Antibody-linker-drug conjugate, preparation method therefor, and anticancer drug composition containing same
09814784 · 2017-11-14
Assignee
Inventors
- Young Jun Park (Incheon, KR)
- Jin-kyo Jeong (Incheon, KR)
- Young Mi Choi (Incheon, KR)
- Minseob Lee (Incheon, KR)
- Yeon Jung Kim (Incheon, KR)
- Kyoung Suk Kim (Incheon, KR)
- Joon hun Choi (Incheon, KR)
- Jin Seo Lee (Incheon, KR)
- Eun Joo Cho (Incheon, KR)
- Hyunnam Song (Incheon, KR)
- Sung Jun Park (Incheon, KR)
- Jong-hyoup Lee (Incheon, KR)
- Matthew Sangyup Lee (Incheon, KR)
- In-Suk Lee (Incheon, KR)
- Joon woo Kim (Incheon, KR)
- Seung Suh Hong (Incheon, KR)
Cpc classification
A61K47/6889
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
A61K47/6829
HUMAN NECESSITIES
A61K47/6811
HUMAN NECESSITIES
A61K47/6855
HUMAN NECESSITIES
C07D403/12
CHEMISTRY; METALLURGY
A61K47/6851
HUMAN NECESSITIES
C07D207/416
CHEMISTRY; METALLURGY
International classification
C07D403/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to an antibody-linker-drug conjugate in which an antibody and a cytotoxic drug are conjugated through an enzyme cleavable peptide linker capable of directly binding to a lysine residue of an antibody, a preparation method therefor, and an anticancer drug composition containing the same as an active ingredient.
Claims
1. An antibody-linker-drug conjugate, wherein the linker-drug moiety is directly bonded to a lysine residue of the antibody, represented by Chemical Formula I-1, or a pharmaceutically acceptable salt thereof: ##STR00214## wherein, Ab is an antibody, X is C.sub.1-C.sub.8 alkyl or C.sub.3-C.sub.6 cycloalkyl, R.sub.1 is a hydroxy, C.sub.1-C.sub.4 alkoxy, or oxo (═O), R.sub.2 is a hydrogen atom or C.sub.1-C.sub.4 alkyl, R.sub.3 is a hydrogen atom, hydroxy, C.sub.1-C.sub.4 alkoxy, amino, oxo (═O), or hydroxyimino (═N—OH), R.sub.4 is aryl, and n is an integer ranging from 1 to 5.
2. The antibody-linker-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody immunospecifically binding to a cancer cell antigen, X is C.sub.1-C.sub.8 alkyl or C.sub.3-C.sub.6 cycloalkyl, R.sub.1 is a hydroxy, or C.sub.1-C.sub.4 alkoxy, R.sub.2 is a hydrogen atom or C.sub.1-C.sub.4 alkyl, R.sub.3 is a hydrogen atom, oxo (═O) or hydroxyimino (═N—OH), R.sub.4 is phenyl unsubstituted or substituted with at least one substituent selected from the group consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, and a halogen atom.
3. The antibody-linker-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein: Ab is trastuzumab, X is methyl, n-propyl, n-pentyl, n-octyl, or cyclopropyl, R.sub.1 is a, hydroxy, or methoxy, R.sub.2 is a hydrogen atom or methyl, R.sub.3 is a hydrogen atom, oxo (═O) or hydroxyimino (═N—OH), and R.sub.4 is phenyl unsubstituted or substituted with at least one substituent selected from the group consisting of methyl, methoxy, and a halogen atom.
4. The antibody-linker-drug conjugate of claim 1, being selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt thereof: ##STR00215## wherein, n is an integer ranging from 1 to 5.
5. The antibody-linker-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the antibody-linker-drug conjugate has a normal antibody structure at a rate of 98% or higher.
6. An antibody-linker-drug conjugate, wherein the linker-drug moiety is directly bonded to a lysine residue of the antibody, represented by Chemical Formula I-3, or a pharmaceutically acceptable salt thereof: ##STR00216## wherein, Ab is an antibody, X is C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloalkyl, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently a hydrogen atom, or C.sub.1-C.sub.4 alkyl, R.sub.5 is a hydrogen atom, hydroxy, C.sub.1-C.sub.4 alkoxy, amino, oxo (═O), or hydroxyimino (═N—OH), Ar is aryl, Y is a nitrogen atom or an oxygen atom, R.sub.6 is a hydrogen atom or C.sub.1-C.sub.4 alkyl when Y is a nitrogen atom; or is absent when Y is an oxygen atom, and n is an integer ranging from 1 to 5.
7. The antibody-linker-drug conjugate of claim 6, or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody immunospecifically binding to a cancer cell antigen, X is C.sub.1-C.sub.8 alkyl or C.sub.3-C.sub.6 cycloalkyl, R.sub.1, R.sub.2, and R.sub.4 are each independently a hydrogen atom or C.sub.1-C.sub.4 alkyl, R.sub.3 is C.sub.1-C.sub.4 alkyl, R.sub.5 is a hydrogen atom, amino, oxo (═O), or hydroxyimino (═N—OH), Ar is phenyl or naphthyl unsubstituted or substituted with at least one substituent selected from the group consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, and a halogen atom, Y is a nitrogen atom or an oxygen atom, and R.sub.6 is a hydrogen atom or C.sub.1-C.sub.4 alkyl when Y is a nitrogen atom, or is absent when Y is an oxygen atom.
8. The antibody-linker-drug conjugate of claim 6, or a pharmaceutically acceptable salt thereof, wherein: Ab is trastuzumab, X is methyl, n-propyl, n-pentyl, n-octyl, or cyclopropyl, R.sub.1, R.sub.2, and R.sub.3 are each methyl, R.sub.4 is a hydrogen atom, or methyl, R.sub.5 is a hydrogen atom, oxo (═O), or hydroxyimino (═N—OH), Ar is phenyl or naphthyl unsubstituted or substituted with at least one substituent selected from the group consisting of methyl, methoxy, and a halogen atom, Y is a nitrogen atom or an oxygen atom, and R.sub.6 is a hydrogen atom, or methyl when Y is a nitrogen atom, or is absent when Y is an oxygen atom.
9. The antibody-linker-drug conjugate of claim 6, being selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt thereof: ##STR00217## ##STR00218## ##STR00219## wherein, n is an integer ranging from 1 to 5.
10. The antibody-linker-drug conjugate of claim 6, or a pharmaceutically acceptable salt thereof, wherein the antibody-linker-drug conjugate has a normal antibody structure at a rate of 90% or higher.
11. A method for preparing an antibody-linker-drug conjugate represented by Chemical Formula I-1, comprising the steps of: (i) condensating a compound of the following Chemical Formula II-1 with a dolastatin 10derivative of the following Chemical Formula III-1 to give a compound of the following Chemical Formula IV-1; (ii) subjecting the compound of the following Chemical Formula IV-1 to an addition reaction with a compound of the following Chemical Formula V to give a compound of the following Chemical Formula VI-1; (iii) condensating the compound of the following Chemical Formula VI-1 with N-hydroxysuccinimide to give a compound of the following Chemical Formula VII-1; and (iv) reacting the compound of the following Chemical Formula VII-1 with an antibody: ##STR00220## ##STR00221## wherein, Ab is an antibody, X is C.sub.1-C.sub.8 alkyl or C.sub.3-C.sub.6 cycloalkyl, R.sub.1 is, hydroxy, C.sub.1-C.sub.4 alkoxy or oxo (═O), R.sub.2 is a hydrogen atom or C.sub.1-C.sub.4 alkyl, R.sub.3 is a hydrogen atom, hydroxy, C.sub.1-C.sub.4 alkoxy, amino, oxo (═O), or hydroxyimino (═N—OH), R.sub.4 is aryl, and n is an integer ranging from 1 to 5.
12. The method of claim 11, wherein the condensation reaction of step (i) is carried out in presence of a condensing agent selected from the group consisting of hydroxybenzotriazole (HOBt), hydroxyazabenzotriazole (HOAt), and hydroxysuccinimide (HOSu), and an organic base selected from the group consisting of pyridine and diisopropylethylamine.
13. The method of claim 11, wherein the condensation reaction of step (iii) is carried out in presence of a condensing agent selected from the group consisting of dicyclohexylcarbodiimide (DCC), and (3-dimethylaminopropyl)carbodiimide (EDC).
14. The method of claim 11, wherein the reaction of step (iv) is carried out in a phosphate buffer having a pH of 6.0 to 8.0 as a reaction solvent.
15. The method of claim 11, wherein the reaction of step (iv) is carried out in a mixed reaction solvent of a phosphate buffer having a pH of 6.0 to 8.0 and an organic solvent.
16. The method of claim 15, wherein the organic solvent amounts to 50% or less of the mixed solvent.
17. The method of claim 11, wherein the compound of Chemical Formula VII-1is used in an amount of 3 to 25 equivalents, based on the antibody, in step (iv).
18. A method for preparing an antibody-linker-drug conjugate represented by Chemical Formula I -3, comprising the steps of: (i) condensating a compound of the following Chemical Formula II-1 with a dolastatin 10 derivative of the following Chemical Formula III-3 to give a compound of the following Chemical Formula IV-3; (ii) subjecting the compound of the following Chemical Formula IV-3 to an addition reaction with a compound of the following Chemical Formula V to give a compound of the following Chemical Formula VI-3; (iii) condensating the compound of the following Chemical Formula VI-3 with N-hydroxysuccinimide to give a compound of the following Chemical Formula VII-3; and (iv) reacting the compound of the following Chemical Formula VII-3 with an antibody: ##STR00222## ##STR00223## wherein, Ab is an antibody, X is C.sub.1-C.sub.8 alkyl, or C.sub.3-C.sub.6 cycloalkyl, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently a hydrogen atom, or C.sub.1-C.sub.4 alkyl, R.sub.5 is a hydrogen atom, hydroxy, C.sub.1-C.sub.4 alkoxy, amino, oxo (═O), or hydroxyimino (═N—OH), Ar is aryl, Y is a nitrogen atom or an oxygen atom, R.sub.6 is a hydrogen atom or C.sub.1-C.sub.4 alkyl when Y is a nitrogen atom; or is absent when Y is an oxygen atom, and n is an integer ranging from 1 to 5.
19. The method of claim 18, wherein the condensation reaction of step (i) is carried out in presence of a condensing agent selected from the group consisting of hydroxybenzotriazole (HOBt), hydroxyazabenzotriazole (HOAt), and hydroxysuccinimide (HOSu), and an organic base selected from the group consisting of pyridine and diisopropylethylamine.
20. The method of claim 18, wherein the condensation reaction of step (iii) is carried out in presence of a condensing agent selected from the group consisting of dicyclohexylcarbodiimide (DCC), and (3-dimethylaminopropyl)carbodiimide (EDC).
21. The method of claim 18, wherein the reaction of step (iv) is carried out in a phosphate buffer having a pH of 6.0 to 8.0 as a reaction solvent.
22. The method of claim 18, wherein the reaction of step (iv) is carried out in a mixed reaction solvent of a phosphate buffer having a pH of 6.0 to 8.0 and an organic solvent.
23. The method of claim 22, wherein the organic solvent amounts to 50% or less of the mixed solvent.
24. The method of claim 18, wherein the compound of Chemical Formula VII-3 is used in an amount of 3 to 25 equivalents, based on the antibody, in step (iv).
Description
DESCRIPTION OF DRAWINGS
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MODE FOR INVENTION
(30) A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as limiting the present invention.
PREPARATION EXAMPLE 1
Preparation of Compounds of Chemical Formula II-1 and II-2
PREPARATION EXAMPLE 1-1
Preparation of Compound (II-1)
(31) ##STR00041##
(32) Under an argon stream, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-(4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (5.0 g, 8.7 mmol, Dubowchik et al., Bioconjugate Chem., 2002, 13 (4), pp 855-869) was dissolved in 60 mL of anhydrous dimethylformamide, added with N, N-diisopropylethylamine (3.0 mL, 17.4 mmol), and cooled to 0° C. To the mixture was added at once bis(4-nitrophenyl) carbonate (7.94 g, 26.1 mmol), followed by stirring at room temperature for 15 hrs. After completion of the reaction, the reaction mixture was concentrated in a vacuum, and the concentrate was purified by silica gel column chromatography to obtain the title compound (3.65 g, 57%) as a pale yellow solid.
(33) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.83 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3 H), 1.09 (t, J=7.2 Hz, 1 H), 1.19 (m, 2 H), 1.34-1.76 (m, 7 H), 1.96 (m, 1 H), 2.15 (m, 2 H), 2.99 (m, 2 H), 3.37 (m, 2 H), 4.19 (t, J=7.8 Hz, 1 H), 4.39 (m, 1 H), 5.24 (s, 2 H), 5.41 (s, 2 H), 5.97 (brt, J=5.6 Hz, 1 H), 7.00 (s, 2H), 7.41 (d, J=8.4 Hz, 2 H), 7.57 (d, J=7.2 Hz, 2H), 7.65 (d, J=8.4 Hz, 2 H), 7.80 (d, J=8.4 Hz, 1 H), 8.09 (d, J=7.2 Hz, 1 H), 8.31 (d, J=7.2 Hz, 2H), 10.05 (brs, 1 H)
PREPARATION EXAMPLE 1-2
Preparation of Compound (II-3)
(34) ##STR00042##
(35) Under an argon stream, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (5.0 g, 8.7 mmol, Dubowchik et al., Bioconjugate Chem., 2002, 13 (4), pp 855-869) was dissolved in 60 mL of anhydrous dimethylformamide, added with N, N-diisopropylethylamine (3.0 mL, 17.4 mmol), and then cooled to 0° C. To the mixture was added at once bis(4-nitrophenyl) carbonate (7.94 g, 26.1 mmol), followed by stirring at room temperature for 15 hrs. After completion of the reaction, the reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 57%) as a pale yellow solid.
(36) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.83 (d, J=6.8 Hz, 3 H), 0.86 (d, J=6.8 Hz, 3 H), 1.09 (t, J=7.2 Hz, 1 H), 1.19 (m, 2 H), 1.34-1.76 (m, 7 H), 1.96 (m, 1 H), 2.15 (m, 2 H), 2.99 (m, 2 H), 3.37 (m, 2 H), 4.19 (t, J=7.8 Hz, 1 H), 4.39 (m, 1 H), 5.24 (s, 2 H), 5.41 (s, 2 H), 5.97 (brt, J=5.6 Hz, 1 H), 7.00 (s, 2H), 7.41 (d, J=8.4 Hz, 2 H), 7.57 (d, J=7.2 Hz, 2 H), 7.65 (d, J=8.4 Hz, 2 H), 7.80 (d, J=8.4 Hz, 1 H), 8.09 (d, J=7.2 Hz, 1 H), 8.31 (d, J=7.2 Hz, 2 H), 10.05 (brs, 1 H)
(37) Under an argon stream, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 4.95 mmol) was dissolved in 90 mL of anhydrous dimethylformamide, and stirred, together with t-butyl(2-aminoethyl)(methyl)carbamate (0.86 g, 4.95 mmol), at 20-25° C. for 2 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-3) (3.8 g, 99%).
(38) LC-MS m/z: 773.5 [M+H].sup.+
(39) To a solution of the amino-protected derivative of compound (II-3) (146 mg, 0.186 mmol) in 5 mL of dichloromethane was dropwise added 2 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 2 hrs. After completion of the reaction, the reaction solvent was removed by vacuum concentration, and then trifluoroacetic acid was completely removed by adding 5 mL of toluene twice to obtain a concentrated TFA salt of the title compound.
PREPARATION EXAMPLE 1-3
Preparation of Compound (II-4)
(40) ##STR00043##
(41) Under an argon stream, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (6.8 g, 9.22 mmol) was dissolved in 150 mL of anhydrous dimethylformamide, and stirred, together with t-butyl methyl(2-(methylamino)ethyl)carbamate (1.82 g, 9.68 mmol) at 20-25° C. for 19 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-4) (5.14 g, 71%).
(42) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.78-0.8 (d, 3 H), 0.8-0.83 (d, 3 H), 1.09-1.99 (m, 10 H), 1.33 (s, 9 H), 1.92 (m, 1 H), 2.07-2.19 (m, 2H), 2.66-2.73 (d, 3H), 2.8-2.81 (m, 3H), 2.9-3.0 (m, 2H), 3.37 (m, 2 H), 4.15 (t, 1 H), 4.34 (m, 1 H), 4.94 (s, 2 H), 5.38 (s, 2 H), 5.94 (brt, 1 H), 6.96 (s, 2H), 7.23 (d, 2 H), 7.55 (d, 2 H), 7.77 (d, 1 H), 8.04 (d, 1 H), 9.95 (brs, 1 H)
(43) LC-MS m/z: 787.5 [1\4+H].sup.+
(44) With the exception of using the amino-protected derivative of compound (II-4), the same procedure as in Preparation Example 1-2 was repeated to obtain a concentrated TFA salt of the title compound.
PREPARATION EXAMPLE 1-4
Preparation of Compound (II-5)
(45) ##STR00044##
(46) Under an argon stream, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (6.8 g, 9.22 mmol) was dissolved in 80 mL of anhydrous dimethylformamide, and stirred, together with t-butyl(2-aminoethyl)carbamate (1.63 g, 10.14 mmol), at 20-25° C. for 15 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-5) (4.13 g, 59%).
(47) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.78-0.8 (d, 3 H), 0.8-0.83 (d, 3 H), 1.09-1.99 (m, 10 H), 1.33 (s, 9 H), 1.92 (m, 1 H), 2.07-2.19 (m, 2H), 2.9-3.0 (m, 2H), 3.37 (m, 2 H), 4.15 (t, 1 H), 4.34 (m, 1 H), 4.94 (s, 2 H), 5.38 (s, 2 H), 5.94 (brt, 1 H), 6.76 (m, 1 H), 6.96 (s, 2H), 7.15 (m, 1H), 7.23 (d, 2 H), 7.55 (d, 2 H), 7.77 (d, 1 H), 8.04 (d, 1 H), 9.95 (brs, 1 H)
(48) LC-MS m/z: 759.5 [M+H].sup.+
(49) With the exception of using the amino-protected derivative of compound (II-5), the same procedure as in Preparation Example 1-2 was repeated to obtain a concentrated TFA salt of the title compound.
PREPARATION EXAMPLE 1-5
Preparation of Compound (II-6)
(50) ##STR00045##
(51) Under an argon stream, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.74 g, 1 mmol) was dissolved in 30 mL of anhydrous dimethylformamide, and stirred, together with t-butyl (2-(methylamino)ethyl)carbamate (0.25 g, 1.4 mmol), at 20-25° C. for 15 hrs. After completion of the reaction, the reaction mixture was concentrated to the completion in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-6) (0.36 g, 48%).
(52) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.79 (d, 3 H), 0.82 (d, 3 H), 1.13-1.7 (m, 10 H), 1.32-1.39 (d, 9 H), 1.89-1.93 (m, 1 H), 2.07-2.17 (m, 2 H), 2.79-2.82 (d, 2 H), 2.89-3.02 (m, 5H), 3.14 (d, 1 H), 3.2 (m, 1H), 4.15 (t, 1 H), 4.34 (m, 1 H), 4.9-4.94 (d, 2 H), 5.37 (s, 2 H), 5.941 (brt, 1 H), 6.81-6.85 (m, 1 H), 7.24 (d, 2H), 7.54 (d, 2 H), 7.76 (d, 1 H), 8.04 (d, 1 H), 9.95 (brs, 1 H)
(53) LC-MS m/z: 773.5 [M+H].sup.+
(54) With the exception of using the amino-protected derivative of compound (II-6), the same procedure as in Preparation Example 1-2 was repeated to obtain a concentrated TFA salt of the title compound.
PREPARATION EXAMPLE 2
Preparation of Compounds of Chemical Formulas III-1, III-2, III-3 and III-4
PREPARATION EXAMPLE 2-1
Preparation of Compound (III-5)
(55) ##STR00046##
(56) Under an argon stream, (2R,3R)-3-((S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (0.63 g, 2.2 mmol) was dissolved in 10 mL of anhydrous dimethylformamide, and added with 2-(2,6-difluorophenyl)ethanamine (0.38 g, 2.4 mmol). The temperature was cooled to 0° C. before benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (0.97 g, 2.2 mmol), and diisopropylethylamine (1.0 mL, 6.6 mmol) were sequentially added to the reaction mixture. Stirring was continued at room temperature for 15 hrs. After completion of the reaction, the resulting reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to afford (R)-t-butyl 2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.60 g, 64%).
(57) To a solution of this compound (0.6 g, 1.4 mmol) in 10 mL of dichloromethane was added 1 mL of trifluoroacetic acid, followed by stirring at room temperature for 3 hrs. After completion of the reaction, the reaction solvent was removed by vacuum concentration, and the residual trifluoroacetic acid was completely removed by adding 10 mL of dichloromethane twice to obtain a trifluoroacetic acid salt of (2R,3R)-N-(2,6-difluorophenethyl)-3-methoxy-2-methyl-3-((R)-pyrrolidin-2-yl)propanamide.
(58) This concentrate (TFA salt) was dissolved, together with (5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-1-methyl-2-oxa-4,7,10-triazatetradecan-14-oic acid (0.77 g, 1.4 mmol), in 10 mL of dimethylformamide, and sequentially added with diethyl cyanophosphate (DEPC) (0.23 mL, 1.54 mmol) and triethylamine (1.17 mL, 8.4 mmol) at 0° C. before being stirred at room temperature for 15 hrs. After completion of the reaction, the solvent was removed, and the residue was dissolved in 50 mL of ethylacetate and washed with 30 mL of distilled water. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford an N-terminal amino-protected derivative of compound (III-5) (0.96 g, 80%).
(59) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.75-1.06 (m, 17 H), 1.18-1.38 (m, 5H), 1.71-2.15 (m, 7 H), 2.21-2.52 (m, 4 H), 2.87-2.91 (m, 4 H), 2.99 (s, 3 H), 3.10 (s, 3 H), 3.38 (s, 3 H), 3.43-3.51 (m, 3 H), 3.82 (m, 1 H), 4.09-4.15 (m, 4 H), 4.69 (m, 1H), 5.12-5.23 (m, 2 H), 6.50-6.60 (m, 2 H), 6.82-6.87 (m, 2 H), 7.13-7.17 (m, 1 H), 7.30-7.34 (m, 5 H)
(60) The N-terminal amino-protected derivative of compound (III-5) (0.96 g, 1.1 mmol) was dissolved in a mixture of 18 mL of t-butyl alcohol and 2 mL of water, and stirred for 3 hrs in the presence of 10% palladium carbon (0.1 g) in a hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through a Celite filter, and washed several times with methanol. Then, the solvent was removed in a vacuum to afford the title compound (0.79 g, 99%).
(61) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.78-0.98 (m, 16 H), 1.06 (m, 1 H), 1.16-1.27 (m, 4 H), 1.38 (m, 1 H), 1.65-1.76 (m, 6 H), 1.89-2.02 (m, 4 H), 2.31-2.38 (m, 5 H), 2.71-2.75 (m, 1 H), 2.88-2.89 (m, 2 H), 3.00 (s, 3 H), 3.29 (s, 3 H), 3.41-3.48 (m, 3 H), 3.79-3.82 (m, 1 H), 4.08-4.15 (m, 2 H), 4.72-4.86 (m, 2 H), 6.56 (m, 1 H), 6.79-6.85 (m, 2 H), 7.10-7.15 (m, 1 H), 7.16-7.22 (m, 1 H), 7.55 (d, 1 H)
PREPARATION EXAMPLE 2-2
Preparation of Compound (III-6)
(62) ##STR00047##
(63) With the exception that 2-aminoacetophenone, instead of 2-(2,6-difluorophenyl)ethanamine, was used, the same procedure as in Preparation Example 2-1 was repeated to obtain the N-terminal amino-protected derivative of compound (III-6): 2.36 g (81%).
(64) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.71-1.04 (m, 17 H), 1.31-1.32 (m, 4H), 1.77-1.81 (m, 2 H), 1.92-2.04 (m, 4 H), 2.17-2.61 (m, 4 H), 2.89-2.95 (m, 3 H), 2.99 (s, 3 H), 3.30 (s, 3 H), 3.46 (m, 3 H), 3.43-3.52 (m, 1 H), 3.99-4.01 (m, 1 H), 4.09-4.15 (m, 1 H), 4.24 (m, 1 H), 4.69-4.76 (m, 6 H), 5.09-5.23 (m, 2 H), 6.46-6.49 (m, 1 H), 7.12 (m, 1 H), 7.26-7.34 (m, 5H), 7.50 (t, J=7.6 Hz, 2 H), 7.62 (t, J=7.2 Hz, 1 H), 7.97 (d, J=7.6 Hz, 2 H)
(65) With the exception of using the amino-protected derivative of compound (III-6), the same procedure as in Preparation Example 2-1 was repeated to obtain the title compound: 1.89 g (96%).
(66) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.81-0.85 (m, 3 H), 0.92-1.14 (m, 13H), 1.25-1.43 (m, 5 H), 1.46-1.89 (m, 6 H), 1.92-2.22 (m, 5 H), 2.33 (s, 3 H), 2.45-2.54 (m, 2 H), 2.74 (d, J=4.8 Hz, 1 H), 3.02 (s, 3 H), 3.32 (s, 3 H), 3.43 (d, J=9.2 Hz, 3 H), 3.52-3.84 (m, 2 H), 3.92-4.06 (m, 2 H), 4.75-4.80 (m, 2 H), 4.94-5.23 (m, 1H), 6.76-6.94 (m, 1 H), 7.34-7.43 (m, 4H), 7.60 (m, 1 H)
PREPARATION EXAMPLE 2-3
Preparation of Compound (III-7)
(67) ##STR00048##
(68) With the exception that (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and 2-amino-1-(2-fluoro-4-methoxyphenyl)ethanone were used respectively instead of (2R,3R)-3-((S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and 2-(2,6-difluorophenyl)ethanamine, the same procedure as in Preparation Example 2-1 was repeated to afford the title compound. 0.18 g (89%).
(69) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.80-0.84 (m, 3 H), 0.88-1.03 (m, 17H), 1.32 (d, J=7.2 Hz, 3 H), 1.35-1.39 (m, 2 H), 1.98-2.10 (m, 4 H), 2.35 (s, 3 H), 2.36-2.51 (m, 3 H), 2.74 (m, 1 H), 3.02 (m, 3 H), 3.29 (s, 3 H), 3.34 (s, 3 H), 3.45 (s, 3H), 3.46-3.56 (m, 1 H), 3.87 (s, 3 H), 3.99 (dd, J=7.6 Hz, 2.8 Hz, 1 H), 4.10-4.15 (m, 1 H), 4.25-4.28 (m, 1 H), 4.58-4.64 (m, 2 H), 4.73-4.78 (m, 1 H), 6.67 (m, 1 H), 6.78 (qd, J=10.4 Hz, 2.8 Hz, 1 H), 7.95 (m, 1 H)
PREPARATION EXAMPLE 2-4
Preparation of Compound (III-8)
(70) ##STR00049##
(71) With the exception that (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and (S)-2-amino-1-(4-fluorophenyl)propan-1-one were used respectively instead of (2R,3R)-3-((S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and 2-(2,6-difluorophenyeethanamine, the same procedure as in Preparation Example 2-1 was repeated to afford the title compound. 0.45 g (100%).
(72) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.71-1.03 (m, 20 H), 1.22 (d, 3 H), 1.33-1.37 (m, 4 H), 1.40-1.44 (m, 3 H), 1.60-1.81 (m, 4 H), 1.98 (m, 2 H), 2.26 (m, 2 H), 2.90 (s, 3 H), 3.21 (s, 3 H), 3.32 (s, 3 H), 3.43 (s, 3 H), 4.06-4.15 (m, 2 H), 4.22-4.27 (m, 2 H), 4.64-4.69 (m, 2 H), 5.09-5.13 (m, 2 H), 5.23-5.30 (m, 2 H), 5.50 (m, 1 H), 7.18 (t, 2 H), 8.01-8.05 (m, 2 H)
PREPARATION EXAMPLE 2-5
Preparation of Compound (III-9)
(73) ##STR00050##
(74) With the exception that (R,E)-2-amino-1-(3-methoxyphenyl)propan-1-one oxime was used instead of 2-(2,6-difluorophenyl)ethanamine, the same procedure as in Preparation Example 2-1 was repeated to afford the title compound. 0.17 g (94%).
(75) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-0.98 (m, 20 H), 1.01 (m, 4 H), 1.19-1.27 (m, 4 H), 1.79-1.83 (m, 2 H), 2.21-2.43 (m, 4H), 2.77 (m, 2 H), 3.00 (m, 2 H), 3.29 (s, 6 H), 3.42 (s, 3 H), 3.99-4.14 (m, 3 H), 4.15-4.37 (m, 3 H), 4.70 (m, 1 H), 5.21-5.23 (m, 2 H), 5.36 (m, 1 H), 7.03 (m, 1 H), 7.15 (m, 2 H), 7.49 (m, 1 H)
PREPARATION EXAMPLE 2-6
Preparation of Compound (III-10)
(76) ##STR00051##
(77) With the exception that (2R,3R)-3-((2S,4S)-1-(t-butoxycarbonyl)-4-((t-butyldimethylsilyl)oxy)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid was used instead of (2R,3R)-3-((S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid, the same procedure as in Preparation Example 2-1 was repeated to obtain a raw N-terminal amino- and hydroxy-protected derivative of compound (III-10). 1.0 g (100%).
(78) To a solution of this compound (1.0 g, 1.02 mmol) in 10 mL of dimethylformamide was added 1.0 M tetrabutylammonium fluoride (3.1 mL, 3.1 mmol), followed by stirring for 5 hrs. After completion of the reaction, the solvent was removed, and the residue was dissolved in 50 mL of ethylacetate, and washed with a saturated sodium hydrogen carbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford an N-terminal amino-protected derivative of compound (III-10) (0.75 g, 84%).
(79) ES-MS m/z: 874 [M+H].sup.+
(80) With the exception of using the N-terminal amino-protected derivative of compound (III-10), the same procedure as in Preparation Example 2-1 was repeated to afford the title compound. 0.64 g (100%).
(81) ES-MS m/z: 740 [M+H].sup.+
PREPARATION EXAMPLE 2-7
Preparation of Compound (III-11)
(82) ##STR00052##
(83) Under an argon stream, (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (0.70 g, 2.2 mmol) was dissolved in 10 mL of anhydrous dimethylformamide, and added with (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl (0.47 g, 2.4 mmol). The temperature was cooled to 0° C. before benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (0.97 g, 2.2 mmol) and diisopropylethylamine (1.0 mL, 6.6 mmol) were sequentially added to the reaction mixture. Stirring was continued at room temperature for 15 hrs. After completion of the reaction, the resulting reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to afford (2S,4R)-t-butyl 2-((1R,2R)-3-(((2R)-1-azido-1-(4-fluorophenyl)propan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-methoxypyrrolidine-1-carboxylate (0.61 g, 56%).
(84) LC-MS m/z: 494.2 [M.sup.+].sup.+, 516.3 [M+Na].sup.+
(85) To a solution of this compound (0.61 g, 1.23 mmol) in 10 mL of dichloromethane was added 7 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the reaction solvent was removed by vacuum concentration, and the residual trifluoroacetic acid was completely removed by adding 5 mL of toluene twice, before a further reaction.
(86) This concentrate (TFA salt) was dissolved, together with (3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methyl heptanoic acid (0.53 g, 1.23 mmol), in 5 mL of dimethylformamide, and sequentially added with diethyl cyanophosphate (DEPC) (0.20 mL, 1.35 mmol) and triethylamine (1.56 mL, 11.09 mmol) at 0° C. before being stirred at room temperature for 16 hrs. When the reaction was completed, the solvent was removed, and the residue was dissolved in 20 mL of ethylacetate and extracted with 1 M potassium hydrogen sulfite, water, a saturated sodium hydrogen carbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford an N-terminal amino-protected derivative of compound (III-11) (0.25 g, 25%).
(87) LC-MS m/z: 779.6[M+H].sup.+
(88) The N-terminal amino-protected derivative of compound (III-11) (0.25 g, 0.31 mmol) was dissolved in a mixture of 10 mL of methyl alcohol, and stirred for 16 hrs in the presence of 10% palladium carbon (0.1 g) in a hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through a Celite filter, and washed several times with methanol. Then, the solvent was removed in a vacuum to afford the title compound as a white solid (97 mg, 41%).
(89) LC-MS m/z: 753[M.sup.+].sup.+, 775[M+Na].sup.+
(90) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.72 (t, 3 H), 0.77-1.02 (m, 17 H), 1.01 (d, 3 H), 1.15 (d, 3 H), 1.32 (m, 1 H), 1.78 (brd, 2H), 1.85-2.12 (m, 4 H), 2.18 (S, 6 H), 2.32-2.50 (m, 4H), 3.30 (s, 3H), 3.38 (s, 6H), 3.48 (m, 1H), 4.00-4.15 (m, 4H), 4.32 (m, 2H), 4.85 (m, 2H), 6.60 (s, 1H), 6.88 (s, 1H), 6.99-7.06 (m, 2H), 7.22-7.32 (m, 2H)
PREPARATION EXAMPLE 2-8
Preparation of Compound (III-12)
(91) ##STR00053##
(92) With the exception that (2R,3R)-3-((2S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (92 mg, 0.32 mmol) and (2R)-1-azido-1-phenylpropan-2-amine HCl (68 mg, 0.32 mmol) were respectively used instead of (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl, the same procedure as in Preparation Example 2-7 was repeated to afford the title compound (44 mg, 50%).
(93) MALDI-TOF MS m/z: 731.6 [M+H].sup.+, 753.5 [M+Na].sup.+
(94) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.81 (t, 3 H), 0.88-1.07 (m, 17 H), 1.18 (d, 3 H), 1.21 (d, 3 H), 1.47 (m, 1 H), 1.74 (brd, 2H), 1.87-2.17 (m, 3 H), 2.24 (S, 6 H), 2.25-2.47 (m, 4H), 3.04 (s, 3H), 3.28-3.34 (m, 1H), 3.31 (s, 3H), 3.38 (s, 3H), 3.45 (m, 1H), 3.79 (d, 1H), 3.82 (m, 1H), 4.05-4.12 (m, 4H), 4.24 (m, 2H), 4.75 (m, 2H), 6.73 (s, 1H), 6.95 (d, 1H), 7.24-7.35 (m, 5H)
PREPARATION EXAMPLE 2-9
Preparation of Compound (III-13)
(95) ##STR00054##
(96) With the exception that (2R,3R)-3-((2S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (0.80 g, 2.8 mmol), instead of (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid, and (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl (0.64 g, 2.8 mmol) were used, the same procedure as in Preparation Example 2-7 was repeated to afford the title compound (0.5 g, 79%).
(97) LC-MS m/z: 749.47 [M+H].sup.+, 771.44 [M+Na].sup.+
(98) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.82 (t, 3 H), 0.91-1.1 (m, 17 H), 1.16 (d, 3 H), 1.20 (d, 3 H), 1.35 (m, 1 H), 1.78 (brd, 2H), 1.87-2.10 (m, 4H), 2.24 (S, 6 H), 2.28-2.48 (m, 4H), 3.03 (s, 3H), 3.28-3.34 (m, 1H), 3.31 (s, 3H), 3.38 (s, 3H), 3.48 (m, 1H), 3.82 (d, 1H), 4.03-4.10 (m, 4H), 4.22 (m, 2H), 4.77 (m, 2H), 6.56 (d, 1H), 6.86 (s, 1H), 6.99-7.07 (m, 2H), 7.23-7.35 (m, 2H)
PREPARATION EXAMPLE 2-10
Preparation of Compound (III-14)
(99) ##STR00055##
(100) With the exception that (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-hydroxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (340 mg, 1.12 mmol), instead of (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid, and (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl (258 mg, 1.12 mmol) were used, the same procedure as in Preparation Example 2-7 was repeated to afford the title compound (350 mg, 71%).
(101) LC-MS m/z: 765.46 [M.sup.+].sup.+
(102) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.80 (t, 3 H), 0.90-1.09 (m, 17 H), 1.11 (d, 3 H), 1.25 (d, 3 H), 1.32 (m, 1 H), 1.78 (brd, 2H), 2.04-2.09 (m, 3 H), 2.23 (S, 6 H), 2.35-2.50 (m, 4H), 3.03 (s, 3H), 3.32 (s, 3H), 3.38 (m, 1H), 3.47 (s, 3H), 3.54-3.56 (m, 2H), 3.90 (m, 1H), 4.0-4.32 (m, 6H), 4.75 (m, 2H), 6.53 (s, 1H), 6.76 (d, 1H), 6.99-7.04 (m, 2H), 7.32-7.35 (m, 2H)
PREPARATION EXAMPLE 2-11
Preparation of Compound (III-15)
(103) ##STR00056##
(104) With the exception that (2R,3R)-3-((2S)-1-(t-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (0.17 g, 0.58 mmol) and (2R)-1-phthalimido-1-(4-methoxyphenyl)propan-2-amine HCl (0.2 g, 0.58 mmol) were used respectively instead of (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl, the same procedure as in Preparation Example 2-7 was repeated to afford an amino-protected derivative of compound (III-15) (0.34 g, 94%).
(105) LC-MS m/z: 891.62[M].sup.+
(106) To a solution of the amino-protected derivative of compound (III-15) (0.34 g, 0.38 mmol) in 10 mL of ethanol was added 1 mL of hydrazine hydrate, followed by stirring at room temperature for 16 hrs. After completion of the reaction, the solvent was removed in a vacuum. The residue was purified by silica gel column chromatography to afford the title compound as a white solid (0.11 g, 38%).
(107) LC-MS m/z: 761.56[M.sup.+].sup.+, 783.60[M+Na].sup.+
(108) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.80 (m, 3H), 0.88-1.07 (m, 18H), 1.19 (d, 3 H), 1.15 (m, 1H), 1.22 (m, 1 H), 1.25 (d, 3H), 1.70-1.90 (m, 4H), 2.07 (S, 6 H), 2.26 (s, 3H), 2.28-2.48 (m, 4H), 3.06 (s, 3H), 3.32 (s, 3H), 3.38 (m, 1H), 3.43 (s, 3H), 3.50 (m, 1H), 3.76 (m, 1H), 3.80 (s, 3H), 3.78 (d, 1H), 3.90-4.25 (m, 4H), 4.54 (m, 2H), 6.89 (d, 2H), 7.35 (d, 2H)
PREPARATION EXAMPLE 2-12
Preparation of Compound (III-16)
(109) ##STR00057##
(110) With the exception that (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-hydroxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (0.36 g, 1.17 mmol) and (2R)-1-phthalimido-1-(4-methoxyphenyl)propan-2-amine HCl (0.41 g, 1.17 mmol) were used respectively instead of (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl, the same procedure as in Preparation Example 2-7 was repeated to afford an amino-protected derivative of compound (III-16) (0.59 g, 74%).
(111) LC-MS m/z: 907.54[M].sup.+
(112) With the exception of using the amino-protected derivative of compound (III-16) (0.59 g, 0.65 mmol), the same procedure as in Preparation Example 2-11 was repeated to afford the title compound as a white solid (0.19 g, 38%).
(113) LC-MS m/z: 777.42[M.sup.+].sup.+, 799.39[M+Na].sup.+
(114) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-0.859 (m, 3H), 0.86-1.06 (m, 17H), 1.13 (d, 3H), 1.24-1.40 (m, 6H), 1.62-1.84 (m, 2H), 1.97-2.41 (m, 3H), 2.20-2.32 (m, 7H), 2.36-2.54 (m, 4H), 3.06 (s, 3H), 3.31 (s, 3H), 3.46 (t, 1H), 3.50 (s, 3H), 3.56 (brs, 1H), 3.62 (m, 1H), 3.67 (m, 1H), 3.72-3.77 (m, 1H), 3.80 (s, 3H), 3.97-4.28 (m, 4H), 4.30 (m, 1H), 4.68 (m, 1H), 4.72-4.83 (m, 1H), 6.89 (d, 2H), 7.33 (d, 2H)
PREPARATION EXAMPLE 2-13
Preparation of Compound (III-17)
(115) ##STR00058##
(116) With the exception that (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-hydroxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (1.43 g, 4.7 mmol) and 2-phthalimido-2-(p-tolyl)ethylamine HCl (1.65 g, 5.2 mmol) were used respectively instead of (2R,3R)-3-((2S,4R)-1-(t-butoxycarbonyl)-4-methoxypyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid and (2R)-1-azido-1-(4-fluorophenyl)propan-2-amine HCl, the same procedure as in Preparation Example 2-7 was repeated to afford an amino-protected derivative of compound (III-17) (2.35 g, 78%).
(117) LC-MS m/z: 878[M+1].sup.+
(118) With the exception of using the amino-protected derivative of compound (III-7) (2.08 g, 2.37 mmol), the same procedure as in Preparation Example 2-11 was repeated to afford the title compound as a white solid (1.7 g, 94%).
(119) LC-MS m/z: 748[M+1].sup.+
(120) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-0.84 (m, 3H), 0.91-1.03 (m, 19H), 1.24-1.29 (m, 4H), 1.29-1.40 (m, 1H), 1.90-2.14 (m, 4H), 2.25 (s, 6H), 2.32 (s, 3H), 2.38-2.48 (m, 3H), 3.03 (s, 3H), 3.14 (brd, 1H), 3.24-3.30 (m, 1H), 3.32 (s, 3H), 3.38-3.43 (m, 1H), 3.48 (s, 3H), 3.54-3.62 (m, 3H), 4.04-4.18 (m, 3H), 4.24-4.34 (m, 2H), 4.77-4.90 (m, 2H), 6.37 (m, 1H), 7.14-7.28 (m, 4H)
PREPARATION EXAMPLE 2-14
Preparation of Compound (III-18)
(121) ##STR00059##
(122) Under an argon stream, (2R,3R)-3-((2S,4S)-1-(t-butoxycarbonyl)-4-((t-butyldimethylsilyl)oxy)pyrrolidin-2-yl)-3-methoxy-2-methylpropanic acid (0.92 g, 2.2 mmol) was dissolved in 10 mL of anhydrous dimethylformamide, and added with 2-(2,6-difluorophenyl)ethanamine (0.38 g, 2.4 mmol). The temperature was cooled to 0° C. before benzotriazol-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate (0.97 g, 2.2 mmol) and diisopropylethylamine (1.0 mL, 6.6 mmol) were sequentially added to the reaction mixture. Stirring was continued at room temperature for 15 hrs. After completion of the reaction, the resulting reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to afford (2S,4S)-t-butyl 4-((t-butyldimethylsilyl)oxy)-2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.87 g, 70.9%).
(123) To a solution of this compound (0.87 g, 1.56 mmol) in 10 mL of dichloromethane was added 7 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the reaction solvent was removed by vacuum concentration, and the residual trifluoroacetic acid was completely removed by adding 5 mL of toluene twice, before a further reaction.
(124) This concentrate (TFA salt) was dissolved, together with (3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoic acid (0.67 g, 1.56 mmol), in 5 mL of dimethylformamide, and sequentially added with diethyl cyanophosphate (DEPC) (0.26 mL, 1.56 mmol) and triethylamine (1.09 mL, 7.82 mmol) at room temperature before being stirred at room temperature for 16 hrs. After completion of the reaction, the solvent was removed, and the residue was dissolved in 20 mL of ethylacetate and extracted with 1 M potassium hydrogen sulfite, water, a saturated sodium hydrogen carbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography (ethylacetate: hexane=2:1.fwdarw.ethylacetate) to afford a hydroxy-protected derivative of compound (III-18) (1.14 g, 84%).
(125) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.04 (s, 6 H), 0.81-1.06 (m, 19 H), 1.19 (m, 3 H), 1.46 (m, 2 H), 1.65 (m, 2 H), 1.97-2.17 (m, 3 H), 2.44-2.46 (m, 3 H), 2.71-2.80 (m, 3 H), 2.81-2.89 (m, 1 H), 2.91-2.94 (m, 2 H), 2.71-2.80 (m, 3 H), 3.04 (s, 3H), 3.26-3.33 (m, 2 H), 3.37 (s, 3 H), 3.74 (m, 2 H), 4.28-4.31 (m, 2H), 4.75-4.82 (m, 2 H), 6.18 (m, 1 H), 6.84-6.89 (m, 2 H), 7.15-7.19 (m, 1 H), 7.58 (d, 1 H)
(126) A solution of the hydroxy-protected derivative of compound (III-18) (1.14 g, 1.31 mmol) in 95 mL of tetrahydrofuran was stirred, together with 1.0 M tetrabutylammonium fluoride (4.9 mL, 4.09 mmol), for 5 hrs, followed by terminating the reaction with a saturated aqueous ammonium chloride solution. Extraction with 450 mL of ethylacetate and 300 mL of water was conducted before vacuum concentration. The concentrate was purified by silica gel column chromatography (dichloromethane:methanol=9:1) to afford the title compound (0.45 g, 45%).
(127) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-1.05 (m, 19 H), 1.26 (m, 3 H), 1.45 (m, 2 H), 1.66 (m, 2 H), 1.97-2.17 (m, 3 H), 2.44-2.46 (m, 3 H), 2.71-2.80 (m, 3 H), 2.81-2.89 (m, 1 H), 2.91-2.94 (m, 2 H), 2.71-2.80 (m, 3 H), 3.04 (s, 3 H), 3.26-3.33 (m, 2 H), 3.37 (s, 3 H), 3.74 (m, 2 H), 4.28-4.31 (m, 2 H), 4.75-4.82 (m, 2 H), 6.18 (m, 1 H), 6.84-6.89 (m, 2 H), 7.15-7.19 (m, 1 H), 7.58 (d, 1 H)
(128) LC-MS m/z: 754 [M.sup.+].sup.+
PREPARATION EXAMPLE 2-15
Preparation of Compound (III-19)
(129) ##STR00060##
(130) With the exception that (2S,4S)-t-butyl 4-((t-butyldimethylsilyl)oxy)-2-((1R,2R)-3-(((S)-1-(4-fluorophenyl)-1-oxopropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.44 g, 0.78 mmol), obtained by using (S)-2-amino-1-(4-fluorophenyl)propan-1-one instead of 2-(2,6-difluorophenyl)ethanamine, was used, the same procedure as in Preparation Example 2-14 was repeated to afford the title compound. 0.34 g (58%).
(131) LC-MS m/z: 764 [M.sup.+].sup.+
PREPARATION EXAMPLE 2-16
Preparation of Compound (III-20)
(132) ##STR00061##
(133) With the exception that (2S,4S)-t-butyl 4-azido-2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.33 g, 0.71 mmol) was used instead of (2S,4S)-t-butyl 4-((t-butyldimethylsilyl)oxy)-2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-14 was repeated to afford an amino-protected derivative of compound (III-20) (0.42 g, 76%).
(134) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-1.07 (m, 15 H), 1.20 (d, 2 H), 1.99-2.11 (m, 4 H), 2.18-2.28 (m, 5 H), 2.33-2.45 (m, 4 H), 2.90-3.27 (m, 6 H), 3.32-3.42 (m, 6 H), 3.47-3.54 (m, 2 H), 3.75-3.78 (m, 1 H), 3.85-3.98 (m, 2H), 4.19-4.21 (m, 1 H), 4.39-4.44 (m, 1 H), 4.46-4.80 (m, 1 H), 4.87-4.90 (m, 1 H), 6.17 (m, 1H), 6.84-6.95 (m, 2 H), 7.14-7.26 (m, 1 H)
(135) The N-terminal amino-protected derivative of compound (III-20) (0.24 g, 0.31 mmol) was dissolved in 10 mL of methyl alcohol, and stirred for 14 hrs in the presence of 10% palladium carbon (15 mg) in a hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through a Celite filter, and washed several times with methanol. Then, the solvent was removed in a vacuum to afford the title compound (0.23 g, 99%).
(136) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-1.03 (m, 14 H), 1.21-1.22 (m, 5H), 2.06-2.45 (m, 6 H), 2.89-3.02 (m, 4 H), 3.30-3.44 (m, 5 H), 3.47-3.52 (m, 2 H), 3.67-3.69 (m, 1 H), 3.71-4.39 (m, 3 H), 4.78-4.80 (m, 1 H), 6.17 (m, 1 H), 6.84-6.95 (m, 2 H), 7.14-7.26 (m, 1 H)
(137) LC-MS m/z: 753 [M.sup.+].sup.+, 775 [M+Na].sup.+
PREPARATION EXAMPLE 2-17
Preparation of Compound (III-21)
(138) ##STR00062##
(139) With the exception that (2S,4S)-t-butyl 4-(benzyl(methyl)amino)-2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.13 g, 0.24 mmol) was used instead of (2S,4S)-t-butyl 4-((t-butyldimethylsilyl)oxy)-2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-14 was repeated to afford an amino-protected derivative of compound (III-21) (0.13 g, 64%).
(140) LC-MS m/z: 857.5 [M.sup.+].sup.+
(141) The N-terminal amino-protected derivative of compound (III-21) (0.13 g, 0.15 mmol) was dissolved in 10 mL of methyl alcohol, and stirred for 12 hrs in the presence of 10% palladium hydroxide (66 mg) under a hydrogen pressure (55 psi). After completion of the reaction, the reaction mixture was filtered through a Celite filter, and washed several times with methanol. Then, the solvent was removed in a vacuum to afford the title compound (115 mg, 100%).
(142) LC-MS m/z 767.6[M.sup.+].sup.+, 789.4[M+Na].sup.+
PREPARATION EXAMPLE 2-18
Preparation of Compound (III-22)
(143) ##STR00063##
(144) To a solution of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate (1.28 g, 2.73 mmol) in 30 mL of dichloromethane was dropwise added 10 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the reaction solvent was removed by vacuum concentration, and trifluoroacetic acid was completely removed by adding 5 ml of toluene twice, before a further reaction.
(145) To a solution of (3R,4S,5S)-t-butyl 4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoate (1.33 g, 2.73 mmol) in 30 mL of dichloromethane was dropwise added 10 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the reaction was removed by vacuum concentration, and the trifluoroacetic acid was completely removed by adding 5 ml of toluene twice, before a further reaction.
(146) Both the concentrates (TFA salts) were dissolved in 60 mL of dichloromethane, and sequentially added with diethyl cyanophosphate (DEPC) (0.52 mL, 3.28 mmol) and triethylamine (3.8 mL, 27.32 mmol) at 0° C. before being stirred at room temperature for 16 hrs. After completion of the reaction, the solvent was removed, and the residue was dissolved in 50 mL of ethylacetate and extracted with 1 M potassium hydrogen sulfite, water, a saturated sodium hydrogen carbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography (ethylacetate: hexane=2:1.fwdarw.ethylacetate) to afford the title compound. (1.3 g, 60%).
(147) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.78-0.88 (m, 3 H), 0.91-1.07 (m, 17 H), 1.31-1.37 (m, 5 H), 1.99-2.11 (m, 3 H), 2.28 (s, 6 H), 2.43-2.62 (m, 6 H), 3.06 (s, 3H), 3.11-3.16 (m, 2 H), 3.34 (s, 3 H), 3.56 (s, 3H), 3.57-3.65 (m, 2 H), 3.97 (s, 3H), 4.08-4.09 (brs, 1H), 4.18-4.2 (d, 1H), 4.3 (brs, 1H), 4.39-4.42 (d, 2 H), 4.7-4.72 (t, 2H), 4.76-4.8 (m, 2H), 4.85-4.91 (brs, 1H), 7.03-7.1 (m, 1H), 7.69-7.78 (m, 2H)
(148) LC-MS m/z: 780.8 [M+H].sup.+, 802.8 [M+Na].sup.+
PREPARATION EXAMPLE 2-19
Preparation of Compound (III-23)
(149) ##STR00064##
(150) With the exception that (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(2,4-dimethylphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-18 was repeated to afford the title compound (3.2 g, 89%).
(151) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-0.85 (m, 3 H), 0.91-1.13 (m, 14 H), 1.15-1.19 (m, 3H), 1.35-1.38 (m, 5 H), 2.00-2.10 (m, 3 H), 2.18-2.24 (m, 3H), 2.37 (s, 3 H), 2.39-2.48 (m, 1H), 2.53 (s, 3H), 2.55-2.62 (m, 1H), 2.87 (s, 6H), 2.89 (s, 1H), 3.02 (s, 3H), 3.07-3.17 (m, 2H), 3.32 (s, 3H), 3.52 (s, 3H), 3.51-3.61 (m, 2H), 4.11-4.12 (brs, 1H), 4.16-4.18 (d, 1H), 4.30 (m, 1H), 4.37-4.4 (d, 1H), 4.64-4.66 (t, 2H), 4.69-4.74 (m, 2H), 6.90 (t, 1H), 7.09-7.11 (m, 2H), 7.66-7.68 (d, 2H)
(152) LC-MS m/z: 760.6 [M+H].sup.+, 782.6 [M+Na].sup.+
PREPARATION EXAMPLE 2-20
Preparation of Compound (III-24)
(153) ##STR00065##
(154) With the exception that (2S,4S)-t-butyl 4-hydroxy-2-(1R,2R)-1-methoxy-3-(2-(6-methoxynaphthalen-2-yl)-2-oxoethyl)amino)-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-18 was repeated to afford the title compound (0.17 g, 67.5%).
(155) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.75-0.82 (m, 3H), 0.85-1.07 (m, 17H), 1.26-1.40 (m, 5H), 1.98-2.10 (m, 6H), 2.39-2.48 (m, 3H), 2.58-2.64 (m, 1H), 3.01 (s, 3H), 3.34 (s, 3H), 3.52-3.73 (m, 2H), 3.57 (s, 3H), 4.12 (brs, 1H), 4.19-4.21 (d, 1H), 4.25-4.31 (brs, 1H), 4.41-4.44 (d, 1H), 4.75-4.83 (m, 2H), 4.87-4.94 (m, 3H), 6.91-6.94 (t, 2H), 7.17 (s, 1H), 7.22-7.24 (d, 1H), 7.78-7.92 (m, 2H), 7.96-7.98 (d, 1H), 8.42 (s, 1H)
(156) LC-MS m/z: 812.7 [M+H].sup.+, 834.7 [M+Na].sup.+
PREPARATION EXAMPLE 2-21
Preparation of Compound (III-25)
(157) ##STR00066##
(158) With the exception that (2S,4S)-t-butyl-4-azido-2((1R,2R)-3-((2-(3,4-dimethoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.39 g, 0.77 mmol) was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-(2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-18 was repeated to afford an amino-protected derivative of compound (III-25) (0.56 g, 89%).
(159) LC-MS m/z: 817.5 [M+H].sup.+, 839.5 [M+Na].sup.+
(160) With the exception of using the amino-protected derivative of compound (III-25), the same procedure as in Preparation Example 2-16 was repeated to afford the title compound (0.38 g, 69%).
(161) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-0.82 (t, 3 H), 0.88-1.02 (m, 17 H), 1.23-1.28 (m, 1 H), 1.31-1.34 (m, 3 H), 1.96-2.12 (m, 2 H), 2.25-2.28 (m, 6 H), 2.36-2.52 (m, 2 H), 2.56-2.65 (m, 1 H), 2.98-3.03 (m, 3 H), 3.25-3.33 (m, 5 H), 3.47-3.49 (m, 1 H), 3.51-3.57 (m, 3 H), 3.72-3.73 (m, 1 H), 3.83-3.96 (d, 6 H), 4.09-4.16 (m, 2 H), 4.18-4.24 (m, 2 H), 4.25-4.36 (m, 1 H), 4.68-4.69 (m, 1 H), 4.74-4.88 m, 2 H), 4.82-4.88 (m, 1 H), 6.91-6.93 (d, 1 H), 7.50 (s, 1 H), 7.60-7.63 (d, 1 H)
PREPARATION EXAMPLE 2-22
Preparation of Compound (III-26)
(162) ##STR00067##
(163) With the exception that (2S,4S)-t-butyl-4-azido-2-((1R,2R)-1-methoxy-2-methyl-3-((2-(naphthalen-2-yl)-2-oxoethyl)amino-3-oxopropyl)pyrrolidine-1-carboxylate (0.40 g, 0.81 mmol) was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-(2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-18 was repeated to afford an amino-protected derivative of compound (III-26) (0.60 g, 92%).
(164) LC-MS m/z: 807.5 [M+H].sup.+, 829.5 [M+Na].sup.+
(165) With the exception of using the amino-protected derivative of compound (III-26), the same procedure as in Preparation Example 2-16 was repeated to afford the title compound (0.28 g, 48%).
(166) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.78-0.82 (t, 3 H), 0.87-1.01 (m, 17 H), 1.22-1.28 (m, 1 H), 1.33-1.34 (m, 3 H), 1.80-2.12 (m, 2 H), 2.24-2.28 (m, 6 H), 2.38-2.50 (m, 2 H), 2.58-2.68 (m, 1 H), 2.95-3.05 (m, 3 H), 3.06-3.13 (m, 1 H), 3.20-3.23 (m, 1 H), 3.28-3.40 (m, 3 H), 3.43-3.45 (m, 1 H), 3.48-3.59 (m, 3 H), 3.60-3.67 (m, 1 H), 3.72-3.75 (m, 1 H), 4.04-4.17 (m, 2 H), 2.20-2.30 (m, 1 H), 4.32-4.36 (m, 1 H), 4.73-4.81 (m, 2 H), 4.85-4.86 (m, 1 H), 4.96-5.02 (m, 1 H), 7.57-7.65 (m, 2 H), 7.88-8.01 (m, 4 H), 8.51 (s, 1 H)
PREPARATION EXAMPLE 2-23
Preparation of Compound (III-27)
(167) ##STR00068##
(168) With the exception that (2S,4S)-t-butyl-4-azido-2-((1R,2R)-1-methoxy-3-((2-(3-methoxyphenyl)-2-oxoethyl)amino)-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.30 g, 0.63 mmol) was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-18 was repeated to afford an amino-protected derivative of compound (III-27) (0.50 g, 100%).
(169) LC-MS m/z: 787.4 [M+H].sup.+, 809.5 [M+Na].sup.+
(170) With the exception of using the amino-protected compound of compound (III-27), the same procedure as in Preparation Example 2-16 was repeated to afford the title compound (0.23 g, 48%).
(171) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.78-0.82 (t, 3 H), 0.88-1.02 (m, 17 H), 1.25-1.29 (m, 3 H), 2.07-2.09 (m, 2 H), 2.18-2.31 (m, 6 H), 2.33-2.40 (m, 2 H), 2.52-2.64 (m, 1 H), 2.92-3.08 (m, 3 H), 3.20-3.25 (m, 1 H), 3.28-3.33 (m, 3 H), 3.38-3.40 (m, 1 H), 3.48-3.76 (m, 1 H), 3.78-3.88 (m, 3 H), 4.06-4.16 (m, 2 H), 4.19-4.28 (m, 1 H), 4.29-4.39 (m, 1 H), 4.70-4.71 (m, 1 H), 4.74-4.81 (m, 2 H), 4.88-4.95 (m, 1 H), 6.96-6.99 (d, 1 H), 7.38-7.42 (t, 1 H), 7.47 (s, 1 H), 7.53-7.55 (d, 1 H)
PREPARATION EXAMPLE 2-24
Preparation of Compound (III-28)
(172) ##STR00069##
(173) To a solution of (2S,4S)-t-butyl 4-((t-butyldimethylsilyl)oxy)-2-((1R,2R)-3-(((S)-1-(4-fluorophenyl)-1-oxopropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.87 g, 1.56 mmol) in 10 mL of dichloromethane was dropwise added 7 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the reaction solvent was removed by vacuum concentration, and trifluoroacetic acid was completely removed by adding 5 ml of toluene twice before subsequent reactions.
(174) This concentrate (TFA salt) was dissolved, together with (3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoic acid (0.67 g, 1.56 mmol), in 5 mL of dimethylformamide, and added with diethyl cyanophosphate (DEPC) (0.26 mL, 1.56 mmol) and triethylamine (1.09 mL, 7.82 mmol) at 0° C. before being stirred at room temperature for 16 hrs. After completion of the reaction, the solvent was removed, and the residue was dissolved in 20 mL of ethylacetate and extracted with 1 M potassium hydrogen sulfite, water, a saturated sodium hydrogen carbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography (ethylacetate: hexane=2:1.fwdarw.ethylacetate) to afford a hydroxy-protected compound (1.14 g, 84%).
(175) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.04 (s, 6 H), 0.81-1.06 (m, 19 H), 1.19 (m, 3 H), 1.46 (m, 2 H), 1.65 (m, 2 H), 1.97-2.17 (m, 3 H), 2.44-2.46 (m, 3 H), 2.71-2.80 (m, 3 H), 2.81-2.89 (m, 1 H), 2.91-2.94 (m, 2 H), 2.71-2.80 (m, 3 H) 3.04 (s, 3 H), 3.26-3.33 (m, 2 H), 3.37 (s, 3 H), 3.74 (m, 2 H), 4.28-4.31 (m, 2 H), 4.75-4.82 (m, 2 H), 6.18 (m, 1 H), 6.84-6.89 (m, 2 H), 7.15-7.19 (m, 1 H), 7.58 (d, 1 H)
(176) A solution of the hydroxy-protected derivative of compound (1.14 g, 1.31 mmol) in 95 mL of tetrahydrofuran was stirred, together with 1.0 M tetrabutylammonium fluoride (4.9 mL, 4.09 mmol), for 5 hrs, followed by terminating the reaction with a saturated aqueous ammonium chloride solution. Extraction with 450 mL of ethylacetate and 300 mL of water was conducted before vacuum concentration. The concentrate was purified by silica gel column chromatography (dichloromethane:methanol=9:1) to afford (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((2S,4S)-2-((1R,2R)-3-(((S)-1-(4-fluorophenyl)-1-oxopropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N, 3-dimethylbutanamide (0.45 g, 45%).
(177) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-1.05 (m, 19 H), 1.26 (m, 3 H), 1.45 (m, 2 H), 1.66 (m, 2 H), 1.97-2.17 (m, 3 H), 2.44-2.46 (m, 3H), 2.71-2.80 (m, 3 H), 2.81-2.89 (m, 1 H), 2.91-2.94 (m, 2 H), 2.71-2.80 (m, 3 H), 3.04 (s, 3 H), 3.26-3.33 (m, 2 H), 3.37 (s, 3 H), 3.74 (m, 2 H), 4.28-4.31 (m, 2 H), 4.75-4.82 (m, 2 H), 6.18 (m, 1 H), 6.84-6.89 (m, 2 H), 7.15-7.19 (m, 1 H), 7.58 (d, 1 H)
(178) LC-MS m/z: 754 [M+H].sup.+
(179) A solution of this compound (0.45 g, 0.59 mmol) in 20 mL of dichloromethane was cooled to −50 to −60° C. and added with pyridine (0.29 ml, 3.54 mmol). To the reaction mixture, drops of a solution of p-nitrophenyl chloroformate (0.71 g, 3.54 mmol) in 10 mL of dichloromethane were slowly added over 20 min, followed by stirring at −50 to −60° C. for 3 hrs. When the reaction was completed, 20 mL of dichloromethane was further added to the reaction mixture, which was then extracted with a 0.5 M potassium hydrogen sulfite solution. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford the title compound (0.4 g, 74%).
(180) LC-MS m/z: 929.6 [M+H].sup.+
PREPARATION EXAMPLE 2-25
Preparation of Compound (III-29)
(181) ##STR00070##
(182) To a solution of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate (1.28 g, 2.73 mmol) in 30 mL of dichloromethane was dropwise added 10 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the reaction solvent was removed by vacuum concentration, and trifluoroacetic acid was completely removed by adding 10 ml of toluene twice, before a further reaction.
(183) To a solution of (3R,4S,5S)-t-butyl 4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoate (1.33 g, 2.73 mmol) in 30 mL of dichloromethane was dropwise added 10 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, the solvent was removed by vacuum concentration, and the trifluoroacetic acid was completely removed by adding 10 mL of toluene twice, before the subsequent reaction.
(184) Both the concentrates (TFA salts) were dissolved in 60 mL of dichloromethane, and sequentially added with diethyl cyanophosphate (DEPC) (0.52 mL, 3.28 mmol) and triethylamine (3.8 mL, 27.32 mmol) at 0° C. before being stirred at room temperature for 16 hrs. After completion of the reaction, the solvent was removed, and the residue was dissolved in 50 mL of ethylacetate and extracted with 1 M potassium hydrogen sulfite, water, a saturated sodium hydrogen carbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography (ethylacetate: hexane=2:1.fwdarw.ethylacetate) to afford (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((2S,4S)-2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N, 3-dimethylbutanamide (1.3 g, 60%).
(185) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.78-0.88 (m, 3 H), 0.91-1.07 (m, 17 H), 1.31-1.37 (m, 5 H), 1.99-2.11 (m, 3 H), 2.28 (s, 6 H), 2.43-2.62 (m, 6 H), 3.06 (s, 3H), 3.11-3.16 (m, 2 H), 3.34 (s, 3 H), 3.56 (s, 3H), 3.57-3.65 (m, 2 H), 3.97 (s, 3H), 4.08-4.09 (brs, 1H), 4.18-4.2 (d, 1H), 4.3 (brs, 1H), 4.39-4.42 (d, 2 H), 4.7-4.72 (t, 2 H), 4.76-4.8 (m, 2H), 4.85-4.91 (brs, 1H), 7.03-7.1 (m, 1H), 7.69-7.78 (m, 2H)
(186) LC-MS m/z: 780.8 [M+H].sup.+, 802.8 [M+Na].sup.+
(187) A solution of this compound (1.27 g, 1.63 mmol) in 20 mL of dichloromethane was cooled to −50 to −60° C. and added with pyridine (1.3 ml, 16.28 mmol). To the reaction mixture, drops of a solution of p-nitrophenyl chloroformate (16.28 g, 3.3 mmol) in 10 mL of dichloromethane were slowly added over 20 min, followed by stirring at −50 to −60° C. for 3 hrs. When the reaction was completed, 20 mL of dichloromethane was further added to the reaction mixture, which was then extracted with a 0.5 M potassium hydrogen sulfite solution. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford the title compound (0.8 g, 54%).
(188) LC-MS m/z: 945.7 [M+H].sup.+, 967.7 [M+Na].sup.+
PREPARATION EXAMPLE 2-26
Preparation of Compound (III-30)
(189) ##STR00071##
(190) With the exception that (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(2,4-dimethylphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-25 was repeated to afford (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((2S,4S)-2-((1R,2R)-3-((2-(2,4-dimethylphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N, 3-dimethylbutanamide (3.2 g, 89%).
(191) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-0.85 (m, 3 H), 0.91-1.13 (m, 14 H), 1.15-1.19 (m, 3H), 1.35-1.38 (m, 5 H), 2.00-2.10 (m, 3 H), 2.18-2.24 (m, 3H), 2.37 (s, 3 H), 2.39-2.48 (m, 1H), 2.53 (s, 3H), 2.55-2.62 (m, 1H), 2.87 (s, 6H), 2.89 (s, 1H), 3.02 (s, 3H), 3.07-3.17 (m, 2H), 3.32 (s, 3H), 3.52 (s, 3H), 3.51-3.61 (m, 2H), 4.11-4.12 (brs, 1H), 4.16-4.18 (d, 1H), 4.30 (m, 1H), 4.37-4.4 (d, 1H), 4.64-4.66 (t, 2H), 4.69-4.74 (m, 2H), 6.90 (t, 1H), 7.09-7.11 (m, 2H), 7.66-7.68 (d, 2H)
(192) LC-MS m/z: 760.6 [M+H].sup.+, 782.6 [M+Na].sup.+
(193) With the exception of using this compound, the same procedure as in Preparation Example 2-25 was repeated to afford the title compound (1.6 g, 43%).
(194) LC-MS m/z: 925.6 [M+H].sup.+, 947.6 [M+Na].sup.+
PREPARATION EXAMPLE 2-27
Preparation of Compound (III-31)
(195) ##STR00072##
(196) With the exception that (2S,4S)-t-butyl 4-hydroxy-2-((1R,2R)-1-methoxy-3-((2-(6-methoxynaphthalen-2-yl)-2-oxoethyl)amino)-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-25 was repeated to afford (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S, 5S)-1-((2S,4S)-4-hydroxy-2-((1R,2R)-1-methoxy-3-((2-(6-methoxynaphthalen-2-yl)-2-oxoethyl)amino)-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N, 3-dimethylbutanamide (0.17 g, 67.5%).
(197) .sup.1H NMR (400 MHz, CDCl.sub.3) δ0.75-0.82 (m, 3H), 0.85-1.07 (m, 17H), 1.26-1.40 (m, 5H), 1.98-2.10 (m, 6H), 2.39-2.48 (m, 3H), 2.58-2.64 (m, 1H), 3.01 (s, 3H), 3.34 (s, 3H), 3.52-3.73 (m, 2H), 3.57 (s, 3H), 4.12 (brs, 1H), 4.19-4.21 (d, 1H), 4.25-4.31 (brs, 1H), 4.41-4.44 (d, 1H), 4.75-4.83 (m, 2H), 4.87-4.94 (m, 3H), 6.91-6.94 (t, 2H), 7.17 (s, 1H), 7.22-7.24 (d, 1H), 7.78-7.92 (m, 2H), 7.96-7.98 (d, 1H), 8.42 (s, 1H)
(198) LC-MS m/z: 812.7 [M+H].sup.+, 834.7 [M+Na].sup.+
(199) With the exception of using this compound, the same procedure as in Preparation Example 2-25 was repeated to afford the title compound (0.15 g, 87%).
(200) LC-MS m/z: 977.9 [M+H].sup.+, 999.9 [M+Na].sup.+
PREPARATION EXAMPLE 2-28
Preparation of Compound (III-32)
(201) ##STR00073##
(202) With the exception that (2S,4S)-t-butyl 4-((t-butyldimethylsilyl)oxy)-2-((1R,2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (0.87 g, 70.9%) was used instead of (2S,4S)-t-butyl 2-((1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidine-1-carboxylate, the same procedure as in Preparation Example 2-24 was repeated to afford (2S)-N-((3R,4S)-1-((2S,4S)-2-((1R, 2R)-3-((2,6-difluorophenethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)-4-hydroxypyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamide (0.45 g, 45%).
(203) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.79-1.05 (m, 19 H), 1.26 (m, 3 H), 1.45 (m, 2 H), 1.66 (m, 2 H), 1.97-2.17 (m, 3 H), 2.44-2.46 (m, 3 H), 2.71-2.80 (m, 3 H), 2.81-2.89 (m, 1 H), 2.91-2.94 (m, 2 H), 2.71-2.80 (m, 3 H), 3.04 (s, 3 H), 3.26-3.33 (m, 2 H), 3.37 (s, 3 H), 3.74 (m, 2 H), 4.28-4.31 (m, 2 H), 4.75-4.82 (m, 2 H), 6.18 (m, 1 H), 6.84-6.89 (m, 2 H), 7.15-7.19 (m, 1 H), 7.58 (d, 1 H)
(204) LC-MS m/z: 754 [M.sup.+].sup.+
(205) With the exception of using this compound, the same procedure as in Preparation Example 2-24 was repeated to afford the title compound (0.46 g, 83%).
(206) LC-MS m/z: 920.1 [M+H].sup.+
PREPARATION EXAMPLE 3
Preparation of Compounds of Chemical Formula IV-1, IV-2, IV-3, and IV-4
PREPARATION EXAMPLE 3-1
Preparation of Compound (IV-5)
(207) ##STR00074##
(208) Under an argon stream, compound (II-1) (0.37 g, 0.50 mmol), prepared in Preparation Example 1-1, was added to a solution of compound (III-5) (0.36 g, 0.50 mmol), prepared in Preparation Example 2-1, in 14 mL of anhydrous dimethylformamide. The reaction mixture was cooled to 0° C., and was sequentially added with HOBt (81 mg, 0.60 mmol) and 3.5 mL of anhydrous pyridine before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound as an ivory solid (0.49 g, 74%).
(209) MALDI-TOF MS m/z: 1345.6 [M+Na].sup.+, 1361.6 [M+K].sup.+
PREPARATION EXAMPLE 3-2
Preparation of Compound (IV-6)
(210) ##STR00075##
(211) With the exception that compound (III-6) (0.35 g, 0.50 mmol), prepared in Preparation Example 2-2, was used instead of compound (III-5), prepared in Preparation Example 2-1, the same procedure as in Preparation Example 3-1 was repeated to afford the title compound (0.42 g, 72%).
(212) MALDI-TOF MS m/z: 1322.8 [M+Na].sup.+, 1339.0 [M+K].sup.+
PREPARATION EXAMPLE 3-3
Preparation of Compound (IV-7)
(213) ##STR00076##
(214) With the exception that compound (III-7) (79 mg, 0.10 mmol), prepared in Preparation Example 2-3, was used instead of compound (III-5), prepared in Preparation Example 2-1, the same procedure as in Preparation Example 3-1 was repeated to afford the title compound (97 mg, 78%).
(215) MALDI-TOF MS m/z: 1400.3 [M+Na].sup.+
PREPARATION EXAMPLE 3-4
Preparation of Compound (IV-8)
(216) ##STR00077##
(217) With the exception that compound (III-8) (104 mg, 0.14 mmol), obtained in Preparation Example 2-4, was used instead of compound (III-5), obtained in Preparation Example 2-1, the same procedure as in Preparation Example 3-1 was repeated to afford the title compound (86 mg, 48%).
(218) MALDI-TOF MS m/z: 1386.9 [M+Na].sup.+, 1402.9 [M+K].sup.+
PREPARATION EXAMPLE 3-5
Preparation of Compound (IV-9)
(219) ##STR00078##
(220) With the exception that compound (III-9) (65 mg, 0.085 mmol), obtained in Preparation Example 2-5, was used instead of compound (III-5), obtained in Preparation Example 2-1, the same procedure as in Preparation Example 3-1 was repeated to afford the title compound (43 mg, 41%).
(221) MALDI-TOF MS m/z: 1381.6 [M+Na].sup.+
PREPARATION EXAMPLE 3-6
Preparation of Compound (IV-10)
(222) ##STR00079##
(223) With the exception that compound (III-10) (82 mg, 0.11 mmol), obtained in Preparation Example 2-6, was used instead of compound (III-5), obtained in Preparation Example 2-1, the same procedure as in Preparation Example 3-1 was repeated to afford the title compound (89 mg, 66%).
(224) MALDI-TOF MS m/z: 1360.0 [M+Na].sup.+, 1377.2 [M+K].sup.+
PREPARATION EXAMPLE 3-7
Preparation of Compound (IV-11)
(225) ##STR00080##
(226) Under an argon stream, compound (III-11)(89 mg, 0.116 mmol), obtained in Preparation Example 2-7, was dissolved in 8 mL of anhydrous dimethyl formamide, and added with compound (II-1)(78 mg, 0.106 mmol), obtained in Preparation Example 1-1. The reaction mixture was cooled to 0° C., and was sequentially added with HOBt (17 mg, 0.128 mmol) and 2.0 mL of anhydrous pyridine before being stirred at room temperature for 17 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound (106 mg, 73%).
(227) LC-MS m/z: 1377.8 [M.sup.+].sup.+, 1400.8 [M+Na].sup.+
PREPARATION EXAMPLE 3-8
Preparation of Compound (IV-12)
(228) ##STR00081##
(229) With the exception that compound (III-12)(17 mg, 0.023 mmol), obtained in Preparation Example 2-8, was used instead of compound (III-11), obtained in Preparation Example 2-7, the same procedure as in Preparation Example 3-7 was repeated to afford the title compound (26 mg, 93%).
(230) MALDI-TOF MS m/z: 1351.3 [M+Na].sup.+, 1367.7 [M+K].sup.+
(231) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.81-0.91 (m, 6H), 0.91-1.03 (m, 21H), 1.63 (d, 2H), 1.22 (d, 1H), 1.28 (m, 2H), 1.32 (m, 1H), 1.50-1.63 (m, 6H), 1.68 (m, 2H), 1.82-1.90 (m, 2H), 1.95 (s, 3H), 1.98 (m, 4H), 2.23-2.28 (m, 3H), 2.34 (brd, 1H), 2.40 (s, 6H), 2.58-2.70 (m, 1H), 2.80-2.85 (m, 1H), 2.91-2.98 (m, 1H), 3.12 (s, 3H), 3.15-3.27 (m, 2H), 3.26-3.33 (m, 8H, OCH.sub.3), 3.34 (d, 2H), 3.42-3.48 (m, 5H, OCH.sub.3), 3.52 (m, 1H), 3.7-3.96 (m, 2H), 3.96-4.08 (m, 2H), 4.15 (d, 1H), 4.18 (m, 1H), 4.44-4.54 (m, 2H), 4.66 (d, 1H), 4.54-5.10 (m, 5H), 6.76 (m, 1H), 7.02-7.12 (m, 2H), 7.23-7.28 (m, 2H), 7.30-7.38 (m, 2H), 7.51-7.58 (m, 2H)
PREPARATION EXAMPLE 3-9
Preparation of Compound (IV-13)
(232) ##STR00082##
(233) With the exception that compound (III-13) (478 mg, 0.638 mmol), obtained in Preparation Example 2-9, was used instead of compound (III-11), obtained in Preparation Example 2-7, the same procedure as in Preparation Example 3-7 was repeated to afford the title compound (347 mg, 40%).
(234) LC-MS m/z: 1347 [M.sup.+].sup.+
(235) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.76-0.90 (m, 6H), 0.90-1.12 (m, 18H), 1.16 (m, 4H), 1.19 (m, 3H), 1.28 (m, 3H), 1.28 (m, 3H), 1.40 (m, 2H), 1.50-1.66 (m, 9H), 1.66-1.80 (m, 2H), 1.93 (s, 3H), 2.0-2.14 (m, 4H), 2.26 (m, 3H), 2.38 (d, 6H), 2.44 (m, 1H), 2.50-2.65 (m, 1H), 2.77 (m, 2H), 3.10-3.24 (m, 6H), 3.25-3.36 (m, 7H, OCH.sub.3), 3.45 (t, 3H), 3.58 (m, 1H), 3.66 (m, 1H), 3.82 (m, 1H), 4.06 (m, 1H), 4.14 (m, 2H), 4.26 (m, 1H), 4.36 (m, 1H), 4.48 (m, 2H), 4.63 (m, 1H), 4.69 (m, 3H), 6.78 (s, 2H), 6.97 (m, 1H), 7.08 (m, 1H), 7.25 (m, 2H), 7.34 (m, 2H), 7.54 (m, 2H)
PREPARATION EXAMPLE 3-10
Preparation of Compound (IV-14)
(236) ##STR00083##
(237) With the exception that compound (III-14)(0.39 g, 0.51 mmol), obtained in Preparation Example 2-10, was used instead of compound (III-11), obtained in Preparation Example 2-7, the same procedure as in Preparation Example 3-7 was repeated to afford the title compound (453 mg, 65%).
(238) LC-MS m/z: 1364[M+1].sup.+
(239) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.81-0.91 (m, 6H), 0.91-1.03 (m, 21H), 1.13 (d, 2H), 1.22 (d, 1H), 1.28 (m, 2H), 1.32 (m, 1H), 1.50-1.63 (m, 6H), 1.68 (m, 2H), 1.82-1.90 (m, 2H), 1.95 (s, 3H), 1.98 (m, 4H), 2.23-2.28 (m, 3H), 2.34 (brd, 1H), 2.40 (s, 6H), 2.58-2.70 (m, 1H), 2.80-2.85 (m, 1H), 2.91-2.98 (m, 1H), 3.12 (s, 3H), 3.15-3.27 (m, 2H), 3.27-3.33 (m, 8H, OCH.sub.3), 3.34 (d, 2H), 3.42-3.48 (m, 5H, OCH.sub.3), 3.52 (m, 1H), 3.7-3.96 (m, 2H), 3.96-4.08 (m, 2H), 4.15 (d, 1H), 4.18 (m, 1H), 4.44-4.54 (m, 2H), 4.66 (d, 1H), 4.74-5.10 (m, 5H), 6.76 (m, 1H), 7.02-7.12 (m, 2H), 7.23-7.28 (m, 2H), 7.30-7.38 (m, 2H), 7.51-7.58 (m, 2H)
PREPARATION EXAMPLE 3-11
Preparation of Compound (IV-15)
(240) ##STR00084##
(241) With the exception that compound (III-15)(110 mg, 0.144 mmol), obtained in Preparation Example 2-11, was used instead of compound (III-11), obtained in Preparation Example 2-7, the same procedure as in Preparation Example 3-7 was repeated to afford the title compound (82 mg, 42%).
(242) LC-MS m/z: 1360 [M+1].sup.+
(243) .sup.1H NMR (400 MHz, CD.sub.3OD) δ0.81-0.91 (m, 6H), 0.92-1.05 (m, 19H), 1.3-1.2 (m, 3H), 1.22 (d, 1H), 1.28 (m, 2H), 1.32 (m, 1H), 1.50-1.63 (m, 6H), 1.68 (m, 2H), 1.82-1.90 (m, 2H), 1.95 (s, 3H), 1.98 (m, 3H), 2.23-2.28 (m, 3H), 2.37 (d, 6H), 2.61 (m, 1H), 2.78 (m, 1H), 3.12 (s, 3H), 3.16-3.24 (m, 2H), 3.26-3.32 (m, 15H, OCH.sub.3), 3.32-3.28 (m, 5H, OCH.sub.3), 3.42-3.50 (m, 1H), 3.74 (d, 3H), 3.86 (m, 1H), 3.96-4.08 (m, 2H), 4.14 (d, 1H), 4.18-4.10 (m, 1H), 4.42-4.52 (m, 2H), 4.64-5.10 (m, 5H), 6.76 (d, 2H), 6.87 (m, 2H), 7.24 (m, 4H), 7.55 (m, 2H)
PREPARATION EXAMPLE 3-12
Preparation of Compound (IV-16)
(244) ##STR00085##
(245) With the exception that compound (III-16)(179 mg, 0.23 mmol), obtained in Preparation Example 2-12, was used instead of compound (III-11), obtained in Preparation Example 2-7, the same procedure as in Preparation Example 3-7 was repeated to afford the title compound (268 mg, 85%).
(246) LC-MS m/z: 1375 [M+1]
(247) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.71-0.80 (m, 5H), 0.80-0.98 (m, 18H), 0.98-1.10 (m, 4H), 1.18 (m, 2H), 1.50 (m, 6H), 1.55-1.75 (m, 4H), 1.81-2.00 (m, 8H), 2.05-2.30 (m, 5H), 2.26 (d, 6H), 2.40-2.61 (m, 1H), 2.65-2.80 (m, 2H), 2.90-3.08 (m, 6H), 3.15 (d, 3H), 3.30 (d, 3H), 3.42 (s, 2H), 3.67 (s, 3H), 3.70 (s, 3H), 3.15-3.43 (m, 3H), 3.85-4.05 (m, 4H), 4.18 (brt, 2H), 4.38 (m, 2H), 4.52 (t, 1H), 4.56-4.80 (m, 3H), 4.88 (d, 1H), 5.01 (m, 2H), 5.10 (d, 1H), 6.00 (m, 1H), 6.84-8.20 (m, 10H), 10.0 (d, 1H)
PREPARATION EXAMPLE 3-13
Preparation of Compound (IV-17)
(248) ##STR00086##
(249) With the exception that compound (III-17)(327 mg, 0.431 mmol), obtained in Preparation Example 2-13, was used instead of compound (III-11), obtained in Preparation Example 2-7, the same procedure as in Preparation Example 3-7 was repeated to afford the title compound (440 mg, 75%).
(250) LC-MS m/z: 1346 [M+1].sup.+
(251) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.81-1.02 (m, 23H), 1.13-1.20 (m, 3H), 1.25-1.32 (m, 2H), 1.40 (m, 1H), 1.50-1.63 (m, 6H), 1.72-1.78 (m, 2H), 1.82-1.98 (m, 3H), 1.96 (s, 3H), 2.00-2.09 (m, 4H), 2.15-2.30 (m, 5H), 2.34-2.42 (d, 6H), 2.40-2.60 (m, 3H), 2.75 (m, 1H), 2.98 (m, 1H), 3.05 (m, 3H), 3.16-3.24 (m, 2H), 3.26-3.35 (m, 8H, OCH.sub.3), 3.38-3.48 (m, 6H, OCH.sub.3), 3.52-3.60 (m, 1H), 3.65-3.80 (m, 1H), 3.90-4.16 (m, 5H), 4.48 (m, 1H), 4.63-5.10 (m, 8H), 6.76 (s, 2H), 7.09-7.28 (m, 6H), 7.53 (m, 2H)
PREPARATION EXAMPLE 3-14
Preparation of Compound (IV-18)
(252) ##STR00087##
(253) Under an argon stream, compound (III-18) (0.36 g, 0.48 mmol), obtained in Preparation Example 2-14, was dissolved in 10 mL of anhydrous dimethyl formamide, and added with compound (II-1)(0.32 g, 0.43 mmol), obtained in Preparation Example 1-1. The reaction mixture was cooled to 0° C., and was sequentially added with HOBt (70 mg, 0.52 mmol) and 2.5 mL of anhydrous pyridine before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound as an ivory solid (0.29 g, 51%).
(254) LC-MS m/z: 1353 [M.sup.+].sup.+, 1375 [M+Na].sup.+
PREPARATION EXAMPLE 3-15
Preparation of Compound (IV-19)
(255) ##STR00088##
(256) With the exception that compound (III-19) (0.34 g, 0.45 mmol), obtained in Preparation Example 2-15, was used instead of compound (III-18), obtained in Preparation Example 2-14, the same procedure as in Preparation Example 3-14 was repeated to afford the title compound (0.28 g, 45%).
(257) LC-MS m/z: 1362.8 [M.sup.+].sup.+, 1384.8 [M+Na].sup.+
PREPARATION EXAMPLE 3-16
Preparation of Compound (IV-20)
(258) ##STR00089##
(259) With the exception that compound (III-20) (91 mg, 0.12 mmol), obtained in Preparation Example 3-14, was used instead of compound (III-18), obtained in Preparation Example 2-16, the same procedure as in Preparation Example 3-14 was repeated to afford the title compound (122 mg, 82%).
(260) LC-MS m/z: 1352 [M.sup.+].sup.+
PREPARATION EXAMPLE 3-17
Preparation of Compound (IV-21)
(261) ##STR00090##
(262) With the exception that compound (III-21)(110 mg, 0.14 mmol), obtained in Preparation Example 2-17, was used instead of compound (III-18), obtained in Preparation Example 2-14, the same procedure as in Preparation Example 3-14 was repeated to afford the title compound (153 mg, 86%).
(263) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.70-0.77 (m, 5 H), 0.81-0.97 (m, 17 H), 1.02-1.07 (m, 3 H), 1.16-1.29 (m, 8 H), 1.34-1.40 (m, 1 H), 1.48-1.51 (m, 6 H), 1.54-1.63 (m, 1 H), 1.63-1.79 (m, 2 H), 1.91-1.97 (m, 5 H), 2.03-2.30 (m, 7 H), 2.35-2.44 (m, 1 H), 2.59-2.61 (m, 1 H), 2.61-2.69 (m, 1 H), 2.70-2.84 (m, 4 H), 3.07-3.12 (m, 1 H), 3.12-3.24 (m, 3 H), 3.26-3.40 (m, 3 H), 3.40-3.48 (m, 1 H), 3.62-4.03 (m, 4 H), 4.03-4.25 (m, 3 H), 4.25-4.68 (m, 4 H), 4.88-5.09 (s, 2 H), 5.35-5.47 (s, 2 H), 5.85-6.03 (t, 1 H), 6.85-7.10 (s, 2 H), 7.14-7.40 (m, 2 H), 7.56-7.66 (m, 1 H), 7.74-7.83 (d, 1 H), 7.90-8.22 (m, 3 H), 10.00 (brs, 1 H)
(264) LC-MS m/z: 1366 [M.sup.+].sup.+
PREPARATION EXAMPLE 3-18
Preparation of Compound (IV-22)
(265) ##STR00091##
(266) A solution of compound (III-22) (1.27 g, 1.63 mmol), obtained in Preparation Example 2-18, in 20 mL of dichloromethane was cooled to −50 to −60° C., and added with pyridine (1.3 mL, 16.28 mmol). To the reaction mixture, drops of a solution of p-nitrophenyl chloroformate (16.28 g, 3.3 mmol) in 10 mL of dichloromethane were slowly added over 20 min, followed by stirring at −50 to −60° C. for 3 hrs. When the reaction was completed, 20 mL of dichloromethane was further added to the reaction mixture, which was then extracted with a 0.5 M potassium hydrogen sulfite solution. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford (3S,5S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)-5-(1R,2R)-3-((2-(3-fluoro-4-methoxyphenyl)-2-oxoethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-3-yl (4-nitrophenyl) carbonate (0.8 g, 54%).
(267) LC-MS m/z: 945.7 [M+H].sup.+, 967.7 [M+Na].sup.+
(268) Under an argon stream, this compound (0.3 g, 0.32 mmol) was dissolved, together with t-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.3 g, 0.64 mmol), in 10 mL of anhydrous dimethyl formamide, and added with 4-methylaminopyridine (0.039 g, 0.32 mmol). The reaction mixture was stirred at 20-25° C. for 19 hrs. After completion of the reaction, the reaction mixture was concentrated to the completion in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative (0.1 g, 25%).
(269) LC-MS m/z: 1285.5 [M+H].sup.+
(270) To a solution of this amino-protected compound (0.18 g, 0.14 mmol) in 8 mL of dichloromethane was added 2 mL of trifluoroacetic acid, followed by stirring at 20-25° C. for 3 hrs. When the reaction was completed, trifluoroacetic acid was completely removed by adding 5 ml of toluene twice, before a further reaction.
(271) This concentrate (TFA salt) was dissolved, together with 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1-pyrrol-1-yl)hexanoate, in 10 mL of dimethylformamide, and added with triethylamine (0.1 mL, 0.7 mmol) at room temperature before being stirred at room temperature for 18 hrs. After completion of the reaction, the reaction mixture was concentrated to the completion in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain the title compound (0.14 g, 71%).
(272) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.70-0.78 (m, 6H), 0.81-0.92 (m, 13H), 1.16-1.24 (23H), 1.46-1.51 (m, 4H), 1.84-1.97 (m, 3H), 3.06-3.17 (m, 12H), 3.30-3.38 (m, 15H), 3.84-3.93 (m, 3H), 4.08-4.85 (m, 8H), 4.86-5.04 (m, 1H), 5.10 (s, 2H), 5.40 (s, 2H), 6.03 (t, 1H), 7.00 (s, 1H), 7.15-7.35 (m, 2H), 7.53-7.55 (d, 1H), 7.77-7.98 (m, 2H), 8.10-8.12 (d, 1H), 8.19-8.48 (m, 1H), 9.44-9.49 (brs, 1H), 10.03 (s, 1H)
(273) LC-MS m/z: 1378.7 [M+H].sup.+
PREPARATION EXAMPLE 3-19
Preparation of Compound (IV-23)
(274) ##STR00092##
(275) With the exception that compound (III-23) (0.8 g, 0.865 mmol), obtained in Preparation Example 2-19, was used instead of compound (III-22), obtained in Preparation Example 2-18, the same procedure as in Preparation Example 3-18 was repeated to afford the title compound (0.22 g, 69%).
(276) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.73-0.75 (m, 4H), 0.81-0.92 (m, 11H), 1.16-1.19 (23H), 1.46-1.50 (m, 4H), 1.54-1.73 (m, 2H), 1.90-1.97 (m, 2H), 2.08-2.25 (m, 4H), 2.28-2.38 (m, 4H), 2.94-2.99 (m, 4H), 3.07-3.17 (m, 11H), 3.33-3.38 (m, 12H), 3.95-4.05 (m, 1H), 4.12-4.27 (m, 3H), 4.51-4.67 (m, 1H), 4.87-4.95 (m, 1H), 5.11 (s, 2H), 5.43 (s, 2H), 6.00 (t, 1H), 7.00 (s, 1H), 7.07-7.15 (m, 1H), 7.22-7.24 (d, 1H), 7.32-7.35 (m, H), 7.53-7.56 (d, 1H), 7.61-7.62 (d, 1H), 7.81-7.84 (d, 1H), 8.07-8.13 (m, 1H), 8.28-8.50 (m, 1H), 9.26 (brs, 1H), 9.91 (s, 1H), 10.06 (s, 1H)
(277) LC-MS m/z: 1380.4 [M+H].sup.+
PREPARATION EXAMPLE 3-20
Preparation of Compound (IV-24)
(278) ##STR00093##
(279) With the exception that compound (III-24) (0.44 g, 0.45 mmol), obtained in Preparation Example 2-20, was used instead of compound (III-22), obtained in Preparation Example 2-18, the same procedure as in Preparation Example 3-18 was repeated to afford the title compound (0.16 g, 64%).
(280) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.48-0.50 (d, 1H), 0.58-0.59 (d, 1H), 0.68-0.77 (m, 4H), 0.81-0.92 (m, 14H), 1.16-1.23 (20H), 1.46-1.51 (m, 4H), 1.81-1.88 (m, 3H), 3.06-3.13 (m, 12H), 3.35-3.38 (m, 15H), 3.84-3.92 (m, 4H), 4.08-5.00 (m, 8H), 5.13 (s, 2H), 5.44 (s, 2H), 6.01-6.03 (t, 1H), 7.00 (s, 1H), 7.22-7.28 (m, 1H), 7.33-7.36 (m, 2H), 7.39-7.42 (d, 1H), 7.60-7.65 (d, 2H), 7.82-7.84 (d, 1H), 7.89-7.93 (m, 2H), 8.00-8.03 (d, 1H), 8.11-8.13 (d, 1H), 8.27-8.53 (m, 1H), 8.64 (s, 1H), 9.38 (brs, 1H), 10.06 (s, 1H)
(281) LC-MS m/z: 1410.5 [M+H].sup.+
PREPARATION EXAMPLE 3-21
Preparation of Compound (IV-25)
(282) ##STR00094##
(283) With the exception that compound (III-25) (0.37 g, 0.47 mmol), obtained in Preparation Example 2-21, was used instead of compound (III-22), obtained in Preparation Example 2-18, the same procedure as in Preparation Example 3-14 was repeated to afford the title compound (0.47 g, 72%).
(284) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.57-0.78 (m, 8 H), 0.78-0.98 (m, 15H), 1.04-1.28 (m, 6 H), 1.28-1.39 (m, 1 H), 1.39-1.54 (m, 5 H), 1.54-1.63 (m, 1 H), 1.63-1.84 (m, 3 H), 1.84-2.03 (m, 3 H), 2.03-2.25 (m, 7H), 2.25-2.40 (m, 2 H), 2.53-2.69 (m, 3 H), 2.69-2.83 (m, 1 H), 2.85-3.06 (m, 5 H), 3.09-3.21 (m, 3 H), 3.26-3.42 (m, 3 H), 3.44-3.52 (m, 1 H), 3.74-3.88 (m, 6 H), 3.88-4.05 (m, 3 H), 4.07-4.16 (m, 1 H), 4.16-4.23 (m, 1 H), 4.23-4.34 (m, 1 H), 4.34-4.48 (m, 2 H), 4.48-4.57 (m, 1 H), 4.58-4.73 (m, 2 H), 4.97 (s, 2 H), 5.42 (s, 2 H), 5.93-6.04 (t, 1 H), 6.98-7.04 (s, 1 H), 7.04-7.12 (d, 1 H), 7.25-7.36 (m, 1 H), 7.42-7.48 (s, 1 H), 7.48-7.57 (m, 1 H), 7.57-7.64 (d, 1 H), 7.64-7.72 (m, 1 H), 7.77-7.86 (m, 1 H), 8.01-8.16 (m, 1 H), 10.01 (brs, 1 H), 10.56 (brs, 1 H)
(285) LC-MS m/z: 1389.6 [M.sup.+].sup.+, 1412.6 [M+Na].sup.+
PREPARATION EXAMPLE 3-22
Preparation of Compound (IV-26)
(286) ##STR00095##
(287) With the exception that compound (III-26)(0.28 g, 0.36 mmol), obtained in Preparation Example 2-22, was used instead of compound (III-22), obtained in Preparation Example 2-18, the same procedure as in Preparation Example 3-14 was repeated to afford the title compound (0.24 g, 48%).
(288) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.66-0.78 (m, 5 H), 0.78-1.06 (m, 19 H), 1.07-1.29 (m, 6 H), 1.29-1.38 (m, 1 H), 1.38-1.53 (m, 5 H), 1.53-1.63 (m, 1 H), 1.63-1.85 (m, 2 H), 1.85-2.02 (m, 3 H), 2.02-2.14 (m, 3 H), 2.14-2.27 (m, 6 H), 2.27-2.41 (m, 2 H), 2.56-2.60 (m, 3 H), 2.61-2.83 (m, 1 H), 2.86-3.08 (m, 5 H), 2.08-3.20 (m, 3 H), 3.20-3.27 (m, 1 H), 3.27-3.40 (m, 3 H), 3.45-3.63 (m, 1 H), 3.64-3.88 (m, 1 H), 3.88-4.06 (m, 2 H), 4.06-4.26 (m, 2 H), 4.28-4.46 (m, 1 H), 4.46-4.74 (m, 2 H), 4.74-4.91 (m, 1 H), 4.97 (s, 2 H), 5.43 (s, 2 H), 5.88-6.10 (t, 1 H), 7.01 (s, 1 H), 7.12-7.38 (m, 2 H), 7.38-7.72 (m, 4 H), 7.72-7.92 (m, 2 H), 7.92-8.22 (m, 3 H), 8.25-8.63 (m, 2 H), 8.73 (s, 1 H), 10.06 (brs, 1 H), 10.59 (brs, 1H)
(289) LC-MS m/z: 1379.6 [M.sup.+].sup.+, 1402.5 [M+Na].sup.+
PREPARATION EXAMPLE 3-23
Preparation of Compound (IV-27)
(290) ##STR00096##
(291) With the exception that compound (HI-27) (0.24 g, 0.32 mmol), obtained in Preparation Example 2-18, was used instead of compound (III-22), obtained in Preparation Example 2-18, the same procedure as in Preparation Example 3-14 was repeated to afford the title compound (0.28 g, 64%).
(292) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.59-0.78 (m, 6 H), 0.78-0.97 (m, 15 H), 0.97-1.06 (m, 2 H), 1.06-1.28 (m, 6 H), 1.28-1.39 (m, 1 H), 1.39-1.54 (m, 5 H), 1.54-1.64 (m, 1 H), 1.64-1.84 (m, 3 H), 1.84-2.02 (m, 3 H), 2.05-2.29 (m, 8 H), 2.30-2.42 (m, 2 H), 2.55-2.60 (m, 3 H), 2.60-2.83 (m, 1 H), 2.86-3.06 (m, 5 H), 3.06-3.21 (m, 6 H), 3.21-3.32 (m, 3 H), 3.36-3.44 (m, 1H), 3.44-3.52 (m, 1 H), 3.65-3.74 (m, 1H), 3.74-3.87 (m, 2 H), 3.89-4.05 (m, 2 H), 4.15-4.25 (m, 1 H), 4.31-4.73 (m, 4 H), 4.96 (s, 2 H), 5.43 (s, 2 H), 5.92-6.06 (t, 1 H), 6.71-6.83 (m, 1 H), 6.83-6.94 (m, 1 H), 6.97-7.04 (m, 1 H), 7.16-7.35 (m, 2 H), 7.38-7.67 (m, 3 H), 7.75-7.88 (d, 1 H), 8.00-8.17 (m, 2 H), 10.01 (brs, 1 H), 10.55 (brs, 1 H)
(293) LC-MS m/z: 1361.6 [M.sup.+].sup.+, 1384.5 [M+Na].sup.+
PREPARATION EXAMPLE 3-24
Preparation of Compound (IV-28)
(294) ##STR00097##
(295) Under an argon stream, the TFA salt of compound (II-3) (0.88 mg, 0.11 mmol), obtained in Preparation Example 1-2, was dissolved in 5 mL of anhydrous dimethyl formamide and added with diisopropylethylamine (0.18 mL, 1.03 mmol). To this solution, a solution of compound (III-28) (75 mg, 0.08 mmol), prepared in Preparation Example 2-24, in 5 mL of anhydrous dimethylformamide was dropwise added in an argon atmosphere, followed by stirring at 20-25° C. for 17 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound (135 mg, 100%).
(296) LC-MS m/z: 1462.9 [M+H].sup.+, 1485.9 [M+Na].sup.+
PREPARATION EXAMPLE 3-25
Preparation of Compound (IV-29)
(297) ##STR00098##
(298) With the exception that the TFA salt of compound (II-4) (0.17 g, 0.212 mmol), obtained in Preparation Example 1-3, was used instead of the TFA salt of compound (II-3), obtained in Preparation Example 1-2, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (0.14 g, 44%).
(299) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.69-0.78 (m, 6H), 0.81-0.90 (m, 14H), 1.11-1.64 (m, 3H), 1.18-1.26 (m, 10H), 1.38-1.51 (m, 6H), 1.58-1.62 (m, 1H), 1.63-1.83 (m, 3H), 1.91-1.99 (m, 3H), 2.11-2.26 (m, 7H), 2.35-2.42 (m, 2H), 2.87 (s, 6H), 2.92-3.05 (m, 3H), 3.13-3.18 (m, 4H), 3.32-3.38 (m, 17H), 3.62 (brs, 1H), 3.92-3.99 (m, 4H), 4.07-4.09 (m, 1H), 4.15-4.84 (m, 9H), 4.98 (s, 2H), 5.4 (s, 2H), 5.96-5.99 (t, 1H), 6.99 (s, 1H), 7.22-7.3 (m, 2H), 7.53-7.65 (m, 2H), 7.77-7.80 (m, 2H), 7.84-7.89 (t, 1H), 8.05-8.07 (d, 1H), 8.37-8.43 (m, 1H), 9.97 (s, 1H)
(300) LC-MS m/z: 1492.8 [M+H].sup.+, 1514.7 [M+Na].sup.+
PREPARATION EXAMPLE 3-26
Preparation of Compound (IV-30)
(301) ##STR00099##
(302) With the exception that the TFA salt of compound (II-5) (0.16 g, 0.212 mmol), obtained in Preparation Example 1-4, and compound (III-29) (0.2 g, 0.212 mmol), obtained in Preparation Example 2-25, were used respectively instead of the TFA salt of compound (II-3), obtained in Preparation Example 1-2, and the compound (III-28), obtained in Preparation Example 2-24, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (0.13 g, 40%).
(303) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.69-0.76 (m, 6H), 0.81-0.92 (m, 14H), 1.10-1.20 (m, 4H), 1.25-1.26 (m, 8H), 1.44-1.51 (m, 5H), 1.55-1.65 (m, 1H), 1.66-1.85 (3H), 1.86-2.05 (m, 3H), 2.06-2.27 (m, 6H), 2.28-2.46 m, 2H), 2.84 (s, 1H), 2.87-2.96 (m, 1H), 2.99-3.04 (m, 5H), 3.14-3.18 (m, 4H), 3.32-3.47 (m, 11H), 3.62 (brs, 1H), 3.92-3.93 (d, 3H), 4.00-4.91 (m, 8H), 4.94 (s, 2H), 5.41 (s, 2H), 5.97-6.00 (t, 1H), 7.00 (s, 1H), 7.24-7.32 (m, 3H), 7.58-7.60 (d, 2H), 7.77-7.80 (m, 2H), 7.85-7.89 (t, 1H), 8.05-8.07 (d, 1H), 8.19 (t, 1H), 8.44 (brs, 1H), 9.97 (s, 1H)
(304) LC-MS m/z: 1464.8 [M+H].sup.+, 1487.9 [M+Na].sup.+
PREPARATION EXAMPLE 3-27
Preparation of Compound (IV-31)
(305) ##STR00100##
(306) With the exception that the TFA salt of compound (II-4) (0.26 g, 0.324 mmol), obtained in Preparation Example 1-3, and compound (III-30) (0.2 g, 0.216 mmol), obtained in Preparation Example 2-26, were used respectively instead of the TFA salt of compound (II-3), obtained in Preparation Example 1-2, and the compound (III-28), obtained in Preparation Example 2-24, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (0.2 g, 64%).
(307) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.71-0.75 (m, 6H), 0.80-0.91 (m, 15H), 1.18-1.19 (m, 3H), 1.22-1.27 (m, 9H), 1.43-1.49 (m, 6H), 1.90-1.98 (m, 3H), 2.09-2.23 (m, 3H), 2.25-2.40 (m, 7H), 2.83-2.86 (m, 4H), 2.96-3.01 (m, 4H), 3.15-3.19 (m, 3H), 3.26 (s, 3H), 3.35-3.38 (m, 16H), 3.85-3.96 (m, 3H), 4.11-4.88 (m, 8H), 4.97 (s, 2H), 5.42 (s, 2H), 5.97-5.99 (t, 1H), 6.99-7.01 (m, 1H), 7.06-7.16 (m, 2H), 7.28 (brs, 2H), 7.60-7.69 (m, 2H), 7.81-7.83 (d, 1H), 8.09-8.12 (d, 1H), 8.29 (brs, 1H), 10.09 (s, 1H)
(308) LC-MS m/z: 1472.9 [M+H].sup.+, 1494.9 [M+Na].sup.+
PREPARATION EXAMPLE 3-28
Preparation of Compound (IV-32)
(309) ##STR00101##
(310) With the exception that the TFA salt of compound (I-5) (0.24 g, 0.324 mmol), obtained in Preparation Example 1-4, and compound (III-30) (0.2 g, 0.216 mmol), obtained in Preparation Example 2-24, were used respectively instead of the TFA salt of compound (II-3), obtained in Preparation Example 1-2, and the compound (III-28), obtained in Preparation Example 2-24, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (0.2 g, 67%).
(311) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.71-0.75 (m, 7H), 0.81-0.92 (m, 16H), 1.03-1.04 (m, 2H), 1.11-1.12 (m, 1H), 1.18-1.27 (m, 7H), 1.46-1.49 (m, 5H), 1.90-1.98 (m, 3H), 2.09-2.23 (m, 6H), 2.28-2.38 (m, 7H), 2.96-3.03 (m, 9H), 3.15-3.17 (m, 4H), 3.28 (s, 3H), 3.35-3.38 (m, 14H), 3.62-3.65 (m, 2H), 3.85-4.00 (m, 3H), 4.17-4.88 (m, 8H), 4.94 (s, 2H), 5.43 (s, 2H), 5.88-6.00 (t, 1H), 7.00 (s, 1H), 7.06-7.16 (m, 2H), 7.27-7.29 (m, 3H), 7.58-7.69 (m, 2H), 7.82-7.84 (d, 1H), 8.09-8.12 (d, 1H), 8.29 (brs, 1H), 10.00 (s, 1H)
(312) LC-MS m/z: 1444.9 [M+H].sup.+, 1466.9 [M+Na].sup.+
PREPARATION EXAMPLE 3-29
Preparation of Compound (IV-33)
(313) ##STR00102##
(314) With the exception that the TFA salt of compound (II-5) (0.23 g, 0.307 mmol), obtained in Preparation Example 1-4, and compound (III-31) (0.2 g, 0.205 mmol), obtained in Preparation Example 2-27, were used respectively instead of the TFA salt of compound (II-3), obtained in Preparation Example 1-2, and the compound (III-28), obtained in Preparation Example 2-24, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (0.16 g, 51%).
(315) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.71-0.75 (m, 7H), 0.81-0.92 (m, 16H), 1.03-1.04 (m, 2H), 1.11-1.12 (m, 1H), 1.18-1.27 (m, 7H), 1.46-1.49 (m, 5H), 1.90-1.98 (m, 3H), 2.09-2.23 (m, 6H), 2.28-2.38 (m, 7H), 2.96-3.03 (m, 9H), 3.15-3.17 (m, 4H), 3.28 (s, 3H), 3.35-3.38 (m, 14H), 3.62-3.65 (m, 2H), 3.85-4.00 (m, 3H), 4.17-4.88 (m, 8H), 4.94 (s, 2H), 5.43 (s, 2H), 5.88-6.00 (t, 1H), 7.00 (s, 1H), 7.06-7.16 (m, 2H), 7.27-7.29 (m, 3H), 7.58-7.69 (m, 2H), 7.82-7.84 (d, 1H), 8.09-8.12 (d, 1H), 8.29 (brs, 1H), 10.00 (s, 1H)
(316) LC-MS m/z: 1497.6 [M+H].sup.+, 1519.6 [M+Na].sup.+
PREPARATION EXAMPLE 3-30
Preparation of Compound (IV-34)
(317) ##STR00103##
(318) With the exception that compound (III-32) (1.2 g, 1.294 mmol), obtained in Preparation Example 2-28, instead of the compound (III-28), obtained in Preparation Example 2-24, was used together with the TFA salt of compound (II-3) (1 g, 1.294 mmol), obtained in Preparation Example 1-2, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (1.3 g, 72%).
(319) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.48-0.49 (m, 1H), 0.58-0.59 (m, 2H), 0.64-0.65 (m, 2H), 0.70-0.76 (m, 5H), 0.81-0.92 (m, 16H), 1.11-1.13 (m, 2H), 1.16-1.20 (m, 3H), 1.22-1.28 (m, 9H), 1.44-1.49 (m, 5H), 1.90-1.98 (m, 3H), 2.09-2.23 (m, 7H), 2.96-3.04 (m, 9H), 3.13-3.18 (m, 4H), 3.33-3.38 (m, 14H), 3.60-3.64 (m, 1H), 3.90-3.94 (m, 4H), 3.99-4.88 (m, 8H), 4.94 (s, 2H), 5.42 (s, 2H), 5.86-5.99 (t, 1H), 7.22-7.29 (m, 4H), 7.38-7.42 (m, 1H), 7.58-7.60 (d, 2H), 7.80-7.82 (d, 1H), 7.89-7.96 (m, 2H), 8.00-8.10 (m, 2H), 8.29 (brs, 1H), 8.70 (s, 1H), 10.00 (s, 1H)
(320) LC-MS m/z: 1452.8 [M+H].sup.+, 1474.7 [M+Na].sup.+
PREPARATION EXAMPLE 3-31
Preparation of Compound (IV-35)
(321) ##STR00104##
(322) With the exception that the TFA salt of compound (II-6) (0.18 g, 0.233 mmol), obtained in Preparation Example 1-5, instead of the TFA salt of compound (II-3), obtained in Preparation Example 1-2, was used together with the compound (III-29) (0.2 g, 0.212 mmol), obtained in Preparation Example 2-25, the same procedure as in Preparation Example 3-24 was repeated to afford the title compound (0.16 g, 51%).
(323) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.68-0.76 (m, 6H), 0.81-0.90 (m, 14H), 1.08-1.20 (m, 4H), 1.23-1.27 (m, 9H), 1.44-1.49 (m, 6H), 1.58-1.62 (m, 1H), 1.63-1.83 (m, 3H), 1.91-1.99 (m, 3H), 2.13-2.20 (m, 6H), 2.35-2.42 (m, 2H), 2.85-2.87 (m, 4H), 2.99-3.01 (m, 4H), 3.14-3.17 (m, 4H), 3.28-3.38 (m, 19H), 3.62 (brs, 1H), 3.92-3.94 (m, 3H), 4.07-4.09 (m, 1H), 4.15-4.84 (m, 8H), 4.98 (s, 2H), 5.43 (s, 2H), 5.98-6.00 (t, 1H), 7.01 (s, 1H), 7.22-7.36 (m, 3H), 7.54-7.64 (m, 2H), 7.77-7.97 (m, 2H), 8.08-8.11 (d, 1H), 8.22-8.45 (m, 2H), 10.01 (s, 1H)
(324) LC-MS m/z: 1478.5 [M+H].sup.+, 1500.5 [M+Na].sup.+
PREPARATION EXAMPLE 4
Preparation of Compounds of Chemical Formula VI-1, VI-2, VI-3, and VI-4
PREPARATION EXAMPLE 4-1
Preparation of Compound (VI-5)
(325) ##STR00105##
(326) To a solution of compound (IV-5) (274 mg, 0.21 mmol), prepared in Preparation Example 3-1, in 5.0 mL of acetonitrile were added 4-mercaptobutyric acid (V−1) (126 mg, 1.05 mmol) and 0.5 mL of DMSO, followed by stirring at room temperature for 4 hrs. The reaction mixture was concentrated in a vacuum to afford the title compound (290 mg, 98%).
(327) MALDI-TOF MS m/z: 1465.3 [M+Na].sup.+
PREPARATION EXAMPLE 4-2
Preparation of Compound (VI-6)
(328) ##STR00106##
(329) To a solution of compound (IV-5) (70 mg, 0.053 mmol), obtained in Preparation Example 3-1, in 2.5 mL of acetonitrile were added 2-(1-(mercaptomethyl)cyclopropane)acetic acid (V-2) (44 mg, 0.3 mmol) and 0.7 mL of DMF, followed by stirring at room temperature for 4 hrs. The reaction mixture was concentrated in a vacuum, and the concentrate was purified by silica gel column chromatography to afford the title compound as a solid (60 mg, 77%).
(330) MALDI-TOF MS m/z: 1492.0 [M+Na].sup.+
PREPARATION EXAMPLE 4-3
Preparation of Compound (VI-7)
(331) ##STR00107##
(332) With the exception that 6-mercaptohexanoic acid (V-3) was used instead of 2-(1-(mercaptomethyl)cyclopropane)acetic acid (V-2), the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (54 mg, 69%).
(333) MALDI-TOF MS m/z: 1493.2 [M+Na].sup.+
PREPARATION EXAMPLE 4-4
Preparation of Compound (VI-8)
(334) ##STR00108##
(335) With the exception that 8-mercaptooctanoic acid (V-4) was used instead of 2-(1-(mercaptomethyl)cyclopropane)acetic acid (V-2), the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (66 mg, 84%).
(336) MALDI-TOF MS m/z: 1522.0 [M+Na].sup.+
PREPARATION EXAMPLE 4-5
Preparation of Compound (VI-9)
(337) ##STR00109##
(338) With the exception that 11-mercaptoundecanoic acid (V-5) was used instead of 2-(1-(mercaptomethyl)cyclopropane)acetic acid (V-2), the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (71 mg, 86%).
(339) MALDI-TOF MS m/z: 1563.3 [M+Na].sup.+
PREPARATION EXAMPLE 4-6
Preparation of Compound (VI-10)
(340) ##STR00110##
(341) With the exception that compound (IV-6) (70 mg, 0.054 mmol), obtained in Preparation Example 3-2 was used instead of compound (IV-5), obtained in Preparation Example 3-1, the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (53 mg, 68%).
(342) MALDI-TOF MS m/z: 1469.5 [M+Na].sup.+
PREPARATION EXAMPLE 4-7
Preparation of Compound (VI-11)
(343) ##STR00111##
(344) With the exception that compound (IV-6) (70 mg, 0.054 mmol), obtained in Preparation Example 3-2, and 6-mercaptohexanoic acid (V-3) were used respectively instead of compound (IV-5), obtained in Preparation Example 3-1, and 2-(1-(mercaptomethyl)cyclopropane)acetic acid (V-2), the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (46 mg, 59%).
(345) MALDI-TOF MS m/z: 1471.6 [M+Na].sup.+
PREPARATION EXAMPLE 4-8
Preparation of Compound (VI-12)
(346) ##STR00112##
(347) With the exception that compound (IV-7) (88 mg, 0.064 mmol), obtained in Preparation Example 3-1, was used instead of compound (IV-5), obtained in Preparation Example 3-1, the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (48 mg, 49%).
(348) MALDI-TOF MS m/z: 1548.1 [M+Na].sup.+
PREPARATION EXAMPLE 4-9
Preparation of Compound (VI-13)
(349) ##STR00113##
(350) With the exception that compound (IV-8) (71 mg, 0.052 mmol), obtained in Preparation Example 3-4, was used instead of compound (IV-5), obtained in Preparation Example 3-1, the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (46 mg, 59%).
(351) MALDI-TOF MS m/z: 1529.4 [M+Na].sup.+
PREPARATION EXAMPLE 4-10
Preparation of Compound (VI-14)
(352) ##STR00114##
(353) With the exception that compound (IV-9) (35 mg, 0.026 mmol), obtained in Preparation Example 3-5, was used instead of compound (IV-5), obtained in Preparation Example 3-1, the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (29 mg, 74%).
(354) MALDI-TOF MS m/z: 1528.2 [M+Na].sup.+
PREPARATION EXAMPLE 4-11
Preparation of Compound (VI-15)
(355) ##STR00115##
(356) With the exception that compound (IV-10) (78 mg, 0.058 mmol), obtained in Preparation Example 3-6, was used instead of compound (IV-5), obtained in Preparation Example 3-1, the same procedure as in Preparation Example 4-2 was repeated to afford the title compound (77 mg, 90%).
(357) MALDI-TOF MS m/z: 1508.1 [M+Na].sup.+, 1524.8 [M+K].sup.+
PREPARATION EXAMPLE 4-12
Preparation of Compound (VI-16)
(358) ##STR00116##
(359) To a solution of compound (IV-11)(58 mg, 0.042 mmol), obtained in Preparation Example 3-7, in 5 mL of methyl alcohol and 1.5 mL of anhydrous dimethylformamide was added 2-(1-(mercaptomethyl)cyclopropane)acetic acid (19 mg, 0.126 mmol), followed by stirring at room temperature for 4 hrs. The reaction mixture was concentrated in a vacuum, and the concentrate was purified by silica gel column chromatography to afford the title compound as a solid (60 mg, 93%).
(360) LC-MS m/z: 1523.8 [M.sup.+].sup.+, 1545.8 [M+Na].sup.+
PREPARATION EXAMPLE 4-13
Preparation of Compound (VI-17)
(361) ##STR00117##
(362) With the exception that compound IV-12)(41 mg, 0.031 mmol), obtained in Preparation Example 3-8, was used instead of compound (IV-11), obtained in Preparation Example 3-7, the same procedure as in Preparation Example 4-12 was repeated to afford the title compound (39 mg, 85%).
(363) MALDI-TOF MS m/z: 1496.8 [M+Na].sup.+
PREPARATION EXAMPLE 4-14
Preparation of Compound (VI-18)
(364) ##STR00118##
(365) With the exception that compound (IV-13) (347 mg, 0.257 mmol), obtained in Preparation Example 3-9, and 11-mercaptoundecanoic acid (112 mg, 0.514 mmol) were used respectively instead of compound (IVI-11), obtained in Preparation Example 3-7, and 2-(1-(mercaptomethyl)cyclopropane)acetic acid, the same procedure as in Preparation Example 4-12 was repeated to afford the title compound (266 mg, 66%).
(366) LC-MS m/z: 1567 [M+1].sup.+
(367) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.76-0.90 (m, 10H), 0.90-1.14 (m, 20H), 1.17 (m, 3H), 1.26 (m, 4H), 1.29 (brs, 20H), 1.37 (m, 4H), 1.50-1.64 (m, 14H), 1.68-1.78 (m, 3H), 1.88 (m, 2H), 1.94 (s, 3H), 2.21-2.26 (m, 6H), 2.99-2.48 (m, 10H, N—CH.sub.3*2), 2.70 (m, 1H), 2.84 (m, 1H), 3.07-3.19 (m, 6H), 3.26-3.45 (m, 6H), 3.45 (m, 4H), 3.82 (m, 1H), 4.15 (m, 1H), 4.47 (m, 2H), 4.64 (m, 1H), 4.81 (m, 1H), 4.98 (m, 3H), 6.96 (t, 1H), 7.08 (t, 1H), 7.25 (d, 2H), 7.28-7.40 (m, 2H), 7.53 (d, 2H)
PREPARATION EXAMPLE 4-15
Preparation of Compound (VI-19)
(368) ##STR00119##
(369) With the exception that compound (IV-14)(453 mg, 0.332 mmol), obtained in Preparation Example 3-10 was used instead of compound (IV-11), obtained in Preparation Example 3-7, the same procedure as in Preparation Example 4-12 was repeated to afford the title compound (60 mg, 93%).
(370) LC-MS m/z: 1510 [M+1].sup.+,
(371) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.46-0.68 (m, 5H), 0.82-1.10 (m, 27H), 1.17 (d, 2H), 1.22 (d, 1H), 1.30 (m, 2H), 1.40 (m, 1H), 1.50-1.63 (m, 6H), 1.68-1.80 (m, 2H), 1.82-1.90 (m, 2H), 1.94 (s, 3H), 2.00-2.15 (m, 4H), 2.20-2.35 (m, 5H), 2.42 (s, 6H), 2.38-2.50 (m, 2H), 2.81-2.89 (m, 1H), 2.91-2.98 (m, 2H), 3.08-3.40 (m, 6H), 3.26-3.33 (m, 10H, OCH.sub.3), 3.43 (d, 2H), 3.45-3.48 (m, 5H, OCH.sub.3), 3.52-3.64 (m, 1H), 3.74-3.86 (m, 1H), 3.906-4.10 (m, 3H), 4.15 (d, 1H), 4.24 (m, 1H), 4.44-4.54 (m, 2H), 4.66 (d, 1H), 4.74-5.10 (m, 5H), 7.09-7.12 (m, 2H), 7.23-7.27 (m, 2H), 7.33-7.37 (m, 2H), 7.52-7.59 (m, 2H)
PREPARATION EXAMPLE 4-16
Preparation of Compound (VI-20)
(372) ##STR00120##
(373) With the exception that compound (IV-15)(82 mg, 0.063 mmol), obtained in Preparation Example 3-7, was used instead of compound (IV-11), obtained in Preparation Example 3-7, the same procedure as in Preparation Example 4-12 was repeated to afford the title compound (38 mg, 42%).
(374) LC-MS m/z: 1506 [M+1]
(375) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.42-0.67 (m, 4H), 0.81-1.08 (m, 27H), 1.13-1.20 (m, 4H), 1.29 (m, 2H), 1.40 (m, 1H), 1.50-1.68 (m, 6H), 1.75 (m, 2H), 1.82-1.90 (m, 2H), 1.94 (s, 3H), 2.01-2.18 (m, 4H), 2.23-2.32 (m, 3H), 2.42 (s, 6H), 2.35-2.50 (m, 1H), 2.61 (m, 1H), 2.79-3.00 (m, 3H), 3.10-3.20 (m, 5H), 3.28-3.40 (m, 19H, 2*OCH.sub.3), 3.43-3.48 (m, 3H), 3.60 (m, 1H), 3.74 (d, 3H), 3.85-4.00 (m, 3H), 4.14 (d, 1H), 4.21-4.30 (m, 1H), 4.48 (m, 2H), 4.64-5.10 (m, 5H), 6.86-7.60 (m, 8H)
PREPARATION EXAMPLE 4-17
Preparation of Compound (VI-21)
(376) ##STR00121##
(377) With the exception that compound (IV-16)(268 mg, 0.195 mmol), obtained in Preparation Example 3-12, was used instead of compound (IV-11), obtained in Preparation Example 3-7, the same procedure as in Preparation Example 4-12 was repeated to afford the title compound as a solid (132 mg, 46%).
(378) LC-MS m/z: 1521 [M].sup.+
(379) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.48-0.50 (m, 6H), 0.71-1.08 (m, 27H), 1.16-1.22 (m, 4H), 1.42-1.54 (m, 9H), 1.60 (m, 1H), 1.70 (m, 1H), 1.94 (s, 3H), 2.10-2.32 (m, 8H), 2.32-2.46 (m, 6H), 2.50 (s, 6H), 2.78-3.10 (m, 8H), 3.16 (m, 2H), 3.21 (m, 1H), 3.28-3.40 (m, 6H), 3.62 (s, 1H), 3.70 (s, 2H), 3.86-4.00 (m, 5H), 4.19 (t, 2H), 4.38 (m, 2H), 4.52 (t, 1H), 4.60 (m, 1H), 4.76 (m, 1H), 4.88 (m, 1H), 5.00 (m, 2H), 5.08 (m, 1H), 5.42 (brs, 2H), 5.98 (m, 1H), 6.81 (d, 1H), 7.17 (d, 1H), 7.28 (dd, 1H), 7.50-7.70 (m, 3H), 7.82 (d, 1H), 8.10 (d, 1H), 10.0 (d, 1H)
PREPARATION EXAMPLE 4-18
Preparation of Compound (VI-22)
(380) ##STR00122##
(381) With the exception that compound (IV-17)(440 mg, 0.33 mmol), obtained in Preparation Example 3-13, was used instead of compound (IV-11), obtained in Preparation Example 3-7, the same procedure as in Preparation Example 4-12 was repeated to afford the title compound (296 mg, 60%).
(382) LC-MS m/z: 1492 [M+1].sup.+
(383) .sup.1H NMR (400 MHz, CD.sub.3OD) δ 0.47-0.62 (m, 5H), 0.81-1.02 (m, 26H), 1.13-1.20 (m, 3H), 1.25-1.32 (m, 2H), 1.40 (m, 1H), 1.50-1.68 (m, 6H), 1.72-1.78 (m, 2H), 1.82-1.98 (m, 3H), 1.96 (s, 3H), 2.00-2.09 (m, 4H), 2.25-2.35 (m, 5H), 2.38-2.48 (m, 6H), 2.850-2.90 (m, 3H), 3.05-3.20 (m, 6H), 3.26-3.35 (m, 9H, OCH.sub.3), 3.40-3.48 (m, 6H, OCH.sub.3), 3.50-3.60 (m, 2H), 3.65-3.80 (m, 3H), 3.90-4.18 (m, 5H), 4.48 (m, 1H), 4.64 (m, 1H), 4.70-5.10 (m, 8H), 7.09-7.58 (m, 8H)
PREPARATION EXAMPLE 4-19
Preparation of Compound (VI-23)
(384) ##STR00123##
(385) To a solution of compound (IV-18) (37 mg, 0.027 mmol), obtained in Preparation Example 3-14, in 5 mL of methyl alcohol and 1.5 mL of anhydrous dimethylformamide was added 2-(1-(mercaptomethyl)cyclopropane)acetic acid (12 mg, 0.083 mmol), followed by stirring at room temperature for 4 hrs. The reaction mixture was concentrated in a vacuum, and the concentrate was purified by silica gel column chromatography to afford the title compound as a solid (33 mg, 81%).
(386) LC-MS m/z: 1499 [M.sup.+].sup.+, 1451 [M+Na].sup.+
PREPARATION EXAMPLE 4-20
Preparation of Compound (VI-24)
(387) ##STR00124##
(388) With the exception that compound (IV-19) (69 mg, 0.051 mmol), obtained in Preparation Example 3-15, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (55 mg, 72%).
(389) LC-MS m/z: 1508.9 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-21
Preparation of Compound (VI-25)
(390) ##STR00125##
(391) With the exception that compound (IV-20) (94 mg, 0.07 mmol), obtained in Preparation Example 3-16, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound (99 mg, 94%).
(392) LC-MS m/z: 1498 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-22
Preparation of Compound (VI-26)
(393) ##STR00126##
(394) With the exception that compound (IV-21) (144 mg, 0.105 mmol), obtained in Preparation Example 3-17, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (116 mg, 73%).
(395) LC-MS m/z: 1512 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-23
Preparation of Compound (VI-27)
(396) ##STR00127##
(397) With the exception that compound (IV-22) (0.14 g, 0.099 mmol), obtained in Preparation Example 3-18, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.11 g, 75%).
(398) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.50-0.52 (m, 4H), 0.65-0.76 (m, 6H), 0.82-0.92 (m, 13H), 1.08-1.24 (18H), 1.43-1.49 (m, 6H), 1.54-1.82 (m, 3H), 1.84-1.98 (m, 3H), 2.10-2.19 (m, 6H), 2.79-3.04 (m, 12H), 3.15-3.24 (m, 3H), 3.30-3.44 (m, 15H), 3.87-3.89 (m, 3H), 3.97-4.00 (m, 1H), 4.08-4.86 (m, 8H), 4.86-5.04 (m, 1H), 5.10 (s, 2H), 5.46 (s, 2H), 6.03 (t, 1H), 7.22-7.36 (m, 2H), 7.62-7.65 (d, 1H), 7.77-7.80 (d, 1H), 7.87-7.89 (m, 2H), 8.17-8.23 (m, 1H), 8.48 (brs, 1H), 10.11 (s, 1H)
(399) LC-MS m/z: 1524.4 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-24
Preparation of Compound (VI-28)
(400) ##STR00128##
(401) With the exception that compound (IV-23) (0.2 g, 0.15 mmol), obtained in Preparation Example 3-19, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.06 g, 27%).
(402) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.43-0.52 (m, 6H), 0.69-0.76 (m, 7H), 0.82-0.92 (m, 15H), 1.02-1.06 (m, 7H), 1.11-1.13 (m, 2H), 1.18-1.23 (m, 4H), 1.43-1.47 (m, 6H), 1.54-1.65 (m, 1H), 1.66-1.84 (m, 2H), 1.89-1.98 (m, 3H), 2.09-2.20 (m, 6H), 2.91-3.03 (m, 10H), 3.15-3.22 (m, 5H), 3.33-3.46 (m, 15H), 3.87-4.02 (m, 3H), 4.15-4.95 (m, 8H), 5.08 (s, 2H), 5.44 (s, 2H), 6.03 (t, 1H), 7.07-7.16 (m, 2H), 7.33-7.35 (m, 2H), 7.62-7.70 (d, 2H), 7.85-7.91 (m, 1H), 7.98-8.07 (m, 1H), 8.20 (brs, 1H), 8.28-8.49 (m, 1H), 10.10 (s, 1H)
(403) LC-MS m/z: 1504.4 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-25
Preparation of Compound (VI-29)
(404) ##STR00129##
(405) With the exception that compound (IV-24) (0.1 g, 0.1 mmol), obtained in Preparation Example 3-20, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.08 g, 53%).
(406) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.43-0.47 (m, 5H), 0.48-0.50 (d, 1H), 0.58-0.59 (d, 1H) 0.69-0.76 (m, 3H), 0.82-0.92 (m, 16H), 1.05-1.09 (m, 6H), 1.11-1.13 (m, 2H), 1.16-1.23 (m, 4H), 1.43-1.47 (m, 6H), 1.54-1.65 (m, 1H), 1.66-1.84 (m, 2H), 1.89-1.98 (m, 3H), 2.10-2.20 (m, 8H), 2.31-2.43 (m, 2H), 2.58-2.68 (m, 1H), 2.70-2.82 (m, 3H), 2.87-2.88 (m, 2H), 2.95-3.00 (m, 4H), 3.13-3.22 (m, 4H), 3.31-3.46 (m, 16H), 3.90-3.92 (m, 3H), 3.97-4.00 (m, 1H), 4.06-5.00 (m, 8H), 5.10 (s, 2H), 5.47 (s, 2H), 6.10 (t, 1H), 7.22-7.28 (m, 1H), 7.33-7.43 (m, 2H), 7.55-7.58 (d, 1H), 7.88-7.99 (m, 2H), 8.01-8.03 (d, 1H), 8.22-8.54 (m, 2H), 8.64 (s, 1H), 10.15 (s, 1H)
(407) LC-MS m/z: 1556.4 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-26
Preparation of Compound (VI-30)
(408) ##STR00130##
(409) With the exception that compound (IV-25) (0.46 g, 0.33 mmol), obtained in Preparation Example 3-21, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.49 g, 96%).
(410) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.30-0.58 (m, 6 H), 0.59-0.78 (m, 8 H), 0.78-1.00 (m, 16 H), 1.05-1.32 (m, 6 H), 1.32-1.54 (m, 6 H), 1.54-1.64 (m, 1 H), 1.64-1.84 (m, 3 H), 1.84-2.05 (m, 3 H), 2.06-2.27 (m, 10 H), 2.27-2.43 (m, 4 H), 2.55-2.71 (m, 1 H), 2.71-2.85 (m, 2 H), 2.85-3.07 (m, 7 H), 3.09-3.18 (m, 3 H), 3.18-3.30 (m, 3 H), 3.74-3.88 (m, 6 H), 3.88-4.05 (m, 4 H), 4.06-4.22 (m, 2 H), 4.22-4.43 (m, 2 H), 4.43-4.53 (m, 1 H), 4.59-4.75 (m, 2 H), 4.96 (s, 2 H), 5.43 (s, 2 H), 5.78 (s, 1 H), 5.96-6.10 (d, 1 H), 7.01-7.13 (d, 1 H), 7.21-7.37 (m, 2 H), 7.41-7.48 (s, 1 H), 7.49-7.57 (m, 1 H), 7.57-7.75 (m, 3 H), 8.00-8.11 (m, 1 H), 10.00-10.20 (brs, 1 H)
(411) LC-MS m/z: 1535.5 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-27
Preparation of Compound (VI-31)
(412) ##STR00131##
(413) With the exception that compound (IV-26) (0.24 g, 0.17 mmol), obtained in Preparation Example 3-22, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.19 g, 73%).
(414) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.37-0.58 (m, 9 H), 0.60-0.78 (m, 6 H), 0.78-0.97 (m, 16 H), 1.06-1.37 (m, 7 H), 1.37-1.53 (m, 6 H), 1.54-1.85 (m, 5 H), 1.86-2.03 (m, 3 H), 2.05-2.26 (m, 10 H), 2.26-2.43 (m, 3 H), 2.58-2.67 (m, 1 H), 2.68-2.84 (m, 2 H), 2.85-3.09 (m, 8 H), 3.10-3.24 (m, 6 H), 3.24-3.33 (m, 3 H), 3.62-3.78 (m, 1 H), 3.78-4.05 (m, 3 H), 4.05-4.24 (m, 2 H), 4.27-4.40 (m, 1 H), 4.46-4.61 (m, 1 H), 4.61-4.72 (m, 1 H), 4.72-4.89 (m, 1 H), 5.01 (s, 2 H), 5.44 (s, 2 H), 5.96-6.14 (t, 1 H), 7.21-7.37 (m, 2 H), 7.45-7.56 (m, 1 H), 7.56-7.74 (m, 4 H), 7.93-8.08 (m, 3H), 8.08-8.19 (m, 1 H), 8.28-8.39 (m, 1 H), 8.49-8.61 (m, 1 H), 8.73 (s, 1 H), 10.14 (brs, 1 H)
(415) LC-MS m/z: 1535.5 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-28
Preparation of Compound (VI-32)
(416) ##STR00132##
(417) With the exception that compound (IV-27) (0.21 g, 0.15 mmol), obtained in Preparation Example 3-23, was used instead of compound (IV-18), obtained in Preparation Example 3-14, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.21 g, 91%).
(418) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.34-0.56 (m, 5 H), 0.64-0.78 (m, 6 H), 0.78-0.96 (m, 15 H), 0.96-1.07 (m, 2 H), 1.07-1.27 (m, 5 H), 1.29-1.39 (m, 1 H), 1.39-1.54 (m, 5 H), 1.54-1.65 (m, 1 H), 1.65-1.84 (m, 3 H), 1.84-2.03 (m, 3 H), 2.05-2.31 (m, 10 H), 2.31-2.44 (m, 2 H), 2.44-2.48 (m, 1 H), 2.58-2.61 (m, 2 H), 2.75-2.85 (m, 1 H), 2.86-3.08 (m, 7 H), 3.09-3.15 (m, 1 H), 3.24-3.55 (m, 14 H), 3.64-3.76 (m, 2H), 3.77-3.86 (m, 2 H), 3.87-4.04 (m, 3 H), 4.04-4.14 (m, 2H), 4.14-4.25 (m, 2H), 4.33-4.40 (m, 1 H), 4.40-4.61 (m, 2 H), 4.61-4.76 (m, 1 H), 4.97 (s, 2 H), 5.44 (s, 2 H), 5.98-6.11 (t, 1H), 6.82-6.94 (m, 1 H), 7.16-7.25 (m, 1 H), 7.25-7.37 (m, 2 H), 7.40-7.49 (m, 1 H), 7.49-7.68 (m, 3 H), 7.81-7.99 (m, 1 H), 7.99-8.12 (m, 1 H), 8.24-8.45 (m, 1 H), 10.12 (brs, 1 H)
(419) LC-MS m/z: 1535.5 [M.sup.+].sup.+
PREPARATION EXAMPLE 4-29
Preparation of Compound (VI-33)
(420) ##STR00133##
(421) With the exception that compound (IV-21) (0.28 g, 0.21 mmol), obtained in Preparation Example 3-17, and 6-mercaptohexanoic acid (0.07 g, 0.41 mmol) were used respectively instead of compound (IV-18), obtained in Preparation Example 3-14, and 2-(1-(mercaptomethyl)cyclopropane)acetic acid, the same procedure as in Preparation Example 4-19 was repeated to afford the title compound as a solid (0.26 g, 81%).
(422) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.64-0.79 (m, 5H), 0.79-0.98 (m, 17H), 0.98-1.14 (m, 4H), 1.14-1.28 (m, 5H), 1.28-1.41 (m, 6H), 1.41-1.65 (m, 14H), 1.65-1.79 (m, 2H), 1.83-2.05 (m, 7H), 2.05-2.32 (m, 13 H), 2.32-2.56 (m, 1 H), 2.57-2.88 (m, 10 H), 2.89-3.06 (m, 6 H), 3.06-3.24 (m, 3 H), 3.69-3.86 (m, 3 H), 3.86-4.09 (m, 3 H), 4.09-4.28 (m, 1 H), 4.29-4.48 (m, 2 H), 4.48-4.78 (m, 2 H), 5.04 (s, 2 H), 5.42 (s, 2 H), 6.03 (brs, 1 H), 7.01 (brs, 2 H), 7.14-7.45 (m, 2 H), 7.45-7.70 (m, 1 H), 7.83-8.06 (m, 2 H), 10.08 (brs, 1 H)
(423) LC-MS m/z: 1514.4 [M.sup.+H].sup.+, 1536.4 [M+Na].sup.+
PREPARATION EXAMPLE 4-30
Preparation of Compound (VI-34)
(424) ##STR00134##
(425) To a solution of compound (IV-28)(137 mg, 0.093 mmol), obtained in Preparation Example 3-24, in 5 mL of methyl alcohol and 3 mL of anhydrous dimethylformamide was added 2-(1-(mercaptomethyl)cyclopropane)acetic acid (41 mg, 0.28 mmol), followed by stirring at room temperature for 15 hrs. The reaction mixture was concentrated in a vacuum, and the concentrate was purified by silica gel column chromatography to afford the title compound as a solid (25 mg, 17%).
(426) LC-MS m/z: 1609.3 [M+H].sup.+
PREPARATION EXAMPLE 4-31
Preparation of Compound (VI-35)
(427) ##STR00135##
(428) With the exception that compound (IV-29) (0.19 g, 0.128 mmol), obtained in Preparation Example 3-25, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (0.085 g, 41%).
(429) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.42-0.55 (m, 6H), 0.66-0.75 (m, 8H), 0.81-0.87 (m, 14H), 1.10-1.23 (m, 6H), 1.37-1.49 (m, 6H), 1.54-1.79 (m, 3H), 1.91-1.98 (m, 7H), 2.27 (s, 1H), 2.30-2.37 (m, 1H), 2.38-2.42 (m, 1H), 2.44-2.45 (m, 1H), 2.56-2.64 (m, 2H), 2.75-2.86 (m, 6H), 2.93-2.99 (m, 4H), 3.12-3.24 (m, 4H), 3.31-3.35 (m, 17H), 3.91-4.06 (m, 6H), 4.14-4.86 (m, 8H), 4.98 (s, 2H), 5.43 (s, 2H), 5.97-5.98 (t, 1H), 7.29-7.30 (m, 2H), 7.58-7.60 (d, 2H), 7.77-7.90 (m, 2H), 7.93-8.10 (m, 2H), 8.11-8.13 (d, 2H), 8.46 (brs, 1H), 10.01 (s, 1H), 12.12 (brs, 1H)
(430) LC-MS m/z: 1639.0 [M+H].sup.+, 1661.0 [M+Na].sup.+
PREPARATION EXAMPLE 4-32
Preparation of Compound (VI-36)
(431) ##STR00136##
(432) With the exception that compound (IV-30) (0.18 g, 0.119 mmol), obtained in Preparation Example 3-26, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (0.12 g, 61%).
(433) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.44-0.52 (m, 5H), 0.66-0.75 (m, 7H), 0.82-0.92 (m, 12H), 1.09-1.23 (m, 6H), 1.43-1.47 (m, 5H), 1.54-1.83 (m, 3H), 1.94-1.97 (m, 4H), 2.10-2.22 (m, 7H), 2.25-2.26 (d, 1H), 2.38-2.46 (m, 4H), 2.56-2.67 (m, 2H), 2.78-2.81 (m, 2H), 2.86-2.91 (m, 2H), 2.96-3.04 (m, 5H), 3.10-3.24 (m, 4H), 3.32-3.35 (m, 17H), 3.91-3.93 (m, 3H), 3.97-4.00 (m, 2H), 4.13-4.65 (m, 8H), 4.80-4.86 (m, 2H), 4.94 (s, 2H), 5.44 (s, 2H), 6.00 (t, 1H), 7.22-7.32 (m, 4H), 7.54-7.56 (d, 2H), 7.78-7.81 (d, 1H), 7.85-8.00 (m, 2H), 8.09-8.23 (m, 2H), 8.47 (brs, 1H), 10.04 (s, 1H), 12.12 (brs, 1H)
(434) LC-MS m/z: 1610.8 [M+H].sup.+, 1632.8 [M+Na].sup.+
PREPARATION EXAMPLE 4-33
Preparation of Compound (VI-37)
(435) ##STR00137##
(436) With the exception that compound (IV-31) (0.2 g, 0.138 mmol), obtained in Preparation Example 3-27, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (0.17 g, 77%).
(437) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.00-0.57 (m, 6H), 0.69-0.76 (m, 8H), 0.82-0.87 (m, 14H), 0.95-1.08 (m, 2H), 1.09-1.18 (m, 2H), 1.20-1.24 (m, 2H), 1.43-1.49 (m, 6H), 1.54-1.64 (m, 1H), 1.81-2.05 (m, 4H), 2.20-2.23 (m, 6H), 2.27-2.38 (m, 9H), 2.56-2.69 (m, 1H), 2.70-2.81 (m, 2H), 2.82-2.86 (m, 7H), 2.96-3.00 (m, 5H), 3.15-3.20 (m, 5H), 3.27 (s, 3H), 3.32-3.34 (m, 15H), 3.89-4.00 (m, 3H), 4.14-4.25 (m, 2H), 4.26-4.82 (m, 8H), 4.98 (s, 2H), 5.43 (s, 2H), 6.00-6.03 (t, 1H), 7.06-7.16 (m, 2H), 7.28-7.30 (m, 2H), 7.59-7.69 (m, 3H), 7.84-7.86 (d, 1H), 7.97-8.05 (m, 1H), 8.14-8.15 (m, 1H), 8.23-8.28 (m, 1H), 8.49 (brs, 1H), 10.02 (s, 1H)
(438) LC-MS m/z: 1619.6 [M+H].sup.+, 1641.6 [M+Na].sup.+
PREPARATION EXAMPLE 4-34
Preparation of Compound (VI-38)
(439) ##STR00138##
(440) With the exception that compound (IV-32) (0.2 g, 0.137 mmol), obtained in Preparation Example 3-28, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (0.17 g, 76%).
(441) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.50-0.52 (m, 4H), 0.69-0.76 (m, 8H), 0.82-0.92 (m, 14H), 1.03-1.05 (m, 2H), 1.11-1.12 (m, 1H), 1.18-1.24 (m, 3H), 1.42-1.49 (m, 6H), 1.52-1.64 (m, 1H), 1.66-1.83 (m, 3H), 1.86-2.02 (m, 4H), 2.10-2.20 (m, 6H), 2.28-2.38 (m, 8H), 2.45-2.49 (m, 1H), 2.56-2.68 (m, 1H), 2.78-2.86 (m, 3H), 2.95-3.10 (m, 8H), 3.15-3.17 (m, 4H), 3.28 (s, 3H), 3.34-3.35 (m, 10H), 3.95-4.00 (m, 3H), 4.02-4.88 (m, 8H), 4.94 (s, 2H), 5.43 (s, 2H), 6.03 (t, 1H), 7.06-7.15 (m, 2H), 7.22-7.29 (m, 4H), 7.54-7.69 (m, 3H), 7.89 (brs, 1H), 7.87-7.99 (d, 1H), 8.03-8.05 (d, 1H), 8.19 (brs, 1H), 8.30 (m, 1H), 8.50 (m, 1H), 10.04 (s, 1H)
(442) LC-MS m/z: 1591.7 [M+H].sup.+, 1613.7 [M+Na].sup.+
PREPARATION EXAMPLE 4-35
Preparation of Compound (VI-39)
(443) ##STR00139##
(444) With the exception that compound (IV-33) (0.15 g, 0.099 mmol), obtained in Preparation Example 3-29, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (0.11 g, 70%).
(445) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.41-0.54 (m, 6H), 0.58-0.65 (m, 2H), 0.68-0.76 (m, 3H), 0.82-0.95 (m, 14H), 1.09-1.23 (m, 8H), 1.32-1.49 (m, 5H), 1.59-1.61 (m, 1H), 1.68-1.71 (m, 3H), 1.87-2.04 (m, 3H), 2.09-2.23 (m, 10H), 2.32-2.39 (m, 3H), 2.62-2.68 (m, ′H), 2.74-3.25 (m, 15H), 3.33-3.50 (m, 14H), 3.90-3.97 (m, 3H), 3.99-4.14 (m, 2H), 4.16-4.89 (m, 8H), 4.90 (s, 2H), 5.45 (s, 2H), 6.10 (t, 1H), 7.22-7.28 (m, 3H), 7.38-7.41 (d, 2H), 7.55-7.85 (d, 2H), 7.86-8.01 (m, 2H), 8.03-8.05 (m, 1H), 8.19-8.20 (brs, 1H), 8.29 (brs, 1H), 8.59 (brs, 1H), 8.84 (s, 1H), 10.08 (s, 1H)
(446) LC-MS m/z: 1642.8 [M+H].sup.+, 1664.8 [M+Na].sup.+
PREPARATION EXAMPLE 4-36
Preparation of Compound (VI-40)
(447) ##STR00140##
(448) With the exception that compound (IV-34) (1.3 g, 0.895 mmol), obtained in Preparation Example 3-30, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (1.1 g, 74%).
(449) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.40-0.50 (m, 7H), 0.70-0.77 (m, 8H), 0.82-0.89 (m, 18H), 1.02-0.07 (m, 3H), 1.18-1.29 (m, 3H), 1.44-1.47 (m, 6H), 1.54-1.76 (m, 4H), 1.89-1.94 (m, 5H), 2.10-2.23 (m, 12H), 2.32-2.44 (m, 3H), 2.61-2.64 (m, 2H), 2.74-3.00 (m, 10H), 3.13-3.17 (m, 3H), 3.20-3.33 (m, 10H), 3.90-4.00 (m, 4H), 4.16-4.84 (m, 8H), 4.95 (s, 2H), 5.41 (s, 2H), 6.01-6.02 (t, 1H), 7.01-7.05 (m, 2H), 7.28-7.29 (m, 4H), 7.59-7.60 (d, 2H), 7.84-7.99 (m, 2H), 8.13-8.16 (m, 1H), 8.23 (brs, 1H), 10.04 (s, 1H)
(450) LC-MS m/z: 1598.9 [M+H].sup.+, 1621.9 [M+Na].sup.+
PREPARATION EXAMPLE 4-37
Preparation of Compound (VI-41)
(451) ##STR00141##
(452) With the exception that compound (IV-35) (0.14 g, 0.095 mmol), obtained in Preparation Example 3-31, was used instead of compound (IV-28), obtained in Preparation Example 3-24, the same procedure as in Preparation Example 4-30 was repeated to afford the title compound (0.072 g, 47%).
(453) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.43-0.50 (m, 6H), 0.66-0.76 (m, 8H), 0.82-0.92 (m, 16H), 1.09-1.23 (m, 6H), 1.44-1.48 (m, 6H), 1.65-1.81 (m, 3H), 1.84-1.97 (m, 4H), 2.01-2.30 (m, 10H), 2.34-2.38 (m, 3H), 2.56-2.64 (m, 2H), 2.80-2.88 (m, 5H), 2.91-3.04 (m, 5H), 3.14-3.17 (m, 6H), 3.27-3.39 (m, 17H), 3.92-3.98 (m, 3H), 4.15-4.86 (m, 8H), 4.98 (s, 2H), 5.42 (s, 2H), 6.02-6.03 (t, 1H), 7.21-7.36 (m, 4H), 7.55-7.63 (d, 2H), 7.77-8.01 (m, 4H), 8.03-8.05 (d, 1H), 8.13 (brs, 1H), 8.45 (brs, 1H), 10.01 (s, 1H)
(454) LC-MS m/z: 1624.9 [M+H].sup.+, 1646.9 [M+Na].sup.+
PREPARATION EXAMPLE 5
Preparation of Compounds of Chemical Formulas VII-1, VII-2, VII-3 and VII-4
PREPARATION EXAMPLE 5-1
Preparation of Compound (VII-5)
(455) ##STR00142##
(456) Under an argon stream, compound (VI-5) (290 mg, 0.20 mmol), obtained in Preparation Example 4-1, was dissolved in 10 mL of anhydrous dimethyl formamide, and added with N-hydroxysuccinimide (35 mg, 0.30 mmol). The reaction mixture was cooled to 0° C., and added with EDC.HCl (58 mg, 0.30 mmol) before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out. The residue was dissolved in 50 mL of ethylacetate, acidified with 5 mL of 1 M HCl, and washed with 30 mL of distilled water and 30 mL of saturated brine, sequentially. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford the title compound (226 mg, 73%).
(457) MALDI-TOF MS m/z: 1562.9 [M+Na].sup.+
PREPARATION EXAMPLE 5-2
Preparation of Compound (VII-6)
(458) ##STR00143##
(459) With the exception that compound (VI-6) (60 mg, 0.041 mmol), obtained in Preparation Example 4-2, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (53 mg, 83%).
(460) MALDI-TOF MS m/z: 1586.7 [M+Na].sup.+
PREPARATION EXAMPLE 5-3
Preparation of Compound (VII-7)
(461) ##STR00144##
(462) With the exception that compound (VI-7) (54 mg, 0.037 mmol), obtained in Preparation Example 4-3, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (57 mg, 98%).
(463) MALDI-TOF MS m/z: 1607.4 [M+K].sup.+
PREPARATION EXAMPLE 5-4
Preparation of Compound (VII-8)
(464) ##STR00145##
(465) With the exception that compound (VI-8) (66 mg, 0.044 mmol), obtained in Preparation Example 4-4, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (53 mg, 84%).
(466) MALDI-TOF MS m/z: 1617.0 [M+Na].sup.+
PREPARATION EXAMPLE 5-5
Preparation of Compound (VII-9)
(467) ##STR00146##
(468) With the exception that compound (VI-9) (71 mg, 0.046 mmol), obtained in Preparation Example 4-5, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (66 mg, 88%).
(469) MALDI-TOF MS m/z: 1660.9 [M+Na].sup.+, 1677.1 [M+K].sup.+
PREPARATION EXAMPLE 5-6
Preparation of Compound (VII-10)
(470) ##STR00147##
(471) With the exception that compound (VI-10) (53 mg, 0.037 mmol), obtained in Preparation Example 4-6, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (38 mg, 67%).
(472) MALDI-TOF MS m/z: 1565.5 [M+Na].sup.+
PREPARATION EXAMPLE 5-7
Preparation of Compound (VII-11)
(473) ##STR00148##
(474) With the exception that compound (VI-11) (46 mg, 0.032 mmol), obtained in Preparation Example 4-7, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (30 mg, 61%).
(475) MALDI-TOF MS m/z: 1568.9 [M+Na].sup.+, 1585.0 [M+K].sup.+
PREPARATION EXAMPLE 5-8
Preparation of Compound (VII-12)
(476) ##STR00149##
(477) With the exception that compound (VI-12) (38 mg, 0.025 mmol), obtained in Preparation Example 4-8, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (31 mg, 78%).
(478) MALDI-TOF MS m/z: 1643.7 [M+Na].sup.+, 1660.2 [M+K].sup.+
PREPARATION EXAMPLE 5-9
Preparation of Compound (VII-13)
(479) ##STR00150##
(480) With the exception that compound (VI-13) (34 mg, 0.023 mmol), obtained in Preparation Example 4-9, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (27 mg, 75%).
(481) MALDI-TOF MS m/z: 1627.7 [M+Na].sup.+, 1644.8 [M+K].sup.+
PREPARATION EXAMPLE 5-10
Preparation of Compound (VII-14)
(482) ##STR00151##
(483) With the exception that compound (VI-14) (29 mg, 0.019 mmol), obtained in Preparation Example 4-10, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (8 mg, 27%).
(484) MALDI-TOF MS m/z: 1625.4 [M+Na].sup.+, 1642.0 [M+K].sup.+
PREPARATION EXAMPLE 5-11
Preparation of Compound (VII-15)
(485) ##STR00152##
(486) With the exception that compound (VI-15) (77 mg, 0.052 mmol), obtained in Preparation Example 4-11, was used instead of compound (VI-5), obtained in Preparation Example 4-1, the same procedure as in Preparation Example 5-1 was repeated to afford the title compound as an ivory solid (34 mg, 41%).
(487) MALDI-TOF MS m/z: 1603.8 [M+Na].sup.+, 1621.2 [M+K].sup.+
PREPARATION EXAMPLE 5-12
Preparation of Compound (VII-16)
(488) ##STR00153##
(489) Under an argon stream, compound (VI-16)(60 mg, 0.039 mmol), obtained in Preparation Example 4-12, was dissolved in 3 mL of anhydrous dimethyl formamide, and added with N-hydroxysuccinimide (18.1 mg, 0.157 mmol). The reaction mixture was cooled to 0° C., and added with EDC.HCl (30.2 mg, 0.157 mmol) before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out. The residue was dissolved in 50 mL of ethylacetate, acidified with 5 mL of 1 M HCl, and washed with 30 mL of distilled water and 30 mL of saturated brine, sequentially. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford the title compound (37 mg, 57%).
(490) LC-MS m/z: 1620.8 [M+].sup.+, 1643.7 [M+Na].sup.+
(491) .sup.1H NMR (400 MHz, CDCl3) δ 0.48 (m, 2H), 0.64 (m, 2H), 0.71 (m, 6H), 0.78-0.88 (m, 17H), 0.92-0.97 (m, 4H), 1.02-1.03 (d, 2H), 1.16 (m, 3H), 1.40-1.45 (m, 5H), 1.50-1.78 (m, 4H), 1.82-2.01 (m, 5H), 2.16 (S, 6H), 2.06-2.28 (m, 3H), 2.39 (m, 1H), 2.47 (S, 3H), 2.44-2.50 (m, 1H), 2.60 (m, 1H), 2.70 (m, 1H), 2.77 (S, 3H), 2.82-2.94 (m, 4H), 2.96-3.00 (m, 3H), 3.10-3.17 (m, 7H), 3.18-3.52 (m, 13H), 3.54-3.70 (m, 2H), 3.76 (m, 1H), 3.84-3.99 (m, 3H), 4.00-4.16 (m, 2H), 4.33 (m, 1H), 4.49 (t, 1H), 4.54-4.68 (m, 2H), 4.92 (m, 2H), 5.38 (s, 2H), 5.95 (m, 1H), 7.00-7.06 (m, 2H), 7.22-7.27 (m, 3H), 7.50-7.55 (m, 2H), 7.70-7.78 (m, 1H), 7.98-8.06 (m, 1H), 9.95 (S, 1H)
PREPARATION EXAMPLE 5-13
Preparation of Compound (VII-17)
(492) ##STR00154##
(493) With the exception that compound (VI-17) (39 mg, 0.026 mmol), obtained in Preparation Example 4-13, was used instead of compound (VI-16), obtained in Preparation Example 4-12, the same procedure as in Preparation Example 5-12 was repeated to afford the title compound (32 mg, 78%).
(494) MALDI-TOF MS m/z: 1595.8 [M+Na].sup.+
PREPARATION EXAMPLE 5-14
Preparation of Compound (VII-18)
(495) ##STR00155##
(496) With the exception that compound (VI-18) (266 mg, 0.170 mmol), obtained in Preparation Example 4-14, was used instead of compound (VI-16), obtained in Preparation Example 4-12, the same procedure as in Preparation Example 5-12 was repeated to afford the title compound (37 mg, 57%).
(497) LC-MS m/z: 1590 [M+].sup.+
PREPARATION EXAMPLE 5-15
Preparation of Compound (VII-19)
(498) ##STR00156##
(499) With the exception that compound (VI-19) (247 mg, 0.16 mmol), obtained in Preparation Example 4-15, was used instead of compound (VI-16), obtained in Preparation Example 4-12, the same procedure as in Preparation Example 5-12 was repeated to afford the title compound (197 mg, 77%).
(500) LC-MS m/z: 1607 [M].sup.+
(501) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.54 (m, 1H), 0.65 (m, 3H), 0.70-0.78 (m, 5H), 0.80-1.00 (m, 18H), 1.00-1.08 (m, 6H), 1.20 (m, 3H), 1.47 (m, 5H), 1.60 (m, 2H), 1.94 (m, 5H), 2.08-2.20 (m, 3H), 2.25 (d, 6H), 2.40 (m, 1H), 2.60 (s, 6H), 2.73-3.02 (m, 13H), 2.84-3.42 (m, 17H, 2*OCH3), 3.60 (m, 1H), 3.70 (m, 1H), 3.80-4.06 (m, 3H), 4.20 (t, 2H), 4.38 (m, 2H), 4.53 (t, 1H), 4.66 (m, 2H), 4.78 (m, 1H), 4.88-5.10 (m, 3H), 5.42 (s, 2H), 6.00 (m, 1H), 7.07 (m, 1H), 7.27-7.37 (m, 3H), 7.50-7.58 (m, 2H), 7.65-7.85 (m, 1H), 8.10 (m, 1H), 9.95 (s, 1H)
PREPARATION EXAMPLE 5-16
Preparation of Compound (VII-20)
(502) ##STR00157##
(503) With the exception that compound (VI-20) (38 mg, 0.025 mmol), obtained in Preparation Example 4-16, was used instead of compound (VI-16), obtained in Preparation Example 4-12, the same procedure as in Preparation Example 5-12 was repeated to afford the title compound (25 mg, 62%).
(504) LC-MS m/z: 1603 [M].sup.+
(505) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.48 (m, 1H), 0.62 (m, 2H), 0.65-0.78 (m, 6H), 0.71-1.02 (m, 25H), 1.16 (m, 2H), 1.30-1.52 (m, 5H), 1.53-1.82 (m, 5H), 1.83-2.12 (m, 5H), 2.14 (d, 6H), 2.54 (s, 3H), 2.58-3.02 (m, 14H), 3.10-3.16 (m, 6H), 3.18-3.20 (m, 7H), 3.28-3.70 (m, 11H), 3.72-3.86 (m, 1H), 3.90-4.04 (m, 1H), 4.03-4.29 (m, 2H), 4.32 (m, 1H), 4.42-4.78 (m, 3H), 4.82-4.98 (m, 2H), 5.06 (m, 1H), 5.39 (s, 2H), 5.96 (m, 1H), 6.78 (m, 1H), 7.03-7.26 (m, 4H), 7.58 (m, 2H), 7.79 (d, 1H), 8.00 (d, 1H), 8.07 (d, 1H), 9.95 (S, 1H)
PREPARATION EXAMPLE 5-17
Preparation of Compound (VII-21)
(506) ##STR00158##
(507) With the exception that compound (VI-21) (132 mg, 0.09 mmol), obtained in Preparation Example 4-17, was used instead of compound (VI-16), obtained in Preparation Example 4-12, the same procedure as in Preparation Example 5-12 was repeated to afford the title compound (136 mg, 93%).
(508) LC-MS m/z: 1619 [M].sup.+
(509) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.54 (m, 1H), 0.63-0.68 (m, 3H), 0.70-0.80 (m, 5H), 0.81-0.90 (m, 17H), 1.00-1.1.08 (m, 8H), 1.22 (m, 3H), 1.40-1.54 (m, 6H), 1.56-1.74 (m, 3H), 1.80-2.00 (m, 3H), 2.26-2.30 (m, 7H), 2.51 (m, 6H), 2.56 (s, 1H), 2.64-3.04 (m, 14H), 3.13-3.30 (m, 6H), 3.36 (m, 6H), 3.62 (s, 1H), 3.69 (s, 2H), 3.87-4.04 (m, 4H), 4.19 (t, 2H0, 4.37 (m, 2H), 4.52 (t, 1H), 4.62 (m, 1H), 4.68 (m, 1H), 4.76 (m, 1H), 4.89 (m, 1H), 4.979 m, 2H), 5.08 (d, 1H), 5.42 (s, 2H), 5.98 (m, 1H), 6.81 (d, 1H), 7.17 (d, 1H), 7.30 (m, 1H), 7.60 (m, 3H), 7.82 (d, 1 h), 8.10 (m, 1H), 10.00 (d, 1H),
PREPARATION EXAMPLE 5-18
Preparation of Compound (VII-22)
(510) ##STR00159##
(511) With the exception that compound (VI-22) (296 mg, 0.198 mmol), obtained in Preparation Example 4-18, was used instead of compound (VI-16), obtained in Preparation Example 4-12, the same procedure as in Preparation Example 5-12 was repeated to afford the title compound (132 mg, 83%).
(512) LC-MS m/z: 1589 [M].sup.+
(513) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.48 (m, 1H), 0.60 (m, 2H), 0.71 (m, 5H), 0.71-0.88 (m, 17H), 0.98 (d, 3H), 0.93-1.08 (m, 3H), 1.16 (m, 3H), 1.37-1.45 (m, 5H), 1.50-1.86 (m, 4H), 1.91 (m, 3H), 2.0-2.42 (m, 11H), 2.68 (m, 1H), 2.77 (S, 3H), 2.82-3.02 (m, 5H), 3.10-3.19 (m, 6H), 3.21-3.30 (m, 7H), 3.36-3.50 (m, 16H), 3.54-3.72 (m, 3H), 3.80-4.00 (m, 4H), 4.15 (t, 1H), 4.33 (m, 1H), 4.49-4.71 (m, 3H), 4.86 (m, 2H), 5.04 (m, 1H), 5.39 (s, 2H), 5.96 (m, 1H), 7.03-7.21 (m, 5H), 7.55 (m, 2H), 7.79 (d, 1H), 8.06 (d, 1H) 9.95 (S, 1H)
PREPARATION EXAMPLE 5-19
Preparation of Compound (VII-23)
(514) ##STR00160##
(515) Under an argon stream, compound (VI-23) (33.4 mg, 0.022 mmol), obtained in Preparation Example 4-19, was dissolved in 3 mL of anhydrous dimethyl formamide, and added with N-hydroxysuccinimide (7.7 mg, 0.066 mmol). The reaction mixture was cooled to 0° C., and added with EDC.HCl (12.8 mg, 0.066 mmol) before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out. The residue was dissolved in 50 mL of ethylacetate, acidified with 5 mL of 1 M HCl, and washed with 30 mL of distilled water and 30 mL of saturated brine, sequentially. The organic layer was dried over anhydrous sodium sulfate, and concentrated in a vacuum. The concentrate was purified by silica gel column chromatography to afford the title compound (32 mg, 91%).
(516) LC-MS m/z: 1596 [M.sup.+].sup.+, 1619 [M+Na].sup.+
PREPARATION EXAMPLE 5-20
Preparation of Compound (VII-24)
(517) ##STR00161##
(518) With the exception that compound (VI-24) (55 mg, 0.037 mmol), obtained in Preparation Example 4-20, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (49 mg, 84%).
(519) LC-MS m/z: 1606 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-21
Preparation of Compound (VII-25)
(520) ##STR00162##
(521) With the exception that compound (VI-25) (65 mg, 0.043 mmol), obtained in Preparation Example 4-21, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (56 mg, 82%).
(522) LC-MS m/z: 1592 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-22
Preparation of Compound (VII-26)
(523) ##STR00163##
(524) With the exception that compound (VI-26) (114 mg, 0.075 mmol), obtained in Preparation Example 4-22, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (94 mg, 78%).
(525) LC-MS m/z: 1609 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-23
Preparation of Compound (VII-27)
(526) ##STR00164##
(527) With the exception that compound (VI-27) (91 mg, 0.0597 mmol), obtained in Preparation Example 4-23, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (25 mg, 25%).
(528) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.51-0.53 (m, 1H), 0.62-0.71 (m, 7H), 0.78-0.87 (m, 12H), 1.05-1.17 (m, 4H), 1.38-1.43 (m, 4H), 1.84-1.98 (m, 3H), 2.10-2.14 (m, 5H), 2.79-3.04 (m, 10H), 3.09-3.13 (m, 3H), 3.25-3.36 (m, 10H), 3.38-3.49 (m, 19H), 3.87-3.89 (m, 4H), 3.98-4.00 (m, 2H), 4.12-4.99 (m, 8H), 5.05 (s, 2H), 5.39 (s, 2H), 6.00-6.03 (t, 1H), 7.23-7.31 (m, 3H), 7.56-7.58 (d, 2H), 7.72-7.80 (m, 3H), 8.06-8.08 (d, 2H), 8.16-8.41 (m, 1H), 10.01 (s, 1H)
(529) LC-MS m/z: 1621.5 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-24
Preparation of Compound (VII-28)
(530) ##STR00165##
(531) With the exception that compound (VI-28) (60 mg, 0.0399 mmol), obtained in Preparation Example 4-24, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (14 mg, 23%).
(532) .sup.1H NMR (400 MHz, DMSO-d6) δ 0.51-0.53 (m, 1H), 0.62-0.71 (m, 7H), 0.78-0.87 (m, 12H), 1.05-1.17 (m, 4H), 1.38-1.43 (m, 4H), 1.84-1.98 (m, 3H), 2.14-2.15 (m, 7H), 2.24-2.33 (m, 6H), 2.79-3.04 (m, 10H), 3.09-3.13 (m, 3H), 3.25-3.36 (m, 10H), 3.38-3.49 (m, 19H), 3.87-3.89 (m, 4H), 3.98-4.00 (m, 2H), 4.12-4.99 (m, 8H), 5.05 (s, 2H), 5.39 (s, 2H), 5.97 (t, 1H), 7.02-7.11 (m, 2H), 7.28-7.29 (m, 1H), 7.57-7.65 (d, 2H), 7.88-7.98 (m, 1H), 8.07-8.08 (m, 1H), 8.22-8.44 (m, 1H), 10.00 (s, 1H)
(533) LC-MS m/z: 1601.7 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-25
Preparation of Compound (VII-29)
(534) ##STR00166##
(535) With the exception that compound (VI-29) (83 mg, 0.053 mmol), obtained in Preparation Example 4-25, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (23 mg, 25%).
(536) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 2H), 0.62-0.71 (m, 5H), 0.78-0.87 (m, 9H), 1.05-1.17 (m, 4H), 1.38-1.43 (m, 4H), 1.84-1.98 (m, 3H), 2.14-2.15 (m, 4H), 2.79-3.04 (m, 10H), 3.09-3.13 (m, 3H), 3.25-3.36 (m, 10H), 3.38-3.49 (m, 19H), 3.87-3.89 (m, 4H), 3.98-4.00 (m, 2H), 4.12-4.99 (m, 8H), 5.05 (s, 2H), 5.39 (s, 2H), 5.97 (t, 1H), 7.18-7.24 (m, 1H), 7.29-7.37 (m, 2H), 7.57-7.58 (m, 1H), 7.78-8.07 (m, 5H), 8.22-8.46 (m, 1H), 8.58 (s, 1H), 10.02 (s, 1H)
(537) LC-MS m/z: 1653.4 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-26
Preparation of Compound (VII-30)
(538) ##STR00167##
(539) With the exception that compound (VI-30) (0.48 g, 0.31 mmol), obtained in Preparation Example 4-26, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (0.44 g, 86%).
(540) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.44-0.54 (m, 1 H), 0.55-0.75 (m, 10 H), 0.75-0.96 (m, 17 H), 0.96-1.04 (m, 1 H), 1.04-1.25 (m, 6 H), 1.27-1.51 (m, 6 H), 1.51-1.82 (m, 3 H), 1.82-2.02 (m, 3 H), 2.02-2.21 (m, 6 H), 2.21-2.36 (m, 3 H), 2.52-2.64 (m, 1 H), 2.65-2.75 (m, 4 H), 2.75-2.80 (m, 5 H), 2.80-2.84 (m, 2 H), 2.84-2.93 (m, 4 H), 2.93-3.06 (m, 4 H), 3.06-3.22 (m, 4 H), 3.25-3.28 (m, 3 H), 3.38-3.46 (m, 2 H), 3.48-3.64 (m, 1 H), 3.71-3.86 (m, 7 H), 3.86-4.04 (m, 4 H), 4.04-4.21 (m, 2 H), 4.21-4.31 (m, 1 H), 4.31-4.44 (m, 2 H), 4.44-4.56 (m, 1 H), 4.56-4.71 (m, 2 H), 4.71-4.87 (m, 1 H), 4.93 (s, 2 H), 5.38 (s, 2 H), 5.87-6.01 (t, 1 H), 6.95-7.09 (d, 1 H), 7.18-7.33 (m, 2 H), 7.37-7.54 (m, 1 H), 7.54-7.68 (m, 3 H), 7.71-7.85 (d, 1 H), 8.02-8.12 (d, 1 H), 9.96 (brs, 1 H)
(541) LC-MS m/z: 1633 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-27
Preparation of Compound (VII-31)
(542) ##STR00168##
(543) With the exception that compound (VI-31) (0.18 g, 0.12 mmol), obtained in Preparation Example 4-27, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (0.13 g, 68%).
(544) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.38-0.59 (m, 8 H), 0.60-0.79 (m, 8 H), 0.79-0.98 (m, 16 H), 1.05-1.36 (m, 7 H), 1.36-1.52 (m, 6 H), 1.54-1.86 (m, 5 H), 1.86-2.02 (m, 3 H), 2.04-2.28 (m, 10 H), 2.28-2.44 (m, 3 H), 2.59-2.68 (m, 1 H), 2.69-2.86 (m, 6 H), 2.86-3.09 (m, 8 H), 3.10-3.25 (m, 6 H), 3.25-3.43 (m, 3 H), 3.71-3.86 (m, 1 H), 3.88-4.07 (m, 2 H), 4.07-4.26 (m, 2 H), 4.31-4.43 (m, 1 H), 4.48-4.74 (m, 2 H), 4.74-4.90 (m, 1 H), 4.97 (s, 2 H), 5.41 (m, 2 H), 5.86-6.13 (t, 1H), 7.16-7.43 (m, 2 H), 7.43-7.55 (m, 1 H), 7.56-7.75 (m, 2 H), 7.75-7.92 (m, 1 H), 7.92-8.21 (m, 4 H), 8.21-8.44 (m, 1 H), 8.44-8.66 (m, 1 H), 8.72 (s, 1 H), 9.38-9.82 (brs, 1 H), 10.01 (brs, 1 H)
(545) LC-MS m/z: 1622 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-28
Preparation of Compound (VII-32)
(546) ##STR00169##
(547) With the exception that compound (VI-32) (0.21 g, 0.14 mmol), obtained in Preparation Example 4-28, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (0.19 g, 88%).
(548) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.43-0.59 (m, 2 H), 0.59-0.78 (m, 8 H), 0.79-1.00 (m, 16 H), 1.00-1.08 (m, 3 H), 1.08-1.29 (m, 5 H), 1.29-1.64 (m, 9 H), 1.64-1.73 (m, 3 H), 1.74-2.04 (m, 4 H), 2.04-2.44 (m, 10 H), 2.56-2.64 (m, 2 H), 2.65-2.92 (m, 12 H), 2.92-3.08 (m, 6 H), 3.08-3.23 (m, 5 H), 3.23-3.32 (m, 3 H), 3.42-3.54 (m, 1 H), 3.55-3.64 (m, 1 H), 3.68-3.88 (m, 3 H), 3.88-4.06 (m, 2 H), 4.06-4.26 (m, 2 H), 4.26-4.46 (m, 2 H), 4.46-4.71 (m, 2 H), 4.74-4.88 (m, 1 H), 4.96 (s, 2 H), 5.41 (s, 2 H), 5.90-6.08 (t, 1 H), 6.74-7.04 (m, 1 H), 7.10-7.38 (m, 3 H), 7.38-7.68 (m, 4 H), 7.72-7.88 (d, 1 H), 8.01-8.18 (d, 1 H), 8.18-8.35 (m, 1 H), 10.00 (brs, 1 H)
(549) LC-MS m/z: 1602 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-29
Preparation of Compound (VII-33)
(550) ##STR00170##
(551) With the exception that compound (VI-33) (0.26 g, 0.17 mmol), obtained in Preparation Example 4-29, was used instead of compound (VI-23), obtained in Preparation Example 4-19, the same procedure as in Preparation Example 5-19 was repeated to afford the title compound as an ivory solid (0.14 g, 52%).
(552) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.53-0.94 (m, 28 H), 0.95-1.28 (m, 9 H), 1.28-1.74 (m, 18 H), 1.76-2.25 (m, 13 H), 2.27-2.43 (m, 1 H), 2.54-2.84 (m, 16 H), 2.84-3.03 (m, 5 H), 3.03-3.21 (m, 7 H), 3.60-3.82 (m, 2 H), 3.82-4.00 (m, 3 H), 4.05-4.24 (m, 1 H), 4.29-4.45 (m, 1 H), 4.45-4.78 (m, 2 H), 5.01 (s, 2 H), 5.39 (s, 2 H), 5.90-6.09 (t, 1 H), 6.97 (brs, 2 H), 7.09-7.43 (m, 2 H), 7.43-7.70 (m, 2 H), 7.70-7.87 (m, 1 H), 8.00-7.33 (m, 2 H), 10.01 (brs, 1 H)
(553) LC-MS m/z: 1610 [M.sup.+].sup.+
PREPARATION EXAMPLE 5-30
Preparation of Compound (VII-34)
(554) ##STR00171##
(555) Under an argon stream, compound (VI-34) (25 mg, 0.016 mmol), obtained in Preparation Example 4-30, was dissolved in 3 mL of anhydrous dimethyl formamide, and added with N-hydroxysuccinimide (7.2 mg, 0.062 mmol). The reaction mixture was cooled to 0° C., and added with EDC.HCl (11.9 mg, 0.062 mmol) before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out. The residue was purified by silica gel column chromatography to afford the title compound (20 mg, 76%).
(556) LC-MS m/z: 1706.9 [M+H].sup.+, 1728.9 [M+Na].sup.+
PREPARATION EXAMPLE 5-31
Preparation of Compound (VII-35)
(557) ##STR00172##
(558) With the exception that compound (VI-35) (0.074 g, 0.0452 mmol), obtained in Preparation Example 4-31, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.049 g, 62.4%).
(559) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 1H), 0.63-0.76 (m, 12H), 0.81-0.86 (m, 18H), 1.02-1.04 (d, 4H), 1.11-1.14 (m, 4H), 1.18-1.22 (m, 2H), 1.42-1.49 (m, 6H), 1.94-1.97 (m, 3H), 2.18-2.19 (m, 6H), 2.73-2.89 (m, 13H), 2.93-2.99 (m, 5H), 3.14-3.21 (m, 5H), 3.31-3.39 (m, 17H), 3.91-4.03 (m, 5H), 4.14-4.85 (m, 8H), 4.98 (s, 2H), 5.42 (s, 2H), 5.96-5.98 (t, 1H), 7.27-7.30 (m, 3H), 7.58-7.59 (d, 2H), 7.77-7.87 (m, 2H), 7.94-8.03 (m, 2H), 8.08-8.11 (d, 1H), 8.46 (brs, 1H), 10.01 (s, 1H)
(560) LC-MS m/z: 1735.4 [M+H].sup.+, 1758.4 [M+Na].sup.+
PREPARATION EXAMPLE 5-32
Preparation of Compound (VII-36)
(561) ##STR00173##
(562) With the exception that compound (VI-36) (0.11 g, 0.0664 mmol), obtained in Preparation Example 4-32, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.079 g, 69.6%).
(563) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 1H), 0.63-0.76 (m, 7H), 0.82-0.92 (m, 12H), 1.02-1.04 (d, 1H), 1.09-1.24 (m, 7H), 1.42-1.49 (m, 8H), 1.91-1.99 (m, 4H), 2.11-2.19 (m, 8H), 2.81-2.83 (m, 10H), 2.99-3.14 (m, 6H), 3.17-0.21 (m, 5H), 3.32-3.35 (m, 20H), 3.92-3.93 (m, 4H), 3.97-4.04 (m, 2H), 4.13-4.85 (m, 8H), 4.94 (s, 2H), 5.43 (s, 2H), 6.00-6.03 (t, 1H), 7.27-7.32 (m, 5H), 7.58-7.61 (d, 2H), 7.77-7.96 (m, 3H), 8.10-8.12 (m, 1H), 8.47 (brs, 1H), 10.02 (s, 1H)
(564) LC-MS m/z: 1708.4 [M+H].sup.+, 1730.4 [M+Na].sup.+
PREPARATION EXAMPLE 5-33
Preparation of Compound (VII-37)
(565) ##STR00174##
(566) With the exception that compound (VI-37) (0.16 g, 0.096 mmol), obtained in Preparation Example 4-33, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.12 g, 71%).
(567) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 1H), 0.70-0.75 (m, 8H), 0.82-0.87 (m, 14H), 1.03-1.05 (m, 4H), 1.08-1.14 (m, 1H), 1.18-1.23 (m, 4H), 1.44-1.49 (m, 7H), 2.08-2.20 (m, 10H), 2.28-2.37 (m, 8H), 2.77-2.89 (m, 13H), 2.93-3.00 (m, 6H), 3.14-3.20 (m, 5H), 3.28 (s, 3H), 3.32-3.34 (m, 18H), 3.88-4.04 (m, 4H), 4.08-4.87 (m, 8H), 4.97 (s, 2H), 5.42 (s, 2H), 5.99-6.00 (t, 1H), 7.05-7.15 (m, 2H), 7.28-7.30 (m, 2H), 7.59-7.69 (m, 2H), 7.80-7.83 (d, 1H), 7.97-8.05 (m, 1H), 8.10-8.11 (m, 1H), 8.23-8.28 (m, 1H), 8.49 (brs, 1H), 10.02 (s, 1H)
(568) LC-MS m/z: 1716.5 [M+H].sup.+, 1740.5 [M+Na].sup.+
PREPARATION EXAMPLE 5-34
Preparation of Compound (VII-38)
(569) ##STR00175##
(570) With the exception that compound (VI-38) (0.15 g, 0.093 mmol), obtained in Preparation Example 4-34, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.1 g, 67%).
(571) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 1H), 0.63-0.66 (m, 3H), 0.69-0.76 (m, 8H), 0.82-0.92 (m, 14H), 1.03-1.05 (m, 2H), 1.11-1.12 (m, 1H), 1.18-1.24 (m, 3H), 1.44-1.49 (m, 7H), 2.13-2.20 (m, 9H), 2.28-2.37 (m, 7H), 2.73-2.89 (m, 7H), 2.93-3.03 (m, 9H), 3.15-3.19 (m, 5H), 3.28 (s, 3H), 3.31-3.35 (m, 15H), 3.85-3.87 (m, 1H), 3.95-4.04 (m, 3H), 4.16-4.88 (m, 8H), 4.94 (s, 2H), 5.42 (s, 2H), 5.99-6.02 (t, 1H), 7.06-7.15 (m, 2H), 7.27-7.29 (m, 4H), 7.58-7.63 (m, 3H), 7.81-7.83 (d, 1H), 7.87-8.04 (m, 1H), 8.10-8.11 (d, 1H), 8.30-8.50 (m, 1H), 10.02 (s, 1H)
(572) LC-MS m/z: 1687.6 [M+H].sup.+, 1709.5 [M+Na].sup.+
PREPARATION EXAMPLE 5-35
Preparation of Compound (VII-39)
(573) ##STR00176##
(574) With the exception that compound (VI-39) (0.12 g, 0.073 mmol), obtained in Preparation Example 4-35, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.058 g, 46%).
(575) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.48-0.53 (m, 1H), 0.58-0.59 (m, 1H), 0.64-0.76 (m, 8H), 0.82-0.92 (m, 15H), 1.03-1.04 (d, 2H), 1.11-1.22 (m, 6H), 1.42-1.49 (m, 6H), 1.86-1.97 (m, 3H), 2.11-2.20 (m, 9H), 2.73-2.95 (m, 10H), 2.98-3.13 (m, 6H), 3.14-3.21 (m, 5H), 3.33-3.37 (m, 2H), 3.90-3.92 (m, 4H), 3.98-4.04 (m, 2H), 4.14-4.80 (m, 8H), 4.94 (s, 2H), 5.42 (s, 2H), 6.00-6.01 (t, 1H), 7.22-7.29 (m, 4H), 7.38-7.42 (d, 1H), 7.59-7.61 (d, 2H), 7.81-7.83 (d, 1H), 7.89-7.96 (m, 2H), 8.00-8.03 (d, 1H), 8.09-8.12 (d, 1H), 8.28-8.51 (m, 1H), 8.64 (s, 1H), 10.02 (s, 1H),
(576) LC-MS m/z: 1740.4 [M+H].sup.+, 1762.3 [M+Na].sup.+
PREPARATION EXAMPLE 5-36
Preparation of Compound (VII-40)
(577) ##STR00177##
(578) With the exception that compound (VI-40) (0.1 g, 0.063 mmol), obtained in Preparation Example 4-36, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.072 g, 68%).
(579) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 1H), 0.62-0.69 (m, 2H), 0.70-0.77 (m, 7H), 0.82-0.89 (m, 18H), 1.02-0.07 (m, 3H), 1.18-1.29 (m, 3H), 1.44-1.47 (m, 6H), 2.19-2.21 (m, 10H), 2.61-2.64 (m, 2H), 2.74-3.00 (m, 10H), 3.17-3.18 (m, 7H), 3.25-3.33 (m, 25H), 3.90-4.01 (m, 5H), 4.16-4.84 (m, 8H), 4.95 (s, 2H), 5.40 (s, 2H), 6.01-6.02 (t, 1H), 7.01-7.05 (m, 2H), 7.26-7.29 (m, 4H), 7.58-7.60 (d, 2H), 7.79-7.82 (m, 1H), 7.94-7.99 (m, 2H), 8.08-8.10 (m, 1H), 10.00 (s, 1H)
(580) LC-MS m/z: 1697.2 [M+H].sup.+, 1719.1 [M+Na].sup.+
PREPARATION EXAMPLE 5-37
Preparation of Compound (VII-41)
(581) ##STR00178##
(582) With the exception that compound (VI-41) (0.062 g, 0.038 mmol), obtained in Preparation Example 4-37, was used instead of compound (VI-34), obtained in Preparation Example 4-30, the same procedure as in Preparation Example 5-30 was repeated to afford the title compound (0.037 g, 56%).
(583) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 0.51-0.53 (m, 1H), 0.63-0.77 (m, 13H), 0.81-0.91 (m, 17H), 1.03-1.04 (d, 2H), 1.09-1.22 (m, 6H), 1.42-1.49 (m, 6H), 1.95-1.97 (m, 3H), 2.11-2.19 (m, 7H), 2.77-2.89 (m, 13H), 3.13-3.17 (m, 7H), 3.32-3.36 (m, 18H), 3.92-3.93 (m, 4H), 4.00-4.04 (m, 2H), 4.14-4.85 (m, 8H), 4.99 (s, 2H), 5.42 (s, 2H), 5.88-6.00 (t, 1H), 7.28-7.41 (m, 4H), 7.58-7.65 (d, 2H), 7.77-8.06 (m, 4H), 8.10-8.11 (d, 1H), 8.21-8.45 (m, 1H), 10.00 (s, 1H)
(584) LC-MS m/z: 1722.5 [M+H].sup.+, 1744.3 [M+Na].sup.+
EXAMPLES
Preparation of Antibody-Linker-Drug conjugate of Chemical Formulas I-1, I-2, I-3 and I-4
Example 1
Preparation of Conjugate (I-5)
(585) ##STR00179##
(586) After being subjected to exchange treatment with PBS, pH 7.5 (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl included), 5 mL of a trastuzumab (Herceptin) solution (4.0 g/mL, 27 μM) was further treated with 2.5 ml of PBS, pH 7.5, and 7.5 mL of 0.05 M sodium borate, pH 8.5. Then, the trastuzumab solution was mixed with a solution of compound (VII-5) (2 mg, 0.001 mmol, 10 equivalents), obtained in Preparation Example 5-1, in 3.75 mL of 20% DMSO at 25° C. for 4 hrs while stirring.
(587) When the reaction was completed, the reaction mixture was filtered through a 0.45 μm filter. The filtrate was loaded into a centrifugal filter (Amicon Ultra-15 30K), and concentrated two or three times by use of PBS, pH 7.5, until the organic solvent and the materials that did not react were removed.
(588) The resulting concentrate was loaded into a Sephadex G-25 resin-packed column (30×300 mm) equilibrated with PBS, pH 7.5, and purified by MPLC (280 nm, UV range 0.08, flow rate 10 mL/min) to afford 13.5 mg of the title compound. (1.28 mg/mL, a total of 10.5 mL, 68%). The concentration of the compound thus obtained was determined by measuring absorbance at three wavelengths (280 nm, 320 nm, 350 nm) with the aid of a UV spectrometer.
Example 2
Preparation of Conjugate (I-6)
(589) ##STR00180##
(590) With the exception that compound (VII-6) (4.2 mg, 0.003 mmol, 15 eqs.), obtained in Preparation Example 5-2, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound 12.1 mg (1.1 mg/mL, a total of 11 mL, 45%).
Example 3
Preparation of Conjugate (I-7)
(591) ##STR00181##
(592) With the exception that compound (VII-7) (3.12 mg, 0.002 mmol, 15 eqs.), obtained in Preparation Example 5-3, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 13.7 mg (1.71 mg/mL, a total of 8 mL, 68%).
Example 4
Preparation of Conjugate (I-8)
(593) With the exception that compound (VII-8) (3.20 mg, 0.002 mmol, 15 eqs.), obtained in Preparation Example 5-4, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 13.9 mg (1.54 mg/mL, a total of 9 mL, 68%).
Example 5
Preparation of Conjugate (I-9)
(594) With the exception that compound (VII-9) (3.27 mg, 0.002 mmol, 15 eqs.), obtained in Preparation Example 5-4, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 15.3 mg (1.53 mg/mL, a total of 10 mL, 77%).
Example 6
Preparation of Conjugate (I-10)
(595) ##STR00182##
(596) With the exception that compound (VII-10) (4.17 mg, 0.003 mmol, 15 eqs.), obtained in Preparation Example 5-6, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 14.2 mg (1.29 mg/mL, a total of 11 mL, 53%)
Example 7
Preparation of Conjugate (I-11)
(597) ##STR00183##
(598) With the exception that compound (VII-11) (3.08 mg, 0.002 mmol, 15 eqs.), obtained in Preparation Example 5-7, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 9.5 mg (1.19 mg/mL, a total of 8 mL, 48%).
Example 8
Preparation of Conjugate (I-12)
(599) ##STR00184##
(600) With the exception that compound (VII-12) (5.4 mg, 0.003 mmol, 10 eqs.), obtained in Preparation Example 5-8, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 50 mg (2.5 mg/mL, a total of 20 mL, 100%)
Example 9
Preparation of Conjugate (I-13)
(601) ##STR00185##
(602) With the exception that compound (VII-13) (5.7 mg, 0.004 mmol, 20 eqs.), obtained in Preparation Example 5-9, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 15.2 mg (1.01 mg/mL, a total of 15 mL, 56%).
Example 10
Preparation of Conjugate (I-14)
(603) ##STR00186##
(604) With the exception that compound (VII-14) (4.3 mg, 0.003 mmol, 15 eqs.), obtained in Preparation Example 5-10, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 19.4 mg (1.55 mg/mL, a total of 12.5 mL, 72%).
Example 11
Preparation of Conjugate (I-15)
(605) ##STR00187##
(606) With the exception that compound (VII-15) (2.3 mg, 0.0015 mmol, 10 eqs.), obtained in Preparation Example 5-11, was used instead of compound (VII-5), obtained in Preparation Example 5-1, the same procedure as in Example 1 was repeated to afford the title compound. 20.1 mg (1.6 mg/mL, a total of 12.5 mL, 92%).
Example 12
Preparation of Conjugate (I-16)
(607) ##STR00188##
(608) After being subjected to exchange treatment with PBS, pH 7.5 (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl included), 5 mL of a trastuzumab (Herceptin) solution (5.4 g/mL, 36 μM) was further treated with 2.5 ml of PBS, pH 7.5, and 7.5 mL of 0.05 M sodium borate, pH 8.5. Then, the trastuzumab solution was mixed with a solution of compound (VII-16) (4.4 mg, 0.003 mmol, 15 eqs.), obtained in Preparation Example 5-12, in 3.75 mL of 20% DMSO at 25° C. for 4 hrs while stirring. When the reaction was completed, the reaction mixture was filtered through a 0.45 μm filter. The filtrate was loaded into a centrifugal filter (Amicon Ultra-15 30K), and concentrated two or three times by use of PBS, pH 7.5, until the organic solvent and the materials that did not react were removed. The resulting concentrate was loaded to a Sephadex G-25 resin-packed column (30×300 mm) equilibrated with PBS, pH 7.5, and purified by MPLC (280 nm, UV range 0.08, flow rate 10 mL/min) to afford the title compound. 22.5 mg (1.73 mg/mL, a total of 13 mL, 83%). The concentration of the compound thus obtained was determined by measuring absorbance at three wavelengths (280 nm, 320 nm, 350 nm) with the aid of a UV spectrometer.
Example 13
Preparation of Conjugate (I-17)
(609) ##STR00189##
(610) With the exception that compound (VII-17) (5.6 mg, 0.004 mmol, 20 eqs.), obtained in Preparation Example 5-13, was used instead of compound (VII-16), obtained in Preparation Example 5-13, the same procedure as in Example 12 was repeated to afford the title compound. 25.6 mg (2.05 mg/mL, a total of 12.5 mL, 95%).
Example 14
Preparation of Conjugate (I-18)
(611) ##STR00190##
(612) With the exception that compound (VII-18) (4.0 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-14, was used instead of compound (VII-16), obtained in Preparation Example 5-12, the same procedure as in Example 12 was repeated to afford the title compound. 23.8 mg (1.98 mg/mL, a total of 12 mL, 99%).
Example 15
Preparation of Conjugate (I-19)
(613) ##STR00191##
(614) With the exception that compound (VII-19) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-14, was used instead of compound (VII-16), obtained in Preparation Example 5-12, the same procedure as in Example 12 was repeated to afford the title compound. 20.4 mg (1.86 mg/mL, a total of 11 mL, 82%).
Example 16
Preparation of Conjugate (I-20)
(615) ##STR00192##
(616) With the exception that compound (VII-20) (3.8 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-16, was used instead of compound (VII-16), obtained in Preparation Example 5-12, the same procedure as in Example 12 was repeated to afford the title compound. 18.9 mg (1.72 mg/mL, a total of 11 mL, 79%).
Example 17
Preparation of Conjugate (I-21)
(617) ##STR00193##
(618) With the exception that compound (VII-21) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-17, was used instead of compound (VII-16), obtained in Preparation Example 5-12, the same procedure as in Example 12 was repeated to afford the title compound. 19.8 mg (1.8 mg/mL, a total of 11 mL, 80%).
Example 18
Preparation of Conjugate (I-22)
(619) ##STR00194##
(620) With the exception that compound (VII-22) (3.8 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-18, was used instead of compound (VII-16), obtained in Preparation Example 5-12, the same procedure as in Example 12 was repeated to afford the title compound. 13.0 mg (1.18 mg/mL, a total of 11 mL, 54%).
Example 19
Preparation of Conjugate (I-23)
(621) ##STR00195##
(622) After being subjected to exchange treatment with PBS, pH 7.5 (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl included), 5 mL of a trastuzumab (Herceptin) solution (5.4 g/mL, 36 μM) was further treated with 2.5 ml of PBS, pH 7.5, and 7.5 mL of 0.05 M sodium borate, pH 8.5. Then, the trastuzumab solution was mixed with a solution of compound (VII-23) (5.7 mg, 0.0036 mmol, 20 eqs.), obtained in Preparation Example 5-19, in 3.75 mL of 20% DMSO at 25° C. for 4 hrs while stifling. When the reaction was completed, the reaction mixture was filtered through a 0.45 μm filter. The filtrate was loaded into a centrifugal filter (Amicon Ultra-15 30K), and concentrated two or three times by use of PBS, pH 7.5, until the organic solvent and the materials that did not react were removed. The resulting concentrate was loaded to a Sephadex G-25 resin-packed column (30×300 mm) equilibrated with PBS, pH 7.5, and purified by MPLC (280 nm, UV range 0.08, flow rate 10 mL/min) to afford the title compound. 16.9 mg (1.21 mg/mL, a total of 15 mL, 67%). The concentration of the compound thus obtained was determined by measuring absorbance at three wavelengths (280 nm, 320 nm, 350 nm) with the aid of a UV spectrometer.
Example 20
Preparation of Conjugate (I-24)
(623) ##STR00196##
(624) With the exception that compound (VII-24) (4.3 mg, 0.0027 mmol, 15 eqs.), obtained in Preparation Example 5-20, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 19.2 mg (1.54 mg/mL, a total of 11.5 mL, 71%).
Example 21
Preparation of Conjugate (I-25)
(625) ##STR00197##
(626) With the exception that compound (VII-25) (5.74 mg, 0.0036 mmol, 20 eqs.), obtained in Preparation Example 5-20, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 14.7 mg (1.23 mg/mL, a total of 12 mL, 54%).
Example 22
Preparation of Conjugate (I-26)
(627) ##STR00198##
(628) With the exception that compound (VII-26) (4.38 mg, 0.0027 mmol, 15 eqs.), obtained in Preparation Example 5-20, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 23.7 mg (1.58 mg/mL, a total of 14 mL, 88%).
Example 23
Preparation of Conjugate (I-27)
(629) ##STR00199##
(630) With the exception that compound (VII-27) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-23, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 21.1 mg (1.15 mg/mL, a total of 14 mL, 85%).
Example 24
Preparation of Conjugate (I-28)
(631) ##STR00200##
(632) With the exception that compound (VII-28) (3.9 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-24, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 17.3 mg (1.58 mg/mL, a total of 13 mL, 70%)
Example 25
Preparation of Conjugate (I-29)
(633) ##STR00201##
(634) With the exception that compound (VII-29) (4.1 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-25, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 9.2 mg (0.92 mg/mL, a total of 10 mL, 24.8%)
Example 26
Preparation of Conjugate (I-30)
(635) ##STR00202##
(636) With the exception that compound (VII-30) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-26, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 16.7 mg (1.19 mg/mL, a total of 14 mL, 67%).
Example 27
Preparation of Conjugate (I-31)
(637) ##STR00203##
(638) With the exception that compound (VII-31) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-27, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 21 mg (1.61 mg/mL, a total of 13 mL, 85%).
Example 28
Preparation of Conjugate (I-32)
(639) ##STR00204##
(640) With the exception that compound (VII-32) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-27, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 12.3 mg (0.88 mg/mL, a total of 14 mL, 50%).
Example 29
Preparation of Conjugate (I-33)
(641) ##STR00205##
(642) With the exception that compound (VII-33) (4.0 mg, 0.0025 mmol, 15 eqs.), obtained in Preparation Example 5-29, was used instead of compound (VII-23), obtained in Preparation Example 5-19, the same procedure as in Example 19 was repeated to afford the title compound. 15.3 mg (1.18 mg/mL, a total of 13 mL, 62%).
Example 30
Preparation of Conjugate (I-34)
(643) ##STR00206##
(644) After being subjected to exchange treatment with PBS, pH 7.5 (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl included), 5 mL of a trastuzumab (Herceptin) solution (5.4 g/mL, 36 μM) was further treated with 2.5 ml of PBS, pH 7.5, and 7.5 mL of 0.05 M sodium borate, pH 8.5. Then, the trastuzumab solution was mixed with a solution of compound (VII-34) (5.7 mg, 0.0036 mmol, 20 eqs.), obtained in Preparation Example 5-30, in 3.75 mL of 20% DMSO at 25° C. for 18 hrs while stirring. When the reaction was completed, the reaction mixture was filtered through a 0.45 μm filter. The filtrate was loaded into a centrifugal filter (Amicon Ultra-15 30K), and concentrated two or three times by use of PBS, pH 7.5, until the organic solvent and the materials that did not react were removed. The resulting concentrate was loaded into a Sephadex G-25 resin-packed column (30×300 mm) equilibrated with PBS, pH 7.5, and purified by MPLC (280 nm, UV range 0.08, flow rate 10 mL/min) to afford the title compound. 26.4 mg (2.20 mg/mL, a total of 12 mL, 98%). The concentration of the compound thus obtained was determined by measuring absorbance at three wavelengths (280 nm, 320 nm, 350 nm) with the aid of a LTV spectrometer.
Example 31
Preparation of Conjugate (I-35)
(645) ##STR00207##
(646) With the exception that compound (VII-35) (4.2 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-31, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 14.5 mg (1.21 mg/mL, a total of 12 mL, 61%).
Example 32
Preparation of Conjugate (I-36)
(647) ##STR00208##
(648) With the exception that compound (VII-36) (4.2 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-32, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 17.9 mg (1.63 mg/mL, a total of 11 mL, 75%).
Example 33
Preparation of Conjugate (I-37)
(649) ##STR00209##
(650) With the exception that compound (VII-37) (4.1 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-33, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 11.8 mg (1.48 mg/mL, a total of 8 mL, 50%).
Example 34
Preparation of Conjugate (I-38)
(651) ##STR00210##
(652) With the exception that compound (VII-38) (4.0 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-34, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 15.8 mg (1.21 mg/mL, a total of 13 mL, 66%).
Example 35
Preparation of Conjugate (I-39)
(653) ##STR00211##
(654) With the exception that compound (VII-39) (4.2 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-35, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 18.3 mg (1.52 mg/mL, a total of 12 mL, 77%).
Example 36
Preparation of Conjugate (I-40)
(655) ##STR00212##
(656) With the exception that compound (VII-40) (4.6 mg, 0.0027 mmol, 15 eqs.), obtained in Preparation Example 5-36, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 12.7 mg (1.15 mg/mL, a total of 11 mL, 47%).
Example 37
Preparation of Conjugate (I-41)
(657) ##STR00213##
(658) With the exception that compound (VII-41) (4.1 mg, 0.0024 mmol, 15 eqs.), obtained in Preparation Example 5-37, was used instead of compound (VII-34), obtained in Preparation Example 5-30, the same procedure as in Example 30 was repeated to afford the title compound. 16.1 mg (1.24 mg/mL, a total of 13 mL, 67%).
Test Example 1
DAR Determination by Protein Mass Spectrometry
(659) From each of the antibody-linker-drug conjugates prepared in the Preparation Examples, N-linked glycan were removed by treatment with the endoglycosidic enzyme PNGase F at 37° C. for 15±3 hrs. The deglycosylated antibody-linker-drug conjugates were analyzed by an LC-ESI-MS system including Agilent 1200 HPLC and Agilent 6530 Q-TOF. The chromatographic separation of antibody-linker-drug conjugates was accomplished on an Agilent Zorbax column (300SB-C18, 2.1 mm×75 mm, 5 μm) using a gradient elution of 0.1% formic acid in water solution/0.1% formic acid in acetonitrile solution, and m/z (mass to charge) data were obtained at a scan rate of 1 spectrum per second within a range of 900 to 4000 m/z, and subjected to deconvolution, in which molecular weights and the % (percentage) abundance of each peak to integrated peaks (D0, D1, D2 . . . Dn) are used to calculate DAR.
(660) Relative abundance of each peak was calculated according to the following equation. % Relative abundance of each peak was calculated by dividing the abundance of each peak by the total abundance of peaks.
(661)
(662) The DAR of antibody-linker-drug conjugates was calculated using the % relative abundance according to the following equation, and the results are given in Tables 1 to 4.
(663)
(664) TABLE-US-00001 TABLE 1 D0 D1 D2 D3 D4 D5 D6 D7 D8 Sum Conjugate (I-6) Mass 145179.9 146631.2 148080.8 149535.2 150983.3 152436.5 153888.3 (Da) Area 3080 5170 6730 5764 4659 2430 1059 28892 % Area 10.7 17.9 23.3 20.0 16.1 8.4 3.7 100 DAR 2.53 Conjugate (I-10) Mass 145170.3 146616.1 148043.7 149473.9 150904.2 152340.0 153771.2 155201.9 156631.2 (Da) Area 474 2570 5670 6155 6765 5567 3940 2430 655 34226 % Area 1.4 7.5 16.6 18 19.8 16.3 11.5 7.1 1.9 100 DAR 3.89 Conjugate (I-11) Mass 145173.9 146631.36 148048.1 149481.0 150912.6 152348.0 153781.7 155215.2 156641.7 (Da) Area 330 1788 4947 6111 6769 7133 4975 3005 784 35842 % Area 0.9 5.0 13.8 17.0 18.9 19.9 13.9 8.4 2.2 100 DAR 4.18
(665) TABLE-US-00002 TABLE 2 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Conjugate (I-16) Mass 145183.4 146693.5 148201.3 149712.0 151221.6 152733.2 154245.0 155747.8 157256.0 158782.5 (Da) Area 102 1424 3455 4709 5143 4244 3996 2766 1683 762 28284 % Area 0.4 5.0 12.2 16.6 18.2 15.0 14.1 9.8 6.0 2.7 100 DAR 4.52 Conjugate (I-19) Mass 145180.0 146700.0 148195.7 149700.2 151191.8 152675.1 154214.2 (Da) Area 0 659 2225 3104 2632 1969 1031 11620 % Area 0 5.7 19.1 26.7 22.7 16.9 8.9 100 DAR 3.53 Conjugate (I-20) Mass 145180.0 146682.2 148171.4 149661.6 151149.2 152635.6 154120.6 155616.1 (Da) Area 0 1192 4051 5884 5670 4260 2553 1374 24984 % Area 0 4.8 16.2 23.6 22.7 17.1 10.2 5.5 100 DAR 3.84 Conjugate (I-21) Mass 145180 146696.3 148185.3 149697.7 151205.5 152699.3 154228.8 155742.3 (Da) Area 0 204 832 1650 1996 906 480 135 6203 % Area 0 3.3 13.4 26.6 32.2 14.6 7.7 2.2 100 DAR 3.73 Conjugate (I-22) Mass 145180 146696.3 148150.3 149640.7 151168 152613.7 15087.2 15554.7 157093.4 158600 (Da) Area 0 204 1040 1925 2478 2635 1932 886 929 328 12333 % Area 0 1.7 8.2 15.6 20.1 21.4 15.7 7.2 7.5 2.7 100 DAR 4.81
(666) TABLE-US-00003 TABLE 3 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Conjugate (I-23) Mass 145188.9 146664.0 148148.6 149631.3 151108.4 152593.1 154080.0 155567.8 (Da) Area 2036 5337 7002 5759 4434 2120 1133 207 28028 % Area 7.3 19.0 25.0 20.0 15.8 7.6 4.0 0.7 100 DAR 2.56 Conjugate (I-24) Mass 145188.9 146664.0 148148.6 149631.3 151108.4 152593.1 154080.0 (Da) Area 2036 5337 7002 5759 4434 2120 1133 27821 % Area 7.3 19.2 25.2 20.7 15.9 7.6 4.1 100 DAR 2.58 Conjugate (I-25) Mass 148184.2 146662.2 148139.3 149624.8 151109.1 152587.6 154068.8 155555.1 158517.7 158517.7 (Da) Area 977 4474 8992 9929 9912 7176 4842 2313 977 240 49832 % Area 2.0 9.0 18.0 19.9 19.9 14.4 9.7 4.6 2.0 0.5 100 DAR 3.67 Conjugate (I-26) Mass 145188.0 146689.5 148201.7 149713.7 151226.1 152731.6 154241.5 155753.7 157258.4 158773.3 (Da) Area 136 1285 3717 5699 5756 5280 4541 3287 2348 955 33004 % Area 0.4 3.9 11.3 17.3 17.4 16.0 13.8 10.0 7.1 2.9 100 DAR 4.63 Conjugate (I-27) Mass 145182.4 146692.2 148195.4 149705.6 151213.7 152718.3 154222.6 155731.2 (Da) Area 2008 3562 5553 4675 3576 1662 1105 456 22597 % Area 8.9 15.8 24.6 20.7 15.8 7.4 4.9 2.0 100 DAR 2.71 Conjugate (I-28) Mass 145185.4 146672.2 148158.1 149641.9 151128.8 152626.0 154108.0 (Da) Area 789 4780 5411 3780 3281 1342 428 19811 % Area 4.0 24.1 27.3 19.1 16.6 6.8 2.2 100 DAR 2.49 Conjugate (I-30) Mass 145180.0 146711.4 148226.3 149742.3 151267.9 152788.8 154311.3 155837.3 (Da) Area 0 144 968 1227 2001 1550 519 153 6562 % Area 0 2.2 14.8 18.7 30.5 23.6 7.9 2.3 100 DAR 3.92 Conjugate (I-31) Mass 145181.5 146691.4 148203.8 149717.6 151228.5 (Da) Area 6412 9396 6548 3337 1388 % Area 23.7 34.7 24.2 12.3 5.1 DAR 1.41 Conjugate (I-32) Mass 145185.5 146679.8 148182.7 149670.4 151166.1 152687.2 154165.9 155657.3 157164.6 (Da) Area 240 696 1841 2965 3326 3067 2007 897 260 15299 % Area 1.6 4.5 12.0 19.4 21.7 20.0 13.1 5.9 1.7 100 DAR 4.07 Conjugate (I-33) Mass 145177.6 146681.5 148183.2 149668.3 151168.4 152663.0 154161.2 155656.4 157868.3 (Da) Area 149 432 1535 2946 4174 3528 2278 1803 375 17220 % Area 0.9 2.5 8.9 17.1 24.2 20.5 13.2 10.5 2.2 100 DAR 4.41
(667) TABLE-US-00004 TABLE 4 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 Sum Conjugate (I-34) Mass 145177.7 146777.9 148378.3 149972.4 151563.0 153163.7 (Da) Area 5864 10420 8579 5125 1867 849 32704 % Area 17.9 31.9 26.2 15.7 5.7 2.6 100 DAR 1.67 Conjugate (I-35) Mass 145180.0 146805.8 148427.5 150051.7 151669.0 153291.4 154912.5 156539.5 158132.1 159784.1 (Da) Area 0 1791 7950 11577 11722 9767 7214 4651 1852 949 57473 % Area 0 3.1 13.8 20.1 20.4 17.0 12.6 8.1 3.2 1.7 100 DAR 4.3 Conjugate (I-36) Mass 145180.0 146784.9 148374.9 149964.2 151552.3 153144.0 154742.7 156334.3 157940.4 159524.4 (Da) Area 0 232 2467 5116 6483 4829 3222 1457 716 356 24878 % Area 0 0.9 9.9 20.6 26.1 19.4 13.0 5.9 2.9 1.4 100 DAR 4.38 Conjugate (I-37) Mass 145180.0 146787.7 148387.9 149985.8 151588.4 153191.5 154798.6 156392.1 157995.3 (Da) Area 0 3495 7843 10015 8988 6940 4126 1274 389 43070 % Area 0 8.1 18.2 23.3 20.9 16.1 9.6 3.0 0.9 100 DAR 3.64 Conjugate (I-38) Mass 145180.0 146759.0 148331.1 149903.0 151476.4 153055.9 154631.0 156203.6 (Da) Area 0 2221 7557 9075 7933 5512 3044 968 % Area 0 3.1 20.8 25.0 21.8 15.2 8.4 2.7 DAR 3.55 Conjugate (I-39) Mass 145187.1 146810.7 148434.4 150059.8 151687.1 153320.3 154948.4 (Da) Area 1463 5565 11616 11593 6224 2476 710 39647 % Area 3.7 14.0 29.3 29.2 15.7 6.2 1.8 100 DAR 2.65 Conjugate (I-40) Mass 145180.0 146770.5 148351.3 149928.2 151506.8 153095.0 154671.9 156259.1 157840.3 159429.5 (Da) Area 0 1848 6545 10020 11504 9447 6600 4246 2260 661 53131 % Area 0 3.5 12.3 18.9 21.6 17.8 12.4 8.0 4.3 1.3 100 DAR 4.36 Conjugate (I-41) Mass 145180.0 146792.1 148401.0 150007.0 151601.6 153213.6 154823.1 (Da) Area 0 236 1671 3110 3322 2200 1234 11773 % Area 0 2.0 14.2 26.4 28.2 18.7 105 100 DAR 3.79
Test Example 2
SEC-HPLC Analysis
(668) Using size exclusion chromatography (SEC-HPLC), examination was made to see whether the antibody was abnormally fragmented or aggregated during the coupling of the antibody with the drug. Having negative influences on antibody's intrinsic properties including antigen-specific affinity, in vivo pharmacokinetics, etc., such abnormal protein structures may be used as an index indirectly indicative of the quality of prepared antibody-linker-drug conjugates.
(669) The antibody-linker-drug conjugates prepared in the Preparation Examples were analyzed for normal antibody structure ratios, using SEC-HPLC. The results are summarized in Tables 5 to 8, below.
(670) TABLE-US-00005 TABLE 5 Sample Main peak .sup.1) (%) HMW .sup.2) (%) LMW .sup.3) (%) Pre-coupled antibody 99.16 0.65 0.19 Conjugate (I-6) 98.55 1.07 0.38 Conjugate (I-10) 98.21 1.40 0.39 Conjugate (I-11) 98.42 1.16 0.42 .sup.1) Main peak: ratio of materials corresponding to the size of normal antibody .sup.2) HMW (High molecular weight): ratio of materials larger than normal antibody due to aggregation .sup.3) LMW (Low molecular weight): ratio of materials smaller than normal antibody due to fragmentation
(671) The antibody-linker-drug conjugates prepared in Examples 2, 6 and 7 were observed to have a normal antibody structure ratio of 98.21 to 98.55%, which was comparable with that of the pre-coupled antibody (99.16%).
(672) TABLE-US-00006 TABLE 6 Sample Main peak .sup.1) (%) HMW .sup.2) (%) LMW .sup.3) (%) Pre-coupled antibody 99.48 0.27 0.25 Conjugate (I-16) 99.24 0.40 0.35 Conjugate (I-17) 99.25 0.38 0.32 Conjugate (I-18) 98.44 1.38 0.18 Conjugate (I-19) 93.56 6.25 0.18 Conjugate (I-20) 94.06 5.73 0.20 Conjugate (I-21) 97.27 2.52 0.20 Conjugate (I-22) 92.43 7.37 0.19 .sup.1) Main peak: ratio of materials corresponding to the size of normal antibody .sup.2) HMW (High molecular weight): ratio of materials larger than normal antibody due to aggregation .sup.3) LMW (Low molecular weight): ratio of materials smaller than normal antibody due to fragmentation
(673) The antibody-linker-drug conjugates prepared in Examples 12 to 18 were observed to have a normal antibody structure ratio of 92.43 to 99.25%, which was comparable with that of the pre-coupled antibody (99.48%).
(674) TABLE-US-00007 TABLE 7 Sample Main peak .sup.1) (%) HMW .sup.2) (%) LMW .sup.3) (%) Pre-coupled antibody 99.48 0.27 0.25 Conjugate (I-23) 99.13 0.53 0.34 Conjugate (I-24) 97.77 1.71 0.52 Conjugate (I-25) 96.94 2.71 0.35 Conjugate (I-26) 98.39 1.30 0.31 Conjugate (I-27) 97.12 2.63 0.24 Conjugate (I-28) 97.44 2.32 0.24 Conjugate (I-29) 97.07 2.68 0.25 Conjugate (I-30) 90.68 9.08 0.24 Conjugate (I-31) 93.80 6.00 0.20 Conjugate (I-32) 94.27 5.51 0.22 Conjugate (I-33) 97.09 2.60 0.32 .sup.1) Main peak: ratio of materials corresponding to the size of normal antibody .sup.2) HMW (High molecular weight): ratio of materials larger than normal antibody due to aggregation .sup.3) LMW (Low molecular weight): ratio of materials smaller than normal antibody due to fragmentation
(675) The antibody-linker-drug conjugates prepared in Examples 19 to 29 were observed to have a normal antibody structure ratio of 90.68 to 99.13%, which was comparable with that of the pre-coupled antibody (99.48%).
(676) TABLE-US-00008 TABLE 8 Sample Main peak .sup.1) (%) HMW .sup.2) (%) LMW .sup.3) (%) Pre-coupled antibody 99.48 0.27 0.25 Conjugate (I-34) 98.63 1.02 0.35 Conjugate (I-35) 98.93 0.77 0.29 Conjugate (I-36) 98.33 1.43 0.24 Conjugate (I-37) 98.85 0.87 0.28 Conjugate (I-38) 98.31 1.44 0.25 Conjugate (I-39) 97.00 2.75 0.24 Conjugate (I-40) 98.15 1.49 0.36 Conjugate (I-41) 97.61 2.15 0.24 .sup.1) Main peak: ratio of materials corresponding to the size of normal antibody .sup.2) HMW (High molecular weight): ratio of materials larger than normal antibody due to aggregation .sup.3) LMW (Low molecular weight): ratio of materials smaller than normal antibody due to fragmentation
(677) The antibody-linker-drug conjugates prepared in Examples 30 to 37 were observed to have a normal antibody structure ratio of 97.00 to 98.93, which was comparable with that of the pre-coupled antibody (99.48%).
Test Example 3
IEF Analysis
(678) Using isoelectric focusing (IEF) analysis, the antibody-linker-drug conjugates (I-6), (I-8), (I-10) and (I-11), prepared respectively in Preparation Examples 2, 4, 6 and 7, were analyzed for isoelectric point (pI) values, and the results are listed in Table 9, below.
(679) TABLE-US-00009 TABLE 9 Lane 2 9 Pre- 3 4 5 6 7 8 Pre- coupled Conjugate Conjugate Conjugate Conjugate Conjugate Conjugate coupled Sample antibody (I-8) (I-6) (I-11) (I-11) (I-10) (I-10) antibody Band 1 8.96 8.87 8.90 8.78 8.80 8.78 8.78 8.98 Band 2 8.85 8.78 8.82 8.71 8.72 8.70 8.69 8.86 Band 3 8.76 8.69 8.74 8.62 8.62 8.6 8.59 8.77 Band 4 8.68 8.59 8.63 8.51 8.51 8.49 8.48 8.68 Band 5 — 8.48 8.52 8.39 8.4 8.38 8.37 — Band 6 — — 8.41 8.29 8.3 8.27 8.26 — Band 7 — — — 8.21 8.2 8.18 8.17 — * Lane 1 and 10 are pI marker.
(680) Coincident with the DAR results, as can be seen in Table 9, lower pI bands were detected from Conjugates (I-10) and (I-11) with more drugs coupled thereto. That is, the number of drugs coupled to the antibody increased.
Test Example 4
In Vitro Cytotoxicity Assay
(681) The antibody-linker-drug conjugates were analyzed for anticancer potency by evaluating antiproliferative activity against the HER-2-overexpressing breast cancer cell line (BT-474) in vitro. For a control, trastuzumab, which was employed for the preparation of the antibody-linker-drug conjugates, was used alone. Trastuzumab is a monoclonal antibody against the HER-2 antigen specifically expressed in breast cancer cells.
(682) BT-474 cells were seeded at a density of 1×10.sup.4 cells/well into 96-well microplates, and incubated for 5 days with various concentrations of the control trastuzumab or the antibody-linker-drug conjugates prepared in the Preparation Examples. The number of viable cells after 5 days incubation was indirectly evaluated by measuring absorbance via colorimetric reagent (CCK-8). The correlation of concentration and potency was fitted as 4-parameter logistic curve and the relative potency was evaluated with the EC.sub.50 (effective concentration of 50%) in the curve fit. The results are given in
(683) As seen in Table 10, the antibody-linker-drug conjugates prepared in Examples 2, 6 and 7 decreased in EC.sub.50 by 13.2 to 17.8 times, compared to the control trastuzumab, indicating that the antibody-linker-drug conjugates of the present invention can exert the same efficacy as the control even at far smaller doses, with the consequent decrease of side effects.
(684) In addition, the antibody-linker-drug conjugates prepared in Preparation Examples 2, 4, 6 and 7, as depicted in quartic curves of
(685) TABLE-US-00010 TABLE 10 EC.sub.50 EC.sub.50 EC.sub.50 Relative (1.sup.st) (2.sup.nd) (Mean) Potency Sample (ng/ml) (ng/ml) (ng/ml) (fold) trastuzumab 125 — 125 1 Conjugate (I-6) 9.47 — 9.47 13.2 trastuzumab 88.2 — 88.2 1 Conjugate (I-10) 6.28 6.28 6.28 14.0 trastuzumab 85.1 — 85.1 1 Conjugate (I-11) 4.66 4.88 4.77 17.8
(686) As is understood from the data of Table 11, the antibody-linker-drug conjugates prepared in Examples 1 to 7 decreased in EC.sub.50 by 7.7 to 34.2 times, compared to the control trastuzumab, indicating that the antibody-linker-drug conjugates of the present invention can exert the same efficacy as that of the control even at far smaller doses, with the consequent decrease of side effects.
(687) In addition, the antibody-linker-drug conjugates prepared in Preparation Examples 1 to 7, as depicted in 4-parameter logistic curves of
(688) TABLE-US-00011 TABLE 11 Sample EC.sub.50 (ng/ml) Relative Potency (fold) trastuzumab 98.6 1 Conjugate(I-16) 2.88 34.2 trastuzumab 110 1 Conjugate(I-18) 14.3 7.7 trastuzumab 96.6 1 Conjugate(I-19) 5.18 18.6 trastuzumab 92.3 1 Conjugate(I-20) 5.72 16.1 trastuzumab 96.6 1 Conjugate(I-21) 7.09 13.6 trastuzumab 92.3 1 Conjugate(I-22) 6.7 13.8
(689) As seen in Table 12, the antibody-linker-drug conjugates prepared in Examples 19 to 29 decreased in EC.sub.50 by 0.5 to 33.6 times, compared to the control trastuzumab. For the antibody-linker-drug conjugates (I-23), (I-25), (1-26), and (I-33), EC.sub.50 values were detected at levels 20- or higher fold lower than that of the control, indicating that the antibody-linker-drug conjugates can exert the same efficacy as that of the control even at 20- or higher fold smaller doses, with the consequent decrease of side effects.
(690) In addition, the antibody-linker-drug conjugates prepared in Preparation Examples 19 to 29, as depicted in 4-parameter logistic curves of
(691) TABLE-US-00012 TABLE 12 Sample EC.sub.50 (ng/mL) Relative Potency (fold) trastuzumab 102 1 Conjugate (I-23) 4.73 21.6 trastuzumab 106 1 Conjugate (I-24) 68.4 1.5 trastuzumab 107 1 Conjugate (I-25) 4.75 22.2 trastuzumab 101 1 Conjugate (I-26) 4.17 24.2 trastuzumab 97.9 1 Conjugate (I-32) 12.7 7.7 trastuzumab 97 1 Conjugate (I-33) 2.89 33.6
(692) As is apparent from the data of Table 10, the antibody-linker-drug conjugates prepared in Examples 30 to 36 decreased in EC.sub.50 by 8.9 to 22.6 times, compared to the control trastuzumab, indicating that the antibody-linker-drug conjugates of the present invention can exert the same efficacy as that of the control even at far smaller doses, with the consequent decrease of side effects.
(693) In addition, the antibody-linker-drug conjugates prepared in Preparation Examples 30 to 36, as depicted in quartic curves of
(694) TABLE-US-00013 TABLE 13 Sample EC.sub.50 (ng/ml) Relative Potency (fold) trastuzumab 104 1 Conjugate(I-34) 11.3 9.2 trastuzumab 104 1 Conjugate(I-35) 10.3 10.1 trastuzumab 104 1 Conjugate(I-36) 5.74 18.1 trastuzumab 116 1 Conjugate(I-37) 13 8.9 trastuzumab 116 1 Conjugate(I-38) 5.13 22.6 trastuzumab 94.7 1 Conjugate(I-39) 7 13.5 trastuzumab 94.7 1 Conjugate(I-40) 4.42 21.4
Test Example 5
Pharmacokinetic (PK) Assay
(695) The antibody-linker-drug conjugates (I-6) and (I-8), respectively prepared in Preparation Examples 2 and 4, and the control trastuzumab were each intravenously injected once at a dose of 20 mg/kg to Balb/c Slc-nu mice, and assayed for pharmacokinetics (PK) for one month.
(696) The antibody-linker-drug conjugates (I-8) and (I-6) were observed to have the half-life of 8.3 and 7.9 days, respectively, with no significant differences from the control trastuzumab's half-life, 8.6 days, implying that the cytotoxic drug is not directly coupled to the neonatal Fc region (FcRn), a site affecting the PK of the antibody itself, during the conjugation.
(697) TABLE-US-00014 TABLE 14 Parameter Conjugate (I-8) Conjugate (I-6) trastuzumab AUC.sub.all (hr*μg/mL) 507280 575495 607582 C.sub.0 (μg/mL) 375.2 403.7 379.9 T.sub.1/2 (day) .sup.1) 8.3 7.9 8.6 .sup.1) User specified lambda-z range (4 m~27 day, 9 points)
Test Example 6
Efficacy Study in Animal Model
(698) For an animal model efficacy test, the antibody-linker-drug conjugates (I-6), (I-10), and (I-11), prepared respectively in Preparation Examples 2, 6 and 7, were intravenously injected once at three different doses (1.25, 5, and 20 mg/kg) into nude mice which were previously xenografted HER-2-overexpressing breast cancer cells (BT-474). For 56 days after injection, the volumes and final weights of the tumors were monitored. The phosphate buffered saline (PBS) was used as a negative control while trastuzumab (Herceptin®) was injected at a dose of 20 mg/kg into a positive control. The results are shown in
(699) As is understood from the data of
(700) Separately, the antibody-linker-drug conjugate (I-16), prepared in Preparation Examples 12, was intravenously injected once at three different doses (0.05, 0.5, and 5 mg/kg) into nude mice which were previously xenografted with HER-2-overexpressing breast cancer cells (BT-474). For 28 days after injection, volumes and final weights of the tumors were monitored. The phosphate buffered saline (PBS) was used as a negative control while trastuzumab (Herceptin®) was injected at a dose of 5 mg/kg into a positive control. The results are shown in
(701) As is understood from the data of
(702) In addition, the antibody-linker-drug conjugates (I-23), (1-25), and (I-26), prepared respectively in Preparation Examples 19, 21, and 22, were intravenously injected once at three different doses (0.05, 0.5, and 5 mg/kg) into nude mice which were previously xenografted with HER-2-overexpressing breast cancer cells (BT-474). For 28 days after injection, the volumes and final weights of the tumors were monitored. The phosphate buffered saline (PBS) was used as a negative control while trastuzumab (Herceptin®) was injected at a dose of 5 mg/kg into a positive control. The results are shown in
(703) As is understood from the data of
(704) In another experiment, nude mice xenografted with HER-2-overexpressing breast cancer cell line (BT-474) were intravenously injected once with three difference doses (0.05, 0.5, and 5 mg/kg) of the antibody-linker-drug conjugate (I-34), prepared in Preparation Example 30. For 28 days after injection, the volumes and final weights of the tumors were monitored. The phosphate buffered saline (PBS) was used as a negative control while trastuzumab (Herceptin®) was injected at a dose of 5 mg/kg into a positive control. The results are shown in
(705) As is understood from the data of