2007), Wiley-Interscience).
[00193] [00193] In the case that R1 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group, this step can be carried out, preferably, by the following method.
[00194] [00194] The deprotection of the protecting group to the amino group of the compound represented by formula (G) is not particularly limited, as long as the reaction proceeds. It can be performed using, for example, 1,8-diazabicyclo [5,4,0] -7-undecene, trimethylguanidine, 1,5,7-triazabicyclo [4,4,0] dec-5-ene or 7-methyl -1,5,7-triazabicycles [4,4,0] dec-5-ene, 1,5-diazabicycles [4,3,0] -5-nonene, and can preferably be performed using 1,8-diazabicycles [ 5.4.0] -7-undecene. The amount of 1,8-diazabicyclo [5,4,0] -7-undecene used in this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 equivalents with respect to the compound represented by formula (15).
[00195] [00195] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and tetrahydrofuran can preferably be exemplified.
[00196] [00196] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 1 to 5 hours. Step 11:
[00197] [00197] This step is a step of condensing the compound represented by formula (16) with a compound represented by formula (C), in order to convert it into the compound represented by formula (1). As the compound represented by formula (C), a commercially available product, a compound produced by a known method or a compound produced by a method according to the production method (VII) described below can be used. If X is an oxycarbonyl (2,5-dioxopyrrolidin-1-yl) group, this step can be carried out, preferably, by the method below.
[00198] [00198] The amount of the compound represented by formula (C) used in this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 equivalents with respect to the compound represented by formula (16).
[00199] [00199] This step preferably employs a base. The base used in this step is not particularly limited, as long as the reaction proceeds. Their examples can include triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine and N-methylpiperidine, and triethylamine can be preferably exemplified. The amount of triethylamine used in this step is not limited, as long as the reaction proceeds. It is preferably 0.75 to 6 equivalents with respect to the compound represented by formula (16).
[00200] [00200] This step can preferably also employ pyridinium p-toluenesulfonate. The amount of pyridinium p-toluenesulfonate used in this step is not limited, as long as the reaction proceeds.
[00201] [00201] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide, pyridine and water, and mixed solvents thereof , and a mixed solvent of pyridine, acetonitrile and tetrahydrofuran can be preferably exemplified.
[00202] [00202] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 1.5 to 7 hours.
[00203] [00203] As the compound represented by formula (B) in production methods (I) to (III), a compound produced by the production method (IV) below can preferably be used. Production method (IV)
[00204] [00204] The production method (IV) is a method for converting a compound represented by formula (H) into the compound represented by formula (B) through steps 12 to 15. Next, steps 12 to 15 will be described in detail . [Chemical formula 85]
[00205] [00205] In the scheme, R1 represents an amino group protected with a protecting group and preferably represents an amino group protected with a benzyloxycarbonyl group or a (9H-fluoren-9-ylmethoxy) carbonyl group, R2 represents a protected carboxyl group with a protecting group and preferably represents a carboxyl group protected with a benzyl group, R3 represents an amino group protected with a protecting group, and preferably represents an amino group protected with a (9H-fluoren-9-ylmethoxy group) ) carbonyl and X represents an active ester group or a carboxyl group and preferably represents an (2,5-dioxopyrrolidin-1-yl) oxycarbonyl group. Step 12:
[00206] [00206] This step is a reaction step of a compound represented by the formula (H) with lead tetracetate in order to convert it into a compound represented by the formula (J). As the compound represented by the formula (H), a commercially available product or a compound produced with reference to a method can be used
[00207] [00207] This step can preferably be carried out in the presence of acetic acid or pyridine, and can be more preferably carried out in the presence of acetic acid.
[00208] [00208] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2- pyrrolidone and dimethylsulfoxide, and mixed solvents thereof, and tetrahydrofuran can be preferably exemplified.
[00209] [00209] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 45 to 85 ° C and, more preferably, a temperature that reaches heating to reflux of tetrahydrofuran. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 3 hours. Step 13:
[00210] [00210] This step is a reaction step of the compound represented by the formula (J) with a compound represented by the formula (K) in the presence of an acid or a base, in order to convert it into a compound represented by the formula (L ). The amount of the compound represented by the formula (K) used in this step is not limited, as long as the reaction proceeds. It is preferably 1 to 4 equivalents with respect to the compound represented by formula (J).
[00211] [00211] This step can be performed in the presence of a base or an acid. The base used in this step is preferably an aqueous solution of sodium hydroxide. The amount of the aqueous sodium hydroxide solution used in this step is not limited, as long as the reaction proceeds. IT IS
[00212] [00212] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Their examples can include 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran and 1,4-dioxane, and 1,2-dimethoxyethane can preferably be exemplified.
[00213] [00213] The reaction temperature of this step is not limited, as long as the reaction proceeds. It is preferably -10 to 15 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 6 hours. Step 14:
[00214] [00214] This step is a step of deprotection of the protecting group to the amino group of the compound represented by the formula (L), in order to convert it into a compound represented by the formula (M).
[00215] [00215] Deprotection of the protecting group to the amino group of the compound represented by the formula (L) can be accomplished by a method well known in the art (see, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition (2007), Wiley-Interscience).
[00216] [00216] If R3 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group, this step can be carried out, preferably, by the method below.
[00217] [00217] The deprotection of the protecting group to the amino group of the compound represented by the formula (L) is not particularly limited, as long as the reaction proceeds. It can be performed using, for example, 1.8-
[00218] [00218] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and acetonitrile and N, N-dimethylacetamide can preferably be exemplified.
[00219] [00219] The reaction temperature of this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 2 to 8 hours.
[00220] [00220] The compound represented by the formula (M) can be precipitated from the reaction solution by the formation of a salt with an acid and, preferably, isolated and purified. As a result, by-products, which can be an inhibiting factor for reactions in subsequent steps, can be removed.
[00221] [00221] The acid described above is preferably 1-hydroxybenzotriazole. The 1-hydroxybenzotriazole used in this step can also function as one of the condensing agents in the next step. Likewise, an acid other than 1-hydroxybenzotriazole can be used
[00222] [00222] This step is a step of condensing the compound represented by formula (M) with a compound represented by formula (N), in order to convert it into the compound represented by formula (B). As the compound represented by the formula (N), a commercially available product, a compound produced by a known method or a compound produced by a method according to the production method (VIII) described below can be used. The amount of the compound represented by the formula (N) used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (M).
[00223] [00223] The compound represented by the formula (M) can preferably be derivatized in an active ester and condensed with the compound represented by the formula (N). Derivatization in the active ester can be carried out, for example, using a condensing agent, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD-HCl) or N, N'-dicyclohexylcarbodi -imide (DCC), and reacting with an additive such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide, cyano (hydroxyimino) ethyl acetate or p-nitrophenol, and it can preferably be carried out using 3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1-hydroxybenzotriazole. The amount of 3- (3-dimethylaminopropyl) carbodiimide hydrochloride used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (5). The amount of 1-hydroxybenzotriazole used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (M).
[00224] [00224] In the case where the compound represented by formula (M) is in the form of a salt of 1-hydroxybenzotriazole, this step can preferably be carried out without the addition of fresh 1-hydroxybenzotriazole.
[00225] [00225] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and a mixed solvent of acetonitrile and water can preferably be exemplified.
[00226] [00226] In the case where the compound represented by formula (M) is not isolated in step 14, and that step is carried out continuously from it, the solvent used in step 14 can be used as is as the solvent of that step.
[00227] [00227] the reaction temperature of this step is not limited, as long as the reaction proceeds. It is preferably -10 to 15 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 1.5 to 7 hours.
[00228] [00228] In a more specific aspect, the compound represented by formula (1) can preferably be produced by the following production method (V) or (VI), and used. Production method (V)
[00229] [00229] The production method (V) is a method for converting the compound represented by formula (2) into the compound represented by formula (1) through steps 16 to 22. Next, steps 16 to 22 will be described in detail. [Chemical formula 86]
[00230] [00230] This step is a reaction step of the compound represented by formula (2) with lead tetracetate in order to convert it into the compound represented by formula (3). As the compound represented by formula (2), a commercially available product or a compound produced with reference to a known method can be used. This step can be
[00231] [00231] This step is a reaction step of the compound represented by formula (3) with benzyl glycolate, in the presence of an acid or a base, in order to convert it into the compound represented by formula (4). This step can be performed in the same way as in step 13 of the production method (IV). Step 18:
[00232] [00232] This step is a step of deprotecting the protecting group to the amino group of the compound represented by formula (4), in order to convert it into the compound represented by formula (5). This step can be performed in the same way as in step 14 of the production method (IV). Step 19:
[00233] [00233] This step is a step of condensing the compound represented by formula (5) with the compound represented by formula (6), in order to convert it into the compound represented by formula (7). As the compound represented by formula (6), a commercially available product, a compound produced by a known method or a compound produced by the production method (VIII) described below can be used. This step can be performed in the same way as in step 15 of the production method (IV). Step 20:
[00234] [00234] This step is a step of deprotecting the protecting groups to the amino group and the carboxyl group of the compound represented by formula (7), in order to convert it into the compound represented by formula (8). This step can be performed in the same way as in step 1 of the production method (I). Step 21:
[00235] [00235] This stage is a stage of condensation of the compound
[00236] [00236] This step is a step of condensing the compound represented by formula (10) with the compound represented by formula (11), in order to convert it into the compound represented by formula (1). This step can be performed in the same way as in step 3 of the production method (I). Production method (VI)
[00237] [00237] The production method (VI) is a method for converting the compound represented by formula (2) into the compound represented by formula (1) through steps 23 to 30. Next, steps 23 to 30 will be described in detail. [Chemical formula 87]
[00238] [00238] This step is a reaction step of the compound represented by formula (2) with lead tetracetate in order to convert it into the compound represented by formula (3). As the compound represented by formula (2), a commercially available product or a compound
[00239] [00239] This step is a reaction step of the compound represented by formula (3) with benzyl glycolate, in the presence of an acid or a base, in order to convert it into the compound represented by formula (4). This step can be performed in the same way as in step 13 of the production method (IV). Step 25:
[00240] [00240] This step is a step of deprotecting the protecting group to the amino group of the compound represented by formula (4), in order to convert it into the compound represented by formula (5). This step can be performed in the same way as in step 14 of the production method (IV). Step 26:
[00241] [00241] This step is a step of condensing the compound represented by formula (5) with the compound represented by formula (12), in order to convert it into the compound represented by formula (13). As the compound represented by formula (13), a commercially available product or a compound produced by a known method can be used. This step can be performed in the same way as in step 15 of the production method (IV). Step 27:
[00242] [00242] This step is a step of deprotecting the protecting group to the carboxyl group of the compound represented by formula (13), in order to convert it into the compound represented by formula (14). This step can be performed in the same way as in step 8 of the production method (III). Step 28:
[00243] [00243] This step is a step of condensing the compound represented by the formula (14) with the compound represented by the formula
[00244] [00244] This step is a step of unprotecting the protecting group to the amino group of the compound represented by formula (15), in order to convert it into the compound represented by formula (16). This step can be performed in the same way as in step 10 of the production method (III). Step 30:
[00245] [00245] This step is a step of condensing the compound represented by formula (16) with the compound represented by formula (9), in order to convert it into the compound represented by formula (1). This step can be performed in the same way as in step 11 of the production method (III). Production method (VII)
[00246] [00246] The compound represented by the formula (9) can preferably be produced by the production method (VII) described below, and used. As a result, impurities that could influence the quality of compounds produced in subsequent steps can be suppressed, and this can contribute to obtaining the high quality compound represented by formula (1). [Chemical formula 88] 工程 ３１ Step 31 工程 ３２ Step 32 (17) (18) (9) Step 31:
[00247] [00247] This step is a step of condensing the compound represented by formula (17) with maleic anhydride in order to convert it into the compound represented by formula (18). The amount of maleic anhydride used in this step is not limited, as long as the reaction proceeds. IT IS
[00248] [00248] This step is preferably performed in acetic acid.
[00249] [00249] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably 80 to 120 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 8 to 32 hours. Step 32:
[00250] [00250] This step is a step of condensing the compound represented by formula (18) with N-hydroxysuccinimide in order to convert it into the compound represented by formula (9). The amount of N-hydroxysuccinimide used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (17).
[00251] [00251] The compound represented by formula (18) can be derivatized into an active ester, a mixed acid anhydride or an acid halide etc. and condensed with N-hydroxysuccinimide, or it can preferably be derivatized in an acid halide and condensed with N-hydroxysuccinimide.
[00252] [00252] The derivatization in the acid halide can preferably be carried out using thionyl chloride. The amount of thionyl chloride used in this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 1.5 equivalents with respect to the compound represented by formula (18). In this step, a base is preferably used. The base used in this step is preferably 2,6-lutidine. The amount of 2,6-lutidine used in this step is not limited, as long as the reaction proceeds. It is preferably 1 to 3 equivalents with respect to the compound represented by formula (18).
[00253] [00253] The solvent used in this step is not particularly limited,
[00254] [00254] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably -25 ° C to 0 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 0.5 to 2 hours. Production method (VIII)
[00255] [00255] The compound represented by formula (6) can be produced by the production method (VIII) below, and used. [Chemical formula 89] Step 33 工程 ３３ (23) (24) Step 34 工程 ３４ (6) Step 33:
[00256] [00256] This step is a step of condensing the compound represented by formula (23) with N-hydroxysuccinimide in order to convert it into the compound represented by formula (24). The amount of the compound represented by the formula (23) used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.5 equivalents with respect to the compound represented by formula (23).
[00257] [00257] The compound represented by formula (23) can be
[00258] [00258] The active esterification can preferably be carried out using 3- (3-dimethylaminopropyl) carbodiimide hydrochloride. The amount of 3- (3-dimethylaminopropyl) carbodiimide hydrochloride used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.5 equivalents with respect to the compound represented by formula (23).
[00259] [00259] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and acetonitrile can preferably be exemplified.
[00260] [00260] The reaction temperature of this step is not limited, as long as the reaction proceeds. It is preferably 10 to 40 ° C. The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 2 to 8 hours. Step 34:
[00261] [00261] This step is a step of condensing the compound represented by formula (24) with L-phenylalanine in order to convert it into the compound represented by formula (6). The amount of L-phenylalanine used in this step is not limited, as long as the reaction proceeds. It is preferably 0.7 to 1.3 equivalents with respect to the compound represented by formula (24).
[00262] [00262] This step preferably employs a base. The base
[00263] [00263] The solvent used in this step is not particularly limited, as long as the reaction is not inhibited. Examples may include acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cycle -hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2-butanone, N, N-dimethylformamide, N, N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylsulfoxide and water, and mixed solvents thereof, and a mixed solvent of acetonitrile and water can preferably be exemplified.
[00264] [00264] The reaction temperature for this step is not limited, as long as the reaction proceeds. It is preferably the compound represented by formula (23). The reaction time for this step is not limited, as long as the reaction proceeds. It is preferably 1 to 4 hours. Production method (IX)
[00265] [00265] The compound represented by formula (1) can also be produced by the production method (IX) below, and used. [Chemical formula 90]
[00266] [00266] In the scheme, R1 represents an amino group protected with a protecting group, R2 represents a carboxyl group protected with a protecting group, R3 represents an amino group protected with a protecting group and X represents an active ester group or a carboxyl group and preferably, it represents an (2,5-dioxopyrrolidin-1-yl) oxycarbonyl group.
[00267] [00267] This step is a reaction step of a compound represented by the formula (H) with lead tetracetate in order to convert it into a compound represented by the formula (J). As the compound represented by the formula (H), a commercially available product or a compound produced with reference to a known method can be used. This step can be performed in the same way as in step 12 of the production method (IV). Step 36:
[00268] [00268] This step is a reaction step of the compound represented by the formula (J) with a compound represented by the formula (K) in the presence of an acid or a base, in order to convert it into a compound represented by the formula (L ). This step can be performed in the same way as in step 13 of the production method (IV). Step 37:
[00269] [00269] This step is a step of deprotection of the protecting group to the carboxyl group of the compound represented by the formula (L), in order to convert it into a compound represented by the formula (O).
[00270] [00270] The deprotection of the protecting group to the carboxyl group of the compound represented by the formula (L) can be accomplished by a method well known in the art (see, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition (2007), Wiley-Interscience). Step 38:
[00271] [00271] This step is a step of condensing the compound represented by formula (O) with the compound represented by formula (11), in order to convert it into a compound represented by formula (P).
[00272] [00272] The compound represented by the formula (O) can preferably be derivatized in an active ester and condensed with the
[00273] [00273] This step is a step of deprotection of the protecting group to the amino group of the compound represented by formula (P), in order to convert it into the compound represented by formula (25).
[00274] [00274] The deprotection of the protecting group to the carboxyl group of the compound represented by the formula (P) can be accomplished by a method well known in the art (see, for example, Peter GM Wuts, Theodora W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition (2007), Wiley-Interscience). Step 40:
[00275] [00275] This step is a step of condensing the compound represented by formula (25) with a compound represented by formula (N), in order to convert it into a compound represented by formula (G).
[00276] [00276] The compound represented by the formula (N) can preferably be derivatized in an active ester and condensed with the compound represented by the formula (25). Step 41:
[00277] [00277] This step is a step of unprotecting the protecting group to the amino group of the compound represented by formula (G), in order to convert it into the compound represented by formula (16). This step can be performed in the same way as in step 10 of the production method (III). Step 42:
[00278] [00278] This step is a step of condensing the compound represented by formula (16) with a compound represented by formula (C), in order to convert it into the compound represented by formula (1). This step can be performed in the same way as in step 11 of the production method (III). Antibody for use in the production of an antibody-drug conjugate
[00279] [00279] The antibody for use in the production of the antibody-drug conjugate of the present invention can be derived from any species, and is preferably an antibody derived from human, rat, mouse or rabbit. In cases of being derived from a species other than human, the antibody is preferably chimeric or humanized using a well known technique. The antibody of the present invention can be a polyclonal antibody or a monoclonal antibody, preferably being a monoclonal antibody.
[00280] [00280] The antibody for use in the production of the antibody-drug conjugate of the present invention is an antibody that preferably has the characteristic of being able to target cancer cells, and is preferably an antibody that has, for example, the recognition property of a cancer cell, the property of binding to a cancer cell, the property of internalization in a cancer cell and / or cytocidal activity against cancer cells.
[00281] [00281] The binding activity of the antibody against cancer cells can be confirmed by flow cytometry. The internalization of the antibody in tumor cells can be confirmed using (1) an antibody visualization assay incorporated into cells under a fluorescence microscope by binding a secondary antibody (fluorescently labeled) to the therapeutic antibody (Cell Death and Differentiation ( 2008) 15, 751-761), (2) an assay for measuring the intensity of fluorescence incorporated into cells by binding a secondary antibody (fluorescence-labeled) to the therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay using the binding of an immunotoxin to the therapeutic antibody, in which the toxin is released upon incorporation into cells to inhibit cell growth (Bio Techniques 28: 162 -165, January 2000). Like the immunotoxin, a recombinant protein complex formed
[00282] [00282] The antitumor activity of the antibody can be confirmed in vitro by determining the inhibitory activity against cell growth. For example, a cancer cell line with overexpression of a target protein for the antibody is cultured, and the antibody is added to the culture system in varying concentrations to determine the inhibitory activity against the formation of foci, the formation of colonies and the growth of spheroids. The antitumor activity can be confirmed in vivo, for example, by administering the antibody to a nude mouse, a lineage of transplanted cancer cells with high expression of the target protein and determining a change in the cancer cell.
[00283] [00283] As the conjugated compound in the antibody-drug conjugate exerts an anti-tumor effect, it is preferable, but not essential that the antibody itself has an anti-tumor effect. For the cytotoxic activity of the antitumor compound to be exerted specifically and selectively against cancer cells, it is important and also preferable that the antibody has the internalizing property and migrates to cancer cells.
[00284] [00284] The antibody for use in the production of the antibody-drug conjugate of the present invention can be obtained by a procedure known in the art. For example, the antibody of the present invention can be obtained by a method normally performed in the art, which involves immunizing animals with an antigenic polypeptide and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to humans, and animals can be immunized with an antigen derived from a non-human animal such as a mouse, a rat and the like. In that case, the cross-reactivity of antibodies that bind to the heterologous antigen obtained with human antigens can be tested to screen for an antibody applicable to a human disease.
[00285] [00285] Alternatively, antibody-producing cells that
[00286] [00286] The antigen can be obtained by genetically modifying the host cells to produce a gene that encodes the antigenic protein. Specifically, vectors are prepared that allow expression of the antigen gene and transferred to host cells so that the gene is expressed. The antigen so expressed can be purified. The antibody can also be obtained by a method in which animals are immunized with the genetically modified cells described above that express the antigen or a cell line that expresses the antigen.
[00287] [00287] The antibody for use in the production of the antibody-drug conjugate of the present invention is preferably a recombinant antibody obtained by artificial modification for the purpose of decreasing heterologous antigenicity against humans, such as a chimeric antibody or a humanized antibody, or is preferably a antibody containing only the gene sequence of an antibody derived from a human, that is, a human antibody. Such antibodies can be produced using a known method.
[00288] [00288] Like the chimeric antibody, an antibody can be exemplified in which variable and constant antibody regions are derived from different species, for example, a chimeric antibody in which a variable region derived from mouse or rat is connected to a constant region antibody derived from humans (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).
[00289] [00289] Like the humanized antibody, an antibody obtained integrating only the complementarity determining region (CDR) of
[00290] [00290] Like the human antibody, an antibody generated using a human antibody-producing mouse having a human chromosome fragment including heavy chain and light chain genes from a human antibody (see Tomizuka, K. et al., Nature Genetics (1997 ) 16, pp. 133-143; Kuroiwa, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. Al., Animal Cell Technology: Basic and Applied Aspects, vol. 10, 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, p. 722-727, etc.) can be exemplified. Alternatively, an antibody obtained by phage expression can be exemplified, the antibody being selected from a library of human antibodies (see Wormstone, IM et. Al, Investigative Ophthalmology & Visual Science (2002), 43 (7), p 2301-2308; Carmen, S. et. Al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 ( 3), pp. 427-431, etc.).
[00291] [00291] In the present invention, modified variants of the antibody for use in the production of the antibody-drug conjugate of the present invention are also included. The modified variant refers to a variant obtained by subjecting the antibody according to the present invention to chemical or biological modification. Examples of the chemically modified variant include variants with a chemical group attached to an amino acid backbone, variants with an N-linked or O-linked chemical group
[00292] [00292] In addition, by regulating the modification of a glycan that is bound to the antibody according to the present invention (glycosylation, defucosylation etc.), it is possible to enhance the antibody-dependent cellular cytotoxic activity. As the technique for regulating the modification of an antibody glycan, WO 99/54342, WO 00/61739, WO 02/31140 etc. are known. However, the technique is not limited to them. In the antibody according to the present invention, antibodies in which the modification of a glycan is regulated are also included.
[00293] [00293] It is known that a lysine residue in the terminal carboxyl of the heavy chain of an antibody produced in a cultured mammalian cell is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) in the terminal carboxyl of the heavy chain of an antibody produced in a cultured mammalian cell are deleted and a proline residue recently located in the terminal carboxyl is amidated (Analytical
[00294] [00294] As isotypes of the antibody according to the present invention, for example, IgG (IgG1, IgG2, IgG3, IgG4) can be exemplified, and IgG1 or IgG2 can be exemplified preferably.
[00295] [00295] Examples of antibodies applicable to the production of the antibody-drug conjugate of the present invention may include, but not be limited to, them
[00296] [00296] 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 internalization in cells that express HER2 by binding to HER2.
[00297] Examples of the anti-HER2 antibody may include trastuzumab (U.S. Patent No. 5,821,337) and pertuzumab (International Publication No. WO 01/00245), and trastuzumab can preferably be exemplified.
[00298] [00298] In the present invention, the term "trastuzumab" refers to the humanized anti-HER2 monoclonal antibody that comprises a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 (Figure 1) and a light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2 (Figure 2).
[00299] [00299] 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 internalization in cells that express HER3 by binding to HER3 on the surface of cells that express HER3.
[00300] [00300] Examples of the anti-HER3 antibody may include patritumab
[00301] [00301] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2; EGP-1) and, preferably, has as activity the internalization in cells that express TROP2 by binding to TROP2.
[00302] [00302] Examples of the anti-TROP2 antibody may include hTINA1- H1L1 (International Publication No. WO 2015/098099).
[00303] [00303] In the present invention, the term "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B cell antigen number 7 homologue; PD-L3; CD276) and, preferably, it has the activity of internalizing cells that express B7-H3 by binding to B7-H3.
[00304] [00304] Examples of the anti-B7-H3 antibody may include M30-H1-L4 (International Publication No. WO 2014/057687).
[00305] [00305] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that specifically binds to GPR20 (Receptor 20 coupled to the G protein) and preferably has the activity of internalizing in cells that express GPR20 connection to the GPR20.
[00306] [00306] Examples of the anti-GPR20 antibody may include h046-H4e / L7 (International Publication No. WO 2018/135501). Conjugation between the antibody and the drug-binding intermediate
[00307] [00307] The antibody-drug conjugate of the present invention can be produced by reacting the compound represented by formula (1) and an antibody having a thiol group (alternatively, referred to as a group
[00308] [00308] The crystals of the compound represented by formula (1) of the present invention are preferably dissolved in a solvent to prepare a solution containing the compound represented by formula (1), which can then be used in the reaction. The solvent for use in this step is not particularly limited, as long as the reaction is not inhibited. Preferably, a solvent containing dimethylsulfoxide, dimethylformamide, dimethylacetamide, or N-methylpyrrolidone can be used, and a solvent containing dimethylsulfoxide can be more preferably used.
[00309] [00309] The antibody having a sulfhydryl group can be obtained by a method well known in the art (Hermanson, G. T, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, using 0.3 to 3 molar equivalents of a reducing agent, such as tris (2-carboxyethyl) phosphine hydrochloride (TCEP), by disulfide between chains in the antibody, and reacting with the antibody in a buffer solution containing a chelating agent such as ethylene diaminetetraacetic acid (EDTA), an antibody can be obtained having a sulfhydryl group with disulfides between chains partially or completely reduced in the antibody.
[00310] [00310] Furthermore, with the use of 2 to 20 molar equivalents of the compound represented by formula (1) by antibody having a sulfhydryl group, an antibody-drug conjugate can be produced in which 2 to 8 molecules of the drug are conjugated by antibody molecule.
[00311] [00311] The average number of conjugated molecules of the drug per antibody molecule of the antibody-drug conjugate produced can be determined, for example, by a calculation method based on the UV absorbance measured for the antibody-drug conjugate and for the precursor of conjugation at two wavelengths, 280 nm and 370 nm (UV method), or a calculation method based on quantification by measuring fragments obtained by treating the conjugate
[00312] [00312] The conjugation between the antibody and the linker-drug intermediate (compound represented by formula (1)) and the calculation of the average number of conjugated molecules of the drug per antibody molecule of the antibody-drug conjugate can be performed with reference to the descriptions in International Publication No. WO 2014/057687, International Publication No. WO 2015/098099, International Publication No. WO 2015/115091, International Publication No. WO 2015/155998 and International Publication No. WO 2018/135501, and so on.
[00313] [00313] In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-HER2 antibody.
[00314] [00314] The anti-HER2 antibody is preferably an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence consisting of at amino acid residues 1 to 214 of SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2 .
[00315] [00315] The average number of units of the conjugated linker-drug per antibody molecule in the anti-HER2-drug antibody conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8 , even more preferably 7.5 to 8 and even more preferably close to 8.
[00316] [00316] The anti-HER2-drug antibody conjugate can be produced with reference to the descriptions in International Publication No. WO 2015/115091 and so on, using the crystals of the compound represented by formula (1) produced by the production method of
[00317] [00317] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate is an anti-HER3 antibody.
[00318] [00318] The anti-HER3 antibody is preferably an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4, or a variant of antibody in which a lysine residue in the terminal carboxyl of the antibody heavy chain is deleted.
[00319] [00319] The average number of units of the conjugated linker-drug per antibody molecule in the anti-HER3-drug antibody conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8 , even more preferably 7.5 to 8 and even more preferably close to 8.
[00320] [00320] The anti-HER3-drug antibody conjugate can be produced with reference to the descriptions in International Publication No. WO 2015/155998 and so on, using the crystals of the compound represented by formula (1) produced by the production method of the present invention .
[00321] [00321] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.
[00322] [00322] The anti-TROP2 antibody is preferably an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of an amino acid sequence consisting of in amino acid residues 21 to 234 of SEQ ID NO: 6, or an antibody variant in which a lysine residue in the terminal carboxyl of
[00323] [00323] The average number of units of the conjugated linker-drug per antibody molecule in the anti-TROP2-drug-conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 5, even more preferably 3.5 at 4.5 and even more preferably close to 4.
[00324] [00324] The anti-TROP2 antibody-drug conjugate can be produced with reference to the descriptions in International Publication No. WO 2015/098099 and so on, using the crystals of the compound represented by formula (1) produced by the production method of the present invention .
[00325] [00325] In the present invention, the term "anti-B7-H3-drug antibody conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.
[00326] [00326] The anti-B7-H3 antibody is preferably an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 8, or an antibody variant in which a lysine residue in the terminal carboxyl of the antibody heavy chain is deleted.
[00327] [00327] The average number of units of the conjugated linker-drug per antibody molecule in the anti-B7-H3-drug antibody conjugate 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 close to 4.
[00328] [00328] The anti-B7-H3-drug antibody conjugate can be produced with reference to the descriptions in International Publication No. WO 2014/057687 and so on, using the crystals of the compound
[00329] [00329] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.
[00330] [00330] The anti-GPR20 antibody is preferably an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of an amino acid sequence consisting of in amino acid residues 21 to 234 of SEQ ID NO: 10, or an antibody variant in which a lysine residue in the terminal carboxyl of the antibody heavy chain is deleted.
[00331] [00331] The average number of units of the conjugated linker-drug per antibody molecule in the anti-GPR20 antibody-drug conjugate produced according to the present invention is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8 , even more preferably 7.5 to 8 and even more preferably close to 8.
[00332] [00332] The anti-GPR20 antibody-drug conjugate can be produced with reference to the descriptions in International Publication No. WO 2018/135501 and so on, using the crystals of the compound represented by formula (1) produced by the production method of the present invention . Pharmaceutical compositions
[00333] [00333] The antibody-drug conjugate produced by the present invention can contain at least one pharmaceutically suitable ingredient and be administered. The pharmaceutically suitable ingredient can be properly selected and applied among formulation additives, or the like, which are generally used in the art, according to the dose, the concentration administered, and so on, of the
[00334] [00334] It can be predicted that the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention has a therapeutic effect by applying it as a systemic therapy to patients and, additionally, by applying it locally to cancerous tissues.
[00335] [00335] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can preferably be used for a mammal, and can be more preferably used for a human.
[00336] [00336] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can preferably be used as an injection, can be more preferably used as an aqueous injection or lyophilized injection, and can even more preferably be used as a lyophilized injection.
[00337] [00337] In the case of an aqueous injection, preferably, the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can be diluted with a suitable diluent and then administered intravenously with drip infusion. Examples of the diluent may include glucose solution (preferably 5% glucose solution) and physiological saline.
[00338] [00338] In the case of a lyophilized injection, preferably, the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can be dissolved in water for injections, and then a necessary amount can be diluted with a suitable diluent and then administered intravenously with drip infusion. The
[00339] [00339] Examples of administration route that can be used to administer the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can include intravenous, intradermal, subcutaneous, intramuscular and intraperitoneal routes, and intravenous routes can preferably be exemplified.
[00340] [00340] The antibody-drug conjugate produced by the present invention can be administered to a human at intervals of once a day every 180 days, preferably it can be administered at intervals of once a week, every 2 weeks, every 3 weeks or every 4 weeks and, even more preferably, it can be administered at intervals of once every 3 weeks. In addition, the antibody-drug conjugate produced by the present invention can be administered at a dose between approximately 0.001 and 100 mg / kg per dose and, preferably, can be administered at a dose of 0.8 to 12.4 mg / kg per dose. In the case of an anti-HER2 antibody-drug conjugate, the antibody-drug conjugate produced by the present invention can preferably be administered at a dose of 5.4; 6.4 or 7.4 mg / kg per dose, and more preferably it can be administered at a dose of 5.4 mg / kg or 6.4 mg / kg per dose.
[00341] [00341] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can be used for the treatment of cancer, and can preferably be used for the treatment of at least one type of cancer selected from the group consisting of cancer of breast, gastric cancer (also called gastric adenocarcinoma), colorectal cancer (also called colon and rectal cancer and including colon and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer ), esophageal cancer, salivary gland cancer, junction adenocarcinoma
[00342] [00342] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can be selectively used as an agent for drug therapy, which is a major method for the treatment of cancer and, as a result, can delay the development of
[00343] [00343] In such drug therapy, the pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can be used as an isolated agent and, in addition, it can be used in combination with an additional therapy in adjuvant therapy and can be combined with surgery, radiation therapy, hormonal therapy or the like. Furthermore, it can also be used as an agent for drug therapy in neoadjuvant therapy.
[00344] [00344] In addition to the therapeutic use as described above, for example, a prophylactic effect can be expected, such as suppressing the growth of small metastatic cancer cells and even eliminating them, for the pharmaceutical composition containing the antibody-drug conjugate produced by present invention. For example, an effect of inhibiting and eliminating cancer cells in a body fluid in the course of metastasis or an effect of, for example, inhibiting and eliminating small cancer cells immediately after implantation in any tissue can be expected. Thus, inhibition of cancer metastasis or a prophylactic effect can be expected, particularly after surgical removal of the cancer.
[00345] [00345] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention can be administered in combination with other agents for the treatment of cancer. The effect
[00346] [00346] The present invention is described in more detail below by way of examples. However, the present invention is not limited to these.
[00347] [00347] In the Examples, the terms "NMR-1H" and "NMR-13C" mean "nuclear magnetic resonance spectrum". Within parentheses, CDCl3 means deuterated chloroform which is a measuring solvent, DMSO-d6 means deuterated dimethyl sulfoxide which is a measuring solvent, D2O means deuterium oxide which is a measuring solvent and MeOH-d4 means deuterated methanol which is a solvent measuring TMS (tetramethylsilane) was used as an internal standard. The meanings of multiplicity in 1H NMR are s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and brs = broad singlet. Example 1 2,5-dioxopyrrolidin-1-yl N - [(benzyloxy) carbonyl] glycylglycinate [Chemical formula 91]
[00348] [00348] To a mixture of N - [(benzyloxy) carbonyl] glycylglycine (200.00 g, 0.751 mol) and acetonitrile (2.0 L), were added N-hydroxysuccinimide (95.10 g, 0.826 mol) and hydrochloride 1- (3-dimethylaminopropyl) -3-ethylcarbodimide (172.80 g, 0.901 mol), and the resulting mixture was stirred at room temperature for approximately 4 hours. The reaction solution was cooled to 1 ºC and stirred for approximately 3 hours. The precipitates were filtered, and a powder separated by filtration was washed with acetonitrile (400 ml). The obtained powder was dried under reduced pressure at 40 ° C to obtain 2,5-dioxopyrrolidin-1-yl N- [(benzyloxy) carbonyl] glycylglycinate (221.6 g, 0.610 mol, yield: 81.2%).
[00349] [00349] 1H NMR (400 MHz, DMSO-d6) δ 2.81 (4H, s), δ 3.69 (2H, d, 6.7 Hz), δ 4.28 (2H, d, 6, 1 Hz), δ 5.04 (2H, s), δ 7.29-7.39 (5H, m), δ 7.56 (1H, t, 6.4 Hz), δ 8.55 (1H, t, 5.8 Hz).
[00350] [00350] 13 C NMR (100 MHz, DMSO-d6) δ 25.4; 38.2; 43.3; 65.6; 127.7; 127.8; 128.3; 137.0; 156.5; 166.3; 170.0; 170.0.
[00351] [00351] MS (ESI) (m / z): 364 ([M + H] +). Example 2 N - [(Benzyloxy) carbonyl] glycylglycyl-L-phenylalanine [Chemical formula 92] (6)
[00352] [00352] To a mixture of L-phenylalanine (80.0 g, 0.487 mol), acetonitrile (400 ml) and water (400 ml), were added triethylamine (74.7
[00353] [00353] 1H NMR (400 MHz, DMSO-d6) δ 2.86-2.91 (1H, m), δ 3.03-3.08 (1H, m), δ 3.64-3.78 (4H, m), δ 4.41 - 4.47 (1H, m), δ 5.04 (2H, s), δ 7.18-7.40 (10H, m), δ 7.50 (1H , t, 6.1 Hz), δ 8.05 (1H, t, 5.8 Hz), δ 8.17 (1H, d, 7.9 Hz), δ 12.77 (1H, s).
[00354] [00354] 13 C NMR (100 MHz, DMSO-d6) δ 36.8; 41.6; 43.5; 53.5; 65.5; 126.5; 127.7; 127.8; 128.2; 128.3; 129.1; 137.0; 137.4; 156.5; 168.6; 169.3; 172.7.
[00355] [00355] MS (ESI) (m / z): 412 ([M-H] -). Example 3 ({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methyl acetate [Chemical formula 93] (3)
[00356] [00356] To N - [(9H-fluoren-ylmethoxy) carbonyl] glycylglycine (650.0 g, 1.834 mol), tetrahydrofuran (9.75 L) and acetic acid (1.95 L) were added, and The resulting mixture was dissolved by heating to 40 ° C. Lead tetracetate (1301.3 g, 2.935 mol) was added to it, and the
[00357] [00357] 1H NMR (400 MHz, CDCl3) δ 2.06 (3H, s), δ 3.90 (2H, d, 4.9 Hz), δ 4.23 (1H, t, 6.7 Hz ), δ 4.45 (2H, d, 6.7 Hz), δ 5.25 (2H, d, 7.3 Hz), δ 5.39 (1H, brs), δ 7.05 (1H, brs ), δ 7.30-7.34 (2H, m), δ 7.41 (2H, t, 7.3 Hz), δ 7.59 (2H, d, 7.3 Hz), δ 7.77 (2H, d, 7.3 Hz).
[00358] [00358] 13 C NMR (100 MHz, CDCl 3) δ 20.8; 44.4; 47.0; 63.9; 67.2; 120.0; 125.0; 127.1; 127.7; 141.3; 143.6; 156.6; 169.8; 171.7.
[00359] [00359] MS (ESI) (m / z): 369 ([M + H] +). Example 4 [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] acetate
[00360] [00360] To a mixture of ({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methyl acetate (610.0 g, 1.656 mol) and 1,2-dimethoxyethane (9.15 L ), benzyl glycolate (470 mL, 3.312 mol) was added and the resulting mixture was cooled to 0 to 5 ° C. A 10 mol / L sodium hydroxide solution (162.6 mL, 1.626 mol) was added to it and the resulting mixture was stirred for approximately 1 hour. Acetic acid (47.4 ml) was added to it and the resulting mixture was stirred at 1 ° C for approximately 1 hour. Then, water (2.0 L) was added dropwise, followed by [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] benzyl acetate (0.61 g), and the resulting mixture was stirred at 0 to 5 ° C for approximately 1 hour. Water (4.7 L) was added dropwise to it, and the resulting mixture was stirred at 0 to 5 ° C for approximately 2.5 hours. The precipitates were filtered, and a powder separated by filtration was washed with a cold aqueous solution of 1,2-dimethoxyethane 50% (v / v) (2.44 L). To the obtained wet powder, 1,2-dimethoxyethane (9.15 L) was added and the resulting mixture was dissolved by stirring at room temperature for approximately 30 minutes. Water (3.66 L) was added thereto, followed by [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] benzyl acetate (0.61 g), and The resulting mixture was stirred at room temperature for approximately 1 hour. Water (3.05 L) was added dropwise thereto, and the resulting mixture was stirred at room temperature for approximately 1 hour. After cooling to 0 to 5 ºC and stirring for approximately 1 hour, the
[00361] [00361] 1H NMR (400 MHz, CDCl3) δ 3.82 (2H, d, 4.9 Hz), δ 4.19- 4.22 (3H, m), δ 4.45 (2H, d, 6.7 Hz), δ 4.83 (2H, d, 6.7 Hz), δ 5.15 (2H, s), δ 5.34 (1H, brs), δ 6.95 (1H, brs) , δ 7.29-7.37 (7H, m), δ 7.40 (2H, t, 7.3 Hz), δ 7.58 (2H, d, 7.3 Hz), δ 7.76 ( 2H, d, 7.9 Hz).
[00362] [00362] 13 C NMR (100 MHz, CDCl 3) δ 44.5; 47.1; 66.6; 66.8; 67.1; 70.6; 120.0; 124.9; 127.1; 127.8; 128.4; 128.5; 128.6; 135.2; 141.3; 143.6; 170; 2; 170; 2; 170.4.
[00363] [00363] MS (ESI) (m / z): 475 ([M + H] +). Example 5 Glycylglycyl-L-phenylalanyl-N - [(carboxymethoxy) methyl] glycinamide [Chemical formula 95] (8)
[00364] [00364] To a mixture of [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] benzyl acetate (340.0 g, 0.717 mol) and acetonitrile (10.2 L) , 1,8-diazabicyclo [5.4.0] -7-undecene (53.6 ml, 0.358 mol) was added, and the resulting mixture was stirred at room temperature for approximately 2 hours. After cooling to 0 to 5 ºC, 1-hydroxybenzotriazole monohydrate (132.0 g, 0.862 mol) and N- [(benzyloxy) carbonyl] glycylglycyl-L-phenylalanine (311.0 g, 0.752 mol) were added, followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (158.0 g, 0.824 mol) in divided portions, and the resulting mixture was stirred at 0 to 5 ° C for approximately 1 hour. A solution
[00365] [00365] 1H NMR (400 MHz, D2O) δ 3.03-3.20 (2H, m), δ 3.79-3.96 (6H, m), δ 3.97 (2H, s), δ 4.64 (1H, t, 7.9 Hz), δ 4.67-4.75 (2H, m), δ 7.29- 7.42 (5H, m).
[00366] [00366] 13 C NMR (100 MHz, D2O) δ 37.3; 41.0; 42.7; 43.2; 56.0; 67.3; 70.0; 127.8; 129.4; 129.8; 136.9; 168.4; 171.7; 172.9; 174.4; 178.1.
[00367] [00367] MS (ESI) (m / z): 422 ([M-H] -). Example 6 1H-benzotriazol-1-ol - [(glycylamino) methoxy] benzyl acetate [Chemical formula 96] (5)
[00368] [00368] To a mixture of [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] benzyl acetate (50.00 g, 105.4 mmol) and acetonitrile (1.5 L), 1,8-diazabicyclo [5.4.0] -7-undecene (8.02 g, 52.7 mmol) was added, and the resulting mixture was stirred at room temperature for approximately 4 hours. 1-hydroxybenzotriazole monohydrate (35.51 g, 231.9 mmol) was added thereto in divided portions at room temperature, and the resulting mixture was stirred for approximately 30 minutes. The reaction mixture was cooled to 1 ºC and stirred for approximately 11 hours. The precipitates were filtered, and a powder separated by filtration was
[00369] [00369] 1H NMR (500 MHz, MeOH-d4) δ 3.63-3.68 (2H, brs), δ 4.19- 4.23 (2H, brs), δ 4.79 (2H, s ), δ 5.16-5.20 (2H, brs), δ 7.25-7.38 (7H, m), δ 7.64-7.72 (2H, dd, 17.3 Hz, 7, 8 Hz).
[00370] [00370] 13 C NMR (125 MHz, MeOH-d4) δ 41.9; 66.3; 67.6; 71.0; 112.3; 118.7; 125.24; 125.27; 128.8; 129.25; 129.30; 129.5; 136.9; 144.4; 169.5; 171.8. Example 7 N - [(Benzyloxy) carboxyl] glycylglycyl-L-phenylalanyl-N - {[2- (benzyloxy) -2-oxoethoxy] methyl} glycinamide [Chemical formula 97] (7)
[00371] [00371] To a mixture of N - [(benzyloxy) carbonyl] glycylglycyl-L-phenylalanine (10.99 g, 26.58 mmol), acetonitrile (120 mL) and water (20 mL), 1H-benzotriazole was added -1-ol - [(glycylamino) methoxy] benzyl acetate (10.00 g, 25.81 mmol), and the resulting mixture was cooled to 2 ºC. 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (5.70 g, 29.73 mmol) was added thereto, and the resulting mixture was stirred at 0 to 5 ° C for approximately 3.5 hours. Ethanol (100 ml) and water (150 ml) were added to the reaction solution, and the resulting mixture was stirred at room temperature for 14 hours. Water (130 mL) was added to it in divided portions, and the resulting mixture was stirred for 2 hours, then cooled to 1 ºC and stirred for approximately 1 hour. The precipitates
[00372] [00372] 1H NMR (500 MHz, DMSO-d6) δ 2.79 (1H, dd, 14 Hz, 9.2 Hz), δ 3.06 (1H, dd, 14 Hz, 4.5 Hz), δ 3.55-3.80 (6H, m), δ 4.15 (2H, s), δ 4.51 (1H, ddd, Hz, 9.2 Hz, 8.6 Hz, 4.5 Hz) , δ 4.63 (2H, d, 6.5 Hz), δ 5.03 (2H, s), δ 5.15 (2H, s), δ 7.15-7.40 (15H, m), δ 7.15-7.40 (15H, m), δ 7.50 (1H, t, 6 Hz), δ 8.02 (1H, t, 5.8 Hz), δ 8.15 (1H, d , 8.6 Hz), δ 8.33 (1H, t, 5.8 Hz), δ 8.60 (1H, t, 7 Hz)
[00373] [00373] NMR-13C (125 MHz, DMSO-d6) δ 37.4; 41.9; 42.2; 43.6; 54.2; 64.5; 65.6; 65.7; 69.1; 126.3; 127.77; 127.85; 128.10; 128.14; 128.2; 128.4; 128.5; 129.2; 135.8; 137.0; 137.9; 156.6; 168.9; 169.5; 169.9; 170.2; 171.5. Example 8 Glycylglycyl-L-phenylalanyl-N - [(carboxymethoxy) methyl] glycinamide [Chemical formula 98] (8)
[00374] [00374] To a mixture of N - [(benzyloxy) carboxyl] glycylglycyl-L-phenylalanyl-N - {[2- (benzyloxy) -2-oxoethoxy] methyl} glycinamide (15.0 g, 23.16 mmol), tetrahydrofuran (315 ml) and water (210 ml), 5% palladium-carbon (3.31 g, water content: 54.7%) was added, and the atmosphere was switched to hydrogen. After stirring at room temperature for approximately 2.5 hours, the atmosphere was changed to nitrogen. Palladium-carbon was
[00375] [00375] 1H NMR (400 MHz, D2O) δ 3.03-3.20 (2H, m), δ 3.79-3.96 (6H, m), δ 3.97 (2H, s), δ 4.64 (1H, t, 7.9 Hz), δ 4.67-4.75 (2H, m), δ 7.29- 7.42 (5H, m).
[00376] [00376] 13C NMR (100 MHz, D2O) δ 37.3; 41.0; 42.7; 43.2; 56.0; 67.3; 70.0; 127.8; 129.4; 129.8; 136.9; 168.4; 171.7; 172.9; 174.4; 178.1.
[00377] [00377] MS (ESI) (m / z): 422 ([M-H] -). Example 9 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid [Chemical formula 99] (18)
[00378] [00378] To a solution of 6-aminohexanoic acid (2.5 kg, 19.1 mol) in acetic acid (10 L), a solution of maleic anhydride (1.87 kg, 19.1 mol) in acetic acid ( 10 L) was added dropwise at 25 to 30 ° C over 1 hour, and the resulting mixture was stirred at the same temperature as above for
[00379] [00379] The obtained 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid (1.40 kg, 6.66 mol) was dissolved in a mixed solution of acetic acid (2.1 L) and purified water (1.4 L) at 25 to 30 ºC. To the solution, purified water (0.7 L) was added, and then the resulting mixture was cooled to 20 to 25 ºC and then stirred for 2 hours. To the obtained suspension, purified water (7.0 L) was added dropwise over 1 hour, and the resulting mixture was cooled to 0 to 5 ºC and then stirred for 1 hour. The precipitates were filtered, and a powder separated by filtration was washed with cold water (2.1 L). The obtained powder was dried under reduced pressure at 40 ºC to obtain 6- (2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid (1.27 kg, 6.02 mol, yield : 90.4%).
[00380] [00380] 1H NMR (400 MHz, DMSO-d6) δ 1.18-1.24 (2H, m), δ 1.45-1.52 (4H, m), δ 2.18 (2H, t , 7.5 Hz), δ 3.38 (2H, t, 7.5 Hz), δ 7.01 (2H, s), δ 11.98 (1H, s). Example 10 1- {6 - [(2,5-Dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrole-2,5-dione [Chemical formula 100] (9)
[00381] [00381] To a mixture of 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid (5.0 g, 23.6 mmol), N-hydroxysuccinimide (3, 0 g, 26.0 mmol) and acetonitrile (50 mL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (5.45 g, 28.4 mmol) was added, and the resulting mixture was stirred at room temperature for approximately 3.5 hours. Water (100 ml) and toluene (100 ml) were added thereto, and the resulting mixture was stirred and then separated into organic and aqueous layers. The aqueous layer was removed. The organic layer was washed twice with water (50 ml) and concentrated to 25 ml under reduced pressure. A silica gel cartridge (KP-sil 10 g) was charged with the residue, then toluene: acetone = 9: 1 (100 ml) was passed through, and an eluate was recovered and concentrated to 25 ml under reduced pressure. 1-Butanol (50 mL) was added to the residue, followed by 1- {6 - [(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrol-2,5-dione (10 mg), and the resulting mixture was stirred at room temperature for 1 hour. 1-Butanol (50 ml) was added dropwise thereto, and the resulting mixture was cooled to -10 ° C and stirred. The precipitates were filtered, and a powder separated by filtration was washed with cold 1-butanol (20 ml). The obtained powder was dried under reduced pressure at 40 ºC to obtain 1- {6 - [(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrol-2,5-dione (6, 52 g, 21.1 mmol, yield: 89.4%).
[00382] [00382] 1H NMR (400 MHz, DMSO-d6) δ 1.27-1.35 (2H, m), δ 1.48-1.56 (2H, m), δ 1.59-1.67 (2H, m), δ 2.65 (2H, t, 7.3 Hz), δ 2.81 (4H, s), δ 3.39 (2H, t, 7.0 Hz), δ 7.00 (2H, s).
[00383] [00383] 13 C NMR (100 MHz, DMSO-d6) δ 23.7; 25.1; 25.4; 27.4; 30.0; 36.8; 134.4; 168.9; 170.2; 171.1.
[00384] [00384] MS (ESI) (m / z): 309 ([M + H] +). Example 11 1- {6 - [(2,5-Dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrole-2,5-dione [Chemical formula 101]
[00385] [00385] A mixed solution of 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoic acid (1.1 kg, 5.21 mol), N-hydroxysuccinimide (0, 72 kg, 6.25 mol) and acetonitrile (11 L) was cooled to -15 ºC. 2,6-lutidine (1.34 kg, 12.50 mol) was added to it and then thionyl chloride (0.74 kg, 6.25 mol) was added dropwise at -15 ºC to -10 ºC over 1 hour. Water (11 L) and toluene (11 L) were added thereto, and the resulting mixture was stirred and then separated into organic and aqueous layers. The aqueous layer was removed. The organic layer was washed twice with cold water (11 L) at 0 to 5 ºC and washed with 20% saline solution (11 L) at 0 to 5 ºC, and the organic layer was concentrated to 5.5 L under reduced pressure . Then, toluene (5.5 L) was added to the residue, and the resulting mixture was concentrated again to 5.5 L under reduced pressure. A funnel was packed with neutral silica gel (Silica gel 60N, 3.3 kg), moistened with toluene, then the concentrate was passed through it and the funnel was washed with toluene: acetone = 9: 1 (29 L) to obtain a filtrate. The filtrate obtained was concentrated to 5.5 L under reduced pressure, then 1-butanol (8.8 L) was added to the residue and then the resulting mixture was stirred at 20 to 25 ºC for 16 hours. 1-Butanol (13.2 L) was added dropwise to it, and the resulting mixture was cooled to -15 ºC and stirred for 1 hour. The precipitates were filtered, and a powder separated by filtration was washed with cold 1-butanol (4.4 L). The obtained powder was dried under reduced pressure at 40 ºC to obtain 1- {6 - [(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrol-2,5-dione (1, 45 kg, 4.72 mol, yield: 90.5%).
[00386] [00386] 1H NMR (400 MHz, DMSO-d6) δ 1.27-1.35 (2H, m), δ 1.48-1.56 (2H, m), δ 1.59-1.67 (2H, m), δ 2.65 (2H, t, 7.3 Hz), δ 2.81 (4H, s), δ 3.39 (2H, t, 7.0 Hz), δ 7.00 (2H, s).
[00387] [00387] NMR-13C (100 MHz, DMSO-d6) δ 23.7; 25.1; 25.4; 27.4; 30.0; 36.8; 134.4; 168.9; 170.2; 171.1.
[00388] [00388] MS (ESI) (m / z): 309 ([M + H] +). Example 12 N- [6- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl- N - [(carboxymethoxy) methyl] glycinamide [Chemical formula 102] (10)
[00389] [00389] To a solution of 1- {6 - [(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrole-2,5-dione (291.3 g, 0.945 mol) in acetonitrile (1.8 L), glycylglycyl-L-phenylalanyl-N- [((carboxymethoxy) methyl] glycinamide (200.0 g, 0.472 mol), water (4.2 L) and N, N- di- isopropylethylamine (48.8 g, 0.378 mol), and the resulting mixture was stirred at room temperature for approximately 9 hours. Isopropyl acetate (2.0 L), anhydrous sodium dihydrogen phosphate (400.0 g), and anhydrous disodium hydrogen phosphate (26.0 g) were added to it, and the resulting mixture was stirred and then separated into organic and aqueous layers . The organic layer was removed. Tetrahydrofuran (1.0 L), ethyl acetate (1.0 L) and anhydrous sodium dihydrogen phosphate (160.0 g) were added thereto and the resulting mixture was stirred and then separated into organic and aqueous layers. The aqueous layer was removed. A 10% (w / v) phosphate buffer solution (pH 3.4; 0.6 L) was added to it, and the resulting mixture was stirred. After separating into organic and aqueous layers and removing the aqueous layer, the organic layer was concentrated to 1.0 L under reduced pressure. 1,2-dimethoxyethane (4.0 L) was added to the residue, and the resulting mixture was concentrated
[00390] [00390] 1H NMR (400 MHz, DMSO-d6) δ 1.15-1.23 (2H, m), δ 1.43-1.52 (4H, m), δ 2.11 (2H, t , 7.3 Hz), δ 2.78-2.84 (1H, m), δ 3.04-3.09 (1H, m), δ 3.37 (2H, t, 7.0 Hz) δ 3.61-3.79 (6H, m), δ 3.94 (2H, s), δ 4.47-4.52 (1H, m), δ 4.61 (2H, d, 6.7 Hz ), δ 6.99 (2H, s), δ 7.15-7.27 (5H, m), δ 8.11- 8.15 (2H, m), δ 8.22 (1H, d, 8 , 5 Hz), δ 8.31 (1H, t, 5.8 Hz), δ 8.63 (1H, t, 6.4 Hz).
[00391] [00391] 13C NMR (100 MHz, DMSO-d6) δ 24.6; 25.8; 27.8; 34.9; 37.0; 37.2; 41.9; 42.1; 42.1; 54.2; 65.1; 69.2; 126.2; 128.1; 129.1; 134.4; 137.9; 168.9; 169.5; 169.8; 171.1; 171.4; 171.9; 172.6.
[00392] [00392] MS (ESI) (m / z): 615 ([M-H] -). Example 13 N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1-1,2-dimethoxyethane adduct
[00393] [00393] To a solution of 1- {6 - [(2,5-dioxopyrrolidin-1-yl) oxy] -6-oxohexyl} -1H-pyrrole-2,5-dione (72.8 g, 0.236 mol) in acetonitrile (450.0 mL), glycylglycyl-L-phenylalanyl-N - [(carboxymethoxy) methyl] glycinamide (50.0 g, 0.118 mol), water (1050.0 mL) and N, N-diisopropylethylamine ( 16.5 ml, 0.095 mol) were added, and the resulting mixture was stirred at room temperature for approximately 15 hours. Isopropyl acetate (500.0 ml), anhydrous sodium dihydrogen phosphate (100.0 g) and anhydrous disodium hydrogen phosphate (6.5 g) were added to it, and the resulting mixture was stirred and then separated into organic and aqueous layers. The organic layer was removed. Isopropyl acetate (500.0 ml) was added, and the resulting mixture was stirred and then separated into organic and aqueous layers. The organic layer was removed. 1,2-Dimethoxyethane (250.0 ml), ethyl acetate (250.0 ml), acetonitrile (25.0 ml) and anhydrous sodium dihydrogen phosphate (400.0 g) were added to it, and the resulting mixture was stirred and then separated into organic and aqueous layers. The aqueous layer was removed. Acetonitrile (750.0 mL), water (113.0 mL), sodium chloride (30.0 g), anhydrous sodium dihydrogen phosphate (7.5 g) and phosphoric acid (85%, 1.5 g, 0.012 mol ) were added thereto, and the resulting mixture was stirred and separated into organic and aqueous layers. The aqueous layer was removed. Water (113.0 ml), sodium chloride (30.0 g) and anhydrous sodium dihydrogen phosphate (7.5 g) were added thereto, and the resulting mixture was stirred and separated into organic and aqueous layers. The aqueous layer was removed. Water (113 mL), sodium chloride (30.0 g) and sodium dihydrogen phosphate
[00394] [00394] 1H NMR (500 MHz, DMSO-d6) δ 1.16-1.23 (2H, m), δ 1.44-1.52 (4H, m), δ 2.11 (2H, t , 7.5 Hz), δ 2.79-2.84 (1H, m), δ 3.05-3.09 (1H, m), δ 3.24 (6H, s), δ 3.37 ( 2H, t, 7.3 Hz), δ 3.43 (4H, s), δ 3.56-3.78 (6H, m), δ 3.99 (2H, s), δ 4.48-4 , 52 (1H, m), δ 4.61 (2H, d, 6.5 Hz), δ 7.00 (2H, s), δ 7.16-7.27 (5H, m), δ 8, 02-8.10 (2H, m), δ 8.15 (1H, d, 8.0 Hz), δ 8.32 (1H, t, 6.0 Hz), δ 8.58 (1H, t, 6.8 Hz), δ 12.61 (1H, brs).
[00395] [00395] NMR-13C (100 MHz, DMSO-d6) δ 25.5; 26.8; 28.7; 35.9; 37.9; 38.2; 42.8; 43.0; 43.1; 55.1; 59.0; 65.2; 69.8; 72.0; 127.2; 129.0; 130.1; 135.4; 138.8; 169.8; 170.4; 170.9; 172.0; 172.3; 172.4; 173.6.
[00396] [00396] MS (ESI) (m / z): 615 ([M-H] -). X-ray powder diffraction:
[00397] [00397] The crystals of the title compound were subjected to powder X-ray diffraction, obtained by irradiation with copper Kα radiation. The results are shown in Table 1 and Figure 3. The main peaks were observed at the diffraction angles (2θ) of 19.0 ° and 25.0 °. Table 1 Diffraction angle 2θ (°) Interplanar spacing d (Å) Relative intensity (%) 7.0 12.6 32.0 12.4 7.1 40.9 19.0 4.7 82.9 25.0 3.6 100.0 25.2 3.5 59.5 Example 14 N- [6- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl - N - [(2 - {[(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro -1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-
[00398] [00398] To a (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro methanesulfonate -1H, 12H- benzo [de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-amine dihydrate (gross quantity: 154.6 g, internal content after correction with value 2.95% water content: 150.0 g, 0.282 mol) in tetrahydrofuran (1.8 L), a 5% (w / v) aqueous solution of sodium sulfate (1.5 L) and N-methylmorpholine (28.5 g, 0.282 mol) was added, and the resulting mixture was stirred at 32 ° C for approximately 1 hour. Cyano (hydroxyimino) ethyl acetate (8.0 g, 56.3 mmol), N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl -L- phenylalanyl-N - [(carboxymethoxy) methyl] glycinamide (crude quantity: 232.0 g, internal content after conversion to 1,2-dimethoxyethane 2.5%: 226.2 g, 0.367 mol) and hydrochloride of 1 - (3-dimethylaminopropyl) -3-ethylcarbodiimide (108.2 g, 0.564 mol) was added to it, and the resulting mixture was stirred at 29 to 32 ºC for approximately 1 hour and then separated into organic and aqueous layers. The aqueous layer was removed. Ethyl acetate (1.8 L) and a 5% (v / v) aqueous solution of acetic acid (0.45 L) were added thereto, and the resulting mixture was stirred and separated into organic and aqueous layers. The aqueous layer was removed. Activated carbon (15.0 g, Kyoryoku Shirasagi (manufactured by Osaka Gas Chemicals Co., Ltd.)) was added to the
[00399] [00399] 1H NMR (400 MHz, DMSO-d6) δ 0.87 (3H, t, 7.3 Hz), δ 1.14-1.21 (2H, m), δ 1.41-1, 50 (4H, m), δ 1.78-1.93 (2H, m), δ 2.09 (2H, t, 7.3 Hz), δ 2.13-2.23 (2H, m), δ 2.36 (3H, s), δ 2.74-2.80 (1H, m), δ 3.00-3.04 (1H, m), δ 3.08-3.25 (2H, m ), δ 3.32-3.37 (2H, m), δ 3.56-3.77 (6H, m), δ 4.02 (2H, s), δ 4.44-4.50 (1H , m), δ 4.64 (2H, d, 6.7 Hz), δ 5.17 (2H, d, 5.5 Hz), δ 5.41 (2H, s), δ 5.57-5 , 62 (1H, m), δ 6.51 (1H, s), δ 6.99 (2H, s), δ 7.14- 7.26 (5H, m), δ 7.30 (1H, s ), δ 7.75 (1H, d, 11.0 Hz), δ 8.00 (1H, t, 5.8 Hz), δ 8.06 (1H, t, 5.3 Hz), δ 8, 12 (1H, d, 7.9 Hz), δ 8.29 (1H, t, 5.8 Hz), δ 8.49 (1H, d, 8.5 Hz), δ 8.62 (1H, t , 6.7 Hz).
[00400] [00400] NMR-13C (100 MHz, DMSO-d6) δ 7.6; 10.8; 10.9; 23.5; 24.6; 25.7; 27.7; 30; 2; 30.6; 34.8; 36.9; 37.1; 41.7; 42.0; 44.4; 49.5; 54.1; 65.1; 66.9; 69.7; 72.2; 96.6; 109.6; 109.8; 119.0; 121.5; 123.4; 123.6; 125.3; 126.2; 128.0; 129.0; 134.3; 136.2; 136.3; 137.7; 140.4; 145.0; 147.7; 147.8; 149.9; 152.2; 156.6; 160; 2; 162.7; 168.8; 169.1; 169.3; 170.0; 171.0; 171.3; 172.3; 172.5.
[00401] [00401] MS (ESI) (m / z): 1034 ([M + H] +). X-ray powder diffraction:
[00402] [00402] The crystals of the title compound were subjected to powder X-ray diffraction, obtained by irradiation with copper Kα radiation. The results are shown in Table 2 and Figure 4. The main peaks were observed at the diffraction angles (2θ) of 5.6 °, 15.5 ° and 22.0 °. Table 2 Diffraction angle 2θ (°) Interplanar spacing d (Å) Relative intensity (%) 5.6 15.9 100.0 5.8 15.3 41.6 15.5 5.7 73.6 17.9 5.0 35.0 20.5 4.3 35.1 21.4 4.2 31.4 22.0 4.0 74.9 Example 15 ({N - [(9H-Fluoren-9-ylmethoxy acetate ) carbonyl] glycyl} amino) methyl
[00403] [00403] To a suspension of N-9-fluorenylmethoxycarbonylglycylglycine (2.85 kg, 8.04 mol) in anhydrous tetrahydrofuran (38.0 kg), acetic acid (2.41 kg, 40.1 mol) was added and lead (IV) tetracetate (5.35 kg, 12.0 mol) under nitrogen atmosphere, and the resulting mixture was refluxed for 1.5 hours. After cooling to room temperature, the precipitated solid was filtered off, and the solid thus filtered off was washed with tetrahydrofuran (10.1 kg). The filtrate obtained and the washings were concentrated under reduced pressure until the amount of the liquid was close to 16 L. To the obtained concentrate, ethyl acetate (26 kg), a 10% aqueous citric acid solution (17.1 L) was added ) and 20% saline (5.7 L), and the resulting mixture was stirred and then separated into organic and aqueous layers. The organic layer obtained was separated into organic and aqueous layers and washed with a 10% aqueous citric acid solution (17.1 L), a 9% aqueous sodium bicarbonate solution (28.5 L) and 20% saline solution ( 14.3 L) in that order. To the obtained organic layer, silica gel 60 (5.7 kg) and ethyl acetate (10.3 kg) were added, and the resulting mixture was stirred for 1 hour. Then, a solid was filtered off, and the solid thus filtered off was washed with ethyl acetate (7.7 kg). The filtrate obtained and the washings were concentrated under reduced pressure until the amount of the liquid was close to 5 L. Cyclopentylmethyl ether (24.5 kg) was added to the residue. The resulting mixture was concentrated again under reduced pressure until the amount of the liquid was close to 5 L. To the obtained concentrate, cyclopentyl methyl ether (14.7 kg) was added, and the resulting mixture was stirred at
[00404] [00404] To a suspension of ({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methyl acetate (2.01 kg, 5.46 mol) in anhydrous 1,2-dimethoxyethane (21 kg), benzyl glycolate (1.81 kg, 10.9 mol) was added under nitrogen atmosphere, and the resulting mixture was cooled to approximately 0 ºC. Tris (pentafluorophenyl) borane (142 g, 0.27 mol) was added thereto, and the resulting mixture was stirred at the same temperature as above for 3 hours. Then, ethyl acetate (27.1 kg) and a 10% aqueous solution of potassium bicarbonate were added thereto, and the resulting mixture was warmed to room temperature and separated into organic and aqueous layers. The obtained organic layer was separated into organic and aqueous layers and washed by the addition of 10% saline solution (20.1 L). The obtained organic layer was concentrated under reduced pressure until the amount of the liquid was close to 4 L. Methanol (15.7 kg) was added to the residue. The resulting mixture was concentrated under reduced pressure until the amount of the liquid was close to 4 L. To the obtained concentrate, methanol (7.8 kg) was added. The resulting mixture was
[00405] [00405] To a solution of [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] benzyl acetate (2.28 kg, 4.81 mol) in N, N-dimethylacetamide (15.0 kg), 1,8-diazabicyclo [5.4.0] undec-7-ene (0.37 kg, 2.4 mol) was added under nitrogen atmosphere, and the resulting mixture was stirred at room temperature for 30 minutes. Pyridinium p-toluenesulfonate (0.60 kg, 2.4 mol), 1-hydroxybenzotriazole monohydrate (0.74 kg, 4.8 mol), N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycylglycyl -L-phenylalanine (2.19 kg, 4.37 mol) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.84 kg, 4.37 mol) were added thereto, and the resulting mixture was stirred at room temperature for 3 hours. Ethyl acetate (21.0 kg) and 10% saline (34 L) were added thereto, and the resulting mixture was stirred and then separated into organic and aqueous layers. The obtained organic layer was
[00406] [00406] 1H NMR (400 MHz, DMSO-d6) δ 2.79 (1H, dd, J = 14.0; 9.8 Hz), 3.05 (1H, dd, J = 14.0; 4 , 3 Hz), 3.58-3.79 (6H, m), 4.15 (2H, s), 4.20-4.24 (1H, m), 4.28-4.30 (2H, m), 4.48-4.53 (1H, m), 4.63 (2H, d, J = 6.7 Hz), 5.14 (2H, s), 7.15-7.43 (13H , m), 7.58 (1H, t, J = 6.1 Hz), 7.71 (2H, d, J = 7.3 Hz), 7.89 (2H, d, J = 7.9 Hz ), 8.01 (1H, t, J = 5.5 Hz), 8.15 (1H, d, J = 7.9 Hz), 8.33 (1H, t, J = 5.8 Hz), 8.59 (1H, t, J = 6.4 Hz).
[00407] [00407] NMR-13C (100 MHz, DMSO-d6) δ 37.3; 41.8; 42.1; 43.5; 46.6; 54.1; 64.4; 65.6; 65.7; 69.0; 120.1; 125.2; 126.3; 127.1; 127.6; 128.0;
[00408] [00408] MS (ESI) (m / z): 736 ([M + H] +). Example 18 N - [(9H-Fluoren-9-ylmethoxy) carbonyl] glycylglycyl-L-phenylalanyl-N- [((carboxymethoxy) methyl] glycinamide [Chemical formula 108] (14)
[00409] [00409] To a suspension of [({N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycylglycyl-L-phenylalanyl} amino) methoxy] benzyl acetate (367 g, 0.499 mol) in tetrahydrofuran (5 , 88 kg) and water (1.61 L), a palladium-carbon-ethylenediamine complex (28 g) was added, and the resulting mixture was stirred at room temperature for 1 hour to 3 hours under a hydrogen gas atmosphere under common pressure . The catalyst was filtered off, and the catalyst thus filtered off was washed with tetrahydrofuran (1.63 kg) to obtain a filtrate and washes. The operation described above for reaction and separation of a catalyst by filtration was performed repeatedly 9 times, and the 9 portions obtained from filtrates and washes were combined. The resulting mixture was concentrated under reduced pressure until the amount of the liquid was close to 17 L. To the obtained concentrate, 2-propanol (39 kg) was added, and the concentration operation under reduced pressure until the amount of the liquid was around 17 L was performed repeatedly three times. To the obtained concentrate, ethyl acetate (45 kg) was added, and the resulting mixture was stirred at room temperature for 6 hours. This suspension was stirred more at approximately 5 ºC for 1 hour. The solid
[00410] [00410] 1H NMR (400 MHz, DMSO-d6) δ 2.79 (1H, dd, J = 14.0; 9.8 Hz), 3.06 (1H, dd, J = 13.7; 4 , 6 Hz), 3.58-3.79 (6H, m), 3.98 (2H, s), 4.21 - 4.25 (1H, m), 4.28 - 4.30 (2H, m), 4.48-4.54 (1H, m), 4.61 (2H, d, J = 6.7 Hz), 7.16- 7.20 (1H, m), 7.22-7 , 27 (4H, m), 7.33 (2H, t, J = 7.3 Hz), 7.42 (2H, t, J = 7.3 Hz), 7.59 (1H, t, J = 6.1 Hz), 7.71 (2H, d, J = 7.3 Hz), 7.89 (2H, d, J = 7.3 Hz), 8.03 (1H, t, J = 5, 5 Hz), 8.16 (1H, d, J = 7.9 Hz), 8.33 (1H, t, J = 5.8 Hz), 8.57 (1H, t, J = 6.7 Hz ).
[00411] [00411] 13 C NMR (100 MHz, CDCl 3) δ 37.4; 41.8; 42.1; 43.5; 46.6;
[00412] [00412] MS (ESI) (m / z): 646 ([M + H] +). Example 19 N - [(9H-Fluoren-9-ylmethoxy) carbonyl] glycylglycyl-L-phenylalanyl-N - [(2 - {[(1S, 9S) - 9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide [Chemical formula 109] (15)
[00413] [00413] To a (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro methanesulfonate -1H, 12H- benzo [de] pyran [3 ', 4': 6.7] indolizine [1,2-b] quinolin-1-amine dihydrate (260 g, 0.458 mol) in dimethylsulfoxide (1.8 L) and tetrahydrofuran (1.3 L), triethylamine (55.6 g, 0.549 mol), 1-hydroxybenzotriazole monohydrate (84.2 g, 0.549 mol), N - [(9H-fluoren-9- ylmethoxy) carbonyl] glycylglycyl-L-phenylalanyl-N- [((carboxymethoxy) methyl] glycinamide (325 g, 0.503 mol) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (114 g, 0.595 mol ) were added under a nitrogen atmosphere, and the resulting mixture was stirred at room temperature for 2 hours. Tetrahydrofuran (3.9 L), ethyl acetate (2.6 L) and an 11% aqueous potassium bicarbonate solution (5.2 L) were added thereto, and the resulting mixture was stirred and layered organic and
[00414] [00414] 1H NMR (400 MHz, DMSO-d6) δ 0.86 (3H, t, J = 7.3), 1.79-1.90 (2H, m), 2.11-2.22 (2H, m), 2.37 (3H, s), 2.77 (1H, dd, J = 14.0; 9.8 Hz), 3.02 (1H, dd, J = 13.7; 4 , 6 Hz), 3.07-3.25 (2H, m), 3.58-3.79 (6H, m), 4.02 (2H, s), 4.18-4.23 (1H, m), 4.26-4.30 (2H, m), 4.45-4.54 (1H, m), 4.64 (2H, d, J = 6.7 Hz), 5.17 (2H , dd, J = 23.5; J = 19.2 Hz), 5.40 (2H, s), 5.56-5.61 (1H, m), 6.52 (1H, s), 7, 14-7.43 (10H, m), 7.58 (1H, t, J = 6.1 Hz), 7.68 (2H, d, J = 7.3 Hz), 7.76 (1H, d , J = 11.0 Hz), 7.86 (2H, d, J = 7.3 Hz), 8.02 (1H, t, J = 5.5 Hz),
[00415] [00415] 13C NMR (100 MHz, DMSO-d6) δ 7.7; 10.9; 11.0; 23.1; 23.7; 27.8; 30.3; 31.4; 37.3; 41.8; 42.1; 43.5; 44.6; 46.6; 49.6; 54.2; 55.6; 65.2; 65.8; 67.0; 69.8; 72.3; 82.0; 96.7; 109.7; 109.9; 119.1; 120.0; 121.6; 123.5; 123.7; 125.2; 125.3; 126.3; 127.0; 127.6; 128.1; 129.1; 136.3; 136.4; 137.8; 140.5; 140.7; 143.8; 143.8; 145.1; 147.8; 147.9; 150.0; 152.3; 156.5; 156.7; 160.3; 162.8; 168.9; 169.2; 169.4; 170.2; 171.4; 172.4.
[00416] [00416] MS (ESI) 1063: (M + H) + Example 20 Glycylglycyl-L-phenylalanyl-N - [(2 - {[(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4 -methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizino [1,2-b ] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide [Chemical formula 110] (16)
[00417] [00417] To a suspension of N - [(9H-fluoren-9-ylmethoxy) carbonyl] glycylglycyl-L-phenylalanyl-N - [(2 - {[(1S, 9S) -9-ethyl-5-fluoro-9 - hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizino [1 , 2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide (400 g, 0.376 mol) in dehydrated tetrahydrofuran (8 L), 1,8-diazabicyclo [5.4.0] undec- 7-ene (51.6 g, 0.339 mol) was
[00418] [00418] 1H NMR (400 MHz, DMSO-d6) δ 0.87 (3H, t, J = 7.3), 1.57-1.67 (6H, m), 1.80-1.92 (2H, m), 2.06-2.25 (2H, m), 2.35-2.38 (3H, m), 2.61 2.63 (2H, m), 2.73-2 , 89 (1H, m), 3.00-3.79 (29H, m), 3.80 (1H, dd, J = 16.2; 7.0 Hz), 3.99-4.10 (2H , m), 4.30-4.51 (1H, m), 4.58 (1H, dd, J = 9.8; 6.1 Hz), 4.63-4.69 (1H, m), 5.01 (0.5H, br), 5.15 (1H, t, J = 18.3 Hz), 5.24 (1H, t, J = 18.3 Hz), 5.41 (2H, s ), 5.54-5.62 (1H, m), 6.52 (0.6H, br), 7.11-7.31 (6H, m), 7.75-7.79 (1H, m ), 8.12-8.15 (0.6H, m), 8.22 (0.2H, d, J = 8.5 Hz), 8.36 (0.2H, t, J = 5.8 Hz), 8.52 (0.2H, t, J = 5.5 Hz), 8.66 (0.2H, t, J = 6.4 Hz), 8.93 (0.6H, t, J = 5.5 Hz), 9.10 (1H, dd, J = 20.1; 9.2 Hz), 9.82 (0.6H, br).
[00419] [00419] MS (ESI) 841: (M + H) + Example 21 Preparation of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] seed crystals] glycylglycyl-L-phenylalanyl-N - [(2 - {[(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide [Chemical formula 111]
[00420] [00420] To a suspension of glycylglycyl-L-phenylalanyl-N - [(2 - {[(1S, 9S) - 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 , 3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizino [1,2-b] quinolin-1-yl] amino} - 2-oxoethoxy) methyl] glycinamide (200 mg, 0.24 mmol) in pyridine (0.2 ml), tetrahydrofuran (2.0 ml) and acetonitrile (0.6 ml), pyridinium p-toluenesulfonate was added (120 mg, 0.48 mmol), triethylamine (100 µL, 0.72 mmol) and N-succinimidyl 6-maleimidohexanoate (73 mg, 0.24 mmol), and the resulting mixture was stirred at room temperature for 3 hours . The reaction solution was purified by silica gel flash column chromatography (Biotage AB) [tetrahydrofuran: acetone = 3: 7 to 7: 3 (v / v)] to obtain the title compound as an oil. To 19.5 mg of the oil obtained, acetone (0.4 ml) and 2-butanol (0.2 ml) were added, and the resulting mixture was heated to approximately 60 ° C. The precipitated solid was filtered at room temperature, and the solid separated by filtration was washed with 2-butanol (approximately 0.2 ml) to obtain the title compound (14.3 mg) as a colorless powder. The obtained powder was used as a seed crystal in the next reaction. Example 22 N- [6- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl- N - [(2 - {[(1S, 9S) - 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4 ': 6.7] indolizine [1,2-b] quinolin-1-
[00421] [00421] pyridinium p-toluenesulfonate (209 g, 0.832 mol), N-succinimidyl 6-maleimidohexanoate (128 g, 0.415 mol) and glycylglycyl-L-phenylalanyl-N - [(2 - {[(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4 ': 6.7] indolizine [1,2- b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide (350 g, 0.416 mol), in pyridine (0.35 L), acetonitrile ( 1.1 L) and tetrahydrofuran (3.5 L), were dissolved under a nitrogen atmosphere. To the solution, triethylamine (63.2 g, 0.625 mol) was then added, and the resulting mixture was stirred at room temperature for 3.5 hours. Tetrahydrofuran (3.5 L), a 19% citric acid aqueous solution (3.5 L), ethyl acetate (2.5 L) and 18% saline (2.5 L) were added to it, and the resulting mixture was stirred and then separated into organic and aqueous layers. To the obtained organic layer, a 19% aqueous citric acid solution (2.5 L) and 18% saline solution (2.5 L) were added, and the resulting mixture was stirred and then separated into organic and aqueous layers. The obtained organic layer was separated into organic and aqueous layers and washed with a 22% aqueous solution of potassium bicarbonate (2.1 L) and subsequently with 18% saline solution (1.8 L). The obtained organic layer was added dropwise to a suspension of activated carbon (35 g) in acetonitrile (35 L) prepared in another container, and the
[00422] [00422] To anhydrous sodium sulfate (1.8 g), cyano (hydroxyimino) ethyl acetate (0.16 g, 1.13 mmol) and a mixed solution of purified water and
[00423] [00423] To N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N - [(2 - {[( 1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6.7] indolizine [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide (4.50 g, 4.35 mmol), a mixed solution of acetone and purified water (10.6 ml and 2.9 ml) containing acetic acid
[00424] [00424] The instrumental data was similar to that of the compound described in Example 14. Example 24 N- [6- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L- phenylalanyl- N - [(2 - {[(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide [Chemical formula 114 ]
[00425] [00425] To anhydrous sodium sulfate (1.8 g), cyano (hydroxyimino) ethyl acetate (0.16 g, 1.13 mmol) and a mixed solution of purified water and tetrahydrofuran (24 mL and 18 mL) containing N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N- [(carboxymethoxy) methyl] glycinamide (crude quantity : 5.76 g, internal content after conversion to 12.40% 1,2-dimethoxyethane: 5.05 g, 8.18 mmol), a mixed suspension of purified water and tetrahydrofuran (9 mL and 15 mL) was added ) containing (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H- methanesulfonate benzo [de] pyran [3 ', 4': 6.7] indolizine [1,2-b] quinolin-1-amine (3.0 g, 5.64 mmol) at 20 to 30 ° C. To the mixed solution, tetrahydrofuran (9 ml) and a tetrahydrofuran solution (7.5 ml) containing N-methylmorpholine (0.63 g, 6.23 mmol) were added, and the resulting mixture was stirred at the same time. temperature as above for 15 minutes. Then, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.16 g, 11.27 mmol) and a mixed solution of purified water and tetrahydrofuran (1.5 mL and 1.5 mL ) have been added to it. The mixed solution was stirred at 20 to 30 ºC for 30 minutes or more. After the completion of the reaction was confirmed, the reaction mixture was separated into organic and aqueous layers, and the aqueous layer was removed. The temperature of the organic layer was adjusted to 15 to 25 ºC, then ethyl acetate (36 mL), anhydrous sodium sulfate (1.26 g) and water
[00426] [00426] The instrumental data were similar to those of the
[00427] [00427] SEQ ID NO: 1 - Amino acid sequence of an anti-HER2 antibody heavy chain SEQ ID NO: 2 - Amino acid sequence of an anti-HER2 antibody light chain SEQ ID NO: 3 - Amino acid sequence of an heavy chain of anti-HER3 antibody SEQ ID NO: 4 - amino acid sequence of a light chain of anti-HER3 antibody SEQ ID NO: 5 - amino acid sequence of a heavy chain of anti-TROP2 antibody SEQ ID NO: 6 - sequence amino acid sequence of an anti-TROP2 antibody light chain SEQ ID NO: 7 - Amino acid sequence of an anti-B7-H3 antibody heavy chain SEQ ID NO: 8 - Amino acid sequence of an anti-B7- antibody light chain H3 SEQ ID NO: 9 - Amino acid sequence of an anti-GPR20 antibody heavy chain SEQ ID NO: 10 - Amino acid sequence of an anti-GPR20 antibody light chain

权利要求:
Claims (52)
[1]
1. Crystals, characterized by the fact that the compound represented by formula (1): [Chemical formula 1] (1).
[2]
2. Crystals according to claim 1, characterized by the fact that the crystals show main peaks at the diffraction angles (2θ) of 5.6 ± 0.2 °, 15.5 ± 0.2 ° and 22.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[3]
3. Method for producing crystals of the compound represented by formula (1): [Chemical formula 2] (1), characterized by the fact that it comprises the steps of: preparing a solution in which the compound represented by formula (1) is
2/26 dissolved; and then precipitating crystals of the compound represented by formula (1) from the solution.
[4]
4. Production method according to claim 3, characterized by the fact that the crystals of the compound represented by the formula (1) show main peaks in the diffraction angles (2θ) of 5.6 ± 0.2 °, 15.5 ± 0.2 ° and 22.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[5]
Production method according to claim 3 or 4, characterized in that the solution in which the compound represented by formula (1) is dissolved comprises a lower ketone and a lower alcohol as solvents.
[6]
Production method according to claim 5, characterized by the fact that the lower ketone is acetone.
[7]
Production method according to claim 5, characterized in that the lower ketone is methyl ethyl ketone.
[8]
Production method according to any one of claims 5 to 7, characterized in that the lower alcohol is 1-propanol.
[9]
Production method according to any one of claims 5 to 7, characterized in that the lower alcohol is 2-butanol.
[10]
Production method according to any one of claims 3 to 9, characterized in that it comprises a step of adding a seed crystal to the crystals of the compound represented by formula (1).
[11]
Production method according to any one of claims 3 to 10, characterized in that the compound represented by formula (1) is produced by a production method (I), wherein the production method (I) is a production method
3/26 comprising the steps of: deprotecting protective groups to an amino group and a carboxyl group of a compound represented by formula (B): [Chemical formula 3]
(B) where R1 represents an amino group protected with a protecting group and R2 represents a carboxyl group protected with a protecting group, in order to convert it into the compound represented by formula (8): [Chemical formula 4]
(8); then condense the compound represented by formula (8) with a compound represented by formula (C): [Chemical formula 5]
(C) where X represents an active ester group or a carboxyl group, in order to convert it into the compound represented by formula (10): [Chemical formula 6]
4/26 (10); and then condensing the compound represented by formula (10) with the compound represented by formula (11): [Chemical formula 7] (11) in order to convert it into the compound represented by formula (1): [Chemical formula 8] ( 1) .
[12]
Production method according to any one of claims 3 to 10, characterized in that the compound represented by formula (1) is produced by a production method (II), wherein the production method (II) is a production method that comprises the steps of:
5/26 deprotecting a protecting group to an amino group of a compound represented by formula (B): [Chemical formula 9]
(B) where R1 represents an amino group protected with a protecting group and R2 represents a carboxyl group protected with a protecting group, in order to convert it into a compound represented by formula (D): [Chemical formula 10]
(D) where R2 represents the same meaning as above; then condense the compound represented by formula (D) with a compound represented by formula (C): [Chemical formula 11]
(C) where X represents an active ester group or a carboxyl group, in order to convert it into a compound represented by formula (E): [Chemical formula 12]
6/26
(E) where R2 represents the same meaning as above; then deprotect the protecting group to the carboxyl group of the compound represented by formula (E), in order to convert it into the compound represented by formula (10): [Chemical formula 13]
(10); and then condense the compound represented by formula (10) with the compound represented by formula (11): [Chemical formula 14]
(11)
in order to convert it into the compound represented by formula (1): [Chemical formula 15]
7/26 (1).
[13]
Production method according to claim 11 or 12, characterized in that it comprises the steps of: dissolving the compound represented by formula (10) in a solvent containing 1,2-dimethoxyethane; and then precipitating crystals from a 1,2-dimethoxyethane adduct of the compound represented by formula (10).
[14]
14. Production method according to claim 13, characterized by the fact that the crystals of the adduct 1,2-dimethoxyethane of the compound represented by the formula (10) show main peaks at the diffraction angles (2θ) of 19.0 ± 0 , 2 ° and 25.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[15]
15. Production method according to any one of claims 11 to 14, characterized by the fact that the condensation step of the compound represented by formula (10) and the compound represented by formula (11), in order to convert it into compound represented by formula (1) is carried out in a two-phase system of an aqueous solution of sodium sulfate and tetrahydrofuran.
[16]
Production method according to any one of claims 3 to 10, characterized in that the compound represented by formula (1) is produced by a production method (III), wherein the production method (III) is a production method that comprises the steps of:
8/26 deprotecting a protecting group to a carboxyl group of a compound represented by formula (B): [Chemical formula 16]
(B) where R1 represents an amino group protected with a protecting group and R2 represents a carboxyl group protected with a protecting group, in order to convert it into a compound represented by formula (F): [Chemical formula 17]
(F) where R1 represents the same meaning as above; then condense the compound represented by formula (F) with the compound represented by formula (11): [Chemical formula 18]
(11)
in order to convert it into a compound represented by formula (G): [Chemical formula 19]
9/26
(G)
where R1 represents the same meaning as above; then deprotect the protecting group to the amino group of the compound represented by formula (G), in order to convert it into the compound represented by formula (16): [Chemical formula 20]
(16); and then condense the compound represented by formula (16) with a compound represented by formula (C): [Chemical formula 21]
(C) where X represents an active ester group or a carboxyl group, in order to convert it into the compound represented by formula (1):
10/26 [Chemical formula 22] (1).
[17]
Production method according to any one of claims 11 to 16, characterized in that the compound represented by formula (11) is in the form of a salt of methanesulfonic acid.
[18]
18. Production method according to any one of claims 11 to 16, characterized in that the compound represented by formula (11) is in the form of a salt of methanesulfonic acid m-hydrate, where m is in the range of 0 to 3.
[19]
19. Production method according to any of claims 11 to 16, characterized in that the compound represented by formula (11) is in the form of a dihydrated methanesulfonic acid salt.
[20]
20. Production method according to any one of claims 11 to 19, characterized in that the compound represented by formula (B) is produced by a production method (IV), wherein the production method (IV) is a production method comprising the steps of: reacting a compound represented by formula (H): [Chemical formula 23]
11/26
(H) where R3 represents an amino group protected with a lead tetracetate protecting group in order to convert it into a compound represented by formula (J): [Chemical formula 24]
(J) where R3 represents the same meaning as above; then react the compound represented by formula (J) with a compound represented by formula (K): [Chemical formula 25]
(K) where R2 represents the same meaning as R2 as defined in any of claims 11 to 19, in the presence of an acid or a base, in order to convert it to a compound represented by the formula (L): [ Chemical formula 26]
(L) where R2 and R3 represent the same meaning as above; then deprotect the protecting group to the amino group of the compound represented by the formula (L), in order to convert it into a compound represented by the formula (M):
12/26 [Chemical formula 27] (M) where R2 represents the same meaning as above; and then condensing the compound represented by formula (M) with a compound represented by formula (N): [Chemical formula 28] (N) where R1 represents the same meaning as R1 as defined in any of claims 11 to 19, in order to convert it into the compound represented by formula (B): [Chemical formula 29] (B) where R1 and R2 represent the same meaning as above.
[21]
21. Production method according to claim 20, characterized by the fact that the reaction step of the compound represented by the formula (H) with lead tetracetate, in order to convert it into the compound represented by the formula (J), is performed in the presence of acetic acid.
[22]
22. Production method according to claim 20 or
13/26 21, characterized by the fact that the reaction step of the compound represented by formula (J) with the compound represented by formula (K), in order to convert it into the compound represented by formula (L), is carried out in presence of an aqueous sodium hydroxide solution.
[23]
23. Production method according to claim 20 or 21, characterized by the fact that the reaction step of the compound represented by formula (J) with the compound represented by formula (K), in order to convert it into the compound represented using formula (L), it is carried out in the presence of tris (pentafluorophenyl) borane.
[24]
24. Production method according to any one of claims 20 to 23, characterized in that it comprises an acid addition step to precipitate a salt of the compound represented by the formula (M) and the acid after the deprotection step of the protecting group for the amino group of the compound represented by the formula (L), in order to convert it into the compound represented by the formula (M).
[25]
25. Production method according to claim 24, characterized in that the acid is 1-hydroxybenzotriazole.
[26]
26. Production method according to any one of claims 11 to 25, characterized in that R1 is an amino group protected with a benzyloxycarbonyl group.
[27]
27. Production method according to any one of claims 11 to 25, characterized in that R1 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
[28]
28. Production method according to any one of claims 11 to 27, characterized in that R2 is a carboxyl group protected with a benzyl group.
[29]
29. Production method according to any one of claims 20 to 28, characterized in that R3 is an amino group protected with a (9H-fluoren-9-ylmethoxy) carbonyl group.
14/26
[30]
30. Production method according to any one of claims 11 to 29, characterized in that X is an (2,5-dioxopyrrolidin-1-yl) oxycarbonyl group.
[31]
31. Production method according to any one of claims 3 to 10, characterized in that the compound represented by formula (1) is produced by a production method (V), wherein the production method (V) is a production method comprising the steps of: reacting the compound represented by formula (2): [Chemical formula 30] (2) with lead tetracetate in order to convert it into the compound represented by formula (3): [Chemical formula 31] (3); then react the compound represented by formula (3) with benzyl glycolate, in the presence of an acid or a base, in order to convert it into the compound represented by formula (4): [Chemical formula 32]
15/26
(4); then deprotect a protecting group to an amino group of the compound represented by formula (4), in order to convert it into the compound represented by formula (5): [Chemical formula 33]
(5); then condense the compound represented by formula (5) with the compound represented by formula (6): [Chemical formula 34]
(6) in order to convert it into the compound represented by formula (7): [Chemical formula 35]
(7); then unprotect protecting groups to an amino group and a
16/26 carboxyl group of the compound represented by formula (7), in order to convert it into the compound represented by formula (8): [Chemical formula 36]
(8); then condense the compound represented by formula (8) with the compound represented by formula (9): [Chemical formula 37]
(9) in order to convert it into the compound represented by formula (10): [Chemical formula 38]
(10); and then condense the compound represented by formula (10) with the compound represented by formula (11): [Chemical formula 39]
17/26 (11) in order to convert it into the compound represented by formula (1): [Chemical formula 40] (1).
[32]
32. Production method according to claim 31, characterized in that it comprises the steps of: dissolving the compound represented by formula (10) in a solvent containing 1,2-dimethoxyethane; and then precipitating crystals from a 1,2-dimethoxyethane adduct of the compound represented by formula (10).
[33]
33. Production method according to claim 32, characterized in that the crystals of the adduct 1,2-dimethoxyethane of the compound represented by the formula (10) show main peaks at the diffraction angles (2θ) of 19.0 ± 0 , 2 ° and 25.0 ± 0.2 ° in powder X-ray diffraction, obtained by irradiation with copper Kα radiation.
[34]
34. Production method according to any one of claims 31 to 33, characterized in that the step of condensing the compound represented by formula (10) with the compound represented by formula (11), in order to convert it into compound represented by
18/26 formula (1), is carried out in a biphasic system of an aqueous solution of sodium sulfate and tetrahydrofuran.
[35]
35. Production method according to any one of claims 3 to 10, characterized in that the compound represented by formula (1) is produced by a production method (VI), wherein the production method (VI) is a production method comprising the steps of: reacting the compound represented by formula (2): [Chemical formula 41] (2) with lead tetracetate in order to convert it into the compound represented by formula (3): [Chemical formula 42]
O O
H
O N N O Me
H
O (3); then react the compound represented by formula (3) with benzyl glycolate, in the presence of an acid or a base, in order to convert it into the compound represented by formula (4): [Chemical formula 43] (4); So
19/26 deprotecting a protecting group to an amino group of the compound represented by formula (4), in order to convert it into the compound represented by formula (5): [Chemical formula 44]
(5); then condense the compound represented by formula (5) with the compound represented by formula (12): [Chemical formula 45]
(12) in order to convert it into the compound represented by formula (13): [Chemical formula 46]
(13); then deprotect a protecting group to a carboxyl group of the compound represented by formula (13), in order to convert it into the compound represented by formula (14): [Chemical formula 47]
20/26
(14); then condense the compound represented by formula (14) with the compound represented by formula (11): [Chemical formula 48]
(11)
in order to convert it into the compound represented by formula (15): [Chemical formula 49]
(15); then deprotect a protecting group to an amino group of the compound represented by formula (15), in order to convert it into the compound represented by formula (16): [Chemical formula 50]
21/26 (16); and then condensing the compound represented by formula (16) with the compound represented by formula (9): [Chemical formula 51] (9) in order to convert it into the compound represented by formula (1): [Chemical formula 52] ( 1) .
[36]
36. Production method according to any one of claims 31 to 35, characterized in that the reaction step of the compound represented by formula (2) with lead tetracetate in order to convert it into the compound represented by formula (3 ) is performed in
22/26 presence of acetic acid.
[37]
37. Production method according to any one of claims 31 to 36, characterized in that the step of converting the compound represented by formula (3) into the compound represented by formula (4) is carried out in the presence of an aqueous solution of sodium hydroxide.
[38]
38. Production method according to any one of claims 31 to 36, characterized in that the step of converting the compound represented by formula (3) into the compound represented by formula (4) is carried out in the presence of tris (pentafluorophenyl) borane.
[39]
39. Production method according to any one of claims 31 to 38, characterized in that it comprises an acid addition step to precipitate a salt of the compound represented by formula (5) and the acid after the deprotection step of the protecting group for the amino group of the compound represented by formula (4), in order to convert it into the compound represented by formula (5).
[40]
40. Production method according to claim 39, characterized in that the acid is 1-hydroxybenzotriazole.
[41]
41. Production method according to any one of claims 31 to 40, characterized in that the compound represented by formula (6) is produced by a method comprising the steps of: condensing the compound represented by formula (23): [Chemical formula 53] (23) with N-hydroxysuccinimide in order to convert it into the compound represented by formula (24):
23/26 [Chemical formula 54] (24); and then condensing the compound represented by formula (24) with L-phenylalanine in order to convert it to the compound represented by formula (6).
[42]
42. Production method according to any one of claims 31 to 41, characterized in that the compound represented by formula (9) is produced by a method comprising the steps of: reacting the compound represented by formula (17): [Chemical formula 55] (17) with maleic anhydride in order to convert it into the compound represented by formula (18): [Chemical formula 56] (18); and then add thionyl chloride to the compound represented by formula (18) and a mixed solution containing N-hydroxysuccinimide and 2,6-lutidine in order to convert it to the compound represented by formula (9).
[43]
43. Production method according to any one of claims 31 to 42, characterized in that the compound represented by formula (11) is in the form of an acid salt
24/26 methanesulfonic.
[44]
44. Production method according to any one of claims 31 to 42, characterized in that the compound represented by formula (11) is in the form of a salt of methanesulfonic acid m-hydrate, where m is in the range of 0 to 3.
[45]
45. Production method according to any one of claims 31 to 42, characterized in that the compound represented by formula (11) is in the form of a dihydrated methanesulfonic acid salt.
[46]
46. Production method according to any of claims 3 to 45, characterized in that it does not use chromatography.
[47]
47. Crystals, characterized by the fact that they are from a 1,2-dimethoxyethane adduct of the compound represented by formula (10): [Chemical formula 47] (10).
[48]
48. Crystals according to claim 47, characterized by the fact that the crystals show major peaks at the diffraction angles (2θ) of 19.0 ± 0.2 ° and 25.0 ± 0.2 ° in the diffraction of x of powder, obtained by irradiation with copper Kα radiation.
[49]
49. Salt, characterized by the fact that it is the compound represented by formula (5): [Chemical formula 58]
25/26 (5) and an acid.
[50]
50. Salt according to claim 49, characterized in that the acid is 1-hydroxybenzotriazole.
[51]
51. Method for producing an antibody-drug conjugate, characterized by the fact that a linker-drug is represented by formula (19): [Chemical formula 60] (19) where A represents the connection position with an antibody, is conjugated to the antibody via a thioether bond, where crystals of the compound represented by formula (1): [Chemical formula 59] (1)
26/26 produced by the method as defined in any of claims 3 to 46, are used as starting material, and the method comprises the steps of: i) reducing an antibody; and then ii) adding a solution in which the crystals of the compound represented by formula (1) produced in the method mentioned above is dissolved, to react the solution with the reduced antibody.
[52]
52. Production method according to claim 51, characterized in that the antibody is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody or an anti- GPR20.

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法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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JP2017-167691|2017-08-31|

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JP2017167691|2017-08-31|

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PCT/JP2018/032056|WO2019044947A1|2017-08-31|2018-08-30|Improved method for producing antibody-drug conjugate|

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