ENHANCER OF FERTILIZATION FUNCTION OF SPERM
20220106270 · 2022-04-07
Inventors
- Takaaki Abe (Miyagi, JP)
- Nobuo Yaegashi (Miyagi, JP)
- Masahito Tachibana (Miyagi, JP)
- Kenichiro Hayashi (Okayama, JP)
Cpc classification
C12N2501/999
CHEMISTRY; METALLURGY
C07D209/18
CHEMISTRY; METALLURGY
C07D209/20
CHEMISTRY; METALLURGY
A61K31/405
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
C07D401/06
CHEMISTRY; METALLURGY
International classification
Abstract
An object of the present invention is to provide an agent for enhancing fertilization function of a mammal sperm, which comprises a low molecular compound which can be produced relatively easily and inexpensively as an active ingredient, and a method for enhancing fertilization function of a mammal sperm and a method for preparing a mammal fertilized egg, which use a low molecular compound which can be produced relatively easily and inexpensively. An agent comprising one or more compounds selected from the group consisting of compounds of the following formula (I.sub.0), formula (II), and formula (III), and physiologically acceptable salts thereof when R.sup.3 is OH is used as an agent for enhancing fertilization function of a mammal sperm.
##STR00001##
Claims
1. (canceled)
2. (canceled)
3. A method for enhancing fertilization function of a mammal sperm, comprising step (a) of transferring at least one collected mammal sperm into a physiological aqueous solution containing one or more compounds selected from the group consisting of compounds of the following formula (I.sub.0), formula (II), and formula (III), and physiologically acceptable salts thereof when R.sup.3 is OH: ##STR00132## [wherein R.sup.1 is a benzoylmethyl group whose benzene ring is unsubstituted or a benzoylmethyl group whose benzene ring is substituted by an alkyl group having 1 to 7 carbon atoms, an alkoxyl group having 1 to 7 carbon atoms, fluorine, and/or chlorine; an unsubstituted or fluorine-substituted linear or branched alkyl group having 4 to 6 carbon atoms; or phenyl group- or cyclopentyl group-substituted methylene or ethylene; wherein the phenyl group is optionally further substituted by one or more phenyl groups, Z.sup.1, Z.sup.2, Z.sup.3 and Z.sup.4 are the same or different and each is a hydrogen atom, a halogen atom, a C1 to C6 alkyl group, a C2 to C6 alkenyl group, a C2 to C6 alkynyl group, an organic oxy group represented by OR.sup.8, R.sup.8 is a C1 to C7 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group, Z.sup.5 is a hydrogen atom or a C1 to C6 alkyl group, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms], ##STR00133## [wherein R.sup.6 is hydrogen or a methyl group, X is an alkylene group having 4 to 6 carbon atoms or an ether group having 4 carbon atoms, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms], and ##STR00134## [wherein A is indole or naphthalene, and when A is indole, positions 3 and 5 of the indole are substituted by an acetic acid group and R.sup.7O, respectively, and when A is naphthalene, positions 1 and 7 of the naphthalene are substituted by an acetic acid group and R.sup.7O, respectively, R.sup.7 is an alkyl group having 1 to 5 carbon atoms or a benzyl group, wherein the benzene ring of the benzyl group is optionally substituted by one or more alkyl groups having 1 to 3 carbon atoms or alkoxyl groups having 1 to 3 carbon atoms, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms].
4. The method for enhancing fertilization function according to claim 3, wherein the compound is a compound of the following formula (I-2) or a physiologically acceptable salt thereof: Formula (I-2): ##STR00135##
5. A method for preparing a mammal fertilized egg, comprising step (a) of transferring at least one collected mammal sperm into a physiological aqueous solution containing one or more compounds selected from the group consisting of compounds of the following formula (I.sub.0), formula (II), and formula (III), and physiologically acceptable salts thereof when R.sup.3 is OH: ##STR00136## [wherein R.sup.1 is a benzoylmethyl group whose benzene ring is unsubstituted or a benzoylmethyl group whose benzene ring is substituted by an alkyl group having 1 to 7 carbon atoms, an alkoxyl group having 1 to 7 carbon atoms, fluorine, and/or chlorine: an unsubstituted or fluorine-substituted linear or branched alkyl group having 4 to 6 carbon atoms: or phenyl group- or cyclopentyl group-substituted methylene or ethylene: wherein the phenyl group is optionally further substituted by one or more phenyl groups, Z.sup.1, Z.sup.2, Z.sup.3 and Z.sup.4 are the same or different and each is a hydrogen atom, a halogen atom, a C1 to C6 alkyl group, a C2 to C6 alkenyl group, a C2 to C6 alkynyl group, an organic oxy group represented by OR.sup.8, R.sup.8 is a C1 to C7 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group, Z.sup.5 is a hydrogen atom or a C1 to C6 alkyl group, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms], ##STR00137## [wherein R.sup.6 is hydrogen or a methyl group, X is an alkylene group having 4 to 6 carbon atoms or an ether group having 4 carbon atoms, R.sup.3 is any group selected from OH OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms], and ##STR00138## [wherein A is indole or naphthalene, and when A is indole, positions 3 and 5 of the indole are substituted by an acetic acid group and R.sup.7O, respectively, and when A is naphthalene, positions 1 and 7 of the naphthalene are substituted by an acetic acid group and R.sup.7O, respectively, R.sup.7 is an alkyl group having 1 to 5 carbon atoms or a benzyl group, wherein the benzene ring of the benzyl group is optionally substituted by one or more alkyl groups having 1 to 3 carbon atoms or alkoxyl groups having 1 to 3 carbon atoms, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms]; and step (b-1) of culturing the mammal sperm of after step (a) in a culture medium containing at least one collected mammal ovum.
6. A method for preparing a mammal fertilized egg, comprising step (b-2) of culturing at least one collected mammal sperm and at least one collected mammal ovum in a culture medium containing one or more compounds selected from the group consisting of compounds of the following formula (I.sub.0), formula (II), and formula (III), and physiologically acceptable salts thereof when R.sup.3 is OH: ##STR00139## [wherein R.sup.1 is a benzoylmethyl group whose benzene ring is unsubstituted or a benzoylmethyl group whose benzene ring is substituted by an alkyl group having 1 to 7 carbon atoms, an alkoxyl group having 1 to 7 carbon atoms, fluorine, and/or chlorine; an unsubstituted or fluorine-substituted linear or branched alkyl group having 4 to 6 carbon atoms; or phenyl group- or cyclopentyl group-substituted methylene or ethylene; wherein the phenyl group is optionally further substituted by one or more phenyl groups, Z.sup.1, Z.sup.2, Z.sup.3 and Z.sup.4 are the same or different and each is a hydrogen atom, a halogen atom, a C1 to C6 alkyl group, a C2 to C6 alkenyl group, a C2 to C6 alkynyl group, an organic oxy group represented by OR.sup.8, R.sup.8 is a C1 to C7 alkyl group, a C2 to C6 alkenyl group or a C2 to C6 alkynyl group, Z.sup.5 is a hydrogen atom or a C1 to C6 alkyl group, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms], ##STR00140## [wherein R.sup.6 is hydrogen or a methyl group, X is an alkylene group having 4 to 6 carbon atoms or an ether group having 4 carbon atoms, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms], and ##STR00141## [wherein A is indole or naphthalene, and when A is indole, positions 3 and 5 of the indole are substituted by an acetic acid group and R.sup.7O, respectively, and when A is naphthalene, positions 1 and 7 of the naphthalene are substituted by an acetic acid group and R.sup.7O, respectively, R.sup.7 is an alkyl group having 1 to 5 carbon atoms or a benzyl group, wherein the benzene ring of the benzyl group is optionally substituted by one or more alkyl groups having 1 to 3 carbon atoms or alkoxyl groups having 1 to 3 carbon atoms, R.sup.3 is any group selected from OH, OR.sup.4, NHR.sup.4, and NR.sup.4R.sup.5, and R.sup.4 and R.sup.5 are the same or different and each is a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms].
7. The method for preparing a mammal fertilized egg according to claim 6, wherein the compound is a compound of the following formula (I-2) or a physiologically acceptable salt thereof. ##STR00142##
8. The method for preparing a mammal fertilized egg according to claim 5, wherein the compound is a compound of the following formula (I-2) or a physiologically acceptable salt thereof. ##STR00143##
Description
EXAMPLE 1
[0084] 1. Synthesis of Compound Group of the Present Invention
[0085] Starting materials for synthesis, reaction reagents, etc., for use in methods for synthesizing compounds shown below are general commercially available products. As for reaction solvents and reaction temperatures, a reaction is carried out using a solvent and a temperature usually used for the reaction, unless otherwise specified. Each reaction is carried out in an argon or dried nitrogen atmosphere.
Synthesis of Compound #1]
[0086] 4-Phenyl-2-(4-chloro-1H-indol-3-yl)-4-oxo-butane (compound #1) was synthesized by a method for synthesizing compound #20 mentioned later using 4-chloroindole instead of indole.
[0087] [Synthesis of Compound #2 and Compound #3]
[0088] 4-(4-Chlorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic acid (compound #2) and 3-(1H-indol-3-yl)-1-oxo-1-phenyl-butane (compound #3) were each synthesized according to a method described in Sayed, G. H. et al, “Synthesis and reactions of some β-aroyl-α-(indol-3-yl)propionic acids” Journal of the Chemical Society of Pakistan, 7 (4), 263-72; 1985.
[0089] (Compound #2)
##STR00036##
Synthesis of compound #4
Trans-4-(4-fluorophenyl)-4-oxo-2-butenoic Acid
[0090] ##STR00037##
[0091] In a 50-mL round-bottomed flask filled with nitrogen, fluorobenzene (0.50 g, 5.21 mmol) was dissolved in dichloromethane (20 mL). To the solution, maleic anhydride (0.51 g, 5.20 mmol) and aluminum chloride (1.40 g, 10.49 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was pH-adjusted to 1 by the addition of 1 N hydrochloric acid (10 mL), followed by extraction with ethyl acetate (40 mL) three times. The organic layer was washed with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified by recrystallization (benzene) to obtain trans-4-(4-fluorophenyl)-4-oxo-2-butenoic acid (0.57 g, yield: 56%); Melting point: 114.8 to 119.6° C.; .sup.1H NMR (CDCl.sub.3): δ 8.06 (m, 2H), 7.98 (d, J=15.4 Hz, 1H), 7.21 (m, 2H), 6.90 (d, J=15.4 Hz, 1H); .sup.13C NMR (CDCl.sub.3): δ 187.5, 170.7, 166.3 (d, J.sub.C-F=255.5 Hz), 138.0, 132.8 (d, J.sub.C-F=3.2 Hz), 131.7 (d, J.sub.C-F=9.9 Hz), 131.6, 116.2 (d, J.sub.C-F=22.1 Hz); IR (neat): 2972, 1705, 1665 cm.sup.−1; FAB-MS m/z 195 [M+H].sup.+.
4-(4-Fluorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic Acid (Compound #4)
[0092] ##STR00038##
[0093] In a 30-mL round-bottomed flask, trans-4-(4-fluorophenyl)-4-oxo-2-butenoic acid (0.21 g, 1.08 mmol) was dissolved in benzene (10 mL). To the solution, indole (0.26 g, 2.19 mmol) was added, and the mixture was stirred at 80° C. for 8 hours and stirred until the temperature became room temperature. The solvent in the reaction solution was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (chloroform:methanol=20:1) to obtain 4-(4-fluorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic acid (compound #4) (0.15 g, yield: 47%); Melting point: 161.6 to 166.6° C.; .sup.1H NMR (DMSO-d.sub.6): δ 8.13 (m, 2H), 7.68 (d, J=7.9 Hz, 1H), 7.35 (m, 4H), 7.09 (t, J=7.2 Hz, 1H), 7.00 (t, J=7.1 Hz, 1H), 4.34 (dd, J=10.7, 3.9 Hz, 1H), 4.03 (dd, J=18.1, 10.7 Hz, 1H), 3.34 (dd, J=18.1, 3.9 Hz, 1H); .sup.13C NMR (DMSO-d.sub.6): δ 197.96, 175.61, 166.00 (d, J.sub.C-F=250.0 Hz), 137.16, 134.11, 131.93 (d, J.sub.C-F=10.0 Hz), 127.15, 124.16, 122.07, 119.97, 119.53, 116.6 (d, J.sub.C-F=22.0 Hz), 112.79, 112.42, 42.03, 38.57; IR (neat): 3419, 2925, 1679 cm.sup.−1; HRFAB-MS found m/z 312.1028 [M+H].sup.+, calcd for 312.1036 (C.sub.18H.sub.15FNO.sub.3).
Synthesis of Compound #5
Trans-4-(2,4-difluorophenyl)-4-oxo-2-butenoic Acid
[0094] ##STR00039##
[0095] In a 50-mL round-bottomed flask filled with nitrogen, 1,3-difluorobenzene (0.51 g, 4.47 mmol) was dissolved in dichloromethane (20 mL). To the solution, maleic anhydride (0.43 g, 4.46 mmol) and aluminum chloride (1.20 g, 9.01 mmol) were added, and the mixture was stirred at room temperature for 4 hours and stirred until the temperature became room temperature. The reaction solution was pH-adjusted to 1 by the addition of 1 N hydrochloric acid (10 mL), followed by extraction with ethyl acetate (40 mL) three times. The organic layer was washed with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified by recrystallization from benzene to obtain trans-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (0.57 g, yield: 56%); Melting point: 114.8 to 119.6° C.; H NMR (acetone-d.sub.6): δ 7.98 (m, 1H), 7.71 (dd, J.sub.H-F=15.6, 3.4 Hz, 1H), 7.23 (m, 2H), 6.75 (dd, J.sub.H-F=15.6, 1.2 Hz, 1H); .sup.13C NMR (acetone-d.sub.6): δ 187.2 (d, J.sub.C-F=2.6 Hz), 166.9 (dd, J.sub.C-F=254.5, 12.3 Hz), 166.4, 163.4 (dd, J.sub.C-F=254.5, 12.9 Hz), 140.0 (d, J.sub.C-F=6.1 Hz), 134.0 (dd, J.sub.C-F=10.9, 3.6 Hz), 133.0 (d, J.sub.C-F=1.6 Hz), 123.3 (dd, J.sub.C-F=12.4, 3.6 Hz), 113.4 (dd, J.sub.C-F=21.5, 3.6 Hz), 105.8 (dd, J.sub.C-F=27.3, 26.3 Hz); IR (neat): 2917, 1697, 1661 cm.sup.−1; FAB-MS m/z 213 [M+H].sup.+.
4-(2,4-Difluorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic Acid (Compound #5)
[0096] ##STR00040##
[0097] In a 30-mL round-bottomed flask, trans-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (0.39 g, 1.84 mmol) was dissolved in benzene (10 mL). To the solution, indole (0.43 g, 2.19 mmol) was added, and the mixture was stirred 80° C. for 8 hours and stirred until the temperature became room temperature. The solvent in the reaction solution was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (chloroform:methanol=20:1) to obtain 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxo-butanoic acid (0.15 g, yield: 51%); Melting point: 180.2 to 184.6° C.; .sup.1H NMR (DMSO-d.sub.6): δ 7.98 (m, 1H), 7.65 (d, J=7.9 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.42 (m, 1H), 7.28 (d, J=2.3 Hz, 1H), 7.24 (m, 1H), 7.09 (t, J=7.1 Hz, 1H), 7.01 (t, J=7.5 Hz, 1H), 4.34 (dd, J=10.5, 3.5 Hz, 1H), 3.90 (ddd, J.sub.H-F=18.5, 10.6, 2.4 Hz, 1H), 3.30 (ddd, J.sub.H-F=18.5, 6.1, 3.5 Hz, 1H); .sup.13C NMR (DMSO-d.sub.6): δ 195.2 (d, J.sub.C-F=4.1 Hz), 174.8, 165.2 (d, J.sub.C-F=253.0, 13.4 Hz), 162.2 (d, J.sub.C-F=255.5, 13.4 Hz), 136.4, 132.7 (dd, J.sub.C-F=108, 4.1 Hz), 126.3, 123.3, 122.2 (dd, J.sub.C-F=12.3, 3.6 Hz), 121.4, 119.1, 118.8, 112.6 (dd, J.sub.C-F=21.1, 3.6 Hz), 111.9, 111.8, 105.4 (dd, J.sub.C-F=26.1 Hz), 45.6 (d, J.sub.C-F=6.3 Hz), 37.9; IR (neat): 3382, 2919, 1678 cm.sup.−1; HRFA-MS found m/z 330. 0910 [M+H].sup.+, calcd for 330. 0942 (C.sub.18H.sub.14F.sub.2NO.sub.3).
Synthesis of Compound #6
Trans-4-(2,4-dimethylphenyl)-4-oxo-2-butenoic Acid
[0098] ##STR00041##
[0099] In a 50-mL round-bottomed flask filled with nitrogen, m-xylene (1.00 g, 9.42 mmol) was dissolved in dichloromethane (40 mL). To the solution, maleic anhydride (0.93 g, 9.42 mmol) and aluminum chloride (2.51 g, 18.84 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was pH-adjusted to 1 by the addition of 1 N hydrochloric acid (10 mL), followed by extraction with ethyl acetate (40 mL) three times. The organic layer was washed with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified by recrystallization (benzene) to obtain trans-4-(2,4-dimethylphenyl)-4-oxo-2-butenoic acid (1.49 g, yield: 77%); Melting point: 85.4 to 88.8° C.; .sup.1H NMR (CDCl.sub.3): δ 7.75 (d, J=15.6 Hz, 1H), 7.56 (d, J=8.2 Hz, 1H), 7.10 (m, 2H), 6.70 (d, J=15.6 Hz, 1H), 2.50 (s, 3H), 2.38 (s, 3H); .sup.13C NMR (CDCl.sub.3): δ 192.5, 170.9, 143.1, 141.7, 139.5, 133.6, 133.0, 130.9, 130.0, 126.4, 21.5, 21.2; IR (neat): 2986, 1703, 1667 cm.sup.−1; FAB-MS m/z 205 [M+H].sup.+.
4-(2,4-Dimethylphenyl)-2-(1-propyl-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound #6)
[0100] ##STR00042##
[0101] In a 30-mL round-bottomed flask, trans-4-(2,4-dimethylphenyl)-4-oxo-2-butenoic acid (0.50 g, 2.45 mmol) was dissolved in benzene (10 mL). To the solution, N-propylindole (0.85 g, 4.90 mmol) was added, and the mixture was stirred at 80° C. for 8 hours and stirred until the temperature became room temperature. The solvent in the reaction solution was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (chloroform:acetone=5:1) to obtain 4-(2,4-dimethylphenyl)-2-(1-propyl-1H-indol-3-yl)-4-oxo-butanoic acid (0.98 g, yield: 67%); Melting point: 139 to 141° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.70 (d, J=7.8 Hz, 1H), 7.59 (d, J=7.8 Hz, 1H), 7.28 (d, J=8.2 Hz, 1H), 7.18 (t, J=15.1 Hz, 1H), 7.07 (m, 2H), 6.99 (d, J=8.7 Hz, 2H), 4.56 (dd, J=6.0, 4.1 Hz, 1H), 3.97 (m, 2H), 3.92 (m, 1H), 3.28 (dd, J=17.8, 4.1 Hz, 1H), 2.43 (s, 3H), 2.30 (s, 3H), 1.80 (m, 2H), 0.89 (t, J=14.7, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 200.9, 179.7, 142.3, 138.9, 136.3, 134.1, 132.8, 129.1, 126.7, 126.2, 126.1, 121.7, 119.4, 119.2, 110.6, 109.5, 48.0, 44.0, 38.0, 23.4, 21.5, 21.3, 11.5; IR (neat): 3428, 2923, 1707 cm; FAB-MS m/z 364 [M+H].sup.+.
[0102] 4-Phenyl-2-(1H-5-ethoxyindol-3-yl)-4-oxo-butanoic acid (compound #7) was synthesized in the same way as in compound #20 using 5-ethoxyindole instead of indole.
[0103] Compounds #8, 13 to 15, 17 to 19, and 21 to 25 were each synthesized with methyl N-methoxycarbonylindoleacetate as a key intermediate.
1-Methoxycarbonylindole-3-acetic Acid Methyl Ester
[0104] ##STR00043##
Indole-3-Acetic Acid Methyl Ester
[0105] Indole-3-acetic acid (2.00 g, 11.42 mmol) was dissolved in methanol (40 ml). To this solution, acetyl chloride (0.5 ml, 6.688 mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction was terminated by the addition of a saturated aqueous solution of sodium bicarbonate, followed by extraction with ethyl acetate (50 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain indole-3-acetic acid methyl ester (2.14 g, yield: 99%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.13 (s, 1H), 6.97 (s, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.23 (d, J=7.9 Hz, 1H), 7.10-7.19 (m, 2H), 3.67 (s, 3H), 3.76 (s, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.3, 136.0, 127.1, 123.2, 122.0, 119.5, 118.6, 111.2, 108.0, 51.9, 31.0; IR (neat): 3410, 1730, 1458, 1435, 1337, 1164, 1095, 1011 cm.sup.−1; EI-MS m/z 189 [M].sup.+.
1-Methoxycarbonyl-3-Indoleacetic Acid Methyl Ester
[0106] Methyl indole-3-acetate (2.00 g, 10.57 mmol) was dissolved in dichloromethane (30 ml). To this solution, tetrabutylammonium iodide (TBAI, 30.0 mg, 0.081 mmol) and a 30% aqueous sodium hydroxide solution (24 ml) were added, and the mixture was cooled to 0° C. To the reaction solution, methyl formate chloride (1.96 g, 20.73 mmol) was added, and the mixture was stirred at 0° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction was terminated by the addition of 6 N hydrochloric acid. Water (50 ml) was added thereto, followed by extraction with chloroform (50 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain methyl N-methoxycarbonylindole-3-acetate (2.26 g, yield: 87%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=7.0 Hz, 1H), 7.59 (s, 1H), 7.53 (d, J=7.7 Hz, 1H), 7.35 (t, J=7.5 Hz, 1H), 7.27 (t, J=7.4 Hz, 1H), 4.00 (s, 3H), 3.72 (s, 3H), 3.71 (s, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 171.1, 151.1, 135.2, 129.9, 124.6, 123.8, 122.8, 118.9, 115.0, 113.8, 53.5, 51.9, 30.6; IR (neat): 1746, 1455, 1382, 1258, 1164, 1089, 1018 cm.sup.−1; EI-MS: m/z 247 [M].sup.+.
[0107] Compounds #8 and 9 were each synthesized according to a method described in International Publication No. WO 2010/045451.
Synthesis of Compound #8
2-(N-tert-Butoxycarbonyl-4-piperidinyl)ethanol
[0108] ##STR00044##
[0109] 2-(4-Piperidinyl)ethanol (1.0 g, 7.7 mmol) was dissolved in methanol (50 ml). To this solution, di-tert-butyl carbonate (2.0 g, 9.3 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the solvent was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (hexane:acetone=9:1) to obtain N-tert-butoxycarbonyl-2-(4-piperidinyl)ethanol (1.68 g, yield: 95%).
Ethane 2-(N-tert-butoxycarbonyl-4-piperidinyl)-1-iodide
[0110] ##STR00045##
[0111] Triphenylphosphine (2.56 g, 9.760 mmol) and imidazole (0.66 g, 9.694 mmol) were dissolved in dichloromethane (15 ml), and the solution was stirred for 5 minutes. Then, iodine (2.47 g, 9.732 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (4 ml) solution of N—N-tert-butoxycarbonyl-2-(4-piperidinyl)ethanol (1.49 g, 6.497 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=9:1) to obtain ethane N-tert-butoxycarbonyl-2-(4-piperidinyl)-1-iodide (2.13 g, yield: 96%).
α-[2-(N-tert-Butoxycarbonyl-4-piperidinyl)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0112] ##STR00046##
[0113] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (500 mg, 2.022 mmol) and hexamethylphosphoric triamide (HMPA, 1.81 g, 10.11 mmol) were dissolved in tetrahydrofuran (4 ml), and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide (LDA) in cyclohexane (2.16 ml, 1.6 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (2 ml) solution of ethane 2-(N-tert-butoxycarbonyl-4-piperidinyl)-1-iodide (686 mg, 2.022 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (15 ml), followed by extraction with ethyl acetate (15 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain α-2-(N-tert-butoxycarbonyl-4-piperidinyl)-ethyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester (626 mg, yield: 68%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.19 (m, 1H), 7.61 (d, J=7.8 Hz, 1H), 7.56 (s, 1H), 7.35 (t, J=7.7 Hz, 1H), 7.25-7.30 (m, 1H), 3.79-4.15 (m, 5H), 3.77 (t, J=7.6 Hz, 1H), 3.68 (s, 3H), 2.65 (m, 2H), 2.05 (m, 2H), 1.65 (m, 2H), 1.25-1.50 (m, 12H), 1.05-1.19 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.9, 168.0, 154.8, 135.4, 129.3, 124.8, 123.1, 122.9, 119.2, 119.2, 115.2, 79.1, 53.7, 53.0, 52.1, 48.9, 43.7, 42.7, 35.9, 34.3, 32.0, 29.5, 28.4; FAB-MS: m/z 459 [M+H].sup.+.
α-[2-(1-Acetyl-4-piperidinyl)-ethyl]-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0114] ##STR00047##
[0115] α-[2-(N-tert-Butoxycarbonyl-4-piperidinyl)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (100 mg, 0.218 mmol) was dissolved in dichloromethane (2 ml). To the solution, trifluoroacetic acid (1.0 ml, 13.07 mmol) was added, and the mixture was stirred at room temperature for 5 minutes. The reaction solution was added dropwise to a 10% aqueous sodium carbonate solution (10 mL) to terminate the reaction. This solution was subjected to extraction with ethyl acetate (10 mL) three times. The organic layer was washed twice with saturated saline (10 mL) and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain α-[2-(4-piperidinyl)-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (74.1 mg). This compound (74.1 mg, 0.207 mmol) was dissolved in tetrahydrofuran (3 mL). To the solution, triethylamine (0.2 mL) and acetyl chloride (10 mg) were added, and the mixture was stirred at room temperature for 1.5 hours. The reaction was terminated by the addition of a saturated aqueous solution of ammonium chloride (10 mL), followed by extraction with ethyl acetate (10 mL) three times. The organic layer was washed twice with saturated saline (10 mL) and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:acetone=9:1) to obtain α-[2-(1-acetyl-4-piperidinyl)-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (53.9 mg, yield: 65%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=6.7 Hz, 1H), 7.61 (d, J=7.8 Hz, 1H), 7.56 (s, 1H), 7.35 (t, J=8.4 Hz, 1H), 7.25-7.28 (m, 1H), 4.57 (d, J=12.8 Hz, 1H), 4.03 (s, 3H), 3.73-3.79 (m, 2H), 3.68 (s, 3H), 2.99 (t, J=12.9 Hz, 1H), 2.50 (t, J=12.6 Hz, 1H), 1.91-2.19 (m, 5H), 1.73 (t, J=10.4 Hz, 2H), 1.49 (m, 1H), 1.26-1.32 (m, 2H), 1.05-1.12 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.8, 168.7, 151.2, 135.4, 129.3, 124.8, 122.9, 119.2, 119.1, 115.2, 53.7, 52.1, 46.6, 42.7, 41.7, 35.9, 34.2, 32.5, 31.6, 29.2, 21.4; FAB-MS: m/z 401 [M+H].sup.+.
α-2-(1-Acetyl-4-piperidinyl)-ethyl-3-indoleacetic Acid (Compound #8)
[0116] ##STR00048##
[0117] α-2-(1-Acetyl-4-piperidinyl)-ethyl-N-methoxycarbonyl-3-indoleacetic acid methyl ester (48.0 mg, 0.120 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:acetone=3:2) to obtain α-2-(1-acetyl-4-piperidinyl)-ethyl-3-indoleacetic acid (compound #8) (25.5 mg, yield: 65%: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.54 (s, 1H), 7.67 (d, J=7.9 Hz, 1H), 7.31 (d, J=8.0 Hz, 1H), 7.16 (t, J=7.7 Hz, 1H), 7.07-7.11 (m, 2H), 4.48 (d, J=12.7 Hz, 1H), 3.81 (t, J=7.5 Hz, 1H), 3.66 (d, J=13.2 Hz, 1H), 2.89 (t, J=12.5 Hz, 1H), 2.43 (t, J=12.6 Hz, 1H), 1.86-2.17 (m, 5H), 1.62 (t, J=16.5 Hz, 2H), 1.41 (m, 1H), 1.22-1.28 (m, 2H), 0.93-1.01 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 178.8, 169.3, 136.2, 126.5, 122.3, 122.0, 119.5, 119.1, 113.3, 111.4, 46.7, 43.1, 42.0, 35.7, 34.2, 32.5, 31.6, 29.7, 21.3; IR (neat): 3410, 1699, 1454, 1271 cm.sup.−1; FAB-MS: m/z 329 [M+H].sup.+.
[0118] α-2-(1-Acetyl-4-piperidinyl)-methyl-3-indoleacetic acid (compound #9) was synthesized by the same approach as in compound #8 using N-tert-butoxycarbonyl-4-piperidinylmethanol instead of 2-(N-tert-butoxycarbonyl-4-piperidinyl)ethanol.
Synthesis of Compound #10
α-4-Aminobutyl-N-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0119] ##STR00049##
[0120] To α-(N-tert-butoxycarbonyl-4-amino-1-butyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (150 mg, 0.358 mmol), trifluoroacetic acid (0.4 ml, 5.227 mmol) was added, and the mixture was stirred at room temperature. After 5 minutes, the reaction solution was added dropwise to an aqueous sodium bicarbonate solution to terminate the reaction. Water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain α-4-aminobutyl-N-methoxycarbonyl-3-indoleacetic acid methyl ester.
α-[N-(1-Acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-N-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0121] ##STR00050##
[0122] α-4-Aminobutyl-N-methoxycarbonyl-3-indoleacetic acid methyl ester (150 mg, 0.493 mmol) was dissolved in tetrahydrofuran (3 ml). To this solution, N-acetyl-L-proline (116 mg, 0.738 mmol), N-hydroxysuccinimide (85.0 mg, 0.739 mmol), dicyclohexylcarbodiimide (152 mg, 0.737 mmol), and 4-N,N-dimethylaminopyridine (72.0 mg, 0.589 mmol) were added, and the mixture was stirred at room temperature for 7 hours. The reaction was terminated with a saturated aqueous solution of ammonium chloride, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:acetone=7:3) to obtain α-[N-(1-acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-N-methoxycarbonyl-3-indoleacetic acid methyl ester (107 mg, yield: 49%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.17 (d, J=7.1 Hz, 1H), 7.61 (d, J=7.7 Hz, 1H), 7.55 (s, 1H), 7.33 (t, J=7.8 Hz, 1H), 7.25 (t, J=7.4 Hz, 1H), 7.18 (s, 1H), 4.50 (d, J=7.3 Hz, 1H), 4.02 (s, 3H), 3.80 (t, J=7.6 Hz, 1H), 3.67 (s, 3H), 3.36-3.58 (m, 2H), 3.10-3.26 (m, 2H), 1.76-2.40 (m, 9H), 1.49-1.56 (m, 2H), 1.33-1.38 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.8, 171.0, 170.8, 151.1, 135.3, 129.2, 124.6, 122.9, 122.8, 119.2, 119.1, 115.0, 59.4, 53.6, 51.9, 48.1, 42.3, 38.9, 31.5, 29.0, 27.2, 24.8, 24.7, 22.3; FAB-MS: m/z 458 [M+H].sup.+.
α-[N-(1-Acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-3-indoleacetic Acid (Compound #10)
[0123] ##STR00051##
[0124] α-[N-(1-Acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-N-methoxycarbonyl-3-indoleacetic acid methyl ester (80.0 mg, 0.175 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain α-[N-(1-acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-3-indoleacetic acid (compound #10) (63.6 mg, yield: 94%): .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 10.21 (s, 1H), 8.03 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 7.27 (s, 1H), 7.09 (t, J=7.3 Hz, 1H), 7.01 (t, J=7.6 Hz, 1H), 4.35 (d, J=7.2 Hz, 1H), 3.85 (t, J=7.6 Hz, 1H), 3.53 (m, 1H), 3.40-3.46 (m, 1H), 3.23 (m, 1H), 3.10-3.17 (m, 1H), 1.85-2.14 (m, 9H), 1.36-1.50 (m, 4H); .sup.13C NMR (100 MHz, acetone-d.sub.6): δ 175.8, 172.1, 170.3, 137.3, 127.5, 123.3, 121.9, 119.7, 119.3, 114.1, 112.0, 60.5, 48.3, 43.3, 39.2, 32.9, 32.5, 25.4, 25.1, 22.2; IR (Neat): 3300, 1634, 1456, 1245 cm; FAB-MS: m/z 386 [M+H].sup.+.
Synthesis of Compound #11
α-[2-(2-Aminoethoxy)-ethyl]-N-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0125] ##STR00052##
[0126] To α-[N-tert-butoxycarbonyl-(2-aminoethoxyethyl)]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (140 mg, 0.322 mmol), trifluoroacetic acid (0.3 ml, 3.920 mmol) was added, and the mixture was stirred at room temperature. After 5 minutes, the reaction solution was added dropwise to an aqueous sodium bicarbonate solution to terminate the reaction. Water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain α-[2-(2-aminoethoxy)-ethyl]-N-methoxycarbonyl-3-indoleacetic acid methyl ester (80.0 mg, yield: 74%).
α-{N-(1-Acetylpyrrolidine-2-carbonyl)-[2-(2-aminoethoxy)-ethyl]}-N-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0127] ##STR00053##
[0128] α-[2-(2-Aminoethoxy)-ethyl]-N-methoxycarbonyl-3-indoleacetic acid methyl ester (80.0 mg, 0.239 mmol) was dissolved in tetrahydrofuran (3 ml). To this solution, N-acetyl-L-proline (56.4 mg, 0.359 mmol), N-hydroxysuccinimide (41.2 mg, 0.358 mmol), dicyclohexylcarbodiimide (74.0 mg, 0.359 mmol), and 4-N,N-dimethylaminopyridine (35.0 mg, 0.286 mmol) were added, and the mixture was stirred at room temperature for 7 hours. The reaction was terminated with a saturated aqueous solution of ammonium chloride, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:acetone=7:3) to obtain α-[N-(1-acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-N-methoxycarbonyl-3-indoleacetic acid methyl ester (76.1 mg, yield: 67%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=7.1 Hz, 1H), 7.57-7.66 (m, 2H), 7.35 (t, J=7.7 Hz, 1H), 7.25-7.28 (m, 2H), 4.56 (t, J=8.3 Hz, 1H), 4.09 (t, J=7.6 Hz, 1H), 4.03 (s, 3H), 3.68 (s, 3H), 3.59 (t, J=9.0 Hz, 1H), 3.32-3.52 (m, 7H), 2.36-2.48 (m, 2H), 1.84-2.18 (m, 7H), 1.49-1.56 (m, 2H), 1.33-1.38 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 174.2, 171.5, 170.8, 151.1, 135.5, 129.3, 124.8, 123.1, 123.0, 119.4, 118.9, 115.2, 69.4, 68.4, 59.2, 53.8, 52.2, 48.2, 39.5, 39.2, 32.2, 27.8, 25.0, 22.5; FAB-MS: m/z 474 [M+H].sup.+.
α-[N-(1-Acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-3-indoleacetic Acid (Compound #11)
[0129] ##STR00054##
[0130] α-[N-(1-Acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-N-methoxycarbonyl-3-indoleacetic acid methyl ester (60.0 mg, 0.127 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain α-[N-(1-acetylpyrrolidine-2-carbonyl)-4-aminobutyl]-3-indoleacetic acid (compound #11) (36.6 mg, yield: 72%): .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 8.48 (d, J=13.4 Hz, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.34 (d, J=8.1 Hz, 1H), 7.09-7.21 (m, 3H), 4.67 (t, J=8.3 Hz, 1H), 4.40-4.11 (m, 1H), 3.18-3.76 (m, 8H), 2.46-2.67 (m, 4H), 1.86-2.22 (m, 7H); .sup.13C NMR (100 MHz, acetone-d.sub.6): δ 178.0, 171.6, 171.2, 136.1, 126.5, 122.3, 122.0, 119.4, 118.9, 113.7, 111.2, 69.3, 68.6, 60.0, 48.5, 41.2, 39.9, 33.7, 29.1, 24.8, 22.3; IR (Neat): 3317, 1634, 1456, 1247, 1119 cm.sup.−1; FAB-MS: m/z 402 [M+H].sup.+.
Synthesis of compound #12
α-(N-tert-Butoxycarbonyl-6-amino-1-hexyl)-α-(1-naphthyl)-acetic Acid Methyl Ester
[0131] ##STR00055##
[0132] α-(1-Naphthyl)-acetic acid methyl ester (150 mg, 0.75 mmol) was dissolved in tetrahydrofuran. To the solution, hexamethylphosphoramide (HMPA, 671 mg, 3.75 mmol) was added, and the mixture was cooled to −78° C. To this solution, lithium diisopropylamide (1.5 M solution in cyclohexane, 0.75 ml, 1 mmol) was added dropwise, and the mixture was stirred at −78° C. for 30 minutes. Then, a tetrahydrofuran solution (2 mL) of N-tert-butoxycarbonyl-6-amino-1-iodohexane (270 mg, 0.82 mmol) was added dropwise thereto, and the mixture was stirred at −78° C. for 1 hour. The temperature of the reaction solution was raised to 0° C. over 15 minutes, and then, water (50 mL) was added to the solution, followed by extraction with ethyl acetate (50 mL) twice. The organic layer was washed with a saturated ammonium chloride solution (20 mL) and subsequently saline (20 mL) and then dehydrated over sodium sulfate to dryness under reduced pressure. The reaction product was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain α-(N-tert-butoxycarbonyl-6-amino-1-hexyl)-α-(1-naphthyl)-acetic acid methyl ester (271 mg, yield: 91%): .sup.1H NMR (400 MHz, CDCl.sub.3) 8.11 (d, J=8.5 Hz, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.40-7.54 (m, 4H), 4.71 (s, 1H), 4.36 (t, J=7.8 Hz, 1H), 3.61 (s, 3H), 3.04 (m, 2H), 2.07 (m, 2H), 1.24-1.48 (m, 17H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 174.7, 155.9, 135.3, 133.8, 131.3, 128.8, 127.5, 126.1, 125.4, 125.3, 124.6, 122.8, 78.7, 51.8, 46.5, 40.3, 32.9, 29.7, 28.9, 28.2, 27.6, 26.3; FAB-MS: m/z 400 [M+H].sup.+.
α-(N-tert-Butoxycarbonyl-6-amino-1-hexyl)-α-(1-naphthyl)-acetic Acid (Compound #12)
[0133] ##STR00056##
[0134] α-(N-tert-Butoxycarbonyl-6-amino-1-hexyl)-α-(1-naphthyl)-acetic acid methyl ester (100 mg, 0.25 mmol) was dissolved in a mixed solution of methanol and an aqueous sodium hydroxide solution (2 N aqueous sodium hydroxide solution:methanol=1:4, 5 mL), and the solution was heated at 50° C. for 1 hour. The reaction solution was pH-adjusted to 3.5 with 6 N hydrochloric acid, and methanol was removed by distillation under reduced pressure. To this solution, water (15 mL) was added, followed by extraction with ethyl acetate (50 mL) twice. The organic layer was washed with a saturated ammonium chloride solution (20 mL) and subsequently saline (20 mL) and then dehydrated over sodium sulfate to dryness under reduced pressure. The reaction product was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(N-tert-butoxycarbonyl-6-amino-1-hexyl)-α-(1-naphthyl)-acetic acid (compound #12) (90 mg, yield: 93%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.13 (d, J=8.4 Hz, 1H), 7.84 (d, J=7.9 Hz, 1H), 7.75 (d, J=8.1 Hz, 1H), 7.41-7.53 (m, 4H), 4.56 (s, 1H), 4.35 (t, J=7.4 Hz, 1H), 3.03 (m, 2H), 2.05 (m, 2H), 1.22-1.46 (m, 17H); .sup.13C NMR (100 MHz, CDCl.sub.3) 179.0, 156.0, 135.1, 133.9, 131.6, 128.9, 127.7, 126.2, 125.5, 125.4, 124.9, 123.1, 79.0, 46.6, 40.4, 32.7, 29.8, 29.0, 28.3, 27.7, 26.4; IR (neat): 3417, 1705, 1457, 1268, 1099 cm; FAB-MS: m/z 386 [M+H].sup.+.
Synthesis of Compound #13
N-tert-Butoxycarbonyl-6-amino-1-hexanol
[0135] ##STR00057##
[0136] 6-Amino-1-hexanol (1.0 g, 8.533 mmol) was dissolved in methanol (10 ml). To this solution, di-tert-butyl carbonate (1.86 g, 8.522 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the solvent was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (hexane:acetone=9:1) to obtain N-tert-butoxycarbonyl-6-aminohexanol (1.80 g, yield: 97%).
N-tert-Butoxycarbonyl-6-amino-1-iodohexane
[0137] ##STR00058##
[0138] Triphenylphosphine (2.35 g, 8.96 mmol) and imidazole (0.61 g, 8.96 mmol) were dissolved in dichloromethane (15 ml), and the solution was stirred for 5 minutes. Then, iodine (2.28 g, 8.98 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (4 ml) solution of N-tert-butoxycarbonyl-6-aminohexanol (1.3 g, 5.98 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=9:1) to obtain N-tert-butoxycarbonyl-6-amino-1-iodohexane (1.67 g, yield: 86%).
[0139] α-Methyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester was synthesized according to a method described in Katayama M, Kato Y, Marumo S. “Synthesis, absolute configuration and biological activity of both enantiomers of 2-(5,6-dichloro-3-indolyl)propionic acid: new dichloroindole auxins” Bioscience, Biotechnology, and Biochemistry, 65 (2), 270-276; 2001.
α-(N-tert-Butoxycarbonyl-6-amino-1-hexyl)-α-methyl-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0140] ##STR00059##
[0141] In a nitrogen atmosphere, α-methyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester (83.8 mg, 0.321 mmol) was dissolved in tetrahydrofuran (2 ml), and the solution was cooled to −78° C. This solution was slowly added dropwise to a 1.0 M solution of lithium bistrimethylsilylamide (LHMDS) in tetrahydrofuran (0.69 ml, 1.5 eq), and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of N-tert-butoxycarbonyl-6-amino-1-iodohexane (105 mg, 0.321 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain α-(N-tert-butoxycarbonyl-6-amino-1-hexyl)-α-methyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester (68.6 mg, yield: 46%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.19 (d, J=6.3 Hz, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.48 (s, 1H), 7.32 (t, J=7.5 Hz, 1H), 7.21 (t, J=7.5 Hz, 1H), 4.54 (s, 1H), 4.03 (s, 3H), 3.62 (s, 3H), 3.06 (m, 2H), 2.04-2.12 (m, 2H), 1.61 (s, 3H), 1.17-1.43 (m, 17H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 176.3, 155.9, 151.3, 135.8, 128.6, 124.9, 124.5, 122.8, 122.0, 120.0, 115.2, 78.9, 53.7, 52.1, 45.5, 40.4, 37.2, 29.9, 29.5, 28.3, 26.5, 24.2, 22.5; FAB-MS: m/z 460 [M].sup.+.
α-(N-tert-Butoxycarbonyl-6-amino-1-hexyl)-α-methyl-3-indoleacetic Acid (Compound #13)
[0142] ##STR00060##
[0143] α-(N-tert-Butoxycarbonyl-6-amino-1-hexyl), α-methyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester (60.0 mg, 0.130 mmol) was dissolved in methanol (4.6 ml). To the solution, water (0.4 ml) and potassium hydroxide (1.68 g, 30 mmol) were added, and the mixture was stirred at 70° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (benzene:acetone=85:15) to obtain α-(N-tert-butoxycarbonyl-6-amino-1-hexyl)-α-methyl-3-indoleacetic acid (compound #13) (40.0 mg, yield: 79%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.26 (s, 1H), 7.71 (d, J=8.0 Hz, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.16 (t, J=7.4 Hz, 1H), 7.06 (t, J=7.3 Hz, 1H), 7.04 (s, 1H), 4.52 (s, 1H), 3.03 (m, 2H), 2.08-2.17 (m, 2H), 1.63 (s, 3H), 1.23-1.48 (m, 17H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 181.7, 156.1, 136.7, 125.5, 121.4, 120.4, 119.2, 118.8, 111.3, 79.1, 45.7, 40.5, 37.5, 29.7, 28.5, 26.5, 24.2, 22.6; IR (neat): 3415, 3339, 1699, 1519, 1460, 1369, 1249, 1170 cm.sup.−1;
FAB-MS: m/z 389 [M+H].SUP.+..
Synthesis of Compound #14
2-(N-tert-Butoxycarbonyl-2-aminoethoxy)-ethanol
[0144] ##STR00061##
[0145] 2-(2-Aminoethoxy)-ethanol (1.0 g, 9.511 mmol) was dissolved in methanol (10 ml). To this solution, di-tert-butyl carbonate (2.07 g, 9.485 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the solvent was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (hexane:acetone=3:2) to obtain 2-(N-tert-butoxycarbonyl-2-aminoethoxy)-ethanol (1.78 g, yield: 91%)
2-(N-tert-Butoxycarbonyl-2-aminoethoxy)-1-iodoethane
[0146] ##STR00062##
[0147] Triphenylphosphine (2.87 g, 10.94 mmol) and imidazole (0.75 g, 11.02 mmol) were dissolved in dichloromethane (15 ml), and the solution was stirred for 5 minutes. Then, iodine (2.78 g, 10.95 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (4 ml) solution of 2-(N-tert-butoxycarbonyl-2-aminoethoxy)-ethanol (1.5 g, 7.308 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=85:15) to obtain 2-(N-tert-butoxycarbonyl-2-aminoethoxy)-1-iodoethane (2.19 g, yield: 95%).
α-[2-(N-tert-Butoxycarbonyl-2-aminoethoxy)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0148] ##STR00063##
[0149] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (500 mg, 2.022 mmol) and hexamethylphosphoric triamide (HMPA, 1.81 g, 10.11 mmol) were dissolved in tetrahydrofuran (4 ml), and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide (LDA) in cyclohexane (2.02 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (2 ml) solution of 2-(N-tert-butoxycarbonyl-2-aminoethoxy)-1-iodoethane (637 mg, 2.022 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (15 ml), followed by extraction with ethyl acetate (15 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain α-[2-(N-tert-butoxycarbonyl-2-aminoethoxy)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (645 mg, yield: 79%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=7.0 Hz, 1H), 7.63 (d, J=7.7 Hz, 1H), 7.57 (s, 1H), 7.34 (t, J=7.7 Hz, 1H), 7.26 (t, J=7.3 Hz, 1H), 4.98 (s, 1H), 4.02-4.06 (m, 4H), 3.69 (s, 3H), 3.43-3.51 (m, 4H), 3.30 (m, 2H), 2.29 (m, 2H), 1.45 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.8, 155.9, 151.2, 135.4, 124.8, 123.1, 122.9, 119.2, 118.8, 115.2, 79.1, 69.8, 68.3, 52.7, 52.1, 40.3, 39.3, 32.2, 28.3; FAB-MS: m/z 435 [M+H].sup.+.
α-[2-(N-tert-Butoxycarbonyl-2-aminoethoxy)-1-ethyl]-3-indoleacetic Acid (Compound #14)
[0150] ##STR00064##
[0151] α-[2-(N-tert-Butoxycarbonyl-2-aminoethoxy)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (80.0 mg, 0.184 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain α-[2-(N-tert-butoxycarbonyl-2-aminoethoxy)-1-ethyl]-3-indoleacetic acid (compound #14) (70.2 mg, yield: 87%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.40 (s, 1H), 7.67 (d, J=7.9 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.15 (t, J=7.8 Hz, 1H), 7.08 (t, J=7.3 Hz, 1H), 7.04 (s, 1H), 5.03 (s, 1H), 4.04 (t, J=7.1 Hz, 1H), 3.30-3.46 (m, 4H), 3.23 (m, 2H), 2.26 (m, 2H), 1.44 (s, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 179.2, 156.2, 136.2, 126.4, 122.6, 122.1, 119.5, 119.1, 112.6, 111.3, 79.4, 69.7, 68.5, 40.3, 39.7, 32.3, 28.4; IR (neat): 3406, 3332, 1699, 1520, 1458, 1367, 1252, 1169, 1119 cm; FAB-MS: m/z 385 [M+Na].sup.+.
Synthesis of Compound #15
N-tert-Butoxycarbonyl-4-amino-1-butanol
[0152] ##STR00065##
[0153] 4-Amino-1-butanol (1.0 g, 11.22 mmol) was dissolved in methanol (10 ml). To this solution, di-tert-butyl carbonate (2.53 g, 11.58 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the solvent was distilled off under reduced pressure, and the residue was purified using silica gel column chromatography (hexane:acetone=9:1) to obtain N-tert-butoxycarbonyl-4-amino-1-butanol (1.88 g, yield: 89%).
N-tert-Butoxycarbonyl-4-amino-1-iodobutane
[0154] ##STR00066##
[0155] Triphenylphosphine (3.3 g, 12.58 mmol) and imidazole (0.86 g, 12.63 mmol) were dissolved in dichloromethane (15 ml), and the solution was stirred at 5 minutes. Then, iodine (3.2 g, 12.61 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (4 ml) solution of N-tert-butoxycarbonyl-4-amino-1-butanol (1.6 g, 8.454 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=9:1) to obtain N-tert-butoxycarbonyl-4-amino-1-iodobutane (1.83 g, yield: 72%).
α-(N-tert-Butoxycarbonyl-4-amino-1-butyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0156] ##STR00067##
[0157] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (400 mg, 1.618 mmol) and hexamethylphosphoric triamide (HMPA, 1.45 g, 8.086 mmol) were dissolved in tetrahydrofuran (4 ml), and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide (LDA) in cyclohexane (1.62 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (2 ml) solution of N-tert-butoxycarbonyl-4-amino-1-iodobutane (484 mg, 1.618 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (15 ml), followed by extraction with ethyl acetate (15 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain α-(N-tert-butoxycarbonyl-4-amino-1-butyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (373 mg, yield: 55%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=7.8 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 7.55 (s, 1H), 7.34 (t, J=7.9 Hz, 1H), 7.25 (t, J=7.7 Hz, 1H), 4.59 (s, 1H), 4.02 (s, 3H), 3.80 (t, J=7.6 Hz, 1H), 3.67 (s, 3H), 3.09 (m, 2H), 2.03 (m, 2H), 1.25-1.53 (m, 13H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.9, 155.9, 151.2, 135.5, 129.3, 124.8, 123.0, 122.9, 119.2, 115.2, 78.9, 53.6, 52.0, 42.5, 40.2, 31.7, 29.8, 28.3, 24.8; FAB-MS: m/z 419 [M+H].sup.+.
α-(N-tert-Butoxycarbonyl-4-amino-1-butyl)-3-indoleacetic Acid (Compound #15)
[0158] ##STR00068##
[0159] α-(N-tert-Butoxycarbonyl-4-amino-1-butyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (100 mg, 0.239 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(N-tert-butoxycarbonyl-4-amino-1-butyl)-3-indoleacetic acid (compound #15) (71.8 mg, yield: 87%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.35 (s, 1H), 7.67 (d, J=7.8 Hz, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.15 (t, J=7.7 Hz, 1H), 7.09 (t, J=7.3 Hz, 1H), 7.00 (s, 1H), 4.57 (s, 1H), 3.81 (t, J=7.5 Hz, 1H), 3.02 (m, 2H), 1.97 (m, 2H), 1.23-1.48 (m, 13H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 179.6, 156.1, 136.1, 126.4, 122.3, 122.0, 119.4, 119.1, 113.0, 111.3, 79.3, 42.9, 40.3, 31.9, 29.7, 28.4, 24.7; IR (neat): 3747, 1699, 1520, 1456, 1367, 1250, 1170 cm; FAB-MS: m/z 347 [M+H].sup.+.
Synthesis of Compound #17
2-Ethyl-1-iodobutane
[0160] ##STR00069##
[0161] Triphenylphosphine (1.93 g, 7.358 mmol) and imidazole (0.5 g, 7.344 mmol) were dissolved in dichloromethane (5.0 ml), and the solution was stirred for 5 minutes. Then, iodine (1.86 g, 7.328 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (2.0 ml) solution of 2-ethyl-1-butanol (0.5 g, 5.672 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane) to obtain 2-ethyl-1-iodobutane (0.35 g, yield: 34%).
α-(2-Ethyl-1-butyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0162] ##STR00070##
[0163] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (100 mg, 0.404 mmol) and hexamethylphosphoric triamide (HMPA, 362 mg, 2.020 mmol) were dissolved in tetrahydrofuran (2 ml), and the solution was cooled to −78° C. A 1.0 M solution of lithium bis(trimethylsilyl)amide (LHMDS) in tetrahydrofuran (0.61 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of 2-ethyl-1-iodobutane (85.8 mg, 0.405 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=9:1) to obtain α-(2-ethyl-1-butyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (104 mg, yield: 78%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=7.0 Hz, 1H), 7.64 (d, J=7.8 Hz, 1H), 7.57 (s, 1H), 7.34 (t, J=7.7 Hz, 1H), 7.26 (t, J=7.4 Hz, 1H), 4.01 (s, 3H), 3.93 (t, J=7.8 Hz, 1H), 3.67 (s, 3H), 1.96 (m, 2H), 1.21-1.41 (m, 5H), 0.82-0.88 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 174.3, 151.3, 135.5, 129.5, 124.7, 122.9, 119.7, 119.3, 115.2, 53.7, 52.0, 40.4, 38.0, 35.6, 25.1, 24.9, 10.4, 10.4; IR (neat): 1738, 1455, 1377, 1256, 1164, 1085 cm.sup.−1; EI-MS: m/z 331 [M].sup.+.
α-(2-Ethyl-1-butyl)-3-indoleacetic Acid (Compound #17)
[0164] ##STR00071##
[0165] α-(2-Ethyl-1-butyl)-1-methoxycarbonyl-3-indoleacetic acid (70.0 mg, 0.211 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(2-ethyl-1-butyl)-3-indoleacetic acid (compound #17) (52.4 mg, yield: 96): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.02 (s, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 7.17 (t, J=7.9 Hz, 1H), 7.11 (t, J=7.5 Hz, 1H), 7.08 (s, 1H), 3.97 (t, J=7.8 Hz, 1H), 1.96 (m, 2H), 1.23-1.39 (m, 5H), 0.78-0.84 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 181.1, 136.1, 126.6, 122.2, 122.2, 119.7, 119.3, 113.7, 111.2, 40.6, 37.8, 35.9, 25.0, 25.0, 10.4, 10.4; IR (neat): 3414, 1703, 1458, 1293, 1098 cm.sup.−1; FAB-MS: m/z 260 [M+H].sup.+.
Synthesis of Compound #18
3-Methyl-1-iodopentane
[0166] ##STR00072##
[0167] Triphenylphosphine (1.93 g, 7.358 mmol) and imidazole (0.5 g, 7.344 mmol) were dissolved in dichloromethane (5.0 ml), and the solution was stirred for 5 minutes. Then, iodine (1.86 g, 7.328 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (2.0 ml) solution of 3-methyl-1-pentanol (0.5 g, 5.672 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=98:2) to obtain 3-methyl-1-iodopentane (0.12 mg, yield: 11%).
α-(3-Methyl-1-pentyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0168] ##STR00073##
[0169] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (50.0 mg, 0.202 mmol) and hexamethylphosphoric triamide (HMPA, 181 mg, 1.011 mmol) were dissolved in tetrahydrofuran (1 ml), and the solution was cooled to −78° C. A 1.0 M solution of lithium bis(trimethylsilyl)amide (LHMDS) in tetrahydrofuran (0.30 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of 3-methyl-1-iodopentane (51.5 mg, 0.243 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=12:1) to obtain α-(3-methyl-1-pentyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (25.8 mg, yield: 39%) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=6.7 Hz, 1H), 7.62 (d, J=7.7 Hz, 1H), 7.56 (s, 1H), 7.34 (t, J=7.8 Hz, 1H), 7.26 (t, J=7.2 Hz, 1H), 4.03 (s, 3H), 3.77 (t, J=7.9 Hz, 1H), 3.68 (s, 3H), 2.01 (m, 2H), 1.10-1.39 (m, 5H), 0.82-0.87 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 174.2, 151.3, 135.5, 129.5, 124.8, 122.9, 119.4, 119.3, 115.2, 53.7, 52.0, 42.9, 34.4, 34.2, 29.8, 29.2, 19.1, 11.3; IR (neat): 1741, 1454, 1378, 1254, 1084 cm.sup.−1; EI-MS: m/z 331 [M].sup.+.
α-(3-Methyl-1-pentyl)-3-indoleacetic Acid (Compound #18)
[0170] ##STR00074##
[0171] α-(3-Methyl-1-pentyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (20.0 mg, 0.060 mmol) was dissolved in methanol (1 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.25 ml) was added, and the mixture was stirred at 70° C. for 2.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(3-methyl-1-pentyl)-3-indoleacetic acid (compound #18) (16.8 mg, yield: 89%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.07 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.33 (d, J=8.1 Hz, 1H), 7.19 (t, J=8.0 Hz, 1H), 7.10-7.13 (m, 2H), 3.82 (t, J=6.7 Hz, 1H), 1.97 (m, 2H), 1.10-1.36 (m, 5H), 0.79-0.85 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 180.4, 136.1, 126.6, 122.2, 122.2, 119.7, 119.3, 113.7, 111.2, 43.2, 34.5, 34.3, 30.1, 29.2, 19.1, 11.3; IR (neat): 3418, 1704, 1456, 1294, 1098 cm.sup.−1; EI-MS: m/z 259 [M].sup.+.
Synthesis of Compound #19
2-Methyl-1-iodopentane
[0172] ##STR00075##
[0173] Triphenylphosphine (1.93 g, 7.358 mmol) and imidazole (0.5 g, 7.344 mmol) were dissolved in dichloromethane (5.0 ml), and the solution was stirred for 5 minutes. Then, iodine (1.86 g, 7.328 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (2.0 ml) solution of 2-methyl-1-pentanol (0.5 g, 5.672 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane) to obtain 2-methyl-1-iodopentane (0.56 g, yield: 54%).
α-(2-Methyl-1-pentyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0174] ##STR00076##
[0175] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (100 mg, 0.404 mmol) and hexamethylphosphoric triamide (HMPA, 362 mg, 2.020 mmol) were dissolved in tetrahydrofuran (2 ml), and the solution was cooled to −78° C. A 1.0 M solution of lithium bis(trimethylsilyl)amide (LHMDS) in tetrahydrofuran (0.61 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of 2-methyl-1-iodopentane (85.8 mg, 0.405 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=9:1) to obtain α-(2-methyl-1-pentyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (101 mg, yield: 75%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=5.7 Hz, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.55 (s, 1H), 7.34 (t, J=7.6 Hz, 1H), 7.27 (t, J=7.5 Hz, 1H), 4.03 (s, 3H), 3.91-3.97 (m, 1H), 3.68 (s, 3H), 1.58-2.24 (m, 2H), 1.10-1.50 (m, 5H), 0.83-0.97 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 174.4, 151.2, 135.4, 129.4, 124.7, 122.9, 122.8, 119.9, 119.4, 115.2, 53.7, 52.0, 40.4, 39.6, 39.3, 30.7, 19.8, 19.4, 14.2; IR (neat): 1739, 1456, 1373, 1217, 1087 cm.sup.−1; EI-MS: m/z 331 [M].sup.+.
α-(2-Methyl-1-pentyl)-3-indoleacetic Acid (Compound #19)
[0176] ##STR00077##
[0177] α-(2-Methyl-1-pentyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (70.0 mg, 0.211 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(2-methyl-1-pentyl)-3-indoleacetic acid (compound #19) (51.9 mg, yield: 95%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.12 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 7.17 (t, J=7.4 Hz, 1H), 7.11 (t, J=7.2 Hz, 1H), 7.06 (s, 1H), 3.96-4.02 (m, 1H), 1.60-2.22 (m, 2H), 1.12-1.51 (m, 5H), 0.79-0.94 (m, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 180.9, 136.1, 126.5, 122.3, 122.2, 119.7, 119.3, 113.3, 111.2, 40.7, 39.9, 39.2, 30.3, 19.8, 19.4, 14.3; IR (neat): 3417, 1699, 1457, 1292, 1099 cm.sup.−1; EI-MS: m/z 259 [M].sup.+.
Synthesis of Compound #20
4-Phenyl-2-(1H-indol-3-yl)-4-oxo-butanoic Acid (Compound #20)
[0178] ##STR00078##
[0179] In a 30-mL round-bottomed flask, trans-4-phenyl-4-oxo-2-butenoic acid (1.0 g, 5.65 mmol) was dissolved in benzene (25 mL). To the solution, indole (0.79 g, 6.77 mmol) was added, and the mixture was stirred at 80° C. for 5 hours and stirred until the temperature became room temperature. The solvent in the reaction solution was distilled off under reduced pressure, and the residue was recrystallized from benzene to obtain 4-phenyl-2-(1H-indol-3-yl)-4-oxo-butanoic acid (compound #20) (1.24 g, yield: 75%); Melting point: 149 to 150° C.; .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 10.17 (1H, brs, 1H), 8.05 (2H, d, J=8.2 Hz), 7.80 (1H, d, J=8.3 Hz), 7.57 (1H, t, J=7.8 Hz), 7.51 (2H, dd, J=8.2, 7.8 Hz), 7.41 (1H, d, J=8.2 Hz), 7.37 (1H, s), 7.13 (1H, t, J=8.2 Hz), 7.06 (1H, t, J=8.2 Hz), 4.57 (1H, dd, J=11.0, 4.1 Hz), 4.13 (1H, dd, J=17.8, 11.0 Hz), 3.41 (1H, dd, J=17.8, 4.1 Hz); IR: (neat): 3400, 3055, 1711, 1677, 1453 cm.sup.−1; HRFAB-MS found m/z 294.1143 [M+H].sup.+, calcd for 294.1130 (C.sub.18H.sub.16NO.sub.3).
Synthesis of Compound #21
4,4,5,5,5-Pentafluoro-1-iodopentane
[0180] ##STR00079##
[0181] Triphenylphosphine (1.1 g, 4.211 mmol) and imidazole (0.29 g, 4.211 mmol) were dissolved in dichloromethane (5.0 ml), and the solution was stirred for 5 minutes. Then, iodine (1.07 g, 4.211 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (2.0 ml) solution of 4,4,5,5,5-pentafluoro-1-pentanol (0.5 g, 2.807 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane) to obtain 4,4,5,5,5-pentafluoro-1-iodopentane (0.36 g, yield: 45%).
α-(4,4,5,5,5-Pentafluoro-1-pentyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0182] ##STR00080##
[0183] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (50.0 mg, 0.202 mmol) and hexamethylphosphoric triamide (HMPA, 181 mg, 1.011 mmol) were dissolved in tetrahydrofuran (1 ml), and the solution was cooled to −78° C. A 1.0 M solution of lithium bis(trimethylsilyl)amide (LHMDS) in tetrahydrofuran (0.30 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of 4,4,5,5,5-pentafluoro-1-iodopentane (81.4 mg, 0.283 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=85:15) to obtain α-(4,4,5,5,5-pentafluoro-1-pentyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (59.8 mg, yield: 73%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.19 (d, J=7.6 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 7.57 (s, 1H), 7.36 (t, J=7.5 Hz, 1H), 7.27 (t, J=6.9 Hz, 1H), 4.03 (s, 3H), 3.83 (t, J=7.6 Hz, 1H), 3.69 (s, 3H), 1.98-2.23 (m, 4H), 1.62-1.68 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 173.5, 151.3, 135.5, 129.1, 125.0, 123.1, 123.1, 119.2, 118.6, 115.3, 53.8, 52.2, 42.3, 31.4, 30.6, 30.3, 30.1, 18.6; IR (neat) 1739, 1456, 1378, 1257, 1198 cm.sup.−1; EI-MS: m/z 407 [M].sup.+.
α-(4,4,5,5,5-Pentafluoro-1-pentyl)-3-indoleacetic Acid (Compound #21)
[0184] ##STR00081##
[0185] α-(4,4,5,5,5-Pentafluoro-1-pentyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (55.5 mg, 0.183 mmol) was dissolved in methanol (1 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.25 ml) was added, and the mixture was stirred at 70° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(4,4,5,5,5-pentafluoro-1-pentyl)-3-indoleacetic acid (compound #21) (43.9 mg, yield: 97%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.06 (s, 1H), 7.67 (d, J=7.9 Hz, 1H), 7.33 (d, J=8.1 Hz, 1H), 7.20 (t, J=8.0 Hz, 1H), 7.12 (t, J=7.9 Hz, 1H), 7.09 (s, 1H), 3.87 (t, J=7.5 Hz, 1H), 1.95-2.22 (m, 4H), 1.60-1.67 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 179.9, 136.2, 126.2, 122.4, 122.4, 119.9, 119.1, 112.5, 111.4, 42.7, 31.5, 30.6, 30.3, 30.1, 18.6; IR (neat): 3418, 1704, 1459, 1198 cm; EI-MS: m/z 335 [M].sup.+.
Synthesis of Compound #22
3-(2-Hydroxy-1-ethyl)-1,1′-biphenyl
[0186] ##STR00082##
[0187] 2-(3-Bromophenyl)-1-ethanol (200 mg, 0.995 mmol) was dissolved in a mixed solvent of dimethoxyethane:ethanol (=5:1) (3.0 ml). To the solution, phenylboronic acid (242 mg, 1.985 mmol), a 2 M aqueous sodium carbonate solution (1.5 ml), and tetrakis(triphenylphosphine) palladium(0) (Pd(PPh.sub.3).sub.4, 56.0 mg, 0.048 mmol) were added, and the mixture was stirred for 4 hours under heating to reflux. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and the filtrate was neutralized by the addition of hydrochloric acid, followed by extraction with ethyl acetate (10 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain 3-(2-hydroxy-1-ethyl)-1,1′-biphenyl (172 mg, yield: 87%).
3-(2-Iodo-1-ethyl)-1,1′-biphenyl
[0188] ##STR00083##
[0189] Triphenylphosphine (327 mg, 1.248 mmol) and imidazole (85.0 mg, 1.249 mmol) were dissolved in dichloromethane (3.0 ml), and the solution was stirred for 5 minutes. Then, iodine (317 mg, 1.248 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (0.5 ml) solution of 3-(2-hydroxy-1-ethyl)-1,1′-biphenyl (165 mg, 0.832 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 1 hour. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=98:2) to obtain 3-(2-iodo-1-ethyl)-1,1′-biphenyl (185 mg, yield: 72%).
α-[2-(1,1′-Biphenyl-3-yl)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0190] ##STR00084##
[0191] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (80 mg, 0.324 mmol) and hexamethylphosphoric triamide (HMPA, 290 mg, 1.618 mmol) were dissolved in tetrahydrofuran (2 ml), and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide (LDA) in cyclohexane (0.32 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of 3-(2-iodo-1-ethyl)-1,1′-biphenyl (99.7 mg, 0.324 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=85:15) to obtain α-[2-(1,1′-biphenyl-3-yl)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (132 mg, yield: 96%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.19 (d, J=6.8 Hz, 1H), 7.55-7.58 (m, 4H), 7.31-7.44 (m, 7H), 7.24 (t, J=8.1 Hz, 1H), 7.15 (d, J=7.5 Hz, 1H), 4.02 (s, 3H), 3.86 (t, J=7.5 Hz, 1H), 3.65 (S, 3H), 2.73 (t, J=7.7 Hz, 2H), 2.25-2.58 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.8, 151.2, 141.4, 141.3, 141.1, 135.5, 129.3, 128.8, 128.6, 127.3, 127.2, 127.1, 124.9, 124.8, 123.1, 122.9, 119.3, 118.9, 115.2, 53.7, 52.1, 41.8, 33.7, 33.5; EI-MS: m/z 427 [M].sup.+.
α-[2-(1,1′-Biphenyl-3-yl)-1-ethyl]-3-indoleacetic Acid (Compound #22)
[0192] ##STR00085##
[0193] α-[2-(1,1′-Biphenyl-3-yl)-1-ethyl]-1-methoxycarbonyl-3-indoleacetic acid methyl ester (80.0 mg, 0.187 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-[2-(1,1′-biphenyl-3-yl)-1-ethyl]-3-indoleacetic acid (compound #22) (60.3 mg, yield: 91%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.01 (s, 1H), 7.65 (d, J=7.9 Hz, 1H), 7.53-7.55 (m, 2H), 7.29-7.41 (m, 7H), 7.17 (t, J=7.2 Hz, 1H), 7.07-7.13 (m, 3H), 3.91 (t, J=7.5 Hz, 1H), 2.71 (t, J=7.7 Hz, 2H), 2.39 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 180.3, 141.8, 141.3, 141.2, 136.1, 128.8, 128.7, 127.4, 127.4, 127.2, 126.4, 124.9, 122.4, 122.3, 119.8, 119.3, 112.9, 111.3, 42.2, 33.8, 33.7; IR (neat): 3420, 1699, 1456, 1216, 1097 cm.sup.−1; EI-MS: m/z 355 [M].sup.+.
Synthesis of compound #23
α-(2-Phenyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0194] ##STR00086##
[0195] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (300 mg, 1.213 mmol) and hexamethylphosphoric triamide (HMPA, 1.09 g, 6.067 mmol) were dissolved in tetrahydrofuran (2 ml), and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide (LDA) in cyclohexane (1.21 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (2 ml) solution of 1-bromo-2-phenylethane (292 mg, 1.577 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (10 ml), followed by extraction with ethyl acetate (10 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (benzene) to obtain α-(2-phenyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (228 mg, yield: 54%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=6.0 Hz, 1H), 7.57 (s, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.31 (t, J=7.8 Hz, 1H), 7.13-7.26 (m, 6H), 3.94 (s, 3H), 3.83 (t, J=7.5 Hz, 1H), 3.64 (s, 3H), 2.66 (t, J=7.8 Hz, 2H), 2.35 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.8, 151.2, 140.9, 135.4, 129.3, 128.4, 128.3, 126.0, 124.8, 123.1, 122.9, 119.3, 119.0, 115.2, 53.7, 52.0, 41.8, 33.5; EI-MS: m/z 351 [M].sup.+.
α-(2-Phenyl-1-ethyl)-3-indoleacetic Acid (Compound #23)
[0196] ##STR00087##
[0197] α-(2-Phenyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (150 mg, 0.427 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 1.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(2-phenyl-1-ethyl)-3-indoleacetic acid (compound #23) (85.3 mg, yield: 72%): .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 10.16 (s, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.40 (d, J=8.1 Hz, 1H), 7.09-7.32 (m, 7H), 7.03 (t, J=7.6 Hz, 1H), 3.93 (t, J=7.4 Hz, 1H), 2.67 (t, J=5.4 Hz, 2H), 2.35 (m, 2H); .sup.13C NMR (100 MHz, acetone-d.sub.6): δ 175.4, 142.4, 137.2, 128.8, 128.7, 127.2, 126.2, 123.2, 121.8, 119.4, 119.2, 113.6, 111.8, 42.5, 34.9, 34.1; IR (neat): 3416, 1700, 1457, 1246, 1098 cm.sup.−1;
FAB-MS: m/z 280 [M+H].SUP.+..
Synthesis of Compound #24
2-Cyclopentyl-1-iodoethane
[0198] ##STR00088##
[0199] Triphenylphosphine (1.03 g, 3.942 mmol) and imidazole (0.27 g, 3.937 mmol) were dissolved in dichloromethane (5 ml), and the solution was stirred for 5 minutes. Then, iodine (1.0 g, 3.940 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (1 ml) solution of 2-cyclopentyl-1-ethanol (0.3 g, 2.627 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane) to obtain 2-cyclopentyl-1-iodoethane (0.46 g, yield: 84%).
1-Methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0200] Indole-3-acetic acid methyl ester (2.00 g, 10.57 mmol) was dissolved in dichloromethane (30 ml). To this solution, tetrabutylammonium iodide (TBAI, 30.0 mg, 0.081 mmol) and a 30% aqueous sodium hydroxide solution (24 ml) were added, and the mixture was cooled to 0° C. To the reaction solution, methyl formate chloride (1.96 g, 20.73 mmol) was added, and the mixture was stirred at 0° C. for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction was terminated by the addition of 6 N hydrochloric acid. Water (50 ml) was added thereto, followed by extraction with chloroform (50 ml) three times. The organic layer was washed twice with saturated saline and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was then purified using silica gel column chromatography (hexane:ethyl acetate=8:2) to obtain 1-methoxycarbonyl-3-indoleacetic acid methyl ester (2.26 g, yield: 87%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=7.0 Hz, 1H), 7.59 (s, 1H), 7.53 (d, J=7.7 Hz, 1H), 7.35 (t, J=7.5 Hz, 1H), 7.27 (t, J=7.4 Hz, 1H), 4.00 (s, 3H), 3.72 (s, 3H), 3.71 (s, 2H); .sup.13C-NMR (100 MHz, CDCl.sub.3) 171.1, 151.1, 135.2, 129.9, 124.6, 123.8, 122.8, 118.9, 115.0, 113.8, 53.5, 51.9, 30.6; EI-MS: m/z 247 [M].sup.+
α-(2-Cyclopentyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0201] ##STR00089##
[0202] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (150 mg, 0.607 mmol) and hexamethylphosphoric triamide (544 mg, 3.036 mmol) were dissolved in anhydrous tetrahydrofuran 2 ml, and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide in cyclohexane (0.61 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a anhydrous tetrahydrofuran 1 ml solution of 2-cyclopentyl-1-iodoethane (204 mg, 0.910 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water 5 ml, followed by extraction with ethyl acetate 5 ml three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=95:5) to obtain α-(2-cyclopentyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (151 mg, yield: 72%): .sup.1H NMR (400 MHz, CDCl3): δ 8.18 (d, J=6.8 Hz, 1H), 7.62 (d, J=7.7 Hz, 1H), 7.56 (s, 1H), 7.34 (t, J=7.4 Hz, 1H), 7.26 (t, J=7.3 Hz, 1H), 4.02 (s, 3H), 3.79 (t, J=7.6 Hz, 1H), 3.68 (s, 3H), 2.03 (m, 2H), 1.73-1.77 (m, 3H), 1.48-1.58 (m, 4H), 1.34 (q, J=7.2 Hz, 2H), 1.04-1.07 (m, 2H); .sup.13C-NMR (100 MHz, CDCl3): δ 174.2, 151.3, 135.5, 129.5, 124.7, 122.9, 119.5, 119.3, 115.2, 53.7, 52.0, 42.8, 39.9, 34.1, 32.6, 32.5, 31.4, 25.1; EI-MS: m/z 343 [M].sup.+
α-(2-Cyclopentyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester (Compound #24)
[0203] ##STR00090##
[0204] α-(2-Cyclopentyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (100 mg, 0.291 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-(2-cyclopentyl-1-ethyl)-1-methoxycarbonyl-3-indoleacetic acid methyl ester (compound #24) (78.5 mg, yield: 99%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.19 (s, 1H), 7.69 (d, J=7.9 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.16 (t, J=8.0 Hz, 1H), 7.10 (t, J=7.5 Hz, 1H), 7.06 (s, 1H), 3.83 (t, J=7.6 Hz, 1H), 2.01 (m, 2H), 1.70-1.75 (m, 3H), 1.45-1.55 (m, 4H), 1.34-1.37 (m, 2H), 0.98-1.03 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 180.7, 136.1, 126.5, 122.2, 122.0, 119.5, 119.2, 113.4, 111.2, 43.1, 39.9, 34.1, 32.5, 31.6, 25.1; IR (neat): 3415, 1703, 1457, 1339, 1098 cm.sup.−1; FAB-MS: m/z 294 [M+Na].sup.+.
Synthesis of Compound #25
Cyclopentyliodomethane
[0205] ##STR00091##
[0206] Triphenylphosphine (1.18 g, 4.491 mmol) and imidazole (0.31 g, 4.495 mmol) were dissolved in dichloromethane (5 ml), and the solution was stirred for 5 minutes. Then, iodine (1.14 g, 4.492 mmol) was added thereto, and the mixture was stirred for 10 minutes. A dichloromethane (1 ml) solution of cyclopentylmethanol (0.3 g, 2.995 mmol) was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was filtered through celite, and a 5% aqueous sodium thiosulfate solution was added to the filtrate to remove iodine. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane) to obtain cyclopentyliodomethane (0.53 g, yield: 84%).
α-Cyclopentylmethyl-1-methoxycarbonyl-3-indoleacetic Acid Methyl Ester
[0207] ##STR00092##
[0208] In a nitrogen atmosphere, 1-methoxycarbonyl-3-indoleacetic acid methyl ester (150 mg, 0.607 mmol) and hexamethylphosphoric triamide (HMPA, 544 mg, 3.036 mmol) were dissolved in tetrahydrofuran (2 ml), and the solution was cooled to −78° C. A 1.5 M solution of lithium diisopropylamide (LDA) in cyclohexane (0.61 ml, 1.5 eq) was slowly added dropwise thereto, and the mixture was stirred at −78° C. for 0.5 hours. To this reaction solution, a tetrahydrofuran (1 ml) solution of cyclopentyliodomethane (153 mg, 0.728 mmol) was slowly added dropwise, and the mixture was stirred at −78° C. for 1 hour. After the reaction was confirmed by TLC to be complete, the temperature was adjusted to 0° C., and the reaction was terminated by the addition of water (5 ml), followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=13:1) to obtain α-cyclopentylmethyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester (153 mg, yield: 76%) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.18 (d, J=6.0 Hz, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.57 (s, 1H), 7.32 (t, J=7.4 Hz, 1H), 7.25 (t, J=7.4 Hz, 1H), 3.99 (s, 3H), 3.88 (t, J=7.7 Hz, 1H), 3.67 (s, 3H), 2.05 (m, 2H), 1.76-1.79 (m, 3H), 1.59-1.62 (m, 2H), 1.47-1.50 (m, 2H), 1.12-1.17 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 174.1, 151.1, 135.4, 129.4, 124.6, 122.8, 119.4, 119.2, 115.1, 53.6, 51.9, 41.7, 38.5, 37.9, 32.5, 32.3, 24.9; EI-MS: m/z 329 [M].sup.+.
α-Cyclopentylmethyl-3-indoleacetic Acid (Compound #25)
[0209] ##STR00093##
[0210] α-Cyclopentylmethyl-1-methoxycarbonyl-3-indoleacetic acid methyl ester (100 mg, 0.304 mmol) was dissolved in methanol (2 ml). To this solution, a 2 N aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at 70° C. for 2.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=95:5) to obtain α-cyclopentylmethyl-3-indoleacetic acid (compound #25) (58.3 mg, yield: 75%); .sup.1H NMR (400 MHz, acetone-d.sub.6): δ 10.13 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.38 (d, J=8.1 Hz, 1H), 7.28 (s, 1H), 7.10 (t, J=8.0 Hz, 1H), 7.02 (t, J=7.1 Hz, 1H), 3.73 (t, J=7.7 Hz, 1H), 2.06 (m, 2H), 1.78-1.83 (m, 3H), 1.47-1.61 (m, 4H), 1.17-1.20 (m, 2H); .sup.13C NMR (100 MHz, acetone-d.sub.6): δ 175.8, 137.3, 127.4, 123.1, 121.8, 119.5, 119.2, 114.1, 111.9, 42.4, 39.6, 38.7, 32.9, 32.9, 25.3, 25.3; IR (neat): 3418, 1699, 1456, 1339, 1097 cm.sup.−1; FAB-MS: m/z 258 [M+H].sup.+.
[0211] Compounds #26 to 31 were each synthesized according to a method described in Muro Fumihito et. al. “Discovery of trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic Acid: An Orally Active, Selective Very Late Antigen-4 Antagonist” Journal of Medicinal Chemistry, 52 (24), 7974-7992; 2009.
Synthesis of Compound #26
N-Methyl-3-indoleacetic Acid Methyl Ester
[0212] ##STR00094##
[0213] 3-Indoleacetic acid methyl ester (200 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (3 mL). To the solution, sodium hydride (60 mg) was added. To this solution, methyl iodide (223 mg, 1.58 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=5:1) to obtain N-methyl-3-indoleacetic acid methyl ester (140 mg, yield: 65%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.9 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.23 (dd, J=8.2, 7.9 Hz, 1H), 7.12 (dd, J=8.2, 7.9 Hz, 1H), 7.03 (s, 1H), 3.75 (s, 3H), 3.77 (s, 2H), 3.69 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.6, 136.9, 127.7, 121.7 (2C), 119.26, 118.9, 109.3, 106.8, 51.9, 32.7, 31.0.
N-Methyl-3-indoleacetic Acid (Compound #26)
[0214] ##STR00095##
[0215] N-Methyl-3-indoleacetic acid methyl ester (120 mg, 0.59 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at 50° C. for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain N-methyl-3-indoleacetic acid (compound #26) (108 mg, yield: 96%); .sup.1H NMR (400 MHz, CDCl3): δ 7.59 (d, J=8.0 Hz, 1H), 7.35 (d, J=8.1 Hz, 1H), 7.18 (s, 1H), 7.16 (dd, J=7.0, 6.1 Hz, 1H), 7.04 (dd, J=8.1, 6.7 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 2H). .sup.13C NMR (100 MHz, CDCl.sub.3): 177.6, 136.8, 127.9, 127.5, 121.8, 119.2, 118.9, 109.5, 106.1, 53.7, 31.7.
Synthesis of Compound #27
N-Ethyl-3-indoleacetic Acid Methyl Ester
[0216] ##STR00096##
[0217] 3-Indoleacetic acid methyl ester (200 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (3 mL). To the solution, sodium hydride (60 mg) was added. To this solution, ethyl iodide (246 mg, 1.58 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=5:1) to obtain N-ethyl-3-indoleacetic acid methyl ester (133 mg, yield: 58%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.8 Hz, 1H), 7.31 (d, J=8.3 Hz, 1H), 7.21 (dd, J=8.3, 7.8 Hz, 1H), 7.11 (dd, J=8.3, 7.8 Hz, 1H), 7.09 (s, 1H), 4.11 (q, J=7.3 Hz, 2H), 3.76 (s, 2H), 3.68 (s, 3H), 1.43 (t, J=7.3, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.6, 160.8, 135.9, 127.8, 125.9, 121.6, 119.0, 109.3, 51.9, 40.8, 31.1, 15.4.
N-Ethyl-3-indoleacetic Acid (Compound #27)
[0218] ##STR00097##
[0219] N-Methyl-3-indoleacetic acid methyl ester (120 mg, 0.59 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at 50° C. for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain N-methyl-3-indoleacetic acid (compound #27) (108 mg, yield: 97%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.9 Hz, 1H), 7.40 (d, J=8.2 Hz, 1H), 7.25 (s, 1H), 7.15 (ddd, J=7.5, 7.6 Hz, 1H), 7.04 (ddd, J=7.3, 7.5 Hz, 1H), 4.20 (q, J=7.3 Hz, 2H), 3.74 (s, 2H), 1.39 (t, J=7.3 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.3, 136.8, 129.0, 127.1, 122.0, 119.8, 119.4, 110.1, 108.1, 41.1, 31.9, 15.8.
Synthesis of Compound #28
N-Propyl-3-indoleacetic Acid Methyl Ester
[0220] ##STR00098##
[0221] 3-Indoleacetic acid methyl ester (200 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (3 mL). To the solution, sodium hydride (60 mg) was added. To this solution, propyl iodide (268 mg, 1.58 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=6:1) to obtain N-propyl-3-indoleacetic acid methyl ester (136 mg, yield: 56%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.8 Hz, 1H) 7.31 (d, J=8.3 Hz, 1H) 7.21 (dd, J=8.0, 7.1 Hz, 1H) 7.11 (dd, J=7.7, 6.9 Hz, 1H) 7.08 (s, 1H) 4.04 (t, J=7.1 Hz, 2H) 3.77 (s, 2H) 3.69 (s, 3H) 1.86 (m, 2H) 0.93 (t, J=7.3 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.6, 136.2, 127.70, 126.7, 121.5, 119.0, 119.0, 109.4, 106.6, 51.9, 47.9, 31.1, 23.5, 11.5.
N-Propyl-3-indoleacetic Acid (Compound #28)
[0222] ##STR00099##
[0223] N-Propyl-3-indoleacetic acid methyl ester (120 mg, 0.52 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at 50° C. for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain N-propyl-3-indoleacetic acid (compound #28) (103 mg, yield: 98%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=8.0 Hz, 1H), 7.32 (d, J=8.2 Hz, 1H), 7.21 (dd, J=7.2, 8.0 Hz, 1H), 7.11 (dd, J=7.3, 9.8 Hz, 1H), 7.09 (s, 1H), 4.04 (t, J=7.1, 2H), 3.79 (s, 2H), 1.85 (m, 2H), 0.92 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.5, 136.2, 127.6, 127.0, 121.6, 119.1, 119.0, 109.5, 106.0, 53.7, 31.7, 23.5, 11.5.
Synthesis of Compound #29
N-Butyl-3-indoleacetic Acid Methyl Ester
[0224] ##STR00100##
[0225] 3-Indoleacetic acid methyl ester (200 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (3 mL). To the solution, sodium hydride (60 mg) was added. To this solution, butyl iodide (290 mg, 1.58 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=6:1) to obtain N-butyl-3-indoleacetic acid methyl ester (137 mg, yield: 53%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.8 Hz, 1H), 7.32 (d, J=8.2 Hz, 1H), 7.21 (dd, J=8.5, 9.8 Hz, 1H), 7.11 (dd, J=9.7, 7.4 Hz, 1H), 7.08 (s, 1H), 4.08 (t, J=7.1 Hz, 2H), 3.77 (s, 2H), 3.69 (s, 3H), 1.80 (m, 2H), 1.34 (m, 2H), 0.93 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): 172.6, 136.2, 127.7, 126.7, 121.5, 119.0, 119.0, 109.4, 106.7, 51.9, 46.0, 32.3, 31.1, 20.2, 13.7.
N-Butyl-3-indoleacetic Acid (Compound #29)
[0226] ##STR00101##
[0227] N-Butyl-3-indoleacetic acid methyl ester (120 mg, 0.52 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at 50° C. for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain N-butyl-3-indoleacetic acid (compound #29) (104 mg, yield: 98%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.59 (d, J=7.9 Hz, 1H), 7.31 (d, J=8.2 Hz, 1H), 7.20 (dd, J=7.1, 7.9 Hz, 1H), 7.11 (dd, J=7.3, 7.5 Hz, 1H), 7.07 (s, 1H), 4.06 (t, J=7.2 Hz, 2H), 3.78 (s, 2H), 1.79 (m, 2H), 1.33 (m, 2H), 0.92 (t, J=7.4, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 178.0, 136.1, 127.6, 126.9, 121.6119.10, 119.0, 109.5, 106.0, 53.6, 31.7, 29.1, 20.2, 13.7.
Synthesis of Compound #30
N-Hexyl-3-indoleacetic Acid Methyl Ester
[0228] ##STR00102##
[0229] 3-Indoleacetic acid methyl ester (200 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (3 mL). To the solution, sodium hydride (60 mg) was added. To this solution, hexyl iodide (334 mg, 1.58 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=6:1) to obtain N-hexyl-3-indoleacetic acid methyl ester (147 mg, yield: 51%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.8 Hz, 1H) 7.31, (d, J=8.2 Hz, 1H), 7.20 (ddd, J=8.6, 5.6 Hz, 1H), 7.11 (ddd, J=8.0, 7.3 Hz, 1H), 7.08 (s, 2H), 4.06 (t, J=7.2 Hz, 2H), 3.77 (s, 2H), 3.69 (s, 3H), 1.81 (m, 2H), 1.30 (m, 6H), 0.87 (t, J=6.9 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): 172.6, 136.1, 127.7, 126.7, 121.5, 119.0, 119.0, 109.4, 106.6, 51.9, 46.3, 31.4, 31.1, 30.2, 22.6, 22.5, 14.0.
N-Hexyl-3-indoleacetic Acid (Compound #30)
[0230] ##STR00103##
[0231] N-Hexyl-3-indoleacetic acid methyl ester (120 mg, 0.52 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at 50° C. for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain N-hexyl-3-indoleacetic acid (compound #30) (103 mg, yield: 96%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.59 (d, J=7.9 Hz, 1H), 7.31 (d, J=8.2 Hz, 1H), 7.20 (ddd, J=7.9, 7.3 Hz, 1H), 7.20 (ddd, J=7.4, 7.7 Hz, 1H), 7.07 (1H, s, 1H), 4.05 (t, J=7.2 Hz, 2H), 3.78 (s, 2H), 1.81 (m, 2H), 1.31 (m, 6H), 0.88 (t, J=6.3 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 178.0, 136.1, 127.6, 127.6, 121.6, 119.1, 119.0, 109.5, 106.0, 53.7, 31.7, 29.2, 28.9, 27.0, 23.0, 14.02.
Synthesis of Compound #31
N-Heptyl-3-indoleacetic Acid Methyl Ester
[0232] ##STR00104##
[0233] 3-Indoleacetic acid methyl ester (200 mg, 1.1 mmol) was dissolved in N,N-dimethylformamide (3 mL). To the solution, sodium hydride (60 mg) was added. To this solution, heptyl iodide (358 mg, 1.58 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and water (5 ml) was added thereto, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=6:1) to obtain N-heptyl-3-indoleacetic acid methyl ester (148 mg, yield: 49%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.69 (3H, s), 7.60 (1H, d, J=7.8), 7.31 (1H, d, J=8.2) 7.11 (1H, dd, J=8.2, 6.7), 7.08 (1H, s), 4.06 (2H, t, J=7.1), 3.77 (2H, s) 3.59 (1H, dd, J=8.2, 6.7), 1.82 (2H, m), 1.29 (8H, m), 0.87 (3H, t, J=7.1); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.57, 136.16, 127.70, 126.66, 121.54, 118.98, 118.98, 109.43, 106.64, 51.89, 46.31, 31.67, 31.11, 30.24, 28.89, 26.96, 22.55, 14.02.
N-Heptyl-3-indoleacetic Acid (Compound #31)
[0234] ##STR00105##
[0235] N-Heptyl-3-indoleacetic acid methyl ester (120 mg, 0.52 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at 50° C. for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain N-heptyl-3-indoleacetic acid (compound #31) (180 mg, yield: 95%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.59 (1H, d, J=7.96), 7.31 (1H, d, J=8.17), 7.21 (1H, ddd, J=8.49, 6.73), 7.11 (1H, ddd, J=7.21, 7.29), 7.08 (1H, S), 4.06 (2H, t, J=7.25), 3.79 (2H, s) 1.81 (2H, m) 1.29 (8H, m) 0.87 (3H, t, J=6.83); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.81, 136.10, 127.55, 126.85, 121.62, 119.11, 118.94, 109.49, 105.91, 53.63, 46.32, 30.99, 29.68, 29.16, 26.64, 22.49, 13.99.
[0236] Compounds #33 and 34 were each synthesized with α-(7-hydroxy-1-naphthalenyl)-acetic acid ethyl ester as a key intermediate. The α-(7-hydroxy-1-naphthalenyl)-acetic acid ethyl ester was synthesized according to a method described in E. Tsuda et. al., “Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters” Journal of Biological Chemistry, 286 (3), 2354-2364; 2011.
Synthesis of Compound #33
α-(7-Butoxy-1-naphthalenyl)-acetic Acid Ethyl Ester
[0237] ##STR00106##
[0238] α-(7-Hydroxy-1-naphthalenyl)-acetic acid ethyl ester (90 mg, 0.39 mmol) was dissolved in N,N-dimethylformamide (5 ml). To this solution, 1-iodobutane (107 mg, 0.58 mmol) was added dropwise, then cesium carbonate (127 mg, 0.39 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, water (5 ml) was added to the reaction solution, followed by extraction with ethyl acetate (10 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain α-(7-butoxy-1-naphthalenyl)-acetic acid ethyl ester as a colorless oil (92 mg, yield: 83%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.71 (d, J=8.9 Hz, 1H), 7.35 (d, J=6.9 Hz, 1H), 7.67 (d, J=8.1 Hz, 1H), 7.27 (d, J=2.3 Hz, 1H), 7.25 (dd, J=8.1, 6.9 Hz, 1H), 7.14 (q, J=8.9, 2.3 Hz, 1H), 4.12 (q, J=7.1 Hz, 2H), 4.07 (t, J=6.6 Hz, 2H), 3.97 (s, 2H), 1.82 (m, 2H), 1.53 (m, 2H), 1.19 (t, J=7.1 Hz, 3H), 0.96 (t, J=7.5 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 171.5, 157.4, 133.2, 130.0, 129.3, 129.1, 128.3, 127.6, 123.0, 118.5, 103.2, 67.6, 60.8, 39.5, 31.2, 19.2, 14.1, 13.8; IR (neat): 2958, 1733, 1510, 1459, 1210, 1156 cm.sup.−1; HREI-MS found m/z286.1556 [M].sup.+, calcd for 286.1569 (C.sub.18H.sub.22O.sub.3).
α-(7-Butoxy-1-naphthalenyl)-acetic Acid (Compound #33)
[0239] ##STR00107##
[0240] α-(7-Butoxy-1-naphthalenyl)-acetic acid ethyl ester (75 mg, 0.26 mmol) was dissolved in a mixed solution of tetrahydrofuran:methanol:2 M aqueous sodium hydroxide solution=2:2:1 (1.5 ml), and the solution was stirred at room temperature for 1 hour. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (10 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain α-(7-butoxy-1-naphthalenyl)-acetic acid (compound #33) (67 mg, yield: 98%); Melting point: 102 to 104° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.75 (d, J=8.9 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.34 (d, J=6.9 Hz, 1H), 7.26 (dd, J=8.1, 6.9 Hz, 1H), 7.23 (d, J=2.0 Hz, 1H), 7.16 (q, J=8.9, 2.0 Hz, 1H), 4.05 (t, J=6.5 Hz, 2H), 4.00 (s, 2H), 1.51 (m, 2H), 1.80 (m, 2H), 0.98 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.6, 157.6, 133.2, 130.2, 129.1, 128.6, 127.9 (2C), 123.0, 118.7, 103.1, 67.7, 39.2, 31.2, 19.3, 13.8; IR (neat): 3021, 2931, 1699, 1457, 1138 cm.sup.−1; HREI-MS found m/z 258.1268 [M].sup.+, calcd for 258.1256 (C.sub.16H.sub.18O.sub.3).
Synthesis of Compound #34
α-(7-Pentoxy-1-naphthalenyl)-acetic Acid Ethyl Ester
[0241] ##STR00108##
[0242] α-(7-Hydroxy-1-naphthalenyl)-acetic acid ethyl ester (90 mg, 0.39 mmol) was dissolved in N,N-dimethylformamide (5 ml). To this solution, 1-iodopentane (116 mg, 0.58 mmol) was added dropwise, then cesium carbonate (127 mg, 0.39 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, water (5 ml) was added to the reaction solution, followed by extraction with ethyl acetate (10 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain α-(7-pentoxy-1-naphthalenyl)-acetic acid ethyl ester as a colorless oil (103 mg, yield: 88%): .sup.1H NMR (400 MHz, CDCl.sub.3): δ 1.00 (t, J=7.2 Hz, 3H), 1.26 (t, J=7.1 Hz, 3H), 1.48 (m, 2H), 1.55 (m, 2H), 1.91 (m, 2H), 4.03 (s, 2H), 4.13 (t, J=6.5 Hz, 2H), 4.19 (q, J=7.1 Hz, 2H), 7.20 (dd, J=8.9, 2.5 Hz, 1H), 7.31 (dd, J=8.1, 7.0 Hz, 1H), 7.33 (d, J=2.5 Hz, 1H), 7.41 (d, J=7.0 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.78 (d, J=8.9 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 171.5, 157.4, 133.2, 130.0, 129.3, 129.1, 128.4, 127.6, 123.0, 118.5, 103.2, 67.8, 60.8, 39.6, 28.9, 28.2, 22.4, 14.1, 14.0; IR (neat): 2969, 1734, 1509, 1459, 1160 cm.sup.−1; HREI-MS found m/z 300.1727 [M].sup.+, calcd for 300. 1725 (C.sub.19H.sub.24O.sub.3).
α-(7-Pentoxy-1-naphthalenyl)-acetic Acid (Compound #34)
[0243] ##STR00109##
[0244] α-(7-Pentoxy-1-naphthalenyl)-acetic acid ethyl ester (90 mg, 0.30 mmol) was dissolved in a mixed solution of tetrahydrofuran:methanol:2 M aqueous sodium hydroxide solution=2:2:1 (1.5 ml), and the solution was stirred at room temperature for 1 hour. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (10 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=6:1) to obtain α-(7-pentoxy-1-naphthalenyl)-acetic acid (compound #34) (75 mg, yield: 92%); Melting point: 104 to 106° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.71 (d, J=8.1 Hz, 1H), 7.39 (d, J=6.9 Hz, 1H), 7.26 (t, J=8.1, 6.9 Hz, 1H), 7.21 (d, J=2.1 Hz, 1H), 7.15 (dd, J=8.9, 2.1 Hz, 1H), 4.03 (t, J=6.5 Hz, 2H), 4.00 (s, 2H), 3.87 (d, J=8.9 Hz, 1H), 1.82 (m, 2H), 1.45 (m, 2H), 1.39 (m, 2H), 0.93 (t, J=7.1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.6, 157.6, 133.2, 130.2, 129.1, 128.6, 128.4, 128.0, 123.0, 118.7, 103.1, 68.0, 39.1, 28.9, 28.2, 22.5, 14.0; IR (neat): 3014, 2945, 1689, 1463, 1169 cm.sup.−1; HREI-MS found m/z 272.1378 [M].sup.+, calcd for 272.1412 (C.sub.17H.sub.20O.sub.3).
[0245] Compounds #35 to 37 were each synthesized with 5-hydroxy-3-indoleacetic acid methyl ester as a key intermediate.
5-Hydroxy-3-indoleacetic Acid Methyl Ester
[0246] ##STR00110##
[0247] 5-Hydroxy-3-indoleacetic acid (1.00 g) was dissolved in methanol (25 ml). To the solution, acetyl chloride (1.0 ml) was slowly added dropwise, and the mixture was stirred at room temperature for 2 hours. After the reaction was confirmed by TLC to be complete, the reaction was terminated by the addition of a saturated aqueous solution of sodium bicarbonate, and the solvent was distilled off under reduced pressure. Then, water (20 ml) was added to the residue, followed by extraction with ethyl acetate (50 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=3:2) to obtain 5-hydroxy-3-indoleacetic acid methyl ester (1.05 g, yield: 98%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.20 (s, J=8.7 Hz, 1H), 7.13 (d, J=2.4 Hz, 1H), 7.00 (d, J=2.4 Hz, 1H), 6.78 (dd, J=8.8, 2.4 Hz, 1H), 3.72 (s, 2H), 3.70 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.6, 149.6, 131.4, 127.9, 124.2, 112.1, 111.9, 103.4, 107.8, 52.0, 31.2; IR (neat): 3411, 3000, 2952, 1728, 1459, 1459, 1154 cm.sup.−1; EI-MS m/z [M].sup.+205, 146; HREI-MS found m/z 205.0761 [M].sup.+, calcd for 205.0739 (C.sub.11H.sub.11NO.sub.3).
Synthesis of Compound #35
5-(3,5-Dimethoxybenzyloxy)-3-indoleacetic Acid Methyl Ester
[0248] ##STR00111##
[0249] 5-Hydroxy-3-indoleacetic acid methyl ester (42.9 mg, 0.21 mmol) was dissolved in N,N-dimethylformamide (DMF). To this solution, 3,5-dimethoxybenzyl bromide (82.2 mg, 0.36 mmol) was added dropwise, then tetra-N-butylammonium iodide (83.0 mg, 2.00 mmol) and cesium carbonate (136.37 mg, 0.42 mmol) put aside in another container were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was confirmed by TLC to be complete, the reaction was terminated by the addition of an aqueous sodium bicarbonate solution, followed by extraction with ethyl acetate (50 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=3:2) to obtain 5-(3,5-dimethoxybenzyloxy)-3-indoleacetic acid methyl ester (81.5 mg, yield: 94%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.17 (d, J=2.2 Hz, 1H), 7.12 (d, J=8.7 Hz, 1H), 7.04 (s, 2H), 6.92 (dd, J=8.7, 2.2 Hz, 1H), 6.64 (d, J=2.2, 2H), 6.41 (t, J=2.2 Hz, 1H), 5.13 (s, 2H), 3.78 (s, 6H), 3.72 (s, 2H), 3.67 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.5, 160.9 (2C), 153.2, 140.0, 131.4, 124.0, 127.5, 113.0, 111.9, 107.9, 105.2 (2C), 102.2, 99.8, 70.8, 55.3 (2C), 51.9, 31.2; IR (neat): 3396, 2948, 1734, 1449, 1159 cm.sup.−1.
5-(3,5-Dimethoxybenzyloxy)-3-indoleacetic Acid (Compound #35)
[0250] ##STR00112##
[0251] 5-(3,5-Dimethoxybenzyloxy)-3-indoleacetic acid methyl ester (81.5 mg, 0.23 mmol) was dissolved in tetrahydrofuran (0.5 ml). To this solution, methanol (0.5 ml) and a 2 N aqueous sodium hydroxide solution (0.25 ml) were added, and the mixture was stirred at room temperature for 0.5 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=10:1) to obtain 5-(3,5-dimethoxybenzyloxy)-3-indoleacetic acid (compound #35) (55.2 mg, yield: 100%); Melting point: 146.1 to 148.6° C.; H NMR (400 MHz, CDCl.sub.3): δ 7.19 (d, J=8.8 Hz, 1H), 7.12 (d, J=2.2 Hz, 1H), 7.06 (s, 1H), 6.92 (dd, J=8.8, 2.2 Hz, 1H), 6.68 (d, J=2.2 Hz, 2H), 6.40 (t, J=2.2 Hz, 1H), 5.01 (S, 2H), 3.77 (S, 6H), 3.73 (s, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.5, 160.8 (2C), 153.3, 140.0, 131.4, 127.5, 124.1, 113.1, 112.0, 107.4, 105.3 (2C), 102.2, 99.9, 70.9, 55.3 (2C), 31.1; IR (neat): 3406, 2957, 2926, 1702, 1458, 1155 cm.sup.−1.
Synthesis of Compound #36
5-Methoxy-3-indoleacetic Acid Methyl Ester
[0252] ##STR00113##
[0253] 5-Hydroxy-3-indoleacetic acid methyl ester (99.3 mg, 0.48 mmol) was dissolved in N,N-dimethylformamide (2 ml). To this solution, iodomethane (206.2 mg, 1.45 mmol) was added dropwise, then potassium carbonate (200.8 mg, 1.45 mmol) put aside in another container was added, and the mixture was stirred overnight at room temperature and subsequently stirred at 80° C. for 4 hours. After the reaction was confirmed by TLC to be complete, a 10% aqueous sodium bicarbonate solution (20 ml) was added thereto, followed by extraction with ethyl acetate (50 ml). The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain 5-methoxy-3-indoleacetic acid methyl ester (58.6 mg, yield: 55.2%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.22 (1H, d, J=8.8), 7.11 (d, J=2.3 Hz, 1H), 7.05 (d, J=1.3 Hz, 1H), 6.93 (dd, J=8.8, 2.3 Hz, 1H), 3.70 (s, 3H), 3.85 (s, 3H), 3.74 (s, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.5, 154.2, 131.2, 127.6, 123.8, 112.5, 111.9, 108.1, 100.6, 55.9, 51.9, 31.2; IR (neat): 3403, 2951, 1729, 1486, 1213, 1154 cm.sup.−1; EI-MS m/z [M].sup.+219, 160; HREI-MS found m/z 219.0886 [M].sup.+, calcd for 219.0895 (C.sub.12H.sub.13NO.sub.3).
5-Methoxy-3-indoleacetic Acid (Compound #36)
Synthesis of Compound #36
[0254] ##STR00114##
[0255] 5-Methoxy-3-indoleacetic acid methyl ester (60.0 mg, 0.27 mmol) was dissolved in methanol (2 ml). To the solution, lithium hydroxide (19.7 mg, 0.82 mmol) was added, and the mixture was stirred at room temperature for 3 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain 5-methoxy-3-indoleacetic acid (compound #36) (15.3 mg, yield: 27.2%); Melting point: 147.0 to 149.8° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.28 (d, J=8.8 Hz, 1H), 7.26 (s, 1H), 7.11 (d, J=2.3 Hz, 1H), 6.77 (dd, J=8.8, 2.3 Hz, 1H), 3.80 (s, 3H), 3.71 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.3, 154.8, 132.6, 128.9, 125.2, 112.7, 112.4, 108.8, 101.4, 55.8, 31.5; IR (neat): 3359, 2996, 2851, 1705, 1456, 1137 cm.sup.−1; EI-MS m/z [M].sup.+205 (75%), 160; HREI-MS found m/z 205.0737 [M].sup.+, calcd for 205.0739 (C.sub.11H.sub.11NO.sub.3).
Synthesis of Compound #37
5-Ethoxy-3-indoleacetic Acid Methyl Ester
[0256] ##STR00115##
[0257] 5-Hydroxy-3-indoleacetic acid methyl ester (109.0 mg, 0.53 mmol) was dissolved in N,N-dimethylformamide (2 ml). To this solution, iodoethane (248.74 mg, 1.60 mmol) was added dropwise, then potassium carbonate (220.5 mg, 1.60 mmol) put aside in another container was added, and the mixture was stirred at room temperature for 2 hours and stirred at 80° C. for 4 hours. After the reaction was confirmed by TLC to be complete, a 10% aqueous sodium bicarbonate solution (20 ml) was added thereto, followed by extraction with ethyl acetate (50 ml). The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain 5-ethoxy-3-indoleacetic acid methyl ester (100.7 mg, yield: 81.2%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.86 (q, J=7.0 Hz, 2H), 7.23 (d, J=8.8 Hz, 1H), 7.05 (d, J=2.3 Hz, 1H), 7.12 (d, J=2.0 Hz, 1H), 6.87 (dd, J=8.8, 2.3 Hz, 1H), 3.75 (s, 2H), 3.70 (s, 3H), 1.45 (t, J=7.0 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.5, 153.4, 131.2, 127.6, 123.7, 113.0, 111.8, 108.1, 101.8, 64.2, 52.0, 31.2, 15.0; IR (neat): 3404, 2978, 1729, 1474, 1211, 1154 cm.sup.−1; HREI-MS found m/z 233.1034 [M].sup.+, calcd for 233.1052 (C.sub.13H.sub.15NO.sub.3).
5-Ethoxy-3-indoleacetic Acid (Compound #37)
[0258] ##STR00116##
[0259] 5-Ethoxy-3-indoleacetic acid methyl ester (90.2 mg, 0.27 mmol) was dissolved in methanol (4 ml). To the solution, lithium hydroxide (13.9 mg, 0.58 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain 5-ethoxy-3-indoleacetic acid (compound #37) (83.8 mg, yield: 98.9%); Melting point: 86.0 to 92.7° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.23 (d, J=8.8 Hz, 1H), 7.12 (d, J=1.9 Hz, 1H), 7.04 (d, J=2.3 Hz, 1H), 6.86 (dd, J=8.8, 2.3 Hz, 1H), 4.09 (q, J=7.0 Hz, 2H), 3.80 (s, 2H), 1.42 (t, J=7.0 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.4, 153.5, 131.2, 127.5, 124.0, 113.2, 111.9, 107.7, 101.7, 64.2, 31.1, 15.0; IR (neat): 3354, 3066, 2930, 1695, 1457, 1112 cm.sup.−1; EI-MS m/z [M].sup.+219, 205 (40%), 190, 174, 162 (70%), 160 (50%); HREI-MS found m/z 219.0886 [M].sup.+, calcd for 219.0895 (C.sub.12H.sub.13NO.sub.3).
Synthesis of Compound #38
5-(1-Propoxy)-3-indoleacetic Acid Methyl Ester
[0260] ##STR00117##
[0261] 5-Hydroxy-3-indoleacetic acid methyl ester (108.4 mg, 0.53 mmol) was dissolved in N,N-dimethylformamide (2 ml). To this solution, iodopropane was added dropwise, then potassium carbonate (219.3 mg, 1.59 mmol) put aside in another container was added, and the mixture was stirred at room temperature for 2 hours and stirred at 80° C. for 4 hours. After the reaction was confirmed by TLC to be complete, a 10% aqueous sodium bicarbonate solution (20 ml) was added thereto, followed by extraction with ethyl acetate (50 ml). The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain 5-(1-propoxy)-3-indoleacetic acid methyl ester (78.6 mg, yield: 60.1%); Melting point: 38.6 to 41.0° C.; H NMR (400 MHz, CDCl.sub.3): δ 7.21 (d, J=8.8 Hz, 1H), 7.10 (d, J=2.3 Hz, 1H), 7.05 (d, J=2.3 Hz, 1H), 6.86 (dd, J=8.8, 2.3 Hz, 1H), 4.01 (t, J=6.7 Hz, 2H), 3.74 (s, 2H), 3.70 (s, 3H), 1.82 (m, 2H), 1.07 (t, J=6.7 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.5, 153.6, 131.2, 127.6, 123.7, 113.0, 111.8, 108.0, 101.7, 70.4, 52.0, 31.2, 22.8, 10.6; IR (neat): 3355, 3061, 2961, 1695, 1457, 1126 cm.sup.−1; EI-MS m/z [M].sup.+247 (70%), 188 (30%), 149, 131 (75%); HREI-MS found m/z 247.1225 [M].sup.+, calcd for 247.1208 (C.sub.14H.sub.17NO.sub.3).
5-(1-Propoxy)-3-indoleacetic Acid (Compound #38)
[0262] ##STR00118##
[0263] 5-(1-Propoxy)-3-indoleacetic acid methyl ester (64.3 mg, 0.26 mmol) was dissolved in methanol (2 ml). To the solution, lithium hydroxide (9.35 mg, 0.39 mmol) was added, and the mixture was stirred at room temperature for 4 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain 5-(1-propoxy)-3-indoleacetic acid (compound #38) (59.3 mg, yield: 97.7%); Melting point: 133.6 to 136.8° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.23 (d, J=8.8 Hz, 1H), 7.13 (s, 1H), 7.04 (d, J=2.2 Hz, 1H), 6.87 (dd, J=8.1, 2.2 Hz, 1H), 3.96 (t, J=6.6 Hz, 2H), 3.76 (s, 3H), 1.82 (m, 2H), 1.05 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 177.4, 153.7, 131.2, 127.5, 123.9, 113.2, 111.9, 107.5, 101.7, 70.4, 31.0, 22.8, 10.6, 10.6; IR (neat): 3407, 2954, 1728, 1456, 1213, 1160 cm.sup.−1; EI-MS m/z [M].sup.+233, 191 (50%); HREI-MS found m/z 233.1043 [M].sup.+, calcd for 233.1052 (C.sub.12H.sub.15NO.sub.3).
Synthesis of Compound #39
5-(1-Butoxy)-3-indoleacetic Acid Methyl Ester
[0264] ##STR00119##
[0265] 5-Hydroxy-3-indoleacetic acid methyl ester (108.4 mg, 0.53 mmol) was dissolved in N,N-dimethylformamide (2 ml). To this solution, iodobutane was added dropwise, then potassium carbonate (184.2 mg, 1.33 mmol) put aside in another container was added, and the mixture was stirred at 80° C. for 4 hours. After the reaction was confirmed by TLC to be complete, a 10% aqueous sodium bicarbonate solution (20 ml) was added thereto, followed by extraction with ethyl acetate (50 ml). The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (hexane:ethyl acetate=7:3) to obtain 5-(1-butoxy)-3-indoleacetic acid methyl ester (140.2 mg, yield: 80.5%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.21 (d, J=7.2 Hz, 1H), 7.10 (d, J=2.3 Hz, 1H), 7.05 (d, J=2.3 Hz, 1H), 6.86 (dd, J=8.8, 2.3 Hz, 1H), 4.01 (t, J=6.5 Hz, 2H), 3.74 (s, 2H), 3.70 (s, 3H), 1.82 (m, 2H), 1.52 (m, 2H), 0.98 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 172.5, 153.6, 131.2, 127.6, 123.7, 113.0, 111.8, 108.0, 101.7, 68.5, 51.9, 31.9, 31.2, 19.3, 13.9; IR (neat): 3355, 2957, 1694, 1459, 1127 cm.sup.−1; HREI-MS found m/z 261.137 [M].sup.+, calcd for 261.1365 (C.sub.15H.sub.19NO.sub.3).
5-(1-Butoxy)-3-indoleacetic Acid (Compound #39)
[0266] ##STR00120##
[0267] 5-(1-Butoxy)-3-indoleacetic acid methyl ester (91.0 mg, 0.35 mmol) was dissolved in methanol (2 ml). To the solution, lithium hydroxide (12.5 mg, 0.52 mmol) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction was confirmed by TLC to be complete, the reaction solution was rendered acidic (pH=3 to 4) by the addition of 6 N hydrochloric acid, and the solvent was distilled off under reduced pressure. Water (5 ml) was added to the residue, followed by extraction with ethyl acetate (5 ml) three times. The organic layer was washed twice with saturated saline and dehydrated over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then, the residue was purified using silica gel column chromatography (chloroform:methanol=9:1) to obtain 5-(1-butoxy)-3-indoleacetic acid (compound #39) (43.8 mg, yield: 51.0%); Melting point: 137.8 to 141.1° C.; H NMR (400 MHz, CDCl.sub.3): δ 7.24 (d, J=8.8 Hz, 1H), 7.14 (s, 1H), 7.04 (d, J=2.0 Hz, 1H), 6.87 (dd, J=8.8, 2.0 Hz, 1H), 4.01 (t, J=6.6 Hz, 2H), 3.76 (s, 2H), 1.78 (m, 2H), 1.05 (t, J=7.4 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.3, 153.8, 131.2, 123.9, 113.2, 111.6, 107.5, 101.6, 31.6, 29.7, 19.3, 13.9; IR (neat): 3407, 2954, 1728, 1456, 1213, 1160 cm.sup.−1; EI-MS m/z [M].sup.+247, 191 (60%); HREI-MS found m/z 247.1189 [M].sup.+, calcd for 247.1208 (C.sub.14H.sub.17NO.sub.3).
Synthesis of Compound of Formula (1)
Synthesis of 4-(2,4-difluorophenyl)-2-(6-fluoro-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound (4-1))
[0268] ##STR00121##
[0269] In a 50-mL round-bottomed flask, 6-fluoroindole (485 mg, 3.59 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (508 mg, 2.39 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 7 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was purified using silica gel column chromatography (hexane:acetone=2:1) to obtain 4-(2,4-Difluorophenyl)-2-(6-fluoro-1H-indol-3-yl)-4-oxo-butanoic acid (433 mg, yield: 52%) as a colorless crystal.
[0270] Melting point: 210 to 214° C.;
[0271] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 10.31 (s, 1H), 8.01 (m, 1H), 7.47 (dd, J=8.8, 5.2, 1H), 7.34 (d, J=2.0, 2H), 7.12-7.20 (m, 3H), 6.92 (td, J=9.6, 2.4, 1H), 4.54 (dd, J=10.4, 4.0, 1H), 4.01 (ddd, J=18.8, 10.8, 3.2, 1H), 3.38 (td, J=18.8, 3.2, 1H); .sup.13C-NMR (100 MHz, CDCl.sub.3) δ 195.18, 174.85, 166.28 J.sub.C-F (dd, 254, 13 Hz), 163.42 J.sub.C-F (dd, 254, 13 Hz), 161.74, 159.4, 137.54 J.sub.C-F (d, 13 Hz), 133.47 J.sub.C-F (dd, 11, 3 Hz), 123. 35 J.sub.C-F (d, 4 Hz), 123. 02 J.sub.C-F (dd, 13, 4 Hz), 120.97 J.sub.C-F (d, 11 Hz), 113.51, 112.94 J.sub.C-F (dd, 10, 2 Hz), 108.27 J.sub.C-F (d, 24 Hz), 105.59 J.sub.C-F (t, 27 Hz), 98.27 J.sub.C-F (d, 26 Hz), 46.68 J.sub.C-F (d, 7 Hz), 38.47;
FAB-MS m/z=348 [M+H].sup.+
Synthesis of 4-(2,4-difluorophenyl)-2-(5-fluoro-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound (3-1))
[0272] ##STR00122##
[0273] In a 50-mL round-bottomed flask, 5-fluoroindole (925 mg, 6.85 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (969 mg, 4.57 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 11 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted twice with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from chloroform and ethyl acetate to obtain 4-(2,4-difluorophenyl)-2-(5-fluoro-1H-indol-3-yl)-4-oxo-butanoic acid (1122 mg, yield: 71%) as a colorless crystal.
[0274] Melting point: 207 to 208° C.;
[0275] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.34 (s, 1H), 8.02 (m, 1H), 7.47 (dd, J=10.4, 2.8, 1H), 7.39-7.43 (m, 2H), 7.13-7.21 (m, 2H), 6.93 (td, J=9.2, 2.8, 1H), 4.52 (dd, J=10.4, 3.6, 1H), 4.03 (ddd, 18.4, 10.8, 3.6, 1H), 3.40 (td, 18.4, 3.6, 1H); .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ 195.17, 174.86, 166.52 J.sub.C-F (dd, 254, 13 Hz), 163.58 J.sub.C-F (dd, 254, 13 Hz), 159.52, 157.21, 134.25, 133.46 J.sub.C-F (dd, 11, 4 Hz), 127.66 J.sub.C-F (d, 11 Hz), 123.01 J.sub.C-F (dd, 10, 4 Hz), 113.42 J.sub.C-F (d, 5 Hz), 113.28 J.sub.C-F (d, 10 Hz), 113.51 J.sub.C-F (dd, 21, 4 Hz), 110.56 J.sub.C-F (d, 27 Hz), 105.61 J.sub.C-F (t, 27 Hz), 104.65 J.sub.C-F (d, 24 Hz), 46.68 J.sub.C-F (d, 8 Hz), 38.48;
FAB-MS m/z=348 [M+H].sup.+
Synthesis of 2-(7-chloro-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic Acid (Compound(5-1))
[0276] ##STR00123##
[0277] In a 50-mL round-bottomed flask, 7-chloroindole (1094 mg, 5.16 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (935 mg, 4.41 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 10 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted twice with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from benzene and acetone to obtain 2-(7-chloro-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic acid (1017 mg, yield: 54%) as a colorless crystal.
[0278] Melting point: 225 to 227° C.;
[0279] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.55 (s, 1H), 8.01 (m, 1H), 7.75 (d, J=8.4, 1H), 7.45 (d, J=2.8, 1H), 7.06-7.14 (m, 4H), 7.08 (t, J=7.6, 1H), 4.57 (dd, J=10.4, 3.6, 1H), 4.03 (ddd, 18.4, 10.8, 3.6, 1H), 3.41 (td, 18.4, 3.6, 1H);
[0280] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.06, 74.67, 166.53, J.sub.C-F (dd, 252, 12 Hz), 163.63 J.sub.C-F (dd, 252, 12 Hz), 134. 45, 133.46 J.sub.C-F (dd, 11, 5 Hz), 129.25, 124.97, 122.98 J.sub.C-F (dd, 13, 4 Hz), 121.91, 120.84, 119.03, 117.22, 114.73, 112.99 J.sub.C-F (dd, 21, 3 Hz), 105.61 J.sub.C-F (t, 27 Hz), 46.71 J.sub.C-F (d, 8 Hz), 38.50;
FAB-MS m/z=364 [M+H].sup.+
Synthesis of 2-(5-chloro-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic Acid (Compound(3-2))
[0281] ##STR00124##
[0282] In a 50-mL round-bottomed flask, 5-chloroindole (1000 mg, 6.61 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (935 mg, 4.41 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 7 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted twice with ethyl acetate (50 mL). The organic layer was washed twice with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from benzene and acetone to obtain 2-(5-chloro-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic acid (1084 mg, yield: 63%) as a light yellow crystal.
[0283] Melting point: 236 to 239° C.;
[0284] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.45 (s, 1H), 8.02 (q, J=8.3, 1H), 7.80 (d, J=1.6, 1H), 7.44 (m, 2H), 7.11-7.22 (m, 3H), 4.54 (dd, J=10.4, 3.8, 1H), 4.01 (ddd, J=18.7, 10.7, 3.2, 1H), 3.41 (td, J=18.7, 3.2, 1H);
[0285] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.10, 174.76, 166.52 J.sub.C-F (dd, 253, 12 Hz), 163.62 J.sub.C-F (dd, 253, 12 Hz), 136.06, 133.44 J.sub.C-F (dd, 12, 4 Hz), 128.47, 125.62, 122.96 J.sub.C-F (dd, 13, 4 Hz), 122.48, 119.33, 113.77, 113.12, 112.96 J.sub.C-F (dd, 22, 3 Hz), 105.61 J.sub.C-F (t, 27 Hz), 46.72 J.sub.C-F (d, 8 Hz), 38.35;
FAB-MS m/z=364 [M+H].sup.+
Synthesis of 2-(4-chloro-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic Acid (Compound(2-1))
[0286] ##STR00125##
[0287] In a 50-mL round-bottomed flask, 4-chloroindole (903 mg, 5.98 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (842 mg, 3.97 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 7 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified using silica gel column chromatography (chloroform:methanol=9:1), and the residue was then recrystallized from benzene and acetone to obtain 2-(4-chloro-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic acid (602 mg, yield: 51%) as a colorless crystal.
[0288] Melting point: 203 to 204° C.;
[0289] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.24 (s, 1H), 8.01 (m, 1H), 7.77 (d, J=8.4, 1H), 7.42 (d, J=8.0, 1H), 7.21˜7.03 (m, 4H), 4.57 (dd, J=10.8, 3.6, 1H), 4.03 (ddd 18.8, 10.8, 3.2, 1H), 3.38 (td, 18.8, 3.2, 1H);
[0290] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.28, 174.97, 166.55 J.sub.C-F (dd, 254, 12 Hz), 163.62 J.sub.C-F (dd, 254, 12 Hz), 137.646, 137.49, 133.46 J.sub.C-F (dd, 11, 4 Hz), 127.37, 123.70, 123.54, 123.05 J.sub.C-F (dd, 13, 4 Hz), 122.39, 119.88, 119.79, 112.95 J.sub.C-F (dd, 22, 4 Hz), 105.58 J.sub.C-F (t, 26 Hz), 46.95 J.sub.C-F (d, 8 Hz), 38.47;
FAB-MS m/z=364 [M+H].sup.+
Synthesis of 4-(2,4-difluorophenyl)-2-(5-methyl-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound(3-3))
[0291] ##STR00126##
[0292] In a 50-mL round-bottomed flask, 5-methylindole (171 mg, 1.31 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (185 mg, 0.87 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 7 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from benzene and acetone to obtain 4-(2,4-difluorophenyl)-2-(5-methyl-1H-indol-3-yl)-4-oxo-butanoic acid (200 mg, yield: 67%) as a colorless crystal.
[0293] Melting point: 200 to 202° C.;
[0294] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.10 (s, 1H), 8.01 (m, 1H), 7.54 (s, 1H), 7.20-7.31 (m, 2H), 7.13-7.20 (m, 1H), 7.96 (d, J=6.8, 1H), 4.53 (dd, J=10.6, 3.6, 1H), 4.01 (ddd 18.8, 10.6, 3.2, 1H), 3.36 (td, 18.8, 3.2, 1H), 2.40 (s, 3H);
[0295] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.33, 175.08, 166.54 J.sub.C-F (dd, 254, 12 Hz), 163.49 J.sub.C-F (dd, 254, 12 Hz), 136. 01, 133.45 J.sub.C-F (dd, 11, 4 Hz), 128.59, 127.63, 124.02, 123.72, 123.05 J.sub.C-F (dd, 13, 4 Hz), 119.43, 112.94 J.sub.C-F (dd, 22, 4 Hz), 112.74, 112.03, 105.59 J.sub.C-F (t, 26 Hz), 47.01 J.sub.C-F (d, 7 Hz), 38.45, 21.64;
FAB-MS m/z=344 [M+H].sup.+
Synthesis of 4-(2,4-difluorophenyl)-2-(1-methyl-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound(6-1))
[0296] ##STR00127##
[0297] In a 50-mL round-bottomed flask, 1-methylindole (2512 mg, 19.17 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (2710 mg, 12.78 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 1 hour to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted twice with ethyl acetate (50 mL). The organic layer was washed twice with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from benzene and acetone to obtain 4-(2,4-difluorophenyl)-2-(1-methyl-1H-indol-3-yl)-4-oxo-butanoic acid (3898 mg, yield: 89%) as a colorless crystal.
[0298] Melting point: 192 to 193° C.; .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ 8.00 (m, 1H), 7.75 (d, J=7.6, 1H), 7.37 (d, J=8.4, 1H), 7.12-7.22 (m, 4H), 7.07 (t, J=7.6, 1H), 4.54 (dd, J=10.8, 3.6, 1H), 4.00 (ddd, 18.8, 10.4, 3.6, 1H), 3.79 (s, 3H), 3.36 (td, 18.8, 3.6, 1H);
[0299] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.22, 174.94, 166.52 J.sub.C-F (dd, 253, 13 Hz), 163.57 J.sub.C-F (dd, 253, 13 Hz), 138.08, 133.46 J.sub.C-F (dd, 11, 4 Hz), 128.00, 127.79, 123.03 J.sub.C-F (dd, 13, 4 Hz), 122.36, 120.07, 119.72, 112.94 J.sub.C-F (d, 22, 4 Hz), 112.33, 110.32, 105.60 J.sub.C-F (t, 27 Hz), 47.01 J.sub.C-F (d, 8 Hz), 38.36, 32.72;
FAB-MS m/z=344 [M+H].sup.+
Synthesis of 4-(2,4-difluorophenyl)-2-(7-methoxy-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound(5-2))
[0300] ##STR00128##
[0301] In a 50-mL round-bottomed flask, 7-methoxyindole (1083 mg, 7.36 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (1041 mg, 4.90 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 14 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from benzene and acetone to obtain 4-(2,4-difluorophenyl)-2-(7-methoxy-1H-indol-3-yl)-4-oxo-butanoic acid (1179 mg, yield: 67%) as a colorless crystal.
[0302] Melting point: 181 to 183° C.;
[0303] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.26 (s, 1H), 8.01 (m, 1H), 7.35 (d, J=8.0, 1H), 7.28 (d, J=2.8, 1H), 7.12-7.20 (m, 2H), 6.98 (t, J=7.8, 1H), 6.67 (d, J=7.8, 1H), 4.54 (dd, J=10.8, 3.6, 1H), 4.03 (ddd 18.8, 10.6, 3.3, 1H), 3.92 (s, 3H), 3.36 (td, 18.6, 3.2, 1H);
[0304] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ1195.28, 174.98, 166.54 J.sub.C-F (dd, 252, 12 Hz), 163.48 J.sub.C-F (dd, 252, 12 Hz), 147.35, 133.44 J.sub.C-F (dd, 11, 4 Hz), 128.81, 127.83, 123.22, 123.05 J.sub.C-F (dd, 13, 4 Hz), 120.43, 113.76, 112.94 J.sub.C-F (dd, 22, 4 Hz), 112.7, 105.59 J.sub.C-F (t, 27 Hz), 102.52, 55.52, 46.97 J.sub.C-F (d, 8 Hz), 38.59;
FAB-MS m/z=360 [M+H].sup.+
Synthesis of 4-(2,4-difluorophenyl)-2-(5-methoxy-1H-indol-3-yl)-4-oxo-butanoic Acid (Compound(3-4))
[0305] ##STR00129##
[0306] In a 50-mL round-bottomed flask, 5-methoxyindole (1166 mg, 7.93 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 10 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted twice with ethyl acetate (50 mL). The organic layer was washed twice with saturated saline (30 mL), and dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from benzene and acetone following by recrystallizing from chloroform to obtain 4-(2,4-difluorophenyl)-2-(5-methoxy-1H-indol-3-yl)-4-oxo-butanoic acid (1478 mg, yield: 75%) as a colorless crystal.
[0307] Melting point: 205 to 206° C.;
[0308] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ10.09 (s, 1H), 8.02 (m, 1H), 7.27-7.31 (m, 3H), 7.13-7.20 (m, 2H), 6.79 (dd, J=8.8, 2.4, 1H), 4.52 (dd, J=10.8, 3.6, 1H), 4.00 (ddd, 18.8, 10.4, 3.4, 1H), 3.80 (s, 3H), 3.38 (td, 18.6, 3.4, 1H;
[0309] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.35, 175.03, 166.53, J.sub.C-F (dd, 253, 12 Hz), 163.61 J.sub.C-F (dd, 253, 12 Hz), 154. 84, 133.45 J.sub.C-F (dd, 11, 4 Hz), 132.72, 127.78, 124.22, 123.04 J.sub.C-F (dd, 12, 4 Hz), 112.95 J.sub.C-F (dd, 22, 4 Hz), 112.96, 112.68, 105.61 J.sub.C-F (t, 26 Hz), 101.58, 55.79, 46.9 J.sub.C-F (d, 8 Hz), 38.67;
FAB-MS m/z=360 [M+H].sup.+
Synthesis of 2-(6-benzyloxy-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic Acid (Compound(4-2))
[0310] ##STR00130##
[0311] In a 50-mL round-bottomed flask, 6-benzyloxyindole (1255 mg, 5.62 mmol) was added and (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid (795 mg, 3.74 mmol) was dissolved in benzene (20 mL) and the mixture was heated at 80° C. for 9 hours to reflux. Distilled water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (50 mL). The organic layer was washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product obtained by the concentration was filtrated with a Hirsch funnel, and the residue was recrystallized from chloroform to obtain 2-(6-benzyloxy-1H-indol-3-yl)-4-(2,4-difluorophenyl)-4-oxo-butanoic acid (531 mg, yield: 33%) as a light yellow crystal.
[0312] Melting point: 177 to 178° C.;
[0313] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ 10.0 4 (s, 1H), 8.01 (m, 1H), 7.65 (d, J=8.8, 1H), 7.48 (d, J=7.6, 2H), 7.38 (t, J=7.2, 2H), 7.31 (m, 1H), 7.13-7.20 (m, 3H), 7.03 (d, J=2.0, 1H), 6.83 (dd, J=8.4, 2.0, 1H), 4.51 (dd, J=10.4, 3.6, 1H), 4.01 (ddd, 18.1, 10.4, 3.3, 1H), 3.36 (td, 18.1, 3.3, 1H);
[0314] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ195.29, 175.00, 166.54 J.sub.C-F (dd, 254, 12 Hz), 163.52 J.sub.C-F (dd, 254, 12 Hz), 156.33, 138.85, 138.45, 133.45, J.sub.C-F (dd, 11, 4 Hz), 129.17, 128.37, 128.23, 123.01 J.sub.C-F (dd, 13, 4 Hz), 122.49, 121.97, 120.54, 113.24, 113.06 J.sub.C-F (dd, 21, 4 Hz), 110.73, 105.59 J.sub.C-F (t, 27 Hz), 96.89, 70.70, 46.94 J.sub.C-F (d, 8 Hz), 38.57;
FAB-MS m/z=436 [M+H].sup.+
Reference Example 1
(E)-4-(2,4-Difluorophenyl)-4-oxo-2-butenoic Acid
[0315] ##STR00131##
[0316] (E)-4-(2,4-Difluorophenyl)-4-oxo-2-butenoic acid which is used for the synthesize of the present compounds #1 to 10 was synthesized as follows. Specifically, in a 100-mL round-bottomed flask, 1,3-difluorobenzene (1300 mg, 11.39 mmol) and maleic anhydride (894 mg, 9.12 mmol) was added and dissolved in dichloromethane (40 mL) and stirred by a stirrer machine. While stirring, to the solution, anhydrous aluminum chloride (2279 mg, 17.09 mmol) were added little by little, and the mixture was stirred at room temperature for 6 hours. The reaction solution in the round-bottomed flask was added into iced water (100 mL) to terminate the reaction. Thereafter, the water layer was extracted with ethyl acetate (150 mL), and the organic layer was extracted with saturated saline (100 mL) for 2 times Thereafter, the organic layer was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a solid. The solid was purified by recrystallization from benzene and a little amount of acetone to obtain (E)-4-(2,4-difluorophenyl)-4-oxo-2-butenoic acid as a crystal with light yellow color by yield 48%.
[0317] Melting point: 136.0 to 139.0° C.;
[0318] .sup.1H-NMR (400 MHz, acetone-d.sub.6) δ 7.98 (m, 1H), 7.72 (dd, J=15.6, 3.6, 1H), 7.20-7.28 (m, 2H), 6.75 (d, J=15.6, 2H);
[0319] .sup.13C-NMR (100 MHz, acetone-d.sub.6) δ187.13, 166.86 J.sub.C-F (dd, 254, 12 Hz), 166.35, 163.33 J.sub.C-F (dd, 254, 12 Hz), 139.95 J.sub.C-F (d, 7 Hz), 133.98 J.sub.C-F (dd, 66, 59 Hz), 132.91, 123.22 J.sub.C-F (d, 9 Hz), 113.33 J.sub.C-F (dd, 22, 3 Hz), 105.76, J.sub.C-F (t, 22 Hz)
Example 2
[0320] 2. Confirmation that the Compound Group of the Present Invention has an Effect of Enhancing Fertilization Function of a Mammal Sperm
[0321] The effect of enhancing fertilization function of a mammal sperm was confirmed by a method given below using the compound group of the present invention (compound #5) synthesized in Example 1. Sperm preculture and semination were performed under conditions of 5% CO.sub.2/20% O.sub.2 and 37° C.
[0322] [Method]
[1] Pregnant mare serum gonadotropin (PMSG) was intraperitoneally administered to female C57/B6 mice. Multiple ovulation was induced with human chorionic gonadotropin (hCG) 48 hours after PMSG administration. The mice were euthanized 5 to 17 hours after hCG administration, and their fallopian tubes were recovered.
[2] Male C57/B6 mice were euthanized, and their epididymal tails were then removed and recovered.
[3] Droplets (drops) consisting of 100 μL each of 4 types of culture media (culture medium for semination [TYH culture medium, manufactured by LSI Medience Corp.] containing 0.0001% DMSO, TYH culture medium containing 1 μM compound #5, TYH culture medium containing 10 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and TYH culture medium containing 10 μM FCCP and 1 μM compound #5) were prepared and covered with mineral oil (manufactured by Sigma-Aldrich Corp.). The recovered epididymal tails were transferred into the mineral oil, and the epididymal ducts were damaged with a 1 mL syringe for tuberculin with a 26 G needle (manufactured by Nipro Corp.) while held with tweezers. The obtained sperm masses were transferred to the 4 types of culture media in the mineral oil and cultured before semination (precultured) for 1 hour.
[4] 3 μL each of the sperms precultured in the 4 types of culture media was transferred into 200 μL of drops of TYH culture medium containing a cumulus oocyte complex (COC) using a micropipette (manufactured by Eppendorf AG, Germany) and covered with mineral oil (manufactured by Sigma-Aldrich Corp.), followed by semination.
[5] The presence or absence of a two-cell egg was observed 24 to 28 hours after semination. When the two-cell egg was observed, fertilization was evaluated as being successful and the fertilization rate was measured.
[0323] [Results]
[0324] Semination using sperms precultured in TYH culture medium containing the solvent DMSO for compound #5, i.e., a culture medium free from compound #5, had a fertilization rate of 40%, whereas semination using sperms precultured in a culture medium containing compound #5 had a fertilization rate of 100% (see Table 1).
[0325] The results indicate that the compound group of the present invention (compound #5) has an effect of activating (enhancing) fertilization function of sperms.
TABLE-US-00001 TABLE 1 The number The number of Fertilization Culture medium of ova two-cell eggs rate (%) TYH culture medium 5 2 40 Compound #5-containing 11 11 100 TYH culture medium
[0326] Sperms move forwardly by flagella through the use of ATP produced by mitochondria. The effect of the compound group of the present invention (compound #5) on sperms having decreased fertilization function was studied by inhibiting such mitochondrial function.
[0327] Semination using sperms precultured in a culture medium containing FCCP, an uncoupler, had a fertilization rate of 30%, which was confirmed to be lower than that (47.4%) of semination using sperms precultured in a culture medium free from FCCP (see Table 2). On the other hand, preculture of sperms in a FCCP-containing culture medium supplemented with compound #5 recovered the fertilization rate to 50% (see Table 2).
[0328] The results indicate that the compound group of the present invention (compound #5) has an effect of improving the decreased fertilization function (e.g., forward motion using ATP) of sperms.
TABLE-US-00002 TABLE 2 The number The number of Fertilization Culture medium of ova two-cell eggs rate (%) TYH culture medium 19 9 47.4 FCCP-containing TYH 10 3 30 culture medium Compound #5- and 10 5 50 FCCP-containing TYH culture medium
INDUSTRIAL APPLICABILITY
[0329] The present invention contributes to treatment of infertility.