US 4,971,978 AGrant
Derivatives of D-Glutamic Acid and D-Aspartic Acid
Issue Date:1990-11-20
•8 Claims
Abstract
A compound of the formula: ##STR1## wherein R.sub.1 and R.sub.2 are substituents; R.sub.6 is hydrogen, loweralkyl, cycloalkyl, loweralkenyl, arylalkyl, (substituted aryl)alkyl, or (heterocyclic)alkyl; B is a bond, an alkylene group, an alkenylene group, an alkadienylene group or a heteroatom containing linking group; D is a substituted acyl group or tetrazolyl; Z is CO, C(S) or S(O).sub.2 ; Ar is a heterocyclic group; and n is 1 to 3. Also disclosed are a composition and a method for antagonizing CCK, a composition and method for treating or preventing gastrointestinal, central nervous, appetite regulating and pain regulating systems, a composition and method for treating or preventing hyperinsulinemia, methods of making the compounds and intermediates useful in making the compounds.
Metadata
Inventors
- Alex M. Nadzan
- James F. Kerwin
- Chun W. Lin
Application Information
Application Number:US 2345250
Filing Date:1988-08-22
Priority Date:1987-09-21
Art Unit:121
Classifications
IPC:
C07D21522A61K 3147
Field of Search:
546514156312
Patent Drawings
This patent does not have any drawings.
Description
The following examples will serve to further illustrate preparation of the novel compounds of the invention.
EXAMPLE 1
N-Boc-(.gamma.-benzyl ester)-R-Glu-di-n-pentylamide
To the solution of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (4.0 g, 11.9 mmol) in tetrahydrofuran (THF) (100 mL) was added bis (2-oxo-3-oxazolidinyl)-phosphinic chloride (BOPCl) (3.1 g, 11.9 mmol) and di-n-pentylamine (6 mL, 30 mmol) at room temperature. The reaction mixture was allowed to stir overnight. The solvents were evaporated and the residue dissolved in ethyl acetate (300 mL) and washed with 1N hydrochloric acid (HCl), saturated NaHCO.sub.3 (sodium bicarbonate), H.sub.2 O and brine and dried over magnesium sulfate (MgSO.sub.4). After evaporation of solvents the product was subjected to flash chromatography using ethyl acetate (EtOAc)-hexane (2:8) as the elutant. The pure oily product was isolated in 78.1% yield (4.5 g). [.alpha.].sub.D =-26.1.degree. (c=1.3, methanol (MeOH)). MS(CI) m/e 477(m+H).sup.+, 421, 381, 363, 325, 299, 282. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta.0.9(m,6H), 1.3(m,8H), 1.4(s,9H), 1.6(m,4H), 1.7(m,2H), 2.5(m,2H), 3.1-3.2(m,2H), 3.5(m,2H), 4.6(m,1H), 5.1(s,2H), 5.4(d,1H,J=9 Hz), 7.4(s,5H).
EXAMPLE 2
(.gamma.-Benzyl ester)-R-Glu-di-n-pentylamide hydrochloride
t-Butyloxycarbonyl derivative of example 1, (1.2 g, 2.5 mmol) was dissolved in 5 mL of 4M HCl in dioxane and the mixture was stirred for 30 min. The solvents were evaporated and the residue was triturated several times with ether and the volatiles were evaporated. The product was filtered and washed several times with diethyl ether. Yield 0.9 g (96%). MS(CI) m/e 377(m+H).sup.+, 359, 269, 241. 1H NMR(DMSO.sub.d6, 300 MHz) .delta. 0.8-0.9(m,6H), 1.15-1.35(m,8H), 1.4-1.6(m,4H), 1.90-2.05(m,2H), 2.45-2.70(m,2H), 3.05(m,1H), 3.15(m,1H), 3.30(H.sub.2 O), 3.35-3.55(m,2H), 4.23(m,1H), 5.1(bs,2H), 7.37(bs,5H), 8.2-8.4(bs,2H).
EXAMPLE 3
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu -di-n-pentylamide
To a solution of hydrochloride of example 2 (7 g, 17 mmol) and N-methylmorpholine (NMM) (3.8 mL, 34.5 mmol) in dimethylformamide (DMF) at 0.degree. C. was added indole-2-carboxylic acid (2.75 g, 17 mmol), 1-hydroxybenzotriazole (HOBt) (4.9 g, 36.3 mmol) followed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (3.25 g, 17 mmol). The reaction mixture was allowed to warm to ambient temperature and was stirred overnight. The solvent was concentrated under vacuo and after addition of water, the mixture was extracted successively with 1 N hydrochloric acid, water, saturated solution of sodium bicarbonate and brine. The solution was dried over magnesium sulfate and filtered. Purification was accomplished by crystallization from ethyl acetate and hexane providing 7.55 g (85.6%) of product. [.alpha.].sub.D =+24.00.degree. (c=0.5, MeOH). mp=84.degree.-86.degree. C. MS(CI) m/e 520(m+H).sup.+, 429, 286, 144. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 0.85-0.95(m,6H), 1.2-1.4 (m,8H), 1.50-1.66(m,4H), 2.0(m,1H), 2.20(m,1H), 2.40-2.65(m,2H), 3.1(m,1H), 3.25(m,1H), 3.48-3.62(m,2H), 5.05(d,1H,J=12 Hz), 5.14 (d,1H,J=12 Hz), 5.18(dt,1H,J=3,9 Hz), 6.95(d,1H,J=2 Hz), 7.15(dt,1H,J=1,7.5 Hz), 7.3(m,2H), 7.35(m,5H), 7.4(d,1H,J=7.5 Hz), 7.65(d,1H,J=7.5 Hz), 9.22(bs,1H) Analysis calculated for C.sub.31 H.sub.41 N.sub.3 O.sub.4 H.sub.2 O: C 71.03, H 7.98, N 8.02; Found: C 71.05, H 7.98, N 8.03.
EXAMPLE 4
N-(2'-Indolylcarbonyl)-R-Glu-N.alpha.,N.alpha.-di-n-pentylamide
To a suspension of 0.9 g 10% palladium on carbon (Pd/,C) in methanol (10 mL) and cyclohexadiene (3 mL) was added under nitrogen atmosphere (N.sub.2) a solution of benzyl ester of example 3 (1.3 g, 25 mmol) in methanol by cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with methanol. The filtrate and washings were combined and concentrated under vacuo. The product was purified by flash chromatography using chloroform (CHCl.sub.3)-methanol and ammonium hydroxide (NH.sub.4 OH) 90:10:1 as elutant. Lyophilization provided product in 83.8% yield (0.9 g). mp=78.degree.-79.degree. C. [.alpha.].sub.D =+2.8.degree. (c=1.0, MeOH). MS(FAB) m/e 430(m+H)+, 273, 245, 226. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta.0.8-0.9(m,6H), 1.12-1.38(m,8H), 1.39-1.5(m,4H), 1.56-1.81(m,2H), 1.85-2.0(m,2H), 2.3(t,J=6 Hz,2H), 3.18(m,1H), 3.54-3.62(m, 2H), 4.85(m,1H), 7.03(t,J=7 Hz,1H), 7.18(t,J=7 Hz,1H), 7.25(s,1H), 7.43(d,=9 Hz,1H), 7.61(d,J=9 Hz,1H), 8.8(d,J=8 Hz,1H), 11.6(bs,1H). Analysis calculated for C.sub.24 H.sub.35 N.sub.3 O.sub.4 H.sub.2 O: C 65.77, H 8.18, N 9.59. Found: C 65.99, H 8.01, N 9.76.
EXAMPLE 5
N-(2'-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-di-n-pentylamide
To a solution of hydrochloride of example 2 (0.25 g, 0.61 mmol) and N-methylmorpholine (0.14 mL, 1.2 mmol) in DMF (5 mL) at 0.degree. C. was added HOBt (0.16 g, 1.2 mmol) and quinoline-2-carboxylic acid (0.11 g, 0.61 mmol) followed by EDCI (0.125 g, 0.65 mmol). The reaction mixture was stirred overnight at room temperature. The solvent was evaporated. After addition of water, the mixture was extracted with ethyl acetate. Combined ethyl acetate extracts were washed with 1.0 N hydrochloric acid, water, a saturated solution of sodium bicarbonate and brine. The solution was dried over magnesium sulfate. The ethyl acetate was evaporated and residue was purified by chromatography using hexane and ethyl acetate as elutant. The product was isolated in 33% yield (0.1 g). MS(CI m/e 532(m+H).sup.+, 347, 156, 128,91. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta.0.85-0.95(m,6H), 1.2-1.45(m,8H), 1,6(m,4H), 2.05(m,1H), 2.25(m,1H)) 2.46-2.68(m.2H). 3.1(m,1H), 3.3(m.1H), 3.6(m,2H), 5.12(s,ZH), 5.Z5(dt,J=3,9 Hz,1H), 7.4(m,5H), 7.6(dt,J=1,6 Hz,1H), 7.78(dt,J.TM.1,6 Hz,1H), 7.9(d,J=9 Hz,1H), 8.15(d,J=9 Hz,1H), 8.25(d,J=3 Hz,1H), 8.30(d,J=9 Hz,1H), 9.1(d,J=9 Hz,1H).
EXAMPLE 6
N-(2'-Quinolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
To a suspension of 0.1 g 10% Pd/C in methanol (3 mL) and cyclohexadiene (1 mL) was added under inert atmosphere (N.sub.2) a solution of benzyl ester of example 5 (0.09 g, 0.170 mmol) in methanol by cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with methanol. The filtrate and washings were combined and concentrated in vacuo. The product was purified by flash chromatography using chloroform-methanol and ammonium hydroxide 90:10:1 as elutant. Lyophilization provided product in 60% yield (0.045 g). MS(CI) m/e 442(m+H).sup.+, 332, 286, 257, 156, 128. 1H NMR(CDCl.sub.3,300 MHz) .delta.0.82-0.92(m,6H), 1.2-1.4(m,8H), 1.50-1.68(m,4H), 2.0(m,1H), 2.2(m,1H), 2.5(m,2H), 3.1(m,1H), 3.25(m,1H), 3.48-3.70(m,2H), 5.2(dt,J=3,9 Hz,1H), 7.60(dt,J=1,6 Hz,1H), 7.75(dt,J=1,6 Hz,1H), 7.85(d,J=6 Hz,1H), 8.18(d,J=9 Hz,1H), 8.25(d,J=6 Hz,1H), 8.31(d,J=9 Hz,1H), 9.2(d,J=6 Hz,1H). Analysis calculated for C.sub.25 H.sub.35 N.sub.3 O.sub.4 0.5 H.sub.2 O: C 66.64, H 7.95, N 9.33. Found: C 66.65, H 7.87, N 9.21.
EXAMPLE 7
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-S-Glu-di-n-pentylamide
To a solution of hydrochloride prepared analogously as in example 2 (0.92 g, 2.23 mmol) and N-methylmorpholine (0.5 mL, 4.5 mmol) in DMF at 0.degree. C. was added indole-2-carboxylic acid (0.36 g, 2.23 mmol), HOBt (0.45 g, 3.35 mmol) followed by EDCI (0.44 g, 2.23 mmol). The reaction mixture was allowed to warm to ambient temperature and was stirred overnight. The solvent was concentrated in vacuo. After addition of ethyl acetate and water, the mixture was extracted with 1.0 N hydrochloric acid, water, a saturated solution of sodium bicarbonate and brine. The solution was dried over magnesium sulfate and filtered. Purification was accomplished by crystallization from ethyl acetate and hexane providing 0.8 g (69.1%) of product. mp=83.degree.-84.degree. C. [.alpha.].sub.D =-23.55.degree. (c=1.0, MeOH). MS(CI) m/e 520(m+H).sup.+. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 0.8-0.9(m,6H), 1.2-1.45(m,8H), 1.52-1.6(m,4H), 2.0(m,1H), 2.2(m,1H), 2.4-2.65(m,2H), 3.1(m,1H), 3.25(m,1H), 3.5-3.68(m,2H), 5.1(d,J=4 Hz; 1), 5.15(d,J=12 Hz,1H), 5.2(m,1H), 6.9(d,J=3 Hz,1H), 7.15(t,J=9 Hz,1H), 7.3(m,1H), 7.35(m,6H), 7.4(d,J=9 Hz,1H), 7.65(d,J=9 Hz,1H), 9.2(s,1H).
EXAMPLE 8
N-(2'-Indolylcarbonyl)-S-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
To a suspension of 0.5 g 10% Pd/C in methanol (10 mL) and cyclohexadiene (3 mL) was added under inert atmosphere (N.sub.2) solution of benzyl ester of example 7 (0.8 g, 1.55 mmol) in methanol by cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with methanol. The filtrate and washings were combined and concentrated in vacuo. The product was purified by flash chromatography using chloroform-methanol and ammonium hydroxide 90:10:1 as elutant. Lyophilization provided product in 89.5% yield (0.6 g). mp=76.degree.-74.degree. C. [.alpha.].sub.D =-6.5.degree. (c=0.8, MeOH). MS(CI) m/e 430(m+H).sup.+, 273, 245, 144. .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 0.8-0.9(m,6H), 1.1-1.45(m,8H), 1.48-1.5(m,4H), 1.53-1.75(m,2H), 1.80-2.0(m,2H), 2.3(t,J=6 Hz,2H) 3.0-3.13(m,2H), 4.85(m,1H), 7.03(t,J=9 Hz,1H), 7.18(t,J=9 Hz,1H), 7.25(s,1H), 7.4(d,J=9 Hz,1H), 7.6(d,J=9 Hz,1H), 8.6(d,J=9 Hz,1H), 11.6(s,1H), 12.2(bs,1H). Analysis calculated for C.sub.24 H.sub.35 N.sub.3 O.sub.4 0.33 H.sub.2 O: C 66.19, H 8.25, N 9.65. Found: C 66.39, H 8.23, N 9.65.
EXAMPLE 9
N-(2'-Quinoxalylcarbonyl)-(.gamma.-benzyl ester)-R-Gludi-n-pentylamide
To the stirred solution of hydrochloride of example 2 (0.37 g, 0.9 mmol) and pyridine (0.2 mL, 2 mmol) in anhydrous DMF (2 mL) at 0.degree. C. was added dropwise a solution of 2-quinoxalyl chloride (0.8 g, 0.95 mmol) in DMF (2 mL) over 2 hours. The reaction was monitored by tlc using hexane-ethylacetate mixture in 2:1 ratio. Solvents were evaporated and the residue diluted with ethyl acetate and washed successively with 1.0 N hydrochloric acid, water, a solution of saturated bicarbonate and brine. The solution was dried over magnesium sulfate. The product was purified by chromatography EtOAc/hexane) to provide 0.355 g (74%). MS(CI) m/e 533(m+H).sup.+, 377, 348. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.82-0.98(t,J=6 Hz,6H), 1.23-1.45(m,8H), 1.5-1.7(m,4H), 2.05(m,1H), 2.3(m,1H), 2.5-2.65(m,2H), 3.1(m,1H), 3.3(m,1H), 3.5-3.72(m,2H), 5.15(s,2H), 5.25(dt,J=4,9 Hz,1H), 7.35(m,5H), 7.85(m,2H), 8.15(dd,J=1.5,12 Hz,2H), 8.78(d,J=9 Hz,1H), 9.62(s,1H).
EXAMPLE 10
N-(2'-Quinoxalylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
To a suspension of 0.08 g 10% Pd/C in absolute ethanol (EtOH, 4 mL) and cyclohexadiene (1 mL) was added under inert atmosphere (N.sub.2) solution of benzyl ester of example 9 (0.08 g, 0.15 mmol) in ethanol via cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with ethanol. The filtrate and washings were combined and concentrated under vacuo. The product was purified by flash chromatography using chloroform-methanol and ammonium hydroxide 90:10:1 as elutant. Lyophilization provided product in 90.0% yield (0.06 g). [.alpha.].sub.D =+12.67.degree. (c=0.6, MeOH). MS(CI) m/e 443(m+H).sup.+, 258. .sup.1 H NMR (CDCl.sub.3 '300 MHz) .delta. 0.78-0.98(m.6H), 1.2-1.4(m,8H), 1.48-1.65(m,4H), 1.8(m,1H), Z.Z5(m,1H), 3.05(m,1H), 3.25(m,1H), 3.5-3.75(m.ZH), 4.18(m.2H), 5.3(t.J=9 Hz.1H). 7.8(m,ZH), 8.1(m,2H), 8.9(d.J=9 Hz,1H), 9.5 (s,1H).
EXAMPLE 11
N-(3'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-di-n-pentylamide
To the stirred solution of hydrochloride of example 2 (1.15 g, 2.8 mmol) in DMF at 0.degree. C. and N-methylmorpholine (0.65 mL, 5.6 mmol) was added HOBt (0.75 g, 5.6 mmol) and quinoline-3-carboxylic acid (0.45 g, 2.9 mmol) followed by EDCI (0.57 g, 3.0 mmol). The reaction was allowed to warm to ambient temperature over 2 hours and stirred overnight. Solvent was evaporated and to the residue was added water and ethyl acetate. The organic extracts were washed successively with 1 N hydrochloric acid, water, a solution of saturated sodium bicarbonate and brine. The solution was dried over magnesium sulfate and filtered. The product was purified by chromatography using ethyl acetate and hexane as eluant. Product was obtained in 56.6% yield (0.85 g). MS(CI) m/e 520(m+H).sup.30 , 363, 335, 279. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.85-0.95(m,6H), 1.2-1.42(m,8H), 1.5-1.7(m,4H), 1.88-2.02(m,1H), 2.25(m,1H), 2.6(m,2H), 3.1(m,1H), 3.28(m,1H), 3.6(m,2H), 5.1(s,2H), 5.3(dt,J=3,9 Hz,1H), 7.1(d,J=7.5 Hz,1H), 7.25(m,2H), 7.3(m,5H), 7.5(m,1H), 7.8(d,J=2 Hz,1H), 8.1(m,1H), 8.9(s,1H).
EXAMPLE 12
N-(3'-Indolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
To a suspension of 0.3 g 10% Pd/C in absolute ethanol (5 mL) and cyclohexadiene (2 mL) was added under inert atmosphere (N.sub.2) a solution of benzyl ester of example 11 (0.41 g, 0.79 mmol) in ethanol by cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with ethanol. The filtrate and washings were combined and concentrated under vacuo. The product was purified by flash chromatography using chloroform-methanol and ammonium hydroxide 90:10:1 as elutant. Lyophilization provided product in 65% yield (0.22 g). MS(CI) m/e 430(m+H).sup.+, 269, 245. .sup.1 H NMR (DMSO.sub.d6, 300 MHz) .delta. 0.8-0.9(m,6H), 1.15-1.32(m,8H), 1.4-1.72(m,4H), 1.78-1.9 (m,2H), 2.20(m,2H), 3.1-3.4(m,2H), 3.6(m,2H), 4.95(m,1H), 7.12(m,2H), 7.41(d,J=9 Hz,1H), 7.93(d,J=7 Hz,1H), 8.1(d,J=6 Hz,1H), 8.18(d,J=2 Hz,1H), 11.5(s,1H), 12.15(bs,1H). Analysis calculated for C.sub.24 H.sub.35 N.sub.3 O.sub.4 : C 67.10, H 8.21, N 9.78. Found: C 66.97, H 8.30, N 9.75.
EXAMPLE 13
N-(1', 2',3',4'-Tetrahydroguinoxalyl-2'-carbonyl)-R-Glu-N.sup..alpha., N.sup..alpha. -di-n-pentylamide
To a suspension of 0.3 g 10% Pd/C in absolute ethanol (5 mL) and formic acid (80%) (1 mL) was added under inert atmosphere (N.sub.2) a solution of benzyl ester of example 9 (0.355 g, 0.67 mmol) and 0.15 g ammonium formate in ethanol via cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with methanol. The filtrate and washings were combined and concentrated in vacuo. The product was purified by flash chromatography using chloroform-methanol and ammonium hydroxide 90:10:1 as elutant. Lyophilization provided product in 67% yield (0.2 g . [.alpha.].sub.D =+12.67.degree. (c=.0.6, MeOH). MS(CI) m/e 447(m+H).sup.+. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 0.84-0.96(m,6H), 1.20-1.38(m,8H), 1.42-1.70(m,4H), 1.9-2.02(m,2H), 2.30(m,2H), 3.05(m,1H), 3.18(m,1H), 3.38-3.6(m,4H), 3.7(d,J=3 Hz,1H), 3.73(d,J=3 Hz,1H), 4.1(m,1H), 5.0(dt,J=2,12 Hz,1H), 6.7(d,J=6 Hz,1H), 6.15(d,J=6 Hz,1H), 6.2(m,2H), 7.85(d,J=9 Hz,l). Analysis calculated for C.sub.24 H.sub.38 N.sub.4 O.sub.4 0.9 H.sub.2 O: C 62.29, H 8.64, N 12.11. Found: C 62.03, H 8.31, N 12.47.
EXAMPLE 14
N-(3,-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-di-n-pentylamide
To a solution of hydrochloride of example 2.(0.41 g, 1 mmol) and N-methylmorpholine (0.22 mL, 2 mmol) in DMF at 0.degree. C. was added guinoline-3-carboxylic acid (0.17 g, 1 mmol), HOBt (0.27 g, 2 mmol) followed by EDCI (0.2 g, 1.05 mmol). The reaction mixture was allowed to warm to ambient temperature and then stirred overnight. The solvent was evaporated and water was added to the residue. The mixture was extracted with ethyl acetate. Combined ethyl acetate extracts were washed successively with 1 N hydrochloric acid, water, a saturated solution of sodium bicarbonate, brine and the solution dried over magnesium sulfate then filtered and concentrated in vacuo. Purification by chromatography using ethyl acetate and hexane as elutants provided 0.4 g (75%) of product. MS(CI). m/e 531(m)+, 374, 347, 156, 128, 91. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.85-0.98(m,6H), 1.2-1.42(m,8H), 1.5-1.7(m,4H), 2.02-2.28(m,2H), 2.50(m,2H), 3.15(m,1H), 3.35(m,1H), 3.5-3.62(m,2H), 5.15(s,2H), 5.30(dt,J=3,9 Hz,1H), 7.35(m,5H), 7.65(t,J=7.5 Hz,1H) 7.85(t,J=7.5 Hz,1H), 7.96(d,J=9 Hz,1H), 8.1(d,J=9 Hz,1H), 8.26(d,J=9 Hz,1H), 8.82(s,1H), 9.4(d,J=0.5 Hz,1H).
EXAMPLE 15
N-(3'-Quinolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di n-pentylamide
To a suspension of 0.3 g 10% Pd/C in methanol (10 mL) and cyclohexadiene (3 mL) was added- under inert atmosphere (N.sub.2) a solution of benzyl ester of example 14 (0.4 g, 0.75 mmol) in methanol via cannula. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with methanol. The filtrate and washings were combined and concentrated in vacuo. The product was purified by flash chromatography using chloroform-methanol and ammonium hydroxide 90:10:1 as elutant. Lyophilization provided product in 60% yield (0.2lg). [.sup..alpha.].sub.D =-2.4.degree. (c=0.5, MeOH). MS(FAB) m/e 464(m+Na).sup.+, 442(m+H).sup.+, 285, 257. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.8-0.95(m,6H), 1.18-1.4(m,8H), 1.45-1.7(m,4H), 2.02(m,1H), 2.2(m,1H), 2.54(m,2H), 3.1(m,1H), 3.25 (m,1H), 3 5(m,2H), 5.23(m,1H), 7.55(t,J=9 Hz,1H), 7.75(t,J=9 Hz,1H), 7.8(d,J=9 Hz,1H), 8.08(d,J=9 Hz,2H), 8.62(d,J=2 Hz,1H), 9.3(d,J=2 Hz,1H). Analysis calculated for C.sub.25 H.sub.35 N.sub.3 O.sub.4 0.75 H.sub.2 O C 65.98, H 8.09, N 9.23. Found: C 65.92, H 8.21, N 8.86.
EXAMPLE 16
Boc-(.beta.-benzyl ester)-R-Asp-di-n-pentylamide
OuBoc-(.alpha.-benzyl ester)-R-Aspartic acid (1.06 g, 3.29 mmol) was stirred at 0.degree. C. in 35 mL of methylene chloride (CH.sub.2 Cl.sub.2) under inert atmosphere. Dipentyl amine (7.0 mL) was added to the mixture followed by the addition of BOPCl (1.0 g). The reaction was maintained at 0.degree. C. and when product had formed by tlc the mixture was taken up in ethyl acetate. The organic solvents were washed several times with water then twice with a 1.0 N hydrochloric acid solution, lastly with a saturated sodium bicarbonate solution. The mixture was dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was purified using a chromatotron and ethyl acetate/hexane mixtures as the elutants to provide 974 mg of product. .sup.1 H NMR(CDCl.sub.3,300, MHz) .delta.7.35(m,5H), 5.3(bd,J=9 Hz,1H), 5.13(d,J=12 Hz,1H), 5.08(d,J=12 Hz,1H), 4.95(m,1H), 3.39(m,2H), 3.10-3.30(m,2H), 2.82(dd,J=7,16 Hz,1H), 2.63(dd,J=7,16 Hz,1H), 1.45-1.65(m,4H), 1.42(bs,9H), 1.39(m,8H), 0.89(m,6H).
EXAMPLE 17
(.beta.-Benzyl ester)-R-Asp-di-n-pentylamide hydrochloride
Boc-(.beta.-benzyl ester)-R-Asp-di-n-pentyl amide (974 mg) was dissolved in 3.0 mL of a 4.0 N hydrochloric acid solution in dioxane. The reaction was monitored by tlc for disappearance of starting material. When reaction was complete the mixture was diluted with diethyl ether and hexane and volatiles removed in vacuo. This process was repeated three times leaving a thick clear oil which was used for subsequent reactions.
EXAMPLE 18
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Asp-di-n-pentylamide
Hydrochloride salt of example 17 (270 mg, 0.67 mmol), indole 2-carboxylic acid (129 mg, 0.80 mmol), HOBt (100 mg, 0.74 mmol) and EDCI (165 mg, 0.86 mmol) were suspended in 8 mL of anhydrous dimethylformamide at 0.degree. C. Triethyl amine (TEA) (200 .mu.L) was added and the reaction mixture stirred under nitrogen allowing warming to ambient temperature overnight. The mixture was taken up in ethyl acetate and washed with water, 1.0 N hydrochloric acid solution, and saturated sodium bicarbonate solution. The organic solution was dried over magnesium sulfate, filtered and the residue purified by chromatography utilizing a chromatotron and ethyl acetate/hexane elutant mixtures to provide 223 mg of product. mp=94.degree.5.degree. C. [.alpha.].sub.D =+44.8.degree. (c=0.125, MeOH). MS(CI) m/e 506(m+H).sup.30, 407, 363, 346, 321. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 9.18(bs,1H), 7.63(bd,J=7 5 Hz,1H), 7.42(bd,J=7.5 Hz,1H), 7.32(m,7H), 7.14(dt,J=1.5,7.5 Hz,1H), 6.85(s,1H), 5.48(dt,J=6,9 Hz,1H), 5.12(m,2H-), 3.15-3.50(m,4H), 2.92(dd,J=6,15 Hz,1H), 2.29(dd,J=6,15 Hz,1H), 1.45 1.60(m,4H), 1.2-1.35(m,8H), 0.88(m,6H). C,H,N Analysis calculated for C.sub.30 H.sub.39 N.sub.3 O.sub.4 0.3 EtOAc: C 70.43, H 7.84, N 7.90; Found C 70.31, H 8.08, N 7.67.
EXAMPLE 19
N-(2'-Indolylcarbonyl)-R-Asp-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
To a suspension of 97 mg of 10% Pd on carbon in ethanol under nitrogen atmosphere was added 100 .mu.L of cyclohexadiene. To this mixture was then added 112 mg of the benzyl ester of example 18 and 400 .mu.L of cyclohexadiene. The reaction was monitored by tlc for the disappearance of starting material. When reaction was complete the mixture was filtered through celite and the volatiles removed in vacuo. The residue was lyophilized to provide product. mp=183.degree.-5.degree. C. [.alpha.].sub.D =+27.1.degree. (c=0.17, acetone). MS(FAB) m/e 438(m+Na).sup.+, 416(m+H).sup.+, 373, 317, 273. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 11.63(s,1H), 8.87(d,J=9 Hz,1H), 7.60(d,J=7.5 Hz,1H), 7.42(d,J=7.5 Hz,1H), 7.24(d,J=1.5 Hz,1H), 7.18(dt,J=1.5,7.5 Hz,1H), 7.02(t,J= 7.5 Hz,1H), 5.25(m,1H), 3.2-3.5(bs,H.sub.2 O), 2.84(dd,J=8,16 Hz,1H), 1.58(bs,2H), 1.43(m,2H), 1.15-1.30(m,8H), 0.85(t,J=7.5 Hz,3H), 0.77(t,J=7.5 Hz,3H). C,H,N Analysis calculated for C.sub.23 H.sub.33 N.sub.3 O.sub.4 0.4 H.sub.2 O: C 65.33, H 8.06, N 9.94; Found C 65.32, H 8.16, N 9.72.
EXAMPLE 20
N-(2'-Quinolylcarbonyl)-(.beta.-benzyl ester)-R-Asp-di-n-pentyl amide
(.beta.-Benzyl ester)-R-Asp-dipentyl amide hydrochloride (105 mg, 0.26 mmol), quinoline-2-carboxylic acid (57 mg, 0.33 mmol), HOBt (36 mg) and EDCI (55 mg, 0.28 mmol) were suspended in 5 mL of dimethyl formamide stirring at 0.degree. C. under nitrogen. To this mixture was added 400 .mu.L of triethyl amine and the reaction mixture was allowed to stir overnight while warming to ambient temperature. The reaction mixture was taken up in ethyl acetate and washed with one portion of water, two portions of 1.0 N hydrochloric acid and a saturated sodium bicarbonate solution. The organics were dried over magnesium sulfate, filtered and the volatiles removed in vacuo. The residue was purified by chromatography to provide 99.5 mg of product. MS(EI) m/e 517(m).sup.+, 362, 333, 156, 128, 91. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 8.87(d,J=9.7 Hz,1H), 8.32(d,J=7.5 Hz,1H), 8.25(d,J=9 Hz,1H), 8.12 (bd,J=9 Hz,1H), 7.87(d,J=7.5 Hz,1H), 7.78(dt,J=1.5,9 Hz,1H), 7.62(dt,J=1.5,9 Hz,1H), 7.25-7.35(m,5H), 5.55(m,1H), 5.17(d,J=12 Hz,1H), 5.10(d,J=12 Hz,1H), 3.4(m,3H), 3.25(m,1H), 3.10(dd,J=7.5,16 Hz,1H), 2.84(dd,J=6,16 Hz,1H), 1.55(m,4H), 1.20-1.35(m,8H), 0.88(t,J=7 Hz,3H), 0.80(t,J=7 Hz,3H).
EXAMPLE 21
N-(2.alpha.-Quinolylcarbonyl)-R-Asp-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
The .beta.-Benzyl ester of example 20 (93 mg, 0.18 mmol) in 2 mL of ethanol were added to 87 mg of 10% palladium on carbon stirring in 4 mL of ethanol under a nitrogen atmosphere. Cyclohexadiene (500 .mu.L was added and the reaction was monitored for disappearance of starting material. When this had occurred the reaction mixture was filtered through celite and the residue concentrated and then lyophilized from water to provide product (56 mg). mp=91.degree.-3.degree. C. [.alpha.].sub.D =+1.25.degree. (c.TM..08, MeOH). MS(FAB m/e 472(m+2Na-H).sup.30, 450(m+Na).sup.30, 428(m+H)+. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 9.05(d,J=7.5 Hz,1H), 8.28(d,J=9 Hz,1H), 8.22(d,J=9 Hz,1H), 8.12(bd,J=7.5 Hz,1H), 7.83(bd,J=7.5 Hz,1H), 7.75(bt,J=7.5 Hz,1H), 7.61(bt,J=7.5 Hz,1H), 5.50(dt,J=6,9 Hz,1H), 3.35-3.50(m,3H), 3.25(m,1H), 3.04(dd,J=6,16 Hz,1H), 2.85(dd,J=5,16 Hz,1H), 2.5(bs,1H), 1.60(m,4H), 1.25(m,8H), 0.88(t,J=7.5 Hz,3H), 0.80(t,J=7.5 Hz,3H). C,H,N Analysis calculated for C.sub.24 H.sub.33 N.sub.3 O.sub.4 0.5 H.sub.2 O : C 66.02, H 7.85, N 9.63; Found C 66.18, H 7.81, N 9.49.
EXAMPLE 22
N-(2'-Indolylcarbonyl)-R-Gln-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
Ammonia was bubbled into a solution of N-(2'-indoylcarbonyl)(.gamma.-benzyl ester)-R-Glu-di-n-pentyl amide (50 mg) in methanol at -78.degree. C. and the reaction vessel sealed. Stirring continued over 48 hrs with warming to ambient temperature. The volatiles were evaporated in vacuo and product isolated as a white solid (37 mg). mp=71.degree.-4.degree. C. [.alpha.].sub.D =-2.8.degree. (c=0.5, acetone). MS(FAB) m/e 429(m+H).sup.+, 396, 272, 244. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 11.6(bs,1H), 8.57(bd,J=7.5 Hz,1H), 7.61(d,J=7.5 Hz,1H), 7.41(d,J=7.5 Hz,1H), 7.30(m,2H), 7.17(bt,J=7.5 Hz,1H), 7.03(bt,J=7.5 Hz,1H), 6.82(bs,1H), 4.84(m,1H), 3.35(bm,H20), 3.08(m,1H), 2.20(m,2H), 1.75-1.90(m,2H), 1.70(m,1 H), 1.6(m,1H), 1.45(m,2H), 1.2-1.33(m,8H), 0.86(m,6H). C,H,N Analysis calculated for C.sub.24 H.sub.36 N.sub.4 O.sub.3 0.2 H.sub.2 O: C 66.68, H 8.49, N 12.97; Found C 66.64, H 8.43, N 12.91.
EXAMPLE 23
N-(2'-Indolylcarbonyl)-(.gamma.-methyl ester)-R-Glu-di-n-pentylamide
To a solution of N-(2'-Indolylcarbonyl)-R-Glu-di-n-pentylamide (38 mg) stirring in ethyl acetate at ambient temperature was added an-excess of diazomethane solution in ether. The excess diazomethane was destroyed by acetic acid (HOAc) addition and the product was isolated by concentration and filtration through a plug of silica gel providing 28 mg of methyl ester. mp=93.degree.-4.degree. C. [.alpha.].sub.D =+11.lo (c=0.045, MeOH). MS(CI) m/e 444(m+H)+, 287, 269, 158. .sup.1 H NMR(CDCl ,300 MHz) .delta. 9.12(bs,1H), 7.66(d,J=7.5 Hz,1H), 7.42(bd,J=7.5 Hz,1H), 7.22-7.32(m,2H), 7.13(dt,J=1.5,7.5 Hz,1H), 6.95(m,1H), 5.25(dt,J=4,9 Hz,1H), 3.68(s,3H), 3.58(m,2H), 3.27(m,1H), 3.10(m,1H), 2.40-2.60(m,2H), 2.2(m,1H), 1.9(m,1H), 1.5-1.7(m,4 H), 1.25-1.4(m,8H), 0.91(m,6H). C,H,N Analysis calculated for C.sub.25 H.sub.37 N.sub.3 O.sub.4 0.5 H20: C 66.33, H 8.47, N 9.28. - Found C 66.24, H 8.22, N 9.18.
EXAMPLE 24
N-Boc-(.alpha.-benzyl ester)-R-Glu-benzylamide
To a solution of Boc-(.gamma.-benzyl ester)-R-glutamic acid (258 mg) and BOPCl (250 mg) in methylene chloride (7 mL) at 0.degree. C. and under nitrogen atmosphere was added benzyl amine (90 .mu.L). The reaction mixture was allowed to stir overnight and reach ambient temperatures. The mixture was taken up in ethyl acetate and extracted with two portions of water, two portions of 0.5 N hydrochloric acid, and one of saturated aqueous sodium bicarbonate solution. The solution was dried over magnesium sulfate and filtered. The solution was concentrated and chromatography of the residue provided 65 mg of product. MS(CI) m/e 427(m+H).sup.+, 399, 371. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 7.23-7.46(m,10H), 6.50(bs,1H), 5.23(bs,1H), 5.12(s,2H), 4.43(bd,J=6 Hz,2H), 4.18(m,1H), 2.4-2.6(m,2H), 2.18(m,1H), 1.96(m,1H), 1.41(bs,9H).
EXAMPLE 25
N-(3'-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-benzylamide
Boc-(.gamma.-benzyl ester)-R-Glu-di-n-pentyl amide (58 mg) was treated with 4.0 mL of 4.5 M hydrochloric acid in dioxane at 0.degree. C. until starting material was consumed by tlc analysis. The volatiles were evaporated in vacuo and the residue was stirred with 3-quinolinecarboxylic acid (68 mg), EDCI (74 mg), and HOBt (22 mg) in 4 mL of anhydrous DMF under nitrogen at 0.degree. C. To this reaction was added triethyl amine (75 .mu.L) and the reaction was allowed to warm to ambient temperature overnight. The mixture was taken up in ethyl acetate and extracted with two portions of water, one portion of 1.0 N hydrochloric acid and once with saturated sodium bicarbonate solution. The organic solution was dried over magnesium sulfate and filtered. Removal of the volatiles in vacuo and trituration of the residue with ethyl acetate provided 44 mg of product. mp=160.degree.-62.degree. C. [.alpha.].sub.D =-15.0.degree. (c=0.8, acetone). MS(CI) m/e 482(m+H).sup. +, 374, 347. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 9.25(m,1H), 8.83(m,1H), 8.08(m,2H), 7.89(m,1H), 7.71(m,1H), 7.4(m,10H, 4.87(bs,6H), 4.7(m,1H), 2.58(bt,J=7.5Hz,2H), 2.30(m,1H), 2.20(m,1H). C,H,N Analysis calculated for C.sub.29 H.sub.27 N.sub.3 O.sub.4 0.833 H.sub.2 O: C 70.13, H 5.82, N 8.46; Found C 70.06, H 5.46, N 8.62.
EXAMPLE 26
N-Boc-(.gamma.-benzyl ester)-R-Glu-dibenzylamide
To a solution of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (715 mg) and BOPCl (750 mg) stirring in 15 mL of methylene chloride at 0.degree. C. was added dibenzyl amine (800 mg). The reaction mixture was allowed to stir overnight and warm to ambient temperature. The reaction mixture was taken up in ethyl acetate and extracted twice with 0.5 N hydrochloric acid solution and then with aqueous saturated sodium bicarbonate solution. The solution was dried over magnesium sulfate and filtered. The mixture was concentrated in vacuo and the residue purified by chromatography utilizing ethyl acetate and hexane as the elutants-to provide 571 mg of product. MS(CI) m/e 516(m).sup.+, 460, 443, 416, 369, 325, 292, 236. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 7.35(m,l1H), 7.18(m,4H), 5.45(bd,J=9 Hz,1H), 5.1(m,2H), 4.7-4.9(m,3H), 4.45(d,J=16 Hz,1H), 4.3(d,J=16 Hz,1H), 2.45(m,2H), 2.10(m,1H), 1.82(m,1H), 1.43(bs,9H).
EXAMPLE 27
(.gamma.-Benzyl ester)-R-Glu-dibenzylamide hydrochloride
Boc-(.gamma.-benzyl ester)-R-Glu-dibenzylamide (571 mg) was treated with 7.0 mL of a 4.5 M solution of hydrochloric-acid in dioxane at -20.degree. C. under nitrogen atmosphere. The reaction was stirred 3 hours at which time tlc indicated no more starting material. The mixture was concentrated in vacuo and the residue triturated with diethyl ether. The volatiles again were evaporated in vacuo and diethyl ether added to the residue. Filtration provided the product as a white solid (475 mg).
EXAMPLE 28
N-(2'-Indolylcarbonyl)-(.alpha.-benzyl ester)-R-Glu-dibenzylamide
EDCI (129 mg), HOBt (10 mg), 2-indolecarboxylic acid (117 mg) and hydrochloride salt of example 27 (236 mg) were suspended in 5 mL of anhydrous DMF under nitrogen at -20.degree. C. To this mixture was added triethyl amine 170 .mu.L and reaction mixture stirred overnight allowing the reaction to warm to ambient temperature. The reaction mixture was taken up in ethyl acetate and washed with three volumes of water, once with 1.0 N hydrochloric acid and once with saturated aqueous sodium bicarbonate solution. The solution was dried over magnesium sulfate and filtered. Concentration of the solution in vacuo and chromatography of the residue provided 240 mg of product. Crystalization of a small sample from ethyl acetate/,hexane provided an analytically pure sample. mp=94.degree.-6.degree. C. [.alpha.].sub.D =+4.5.degree. (c=0.11, MeOH). MS(FAB) m/e 560(m+H).sup.+, 363, 335, 234. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 9.05(bs,1H), 7.68(d,J=7.5 Hz,1H), 7.29-7.45(m,14H), 7.20(m,5H), 7.0(m,1H), 5.38(dt,J=3,9 Hz,1H), 5.10(d,J=15 Hz,1H), 5.05(d,J=15 Hz,1H), 4.95(d,J=I5 Hz,1H), 4.82(d,J=15 Hz,1H), 4.45(d,J=1-5 Hz,1H), 4.22(d,J=15 Hz,1H), 2.40-2.60(m,2H), 2.25(m,1H), 2.03(m,1H). C,H,N analysis calculated for C.sub.35 H.sub.33 N.sub.3 O.sub.4 : C 75.11, H 5.95, N 7.51; Found C 75.63, H 6.00, N 7.55.
EXAMPLE 29
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -dibenzylamide
N-(2'-Indolylcarbonyl)-(r-benzyl ester)-R-Glu-dibenzylamide (210 mg) was stirred in 3 mL of methanol and 3.0 mL of 1.0 N sodium hydroxide (NaOH) solution was added at room temperature. The reaction was stirred until tlc analysis indicated the complete disappearance of starting material. The mixture was then concentrated in vacuo and the residue taken up in ethyl acetate and partioned with saturated sodium bicarbonate solution. The aqueous solution was separated and acidified with hydrochloric acid to pH 2.0. This solution was extracted three times with equal portions of ethyl acetate and these extracts were combined and dried over magnesium sulfate. Filtration and evaporation of the volatiles in vacuo provided a residue which when triturated with hexane gave product. mp=111.degree.-3.degree. C. [.alpha.].sub.D =-5.4.degree. (c=0.13, acetone). MS(FAB) m/e 470(m+H).sup.+, 273, 234. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 11.65(bs,1H), 8.75(d,J=7.5 Hz,1H), 7.63(d,J=7.5 Hz,1H), 7.15-7.45(m,14H), 7.05(t,J=7.5 Hz,1H), 5.10(m,1H), 4.90(d,J=16 Hz,1H), 4.71(d,J=15 Hz,1H), 4.50(d,J=16 Hz,1H), 4.17(d,J=15 Hz,1H), 2.37(bt,J=7 Hz,2H), 2.0(m,2H). C,H,N Analysis calculated for C.sub.28 H.sub.27 N.sub.3 O.sub.4 0.4 H.sub.2 O: C 70.53, H 5.88, N 8.82; Found C 70.49, H 5.79, N 8.79.
EXAMPLE 30
Methyl N-Boc-(.gamma.-benzyl ester)-R-Glutamyl-R-phenylglycinate
N-Boc-(.gamma.-benzyl ester)-R-Glutamic acid (692 mg), EDCI (583 mg), HOBt (220 mg), and R-phenylglycine methyl ester hydrochloride (384 mg) were suspended in 15 mL of anhydrous DMF at -20.degree. C. under nitrogen atmosphere. Triethylamine (700 .mu.L) was added and the reaction mixture was allowed to stir overnight with warming to ambient temperature. The mixture was taken up in a large volume of ethyl acetate and extracted with water, 1.0 N solution of hydrochloric acid, and saturated sodium bicarbonate solution. The solution was dried over magnesium sulfate and then filtered. Evaporation of the volatiles in vacuo and chromatography of the residue provided 832 mg of product. MS(FAB m/e 485(m+H).sup.+, 429, 385, 367, 277. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 7.36(bm,l1H), 5.54(d,J=7.5 Hz,1H), 5.23(m,1H), 5.15(m,2H), 4.26(m,1H), 3.72(s,3H), 2.54(dt,J=3,7.5 Hz,2H), 2.18(m,1H), 1.98(m,1H), 1.42(s,9H).
EXAMPLE 31
Methyl N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glutamyl-R-phenylglycinate
Boc-(.gamma.-benzyl ester)-R-Glu-R-phenylglycine methyl ester (832 mg) was treated with 6.0 mL of 4.5 M hydrochloric acid in dioxane at 0.degree. C. When tlc analysis of the reaction mixture indicated complete consumption of the starting material the solvents were removed in vacuo. Several times ether and hexane were added to the residue and solvents evaporated in vacuo. The resulting oily product was used without further purification. Hydrochloride salt (260 mg), 2-indolecarboxylic acid (121 mg), EDCI (163 mg), and HOBt (96 mg) were suspended in 7.0 mL anhydrous DMF at -10.degree. C. and triethyl amine (210 .mu.L) was added. The reaction mixture was stirred and warmed to ambient temperature overnight. The mixture was taken up in a large volume of ethyl acetate and extracted with two portions of water, two portions of 1.0 N hydrochloric acid solution, and saturated sodium bicarbonate solution. The solution was dried over magnesium sulfate and filtered and the volatiles evaporated in vacuo. Chromato-graphy of the residue utilizing ethyl acetate and hexane mixtures as the elutants provided 262 mg of product. mp=177.degree.-8.degree. C. [.alpha.].sub.D =-71.9.degree. (c=0.32, DMSO-MeOH (1/1)). MS(CI) m/e 528(m+H).sup.+, 436, 420, 395, 363, 294, 277. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 9.25(bs,1H), 7.75(m,1H), 7.67(d,J=7 5 Hz,1H), 7.26-7.40(m,13H), 7.14(t,J=7.5 Hz,1H), 6.94(s,1 H), 5.55(d,J=7.5 Hz,1H), 5.12(m.2H), 1.S8{m,1H), 3.74{s,3H), 2.65(m,2H), 2.25(m,2H). C,H,N Analysis calculated for C.sub.30 H.sub.29 N.sub.3 O.sub.6 C 68.28, H 5.54, N 7.97; Found: C 68.29, H 5.66, N 8.08.
EXAMPLE 32
Methyl N-(3'-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-Glutamyl-R-phenylglycinate
Hydrochloride salt used in example 31 (373 mg), 3-quinolinecarboxylic acid (168 mg), EDCI (210 mg) and HOBt (96 mg) were suspended in 9.0 mL of anhydrous DMF stirring at -10.degree. C. under nitrogen atmosphere. Triethyl amine (280 .mu.L) was added and the resulting mixture stirred overnight warming to ambient temperature. The mixture was taken up in a large volume of ethyl acetate and extracted twice with portions of water, twice with 1.0 N hydrochloric acid solutions, and once with saturated sodium bicarbonate. The solution was then dried over magnesium sulfate and filtered. Evaporation of the volatiles and chromatography of the residue utilizing ethyl acetate and hexane mixtures as the elutants provided 367 mg of product. mp=144.degree.-6.degree. C. [.alpha.].sub.D =+69.56.degree. (c=0.11, MeOH). MS(CI) m/e 540(m+H).sup.+, 432, 375, 347, 294. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 9.32(d,J=3 Hz,1H), 8.58(m,1H), 8.15(d,J=7.5 Hz,1H), 7.9(d,J=7.5 Hz,1H), 7.83(m,1H), 7.60(m,3H), 7.34(m,10H), 5.55(d-,J=7 Hz,1H), 5.18(m,2H), 4.83(m,1H), 3.73(s,3H), 2.60-2.85(m,2H), 2.20-2.40(m,2H). C,H,N Analysis calculated for C.sub.31 H.sub.29 N.sub.3 O.sub.6 C 68.99, H 5.42, N 7.79; Found: C 68.88, H 5.45, N 7.78.
EXAMPLE 33
N-(6'-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-di-n-pentyl amide
Hydrochloride salt of example 2 (190 mg), EDCI 138 mg), HOBt (86 mg), and 6-quinolinecarboxylic acid (99.6 mg) were suspended in 6.0 mL of anhydrous DMF at 0.degree. C. under a nitrogen atmosphere. Triethyl amine (165 .mu.L) was added and the reaction mixture stirred overnight with warming to ambient temperature. The reaction mixture was poured into a large volume of ethyl acetate and this solution was extracted twice with water, twice with 1.0 N hydrochloric acid solution, and once with saturated sodium bicarbonate solution. The solution was dried over magnesium sulfate and filtered. Evaporation of the volatiles in vacuo and chromatography of the residue provided 245 mg of product. [.alpha.].sub.D =+16.9.degree. (c=0.61, MeOH). MS(CI m/e 532(m+H).sup.+, 424, 398, 375, 347, 264. 1H NMR(CDCl.sub.3, 300 MHz) .delta. 8.97(dd,J=1.5,5 Hz,1H), 8.30(s,1H), 8.18(dd,J=1.5,9 Hz,1H), 8.10 (bs,2H), 7.71(bd,J=8 Hz,1H), 7.44(m,1H), 7.30-7.35(m,5H), 5.25(dt,J=3.5,9 Hz,1H), 5.14(bs,2H), 3.60m,2H), 3.30(m,1H), 3.12(dt,J=7.5,14 Hz,1H), 2.45-2.70(m,2H), 2.25(m,1H), 2.04(m,1H), 1.68(m,2H), 1.55(m,2H), 1.2-1.4(m,8H), 0.92(m,6H).
EXAMPLE 34
N-(6'-Quinolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
To a suspension of 249 mg of palladium on carbon (10%) in 5 mL of ethanol under nitrogen atmosphere was added 100 .mu.L of cyclohexadiene, followed by 154 mg of benzyl ester of example 33 in 5 mL of ethanol. To this reaction mixture was added an additional 400 .mu.L of cyclohexadiene and the reaction was monitored for consumption of starting material. The reaction mixture was filtered through celite and the volatiles removed in vacuo. Crystallization from ethyl acetate and hexane gave 114 mg of product. mp=146.degree.-8.degree. C. [.alpha.].sub.D =-18.9.degree. (c=.095, acetone) MS(CI) m/e 442(m+H).sup.+, 398, 323, 304, 294, 257, 173, 158. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 8.94(dd,J=1.5,4.5 Hz,1H), 8.27(d,J=1.5 Hz,1H), 8.10-8.18(m,2H), 8.02(d,J=9 Hz,1H), 7.70(d,J= 7.5 Hz,1H), 7.42(dd,J=4.5,8.5 Hz,1H), 5.24(m,1H), 3.45-3.65(m,2H), 3.2-3.35(m,1H), 3.05-3.17(m,1H), 2.55(m,2H), 2.23(m,1H), 2.05(m,1H), 1.65(m,2H), 1.55(m,2H), 1.25-1.40(m,8H), 0.90(m,6H). C,H,N Analysis calculated for C.sub.25 H.sub.35 N.sub.3 O.sub.4 0.33 H.sub.2 O: C 67.08, H 8.04, N 9.39; Found: C 67.10, H 7.96, N 9.34.
EXAMPLE 35
Methyl N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-Glutamyl S-phenylglycinate
N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-glutamic acid (2.13 gram) and methyl-S-phenylglycinate hydrochloride were reacted in a fashion similar to that in example 21 to provide 1.00 grams (33% yield) of product after chromatography. mp=91.degree.-92.degree. C. [.alpha.].sub.D =+54.6.degree. (c=0 52, acetone). MS(FAB) m/e 485(m+H).sup.+, 429, 385, 367, 350, 345, 325, 277. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 7.33(m,10H), 7.23(m,1H), 5.53(d,J=7.5 Hz,1H), 5.25(m,1H), 5.13(s,2H), 4.25(m,1H), 3.72(s,3H), 2.52(m,1H), 2.40(m,1H), 2.14(m,1H), 1.94(m,1H), 1.42(bs,9H).
EXAMPLE 36
Methyl N-(2'-Indolylcarbonyl)-('-benzylester)-R-Glutamyl -S-phenylglycinate
Boc-(.gamma.-benzyl ester)-R-Glu-S-phenylglycine methyl ester (362 mg) was treated with 8.0 mL of 4.5 M hydrochloric acid in dioxane at -10.degree. C. under nitrogen atmosphere. Reaction progress was monitored by tlc and when starting material was consumed the solvents were removed in vacuo. EDCI (212 mg), HOBt (100 mg), and indole-2-carboxylic acid (133 mg) were added to this residue and the contents dissolved in 15 mL of anhydrous DMF at 0.degree. C. Triethyl amine (260 .mu.L) was added and the reaction was allowed to stir overnight under nitrogen warming to ambient temperature. The reaction mixture was poured into ethyl acetate and extracted twice with portions of water, twice with portions of 1.0 N HCl solution and once with saturated sodium bicarbonate solution. The solution was dried over magnesium sulfate, filtered, and concentrated in vacuo. Chromatography of the residue provided 230 mg of the desired product. mp=179.degree.-81.degree. C. [.alpha.].sub.D =+49.4.degree. (c=0.34, MeOH). MS(CI) m/e 528(m+H)+, 437, 420, 395, 294. 1.sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 9.38(bs,1H), 7.8(d,J=7.5 Hz,1H), 7.65(d,J=7.5 Hz,1H), 7.3-7.45(m,13H), 7.14(dt,J=1.5,7.5 Hz,1H), 6.98(m,1H), 5.58(d,J=7.5 Hz,1H), 5.1(s,2H), 4.92(m,1H), 3.71(s,3H), 2.62(m,1H), 2.41(m,1H), 2.15-2.32(m,2H). C,H,N Analysis calculated for C.sub.30 H.sub.29 N.sub.3 O.sub.6 : C 68.28, H 5.54, N 7.97; Found: C 68.38, H 5.63, N 7.74.
EXAMPLE 37
Methyl N-(2,-Indolylcarbonyl)-R-Glutamyl-S-phenylglycinate
To a suspension of 81 mg of palladium (10%) on carbon in 2 mL of ethanol under nitrogen atmosphere were added cyclohexadiene (100 .mu.L) followed by a solution of benzyl ester of example 36 in 2.0 mL of ethanol. An additional 400 .mu.L of cyclohexadiene was added and the reaction stirred at room temperature until starting material was consumed by tlc analysis. The reaction mixture was filtered through celite and the filtrate concentrated in vacuo. The residue was lyophilized to provide 36 mg of final product. mp=133.degree.-9.degree. C. [.alpha.].sub.D =+18.4o (c=0.065, acetone). MS(FAB) m/e 438 (m+H)+, 391, 273, 245. 1H NMR(DMSO.sub.d6,300 MHz) .delta. 11.63(bs,1H), 9.43(bs,1H), 8.92(d,J=7.5 Hz,1H), 7.61(d,J=7.5 Hz,1H), 7.40(m,7H), 7.23(bs,1H), 7.18(t,J=7.5 Hz,1H), 7.03(t,J=7.5 Hz,1H), 5.47(m,1H), 4.55(m,1H), 3.62(s,3H), 3.25-3.50(H.sub.2 O), 2.21(bt,J=7.5 Hz(2H), 1.93(m,2H). C,H,N Analysis calculated for C.sub.23 H.sub.23 N.sub.3 O.sub.6 1.33 H.sub.2 O: C 59.85, H 5.56, N 9.11; Found C 59.76, H 5.16, N 8.74.
EXAMPLE 38
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha. -(2'-biphenylamide)
To a solution of Boc(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol) and 2-aminobiphenyl (423 mg, 2.5 mmol) in THF (15 mL) at 4.degree. C. were added TEA (140 mL, 1.0 mmol) and BOPCl (254 mg, 1.0 mmol) and the reaction was allowed to warm to room temperature overnight. The reaction mixture was poured into EtOAc and extracted with portions of 1 M phosphoric acid (H.sub.3 PO.sub.4); 1 M Na.sub.2 CO.sub.3 (sodium carbonate); H.sub.2 O then dried over MgSO.sub.4. Evaporation of the solvent gave a pink oil. Flash chromatography on silica gel eluted with 8:1 hexanes-ethyl acetate gave: 246 mg, (0.51 mmol, 51% yield). R.sub.f =0.46 (2:1 hexanes-EtOAc); 0.24 (4:1 hexanes-EtOAc). The material above was treated with HCl-dioxane (1.5 mL, 6 mmol) under an N.sub.2 atmosphere for 4 hours at room temperature. The reaction was evaporated in vacuo and then placed under high vacuum overnight. R.sub.f =0.63 (100:60:18:33 EtOAc-pyridine-HOAc-H.sub.2 O). The HCl salt (0.51 mmol), 2-indole carboxylic acid (97mg, 0.60 mmol), HOBt (81 mg, 0.60 mmol) and TEA (167 .mu.L, 1.2 mmol) were dissolved in CH.sub.2 Cl.sub.2 (8 mL) and treated with EDCI (115 mg, 0.66 mmol) at room temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in EtOAc and extracted with successive portions of 1 M H3P04 (3x), 1 M Na.sub.2 CO.sub.3 (3x), H.sub.2 O-brine, then dried over MgS04, filtered and evaporated. The oily residue was crystallized from give 209 mg, (0.394 mmol, 77%, R.sub.f =0.23 (2/1 hexane-EtOAc)). The benzyl ester (190 mg, 0.36 mmol) in MeOH (10 mL) and acetic acid (HOAc, 5 mL) was treated with 10% Pd/C (prewetted with MeOH under N.sub.2 atmosphere, 25 mg) under 1 atmosphere H.sub.2 (balloon) for 1.5 hours at room temperature. The catalyst was filtered, the solvent was evaporated in vacuo and the residue was recrystallized from aqueous ethanol. Yield: 127 mg, (0.29 mmol, 81%). R.sub.f =0.28 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.40 H). mp=136.degree.-42.degree. C. (becomes translucent). [.alpha.].sub.D =+7.5.degree. (1.2, MeOH). MS(FAB+) m/e 442(m+H).sup.30, 464(m+Na).sup.30, 273(-168), 245(-196). .sup.1 H NMR (DMSO.sub.d6,300 MHz) .delta. 1.83-1.96(m,1H), 1.98-2.lO(m,1H), 2.30(brt,2H), 4.50(m,1H;dd(+D.sub.2 O),J=5,9 Hz), 7.05(dt,1H), 7.12-7.40(m,9H), 7.46(d,1H), 7.63(d,J=7 Hz,1H), 7.68(d,J=5 Hz,1H), 8.55(d,J=7 Hz,1H), 9.30(s,1H), 11.6(s,1H), 12.16(brs,1H). Analysis calculated for C.sub.26 H.sub.23 N.sub.3 O.sub.4 H.sub.2 O: C 69.32, H 5.37, N 9.33, Found: C 69.58, H 5.27, N, 9.33.
EXAMPLE 39
Boc-(.gamma.-benzyl ester)-R-Glu-(diphenylmethyl)amide
BOPCl (255 mg, 1.0 mmol) was added to a solution of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol) and diphenylmethylamine (431 mL, 2.5 mmol) in dry THF cooled to. 4.degree. C. The reaction was allowed to attain room temperature overnight. The reaction mixture was poured into EtOAc and extracted successively with 1 M H.sub.3 PO.sub.4 (3x), 5% NaHCO.sub.3 (3x), H.sub.2 O (3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. The crude product was chromatographed on flash silica gel eluted with 4:1 hexanes/ethyl acetate. Yield: 181 mg, (0.36 mmol), 36%. MS(CI m/e 503(m+H).sup.30 , 520(m+NH.sub.4).sup.+, 447(-56), 403(-100)>312-(-191). .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 1.42(s,9H), 1.90 2.03(m,1H), 2.12-2.23(m,1H), 2.37-2.6(m,2H), 4.21(m,1H), 5.12(s,2H), 5.25(brd,J=7 Hz,1H), 6.20(d,J=8 Hz,1H), 7.02(brd,J=7 Hz,1H), 7.20-7.33(m,15H). C,H,N Analysis calculated for C.sub.30 H.sub.34 N.sub.2 O.sub.5 C 71.69, H 6.82, N 5.58; Found: C 71.46, H 6.89, N 5.58.
EXAMPLE 40
(.gamma.-Benzyl ester)-R-Glu-(diphenylmethyl)amide hydrochloride
Boc-(.gamma.-benzyl ester)-R-Glu-(diphenylmethyl)amide (100 mg, 0.20 mmol) was dissolved in 4N HCl-dioxane (1.0 mL, 4 mmol) under N.sub.2. After 2 hours, the solvent was evaporated. R.sub.f .TM.0.21 (18:1 CHCl.sub.3 -EtOH). Product was used directly in the next step. Rf =0.80 (90:10:1, CHCl.sub.3 -MeOH-NH.sub.4 OH).
EXAMPLE 41
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(diphenylmethyl)amide
EDCI (42.1 mg, 0.22 mmol) was added to a cooled (4.degree. C.) solution of indole-2-carboxylic acid (36 mg, 0.22 mmol), hydrochloride salt of example 40 (0.2 mmol), HOBt (30 mg, 0.22 mmol) and TEA (62 mL, 0.44 mmol) in 4 mL dry CH.sub.2 Cl.sub.2. The reaction was allowed to reach ambient temperature. After 3 days, the reaction mixture was poured into EtOAc and extracted successively with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), H.sub.2 O (3x), then dried over MgSO.sub.4, filtered and evaporated. The crude product was recrystallized from EtOAc. Yield: 73 mg, (0.13 mmol), 67%. R.sub.f =0.19 (2:1 hexanes-ethyl acetate). MS(CI) m/e 546(m+H)+, 455(-91), 438(-108). .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 9.07(s, 1H), 7.66(d,1H), 7.45(d,1H), 7.12-7.41(m,19H), 6.45(m,1H), 6.21(dd,1H,d(+D.sub.2 O),J=7 Hz), 5.12(d,J=12 Hz,1H), 5.08(d,J=12 Hz,1H), 4.72-4.80(m,1H), 2.65-2.76(m,1H), 2.42-2.52(m,1H), 2.15-2.32(m,2H).
EXAMPLE 42
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha. -(diphenylmethyl) amide
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(diphenylmethyl) amide (60 mg, 0.11 mmol) was dissolved into MeOH (10 mL) and 1 N NaOH (220 .mu.L) was added at room temperature. After 5 hours, an additional 220 .mu.L of 1 N NaOH was added and the reaction was refrigerated for 3 days. The solvent was evaporated in vacuo. The residue in H.sub.2 O was extracted with EtOAc (3x) then acidified with 6 N HCl and reextracted with EtOAc (3x). The latter EtOAc extractions were combined and washed until neutral to pH paper, then dried over MgSO.sub.4 and evaporated in vacuo. The crude product was recrystallized from aqueous ethanol. R.sub.f =0.35 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH). mp=224.degree.-5.degree. C. [.alpha.]D =-24.4.degree. (c=0.53, MeOH). MS(FAB+m/e 456(m+H)+, [.alpha.].sub.D =-24.4.degree. (c=0.53, MeOH). MS(FAB+) m/e 456 (m+H).sup.30, 911(2m+H)+.sup.1 H NMR(CDCl.sub.3,300 MHz .delta. 1.95-2.07(m,2H), 2.24-2.40(m,2H), 4.62(m,1H), 6.14(d,J=8 Hz,1H), 7.03(dt,1H), 7.18(dt,1H), 7.22-7.36(m,l1H), 7.43(dd,J=7,.ltoreq.1Hz,1H), 7.62(d,J=8 Hz,1H), 8.02(d,J=7 Hz,1H), 8.98(d,J=8 Hz,1H), 11.63(s,1H), 12.04(brs,1H). C,H,N Analysis calculated for: C.sub.27 H.sub.25 N.sub.3 O.sub.4 0.75 H.sub.2 O: C 69.14, H 5.70, N 8.96; Found: C 69.01, H 5.52, N 8.58.
EXAMPLE 43
Boc-(.gamma.-benzyl ester)-R-Glu-(2'-indanyl)amide .
BOPCl (254 mg, 1.0 mmol) was added to a solution of Boc-(.gamma.-benzyl ester)-R-glutamic acid (337 mg, 1.0 mmol), 2-aminoindane (425 mg, 2.5 mmol) and TEA (348 .mu.L, 2.5 mmol) in dry THF at room temperature. The reaction mixture was stirred overnight after which the solvent was removed in vacuo. The residue was dissolved into EtOAc and extracted with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and evaporated in vacuo. The crude product was crystallized from hexanes-CHCl.sub.3. Yield: 335 mg, (0.69 mmol, 69%). R.sub.f =0.26 (2:1 hexanes-ethyl acetate).
EXAMPLE 44
(.gamma.-Benzyl ester)-Rl-Glu-(2'-indanyl)amide hydrochloride
Boc-(.gamma.-benzyl ester)-R-Glu-2'-indanylamide (300 mg, 0.61 mmol) was dissolved in HCl-dioxane (3 mL, 12 mmol) at room temperature under N.sub.2 The solvent was removed after 30 minutes and the crude product was placed under high vacuum overnight.
EXAMPLE 45
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-Glu-(2'-indanyl)amide
EDCI (124 mg, 0.65 mmol) was added to a solution of indole-2-carboxylic acid (105 mg, 0.65 mmol), hydrochloride salt from example 44 (0.61 mmol assumed), HOBt (88 mg, 0.65 mmol) and TEA (181 .mu.L, 1.3 mmol) in dry CH.sub.2 Cl.sub.2 (10 mL). The reaction was stirred overnight then poured into EtOAc and extracted successively with 1 N H.sub.3 PO.sub.4 (3x), 1 M Na2C03 (3x), H.sub.2 O (2x), brine (lx) then dried over MgSO.sub.4, filtered and evaporated in vacuo. The crude product was mixed with EtOAc, cooled to -20.degree. C. and filtered. Yield: 235 mg, (0.47 mmol, 78%). R.sub.f =0.17 (2:1 hexanes-EtOAc).
EXAMPLE 46
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha. -(2'-indanyl)amide
Benzyl ester of example 45 (215 mg, 0.43 mmol) was dissolved into 2:1 MeOH-EtOAc (30 mL) and treated with 10% palladium on carbon (50 mg, prewetted with MeOH under N2) under one atmosphere H.sub.2 (balloon). After 2 hours, the catalyst was filtered and the filtrate was evaporated to dryness in vacuo. The crude product was recrystallized from 80% aqueous ethanol. R.sub.f 0.21 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH). mp=184.degree.-5.degree. C. [.alpha.].sub.D =-60.2.degree. (c=1.13, MeOH). MS(FAB.sym.) m/e 406(m+H).sup.+, 289(-117), 273(-133), 245(-161). .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.85-2.05(m,2H), 2.22-2.39(m,2H), 2.73-2.85(m,2H), 3.13-3.23(m,2H), 4.40-4.52(m,2H), 7.04(t,J=7 Hz,1H), 7.12-7.26 (m,6H), 7.42(d,J=9 Hz,l) 7.62(d,J=8 Hz,1H), 8.38(d,J=7 Hz,1H), 8.48(d,J=8 Hz,1H), 11.59(s,1H), 12.15(brs,1H). C,H,N Analysis calculated for: C.sub.23 H.sub.23 N.sub.3 O.sub.4 : C 68.13, H 5.72, N 10.36; Found: C 67.89, H 5.63, N 10.18.
EXAMPLE 47
Boc-(.gamma.-benzyl ester)-R-Glu-(3'-guinolyl)amide
BOPCl (254 mg, 1.0 mmol) was added to a cooled (4.degree. C.) solution of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol) and 3-aminoquinoline (361 mg, 2.5 mmol) and TEA (153 .mu.L, 1.1 mmol), in dry THF (15 mL). The reaction was allowed to reach ambient temperature. After 3 days, additional TEA (153 .mu.L) and BOPCl (30 mg) was added and stirring continued for 1 day. The solvent was evaporated in vacuo and the residue, dissolved in EtOAc, was extracted successively with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x), then dried over MgSO.sub.4, filtered and evaporated. Yield: 344 mg, (0.74 mmol, 74%). R.sub.f =0.31 (18:1 CHCl.sub.3 -EtOH).
EXAMPLE 48
(.gamma.-Benzyl ester)-R-Glu-(3'-quinolyl)amide
Boc-(.gamma.-benzyl ester)-R-Glu-(3,-quinolyl)amide (334 mg, 0.74 mmol) was mixed with HCl-dioxane (3.7 mL, 15 mmol) under an N.sub.2 atmosphere at ambient temperature. Additional HCl-Dioxane (3.7 mL) was added after 1 hour. After 2 hours, the solvent was evaporated and the residue was placed under high vacuum overnight. R.sub.f =0.10 (18:1 CHCl -EtOH).
EXAMPLE 49
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(3'-quinolyl)amide
EDCI (143 mg, 0.75 mmol) was added to a cooled (4.degree. C.) solution of indole-2-carboxylic acid (121 mg, 0.75 mmol), hydrochloride salt of example 48 (0.74 mmol assumed), HOBt (101 mg, 0.75 mmol), TEA (209 .mu.L, 1.5 mmol) in CH.sub.2 Cl.sub.2 (15 mL). The reaction was allowed to attain ambient temperature overnight. The solvent was evaporated in vacuo and the residue was dissolved in EtOAc and extracted with 1 M H.sub.3 PO4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. The crude product was recrystallized from EtOAc-hexanes to give: 174 mg, (0.34 mmol) 46%. R.sub.f =0.22 (1:2 hexanes-EtOAc). mp=175.degree.-7.degree. C. [.alpha.].sub.D =-3.7.degree. (c=0.83, MeOH). MS CI m/e 507(m+ H).sup.+ 399(-108), 272(-235), 256(-251). .sup.1 H NMR(CDCl.sub.3 300 MHz) .delta. 2.32-2.41(m,2H), 2.58-2.68(m,1H),.2 33-2.43(m,1H), 4.89(brq,1H), 5.12(d,J=14 Hz,1H), 5.17(d,J=14 Hz,1H), 7.08(d,J<1Hz,1H), 7.13(dt,1H), 7.26(s,1H), 7.30(s,5H), 7.41(d,J=8 Hz,1H), 7.48(dt,J=1,8 Hz,1H), 7.60(dt,J=1,7 Hz,1H), 7.68(brt,J=9 Hz,1H), 7.82(d,J=7 Hz,1H), 8.02(d,J=8 Hz,1H), 8.66(d,J=2 Hz,1H), 8.79(d,J=2 Hz,1H), 9.37(s,1H), 9.54(s,1H). C,H,N Analysis calculated for C30H26N404 H20 C 69.89, H 5.28, N 10.87; Found: C 69.78, H 5.22, N 10.88.
EXAMPLE 50
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha.-(3'-quinolyl)amide
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(3'-quinolyl)amide (150mg, 0 30 mmol) was dissolved in MeOH (10 mL) and then treated with 5% palladium on barium sulfate (Pd/BaSO.sub.4) (50 mg) under 1 atmosphere H.sub.2 (balloon). After 4 hours, the catalyst was removed by filtration and the solvent was evaporated in vacuo. The crude product was recrystallized from aqueous methanol to give 69 mg, (0.16 mmol, 55%). [.alpha.].sub.D =-43.8.degree. (c=1.04, 1:1 DMF-MeOH) MS(FAB+) m/e 417(m+H).sup.+ 307(-110), 277(140), 257(-160). .sup.1 H NMR(DMSO300MHz) .delta. 2.03-2.25(m,2H ), 2.35-2.5(m,2H), 4.68(m,1H;dd(D.sub.2 O ),J=5,9 Hz), 7.05(dt,J=1,7 Hz,1H), 7.20(dt.J=1,7 Hz,1H), 7.32(d,J=1Hz,1H), 7.43{ d,J=8 Hz,1H), 7.58(dt,J=1,8 Hz,1H), 7.63-7.68(m,2H), 7.95(dt,J=1,9 Hz,2H), 8.72(d,J=2 Hz,1H), 8.74(d,J=8 Hz,1H), 8.97(d,J=2 Hz,1H), 10.16(s,1H), 11.12(brs,1H), 12.23(brs,1H). C,H,N Analysis calculated for C.sub.23 H.sub.20 N.sub.4 O.sub.4 H.sub.2 O: C 65.63, H 4.90, N 13.31; Found: C 65.84, H4.93, N 13.14.
EXAMPLE 51
Boc-(.gamma.-benzyl ester)-R-Glu-(1'-adamantyl)amide
BOPCl (254 mg, 1.0 mmol) was added to a cooled solution (4.degree. C.) of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol), 1-aminoadamantane (378.3 mg, 2.5 mmol), and TEA (153 .mu.L, 1.1 mmol) in dry THF (15 mL). The reaction was allowed to reach ambient temperature overnight. The solvent was evaporated in vacuo and the residue, dissolved in ethyl acetate, was extracted successively with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and evaporated in vacuo. Yield: 276 mg, (0.59 mmol, 59%). R.sub.f =0.75 (2:1 hexanes-EtOAc). MS(CI) m/e 471(m+H).sup.+ 415(-56), 387(-84), 370(-101). .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 7.33-7.38(m,5H), 5.78(s,1H), 5.22(bdd, J=2,7 Hz,1H), 5.12(s,2H), 4.36-4.49(m,1H), 3.91-4.12(m,2H), 2.37-2.59(m,2H), 2.05-2.12(m,6H), 1.96(bd,J=2 Hz,7H), 1.82-1.92(m,1H), 1.64-1.68(m,9H).
EXAMPLE 52
(.gamma.-Benzyl ester)-R-Glu-(1'-adamantyl)amide hydrochloride
Boc-(.gamma.-benzyl ester)-R-Glu-(1'-adamantyl)amide (276 mg, 0.59 mmol) was mixed with HCl-dioxane (3.0 mL, 12 mmol) under an N.sub.2 atmosphere at ambient temperature. After 30 minutes, the solvent was evaporated and the residue was placed under high vacuum overnight. R.sub.f =0.70 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH).
EXAMPLE 53
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(1'-adamatyl)amide
EDCI (115 mg, 0.6 mmol) was added to a cooled (4.degree. C.) solution of indole-2-carboxylic acid (97 mg, 0.60 mmol), hydrochloride salt of example 52 (0.59 mmol assumed), HOBt (81 mg, 0.6 mmol), and TEA (132 .mu.L, 1.2 mmol) in CH.sub.2 Cl.sub.2 (10 mL). The reaction was allowed to attain ambient temperature overnight. The solvent was evaporated and the residue was dissolved in EtOAc and extracted with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x). The crude product was chromatographed on flash silica gel eluted with a step gradient of 4:1 to 2:1 hexanes-ethyl acetate. Yield: 179 mg, (0.35 mmol), 59%. R.sub.f =0.24 (2:1 hexanes-ethyl acetate).
EXAMPLE 54
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha.-(1,-adamantyl)amide
N-(2'-Indolylcarbonyl)-(.gamma.-benzylester)-R-Glu-(1'-adamantyl)amide (176 mg, 0.35 mmol) was dissolved in MeOH (10 mL) and then treated with 5% Pd/BaSO.sub.4 (100 mg) under H.sub.2 atmosphere (balloon). After 3 hours, the catalyst was removed by filtration and the solvent was evaporated in vacuo. The crude product was recrystallized from aqueous methanol to give: 117 mg, (0.28 mmol, 79%). [.alpha.].sub.D =-11.6.degree. (c=0.98, MeOH). L MS(FAB+) m/e 424(m+H).sup.+ 408(-16) 273(-151), 246(-178). .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.62(brs,6H), 1.93(brs,7H) 2.01(brs,4H), 2.26-2.42(m 2H) 4.43(m,1H +D.sub.2 O:dd J=6,8 Hz), 7.03(dt,J=8 Hz,1), 7.19(dt,J=1,7Hz,1H), 7.23(d,J=2Hz,1H), 7.42(d,J=8Hz, 1H), 7.52(d,J=8 Hz,1H), 8.33(d,J=8 Hz,l), 11.11(brs,1H), 12.17(brs,1H). C,H,N Analysis calculated for C.sub.24 H.sub.29 N.sub.3 O.sub.4 H.sub.2 O: C 67.35, H 6.95, N 9.82; Found: C 67.12, H 6.97, N 9.73.
EXAMPLE 55
Boc-(.gamma.-benzyl ester)-R-Glu-(1',2'-diphenylethyl)amide
BOPCl (254 mg, 1.0 mmol) was added to a cooled solution (4.degree. C.) of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol), 1,2-diphenylethylamine (484 .mu.L, 2.5 mmol), and TEA (153 .mu.L, 1.1 mmol) in dry THF (15 mL). The reaction was allowed to reach ambient temperature overnight. After 3 days, the solvent was evaporated in vacuo and the residue, dissolved in ethyl acetate, was extracted successively with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and evaporated in vacuo. The crude product was recrystallized from hexanes-ethyl acetate. Yield: 277 mg, (0.54 mmol, 54%). R.sub.f =0.46 (2:1 hexanes-ethyl acetate).
EXAMPLE 56
(.gamma.-Benzyl ester)-R-Glu-(1',2'-diphenylethyl)amide hydrochloride
Boc-(.gamma.-benzyl ester)-R-Glu-(1',2'-diphenylethyl)amide (247 mg, 0.48 mmol) was mixed with HCl dioxane (2.5 mL, 10 mmol) under an N.sub.2 atmosphere at ambient temperature. After 1 hour, the solvent was evaporated and the residue was placed under high vacuum overnight. R.sub.f =0.63, 0.69 (90:10:1 CHCl.sub.3 -MeOH-NH.sub.4 OH).
EXAMPLE 57
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(1', 2'-diphenylethyl)amide
EDCI (95 mg, 0.48 mmol) was added to a cooled (4.degree. C.) solution of indole-2-carboxylic acid (77 mg, 0.48 mmol), hydrochloride salt of example 56 (0.48 mmol assumed), HOBt (65 mg, 0.48 mmol), and TEA (139 .mu.L, 1.0 mmol) in anhydrous CH.sub.2 Cl.sub.2 (10 mL). The reaction was allowed to attain ambient temperature overnight. After 3 days, the solvent was evaporated and the residue, dissolved in EtOAc, was extracted with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. Yield: 253 mg, (0.45 mmol, 94%). R.sub.f =0.20 (2:1 hexanes-EtOAc). MS(FAB+) m/e 560(m+H).sup.+ 363(-197).
EXAMPLE 58
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha.-(1', 2'-diphenylethyl)amide
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(1', 2'-diphenyl ethyl)amide (176 mg, 0.35 mmol) was dissolved in MeOH (50 mL) with warming and, after returning to ambient temperature, was treated with 1 N NaOH (560 .mu.L, 0.56 mmol). After 3 hours, the solvent was evaporated in vacuo and the residue, dissolved in EtOAc, was extracted with 1 M H.sub.3 PO.sub.4 (3x), brine (3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. The tlc indicated two slightly resolved spots were present (18:1 CHCl.sub.3 -EtOH). The crude product was chromatographed on flash silica gel eluted with 18:1 CHCl.sub.3 -EtOH to give 71 m9. (0.15 mmol, 51%). A small amount of each isomer was isolated pure and MS(FAB+) showed them to be methyl esters, The combined product was redissolved in MeOH and again treated with 1 N NaOH (0.5 mL, 0.5 mmol) until both methyl esters were consumed (determined by tlc as above). The solvent was then evaporated in vacuo and the residue, dissolved in H.sub.2 O, was acidified with 6 N HCl and extracted into EtOAc (3x). The combined layers were washed with H.sub.2 O until neutral to pH paper then dried over MgSO.sub.4, filtered and evaporated in vacuo. The crude product was chromatographed by Prep HPLC (C18 column; with a gradient of 25-80% CH.sub.3 CN over 10 minutes with 0.05 M NH.sub.4 OAc, pH 4.5, as the aqueous buffer). [.alpha.].sub.D .TM.+7.5.degree. (c=1.07, DMF). mp=218.degree.-219.5.degree. C. MS(FAB+) m/e 470(m+ H).sup.+ 284(-186), 273(-197). .sup.1 H NMR(CD.sub.3 OD,300 MHz): .delta. (1:1 mixture of diastereomers) 1.8-2.1(m,2H), 2.14-2.21(m,1H) 2.31(t,J=7 Hz,1H), 2.95-3.18(m,2H), 4.06-4.14(m,1H), 5.15(t,J=7 Hz,0.5H), 5.22(dd,J=5,10 Hz,0.5H), 6.98-7.47(m,14H), 7.08(d,J=7 Hz,0.5H), 7.63(d,J=7 Hz,0.5H). C,H,N Analysis calculated for C28H27N304 1.5 H.sub.2 O: C 67.73, H 6.09, N 8.46; Found: C 67.59, H 5.77, N 8.32.
EXAMPLE 59
N-(N.sup.i -Methyl-2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Gludi-n-pentylamide
EDCI (96 mg, 0.50 mmol) was added to a cooled (4.degree. C.) solution on N-methylindole-2-carboxylic acid (88 mg, 0.50 mmol), hydrochloride salt of example 2 (206 mg, 0.50 mmol), HOBt (68 mg, 0.50 mmol), and TEA (140 .mu.L, 1.0 mmol) in CH.sub.2 Cl.sub.2 (15 mL). The reaction was allowed to attain ambient temperature overnight. The solvent was evaporated and the residue was dissolved in EtOAc and extracted with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. Yield: 262 mg, (0.49 mmol, 98%). R.sub.f =0.86 (18:1 CHCl.sub.3 -EtOH).
EXAMPLE 60
N-(N.sup.i -Methyl-2'-Indolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -dipentylamide
The benzyl ester of example 59 (245 mg, 0.46 mmol) was dissolved in MeOH (20 mL) and then treated with 5% Pd/BaSO.sub.4 (50 mg) under 1 atmosphere H.sub.2 (balloon). After 2 hours, the catalyst was removed by filtration and the solvent was evaporated in vacuo. The crude product was dried under high vacuum overnight. R.sub.f =0.42 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH). mp=132.degree.-133.degree. C. [.alpha.].sub.D =+8.4.degree. (c=1.09, MeOH). MS(FAs+) m/e 444(m+H).sup.+ 287(-157), 269(-175), 259(-185). .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 0.83-0.87(m,6H), 1.17-1.33(m,8H), 1.41-1.52(m,2H), 1.52-1.70(brm,2H), 1.85-1.94(brm,1H), 2.36(t,J=7 Hz,2H), 3.08(quintet,1H), 3.25-3.50(m,3H), 3.96(s,3H), 4.87(m,1H), 7.11(t,J=7 Hz,1H), 7.22(s,1H), 7.28(dt,J=1,7 Hz,1H), 7.52(d,J=8 Hz,1H), 7.64(d,J=8 Hz,1H), 8.54(d,J=8 Hz,1H), 12.20(bs,1H). C,H,N Analysis calculated for C.sub.25 H.sub.37 N.sub.3 O.sub.4 C 67.69, H 8.41, N 9.47; Found: C 67.52, H 8.34, N 9.43.
EXAMPLE 61
Boc-(.gamma.-benzyl ester)-R-Glu-(9,-fluorenyl)amide
BOPCl (254 mg, 1.0 mmol) was added to a cooled solution (4.degree. C.) of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol), 9-aminofluorene (453 mg, 2.5 mmol), and TEA (153 .mu.L, 1.1 mmol) in dry THF (15 mL). The reaction was allowed to reach ambient temperature overnight after which additional BOPCl (50 mg) and TEA (30 .mu.L) were added. After several hours, the solvent was evaporated in vacuo and the residue was dissolved in EtOAc and extracted successively with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x) then dried over MgSO.sub.4, filtered and evaporated in vacuo. The crude product was reprecipitated from hexanes-ethyl acetate Yield: 163 mg, (0.33 mmol, 33%). R.sub.f =0.41 (2:1 hexanes-EtOAc). MS(CI) m/e 501(m+H).sup.+ 445(-56), 401(-100). .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 7.70(d,J=7 Hz,2H), 7.52(d,J=7 Hz,2H), 7.40(t,J=7 Hz,2H), 7.26-7.32(m,9H), 6.40(bd,J.TM.8 Hz,1H), 6.18(d,J=8 Hz,1H), 5.32(bd,J=6 Hz,1H), 5.10(s,2H), 4.18-4.27(m,1H), 2.45-2.67(m,2H), 2.18-2.30(m,1H), 1.98-2.10 (m,1H), 1.49(s,9H).
EXAMPLE 62
(.gamma.-Benzyl ester)-R-Glu-(9'-fluorenyl)amide hydrochloride
Boc-(.gamma.-benzyl ester)-R-Glu-(9'-fluorenyl)amide (158 mg, 0.32 mmol) was mixed with HCl-dioxane (2.0 mL, 8.0 mmol) under an N.sub.2 atmosphere at ambient temperature. After 90 minutes, the product was precipitated with the addition of ether and the resulting solid collected and dried in vacuo. Yield: 133 mg, (0.31 mmol, 97%). R.sub.f =0.10 (100:60:18:33 EtOAc-pyridine -acetic acid-H.sub.2 O).
EXAMPLE 63
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(9'-fluorenyl)amide
EDCI (69 mg, 0.35 mmol) was added to a cooled (4.degree. C.) solution of indole-2-carboxylic acid (56 mg, 0.35 mmol), hydrochloride salt from example 62 (133 mg, 0.31 mmol), HOBt (47 mg, 0.35 mmol) and TEA (98 .mu.L, 0.7 mmol) in CH.sub.2 Cl.sub.2 (10 mL). The reaction was allowed to attain ambient temperature overnight. The solvent was evaporated and the residue was dissolved in ethyl acetate and extracted with 1 M H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), brine (3x). The crude product was recrystallized from ethyl acetate to give: 115 mg, (0.119 mmol), 57%. R.sub.f =0.62 (1:1 hexanes-ethyl acetate).
EXAMPLE 64
N-(2'-Indolylcarbonyl)-R-Flu-N.sup..alpha. -(9'-fluorenyl)amide
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(9'-fluorenyl) amide (104 mg, 0.18 mmol) was dissolved in 3:1 MeOH-EtOAc (20 mL) and then treated with 5% Pd/BASO.sub.4 (50 mg) under 1 atmosphere H.sub.2 (balloon) overnight. The catalyst was removed by filtration and the solvent was evaporated in vacuo. The crude product was recrystallized from aqueous ethanol R.sub.f =0.15 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH). mp=265.degree.-8.degree. C. [.alpha.].sub.D .TM.-35.2.degree. (c=1.07, 1:1 DMF - MeOH). MS(CI) m/e 454(m+H).sup.+ 436(-18), 273(-181). .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.98-2.17(m,2H), 2.37-2.44(m,2H), 4.52(brq,1H,+D 0: dd,J=5,8 Hz), 6.05(d,J=8 Hz,1H), 7.04(t,J=7 Hz,1H), 7.19(t,J=7 Hz,1H), 7.27(d,J=1Hz,1H), 7.33(d,J=7 Hz,2H), 7.40-7.52(m,5H), 7.62(d,J=8 Hz,1H), 7.36(d,J=7 Hz,2H), 8.53(d,J=7 Hz,1H), 8.70(d,J=8 Hz,1H), 11.63(brs,1H). C,H,N Analysis calculated for: C.sub.27 H.sub.23 N.sub.3 O.sub.4 H.sub.2 O: C 70.12, H 5.23, N 9.09; Found: C 70.17, H 5.17, N 9.05.
EXAMPLE 65
Cbz-(.gamma.-benzyl ester)-R-Glu-(1',3'-diphenyl-5'-pyrazyl)amide
Phosphorous pentachloride (PCl.sub.5) (208 mg, 1.0 mmol) was added in one portion to a solution of Cbz-(.gamma.-benzyl ester)-R-Glutamic acid (371 mg, 1.0 mmol) in CH.sub.2 Cl.sub.2 (10 mL) at -20.degree. C. After 20 minutes, 1,3-diphenyl-5-aminopyrazole (588 mg, 2.5 mmol) was added and the temperature was raised to 0.degree. C. for 2 hours. The reaction mixture was poured into ethyl acetate and extracted successively with 1 N H.sub.3 PO.sub.4 (3x), 1 M Na.sub.2 CO.sub.3 (3x), and H.sub.2 O (3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. The residue was chromatographed on flash silica eluted with a stepped gradient from 4:1 to 2:1 hexanes-ethyl acetate. Yield: 555 mg, (0.94 mmol, 74%). R.sub.f =0.31 (2:1 hexanes-ethyl acetate). .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 8.69(bs,1H), 7.87(dd, J.about.<1,7 Hz,2H), 7.30-7.50(m,18H), 7.05(s,1H), 5.66(bd,J=7 Hz,1H), 5.02-5.11(m,4H), 4.28-4.34(m,1H), 2.58-2.68(m,1H), 2.41-2.52(m,1H), 2.16-2.26(m,1H), 1.94-2.06(m,1H).
EXAMPLE 66
R-Glu-N.sup..alpha. -(1',3'-diphenyl-5'-pyrazyl)amide
Cbz-(.gamma.-benzyl ester)-R-Glu-(1',3'-diphenyl-5,-pyrazyl)amide (417 mg, 0 70 mmol) and 10% Pd/C (100 mg, prewetted with MeOH under an N.sub.2 atmosphere) were combined in ethanol (100 mL) under 1 atmosphere H.sub.2 (balloon) overnight. The mixture was then filtered and rinsed with fresh solvent. Evaporation gave the deprotected product (260 mg, 0.70 mmol, quantitative). R.sub.f =0.23 (8:1:1 butanol-AcOH-H.sub.2 O). The product was used directly in the next step.
EXAMPLE 67
N-(2-Indolylcarbonyl)-R-Glu-N.sup..alpha. -(1',3'-diphenyl-5'-pyrazyl)amide
2-Indolecarboxy-2,4,6-trichlorophenyl ester (255 mg, 0.75 mmol) and R-Glu-(1',3'-diphenyl-5'-pyrazyl)amide (260 mg, 0.70 mmol) were dissolved in dry ethyl acetate (15 mL) and TEA (209 .mu.L, 1.5 mmol) was added at room temperature. After one day, additional TEA (209 .mu.L) was added. After 4 days, the reaction was diluted with ethyl acetate and then extracted successively with 1 M H.sub.3 PO.sub.4 (3x), H.sub.2 O (3x) and brine (lx) then dried over MgSO.sub.4, filtered and evaporated in vacuo. The residue was recrystallized from aqueous ethanol to give: 130 mg, (0.26 mmol, 36%). mp=261.degree.-5.degree. C.(dec). [.alpha.].sub.D =-0.3.degree. (c=1.09, 1:1 DMF-MeOH). MS(FAB+) m/e 508(m+H).sup.+ 365(-143), 290(-218), 262(-216), 236(-272). .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.92-2.12(m,2H), 2.33(brt,J=7 Hz,2H), 4.58(m 1H, dd(+D.sub.2 O),J.about.5,9 Hz), 6.92(s,1H), 7.06(dt,J=1,8 Hz,1H), 7.21(dt,J=1,7 Hz,1H), 7.28(brs,1H), 7.35(brt,2H), 7.42-7.47(m,6H), 7.63(t,J=8 Hz,3H), 7.37(d,J=7 Hz,2H), 8.78(brd,J=6 Hz,1H), 10.28(s,1H), 11.63(s,1H). C,H,N Analysis calculated for C.sub.29 H.sub.25 N.sub.5 O.sub.4 H.sub.2 O C 67.43; H 5.07, N 13.56; Found: C 67.69, H 5.13, N 13.40.
EXAMPLE 68
Boc-(.gamma.-benzyl ester)-R-Glu-(.alpha., .alpha.-.beta.-phenylethyl)amide
To a solution of Boc-(.gamma.-benzyl ester)-R-Glutamic acid (337 mg, 1.0 mmol) and .alpha., .alpha.-.beta.-phenyl ethylamine (373 mg, 2.5 mmol) in dry THF (15 mL) was added BOPCl (254 mg, 1.0 mmol) and TEA (1.1 mmol) and the reaction was stirred for 3 days. After evaporation of the solvent in vacuo, the residue was dissolved in ethyl acetate and extracted successively with 1 M H.sub.3 PO.sub.4 (3x) 5% NaHCO.sub.3 (3x), brine (3x), then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. The oily residue was chromatographed on flash silica gel eluted with 4:1 hexane-ethyl acetate to yield: 303 mg, (0.65 mmol, 65%) as a colorless oil. R.sub.f =0.46 (2:1 hexanes-ethyl acetate). MS(CI) m/e 469(m+H).sup.+ 413(-56), 369(-100), 278(-191). 1H NMR(CDCl.sub.3, 300 MHz) .delta. 1.28(s,3H), 1.33(s,3H), 1.42(s,9H), 1.82-1.94(m,1H), 2.03-2.16(m,1H), 2.35-2.54(m,2H), 2.96(d,J=13 Hz,1H), 3.07(d,J=13 Hz,1H), 4.00(m,1H), 5.12(s,2H), 5.18(brd,1H), 5.80(s,1H), 7.12(dd,J=3,7 Hz,2H), 7.2-7.28(m,3H), 7.35(s,5H).
EXAMPLE 69
(.gamma.-Benzyl ester)-R-Glu-(.alpha., .alpha.-.beta.-phenylethyl)amide hydrochloride
The Boc protected material of example 68 (260 mg, 0.56 mmol) was treated with HCl-dioxane (3 mL, 12 mmol) precooled in an ice bath. An additional 2 mL of HCl-dioxane was added after 2 hours and again at 3 hours at room temperature. After 3.5 hours, the solvent was evaporated and the residue was used directly. MS(CI) m/e 369(m+H).sup.+, 351(-18), 277(-92), 236(-133), 192 (-177). R.sub.f =0.85 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH), 0.46 (100:60:18:33 EtOAc-pyridine-HOAc-H.sub.2 O).
EXAMPLE 70
N-(2'-Indolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-(.alpha., .alpha.-dimethyl-.beta.-phenylethyl)amide
EDCI (115 mg, 0.6 mmol) was added to a solution of hydrochloride salt of example 69 (0.56 mmol assumed), indole-2-carboxylic acid [97 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and TEA (167 .mu.L, 1.2 mmol) in CH.sub.2 Cl.sub.2 (10 mL) cooled to 4.degree. C. in an ice bath. The reaction was allowed to warm to ambient temperature overnight. The solvent was evaporated and the residue dissolved in ethyl acetate. This solution was extracted with portions of 1 M H.sub.3 PO.sub.4, 1 M Na.sub.2 CO.sub.3, H.sub.2 O, then dried over MgSO.sub.4, filtered and the solvent evaporated. The residue was chromatographed on flash silica gel and eluted with 2:1 hexane-ethyl acetate to give 164 mg, (0.32 mmol, 57%). R.sub.f =0.27 (2:1 hexanes-ethyl acetate).
EXAMPLE 71
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha. -(.alpha., .alpha.-.beta.-phenethyl)amide
The benzyl ester of example 70 (164 mg, 0.32 mmol) was treated with 10% Pd/C (50 mg), prewetted with MeOH under an N.sub.2 atm) in 10 mL MeOH under 1 atmosphere H.sub.2 (balloon) for 1 hour. After filtration of the catalyst, the solvent was evaporated in vacuo. The residue was recrystallized from hot 80% aqueous ethanol to give 101 mg, (0.24 mmol 75% yield). mp=109.degree.-114.degree. C. [.alpha.].sub.D =-38.0.degree. MS(FAB+) m/e 422(m+H).sup.+ 273(-149), 245(-177). .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.18(s,3H), 1.26(s,3H), 1.85-2.05(m,2H), 2.21-2.40(m,2H), 2.90(d,J=13 Hz,1H), 3.07(d,J=13 Hz,1H), 4.44(m,1H,dd,+(D20), J=6,9 Hz), 7.05(dt,1H), 7.11-7.22(m,6H), 7.28(brs,1H), 7.41(s,1H), 7.44(d,J=8 Hz,1H), 7.6 (d,J=8 Hz,1H), 8.44(d,1H), 11.63(brs,1H), 12.18(brs,1H). C,H,N Analysis calculated for C.sub.24 H.sub.27 N.sub.3 O.sub.4 0.1 H.sub.2 O: C 64.92, H 6.70, N 9.46; Found: C 64.72, H 6.50, N 9.37.
EXAMPLE 72
N-Cbz-(.gamma.-benzyl ester)-R-Glu-diphenylamide
PCl.sub.5 (208 mg, 1.0 mmol) was added in one portion to a solution of Cbz(.gamma.-benzyl ester)-R-Glutamic acid (371.4 mg, 1.0 mmol) in CH.sub.2 Cl.sub.2 (10'mL) at -20.degree. C. After 20 minutes, diphenylamine (423 mg, 2.5.mmol) was added and the temperature was raised to 0.degree. C. for 2 hours. The reaction mixture was poured into ethyl acetate and extracted successively with 1 N HCl(3x), 1 M Na.sub.2 CO.sub.3 (3x), and H.sub.2 O(3x) then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo., The residue was chromatographed on flash silica eluted with a stepped gradient from 4:1 to 2:1 hexanes-ethyl acetate. Yield: 405 mg, (0.77 mmol, 77%). R.sub.f =0.31 (2:1 hexanes-ethyl acetate). MS(CI) m/e 522(m+H).sup.+ 540(m+NH.sub.4).sup.+.sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 1.84-2.12(m,2H), 2.24-2.47(m,2H), 4.63(m,1H), 5.05(s,2H), 5.08(brs,2H), 5.60(d,J=8 Hz,1H), 7.2-7.5(m,20H).
EXAMPLE 73
R-Glu-N.sup..alpha.,N.sup..alpha. -diphenylamide
Cbz-(.gamma.-benzyl ester)-R-Glu-diphenyl amide (400 mg, 0.77 mmol) and 10% Pd/C (50 mg, prewetted with MeOH under an N.sub.2 atmosphere) were combined in MeOH (15 mL) under 1 atmosphere H.sub.2 (balloon) for 3 hours. The catalyst was then filtered and rinsed with fresh solvent. Evaporation gave the deprotected product (244 mg, 0.75 mmol, 98%). R.sub.f =0.13 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH). The product was used directly in the next step.
EXAMPLE 74
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -diphenylamide
2-Indolecarboxy-2,4,6-trichlorophenyl ester (29 mg, 0.084 mmol) and R-Glu-diphenyl amide (25 mg, 0.084 mmol) were dissolved in dry THF (4 mL) and TEA (22 .mu.L, 0.16 mmol) was added at room temperature. The tlc at 3 hours showed no reaction and an additional 22 .mu.L TEA was added. After 2 days, the THF was evaporated in vacuo and the residue, dissolved in ethyl acetate, was extracted successively with 1 M H.sub.3 PO.sub.4 (3x), H.sub.2 O(3x) and brine (lx) then dried over MgSO.sub.4, filtered and evaporated in vacuo. The crude product was recrystallized from hot ethyl acetate-hexanes to give 12.1 mg, (0.027 mmol, 33% yield). R.sub.f =0.14 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH) MS(FAB+) m/e 442(m+H).sup.+ 273(-168), 245(-196). .sup.1 H NMR(CDCl.sub.3 -CD.sub.3 OD,300 MHz) .delta. 2.03-2.20(m, 2H), 2.38(dd,J=8,15 Hz, 2H), 5.02(dd,J=5,8 Hz,1H), 7.1(dJ<1Hz,1H), 7.13(d,J=7 Hz,1H), 7.25-7.48(m,12H), 7.66(d,J=8 Hz,1H). C,H,N Analysis calculated for C.sub.26 H.sub.23 N.sub.3 O.sub.4 EtOAc: C 69.96, H 5.44, N 9.07; Found: C 69.72, H 5.57, N 8.90.
EXAMPLE 75
N-(3-(3'-Pyridyl)acryloyl)-(.gamma.-benzyl ester)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
Hydrochloride salt (0.23 g, 0.56 mmol) of example 2, EDCI 0.12 g, 0.6 mmol); HOBt (0.075 g, 0.56 mmol) and 3-(3-pyridyl)acrylic acid (0.085 g, 0.56 mmol) were stirred at 0.degree. C. under nitrogen in 10 mL of CH.sub.2 Cl.sub.2) To this mixture was added NMM (0.12 mL, 1.1 mmol). The reaction mixture was stirred overnight and permitted to reach ambient temperature. The reaction mixture was poured into ethyl acetate and extracted twice with portions of 1.0 N hydrochloric acid, and once with saturated sodium bicarbonate solution. The organic solution was dried over magnesium sulfate and filtered. Evaporation of the volatiles in vacuo provided a residue which was purified by chromatography using ethyl acetate/hexane as the elutant provided 0.22 g (78% yield) of product. MS(CI) m/e 508(m+H).sup.+ .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.85-0.96(m,6H), 1.19-1.42(m,8H), 1.48-1.70(m,4H), 1.78-1.95(m,2H), 2.40-2.60(m,2H), 5.13(m,3H), 5.02(d,J=15 Hz,1H), 6.98(d,J=9 Hz,1H), 7.35(m,6H), 7.6(d,J=15 Hz,1H), 7.80(d,J=9 Hz 1H), 8.56(dd,J=1,6 Hz,1H), 8.72(d,J=3 Hz,1H).
EXAMPLE 76
N-(8'-Quinolylsulfonyl)-(.gamma.-benzyl ester)-R-Glu-di-n-pentyl amide
To a mixture of hydrochloride salt (1.0 mmol) of example 2 and triethylamine (2.0 mmol) stirring in 10 mL of methylene chloride at 0.degree. C. was added 8-quinolylsulfonyl chloride (1.0 mmol). The reaction mixture was allowed to warm to ambient temperature and continue stirring overnight. The reaction mixture was poured into ethyl acetate and extracted with 1.0 N hydrochloric acid and saturated sodium bicarbonate solution. The mixture was dried over magnesium sulfate and filtered. Evaporation of the volatiles in vacuo and chromatography of the residue provided product.
EXAMPLE 77
N-(8'-Quinolylsulfonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentyl amide
The product of example 76 was treated with sodium hydroxide solution in methanol as in example 29. Extraction of the acidified solution numerous times with ethyl acetate and subsequent drying, filtration, concentration, and trituration as in example 29 provided product.
EXAMPLE 78
Ethyl N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-Glutamyl (N-benzyl)glycinate
N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-glutamic acid (5.17 grams, 15.3 mmol) and N-benzylglycine ethyl ester (9.12 grams, 47.2 mmol) were stirred in 100 mL of CH.sub.2 Cl.sub.2 at -10.degree. to 0.degree. C. under nitrogen. To this solution was added BOPCl (4.17 grams, 16.4 mmol) and the reaction mixture allowed to slowly warm to room temperature overnight. The mixture was taken up in ethyl acetate and extracted three times with dilute hydrochloric acid, once with saturated sodium bicarbonate solution and dried over magnesium sulfate. The solution was filtered and concentrated in vacuo. The residue was purified by chromatography using ethyl acetate and hexane as elutants to provide 6.59 grams of pure product (84% yield). MS(CI) m/e 513(m+H).sup.+ 322, 305, 276. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 7.25-7.40 m,8H), 7.18-7.25(m,2H), 5.36(m,2H), 5.10(m,2H), 4.95(bd,J=15 Hz,1H), 4.53(bd,J= 15 Hz,1H), 4.15(m,2.5H), 3.70(d,J=17 Hz,0.5H), 2.40-2.60(m,2H), 2.20(m,1H), 1.82(m,1H), 1.43(bs,9H), 1.23(m,3H).
EXAMPLE 79
Ethyl (.gamma.-benzyl ester)-R-Glutamyl(N-benzyl)glycinate hydrochloride
The ethyl ester product of example 78 (3.46 grams, 6.76 mmol) was stirred at 5.degree. C. in 10 mL of 4.5 N hydrochloric acid in dioxane. When the reaction was complete by tlc, diethyl ether. was added and the solvents removed in vacuo. The product was collected via filtration using diethyl ether. Yield 2.96 grams (97%). MS(FAB) m/e 413(m+H).sup.+ 395, 378, 323, 305, 289. .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 8.38(bs 1H) 7.25-7 40( ,10H) 5.11(bs,2H), 4.97(bd,J=17 Hz,0.67H), 4.70(bd,J=17 Hz,0.33H), 4.35-4.55(m,3H), 4.22(m,1H), 4.08(m,2H), 3.69(d,J=17 Hz,1H), 3.35(bs,H.sub.2 O), 2.60-2.70(m,2H), 2.10(m,2H), 1.15(m,3H).
EXAMPLE 80
Ethyl N-(2'-Naphthoyl)-(r-benzyl ester)-R-Glutamyl(N-benzyl) glycinate
The hydrochloride of example 79 (288 mg, 0.64 mmol) was coupled to 2-naphthoic acid in a similar manner to that in example 70 to provide product (322 mg, 88% yield) after purification by chromatography. mp=103.degree.-105.degree. C. [.alpha.].sub.D =-6.8.degree. (c=0.25, MeOH-DMF 1:1). MS(CI) m/e 567(m+H).sup.+ 374, 346. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 8.35(s,1H), 7.85-7.95(m,4H), 7.56(m,2H), 7.20-7.40(m,10H), 5.49(dt,J=3.5,8.5 Hz,0.67H), 5.20(m,0.33H), 5.11(m,2H), 5.03(d,J=16 Hz,0.67H), 4.69(s,0.67H), 4.62(d,J=16 Hz,0.67), 4.10-4.45(m,4H), 3.74(d,J=16 Hz,1H), 2.30-2.70(m,3H), 2.00-2.10(m,1H), 1.23(m,3H). C,H,N analysis calculated for C.sub.34 H.sub.34 N.sub.2 O.sub.6 C 72.07, H 6.05, N 4.94; found C 71.83, H 6.11, N 4.87.
EXAMPLE 81
Ethyl N-(2-Naphthoyl)-R-Glutamyl(N-benzyl)glycinate
The product of example 80 (167 mg) was subjected to hydrogenolysis as in example 21 utilizing cyclohexadiene as the reductant. Product was purified by chromatography over silica with 0.5% acetic acid in ethyl acetate solution and recovered by lyophilization to provide 121 mg (86% yield) of product. mp=56.degree.-62.degree. C. [.alpha.].sub.D =-10.5 (c=0.76, MeOH). MS(CI) m/e 477(m+H).sup.+, 284, 194, 155. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 8.92(bd,J=7.5 Hz,0.33H), 8.83(bd,J=7.5Hz,0.67H), 8.52(bd,J=5.5 Hz,1H), 8.00(m,4H), 7.63(m,2H), 7.46(bd,J=7 Hz,1H), 7.23-7.40(m,4H), 5.12(m,1H), 5.07(bs,0.33H), 4.95(m,0.67H) .4.55(m,2H), 4.19(m,0.67H), 4.06(q,J=7 Hz,l.33H), 3.96(m,0.33H), 3.64(bd,J=17 Hz,0.67H), 2.43(m,l.33H), 2.35(m,0.67H), 1.90-2.10(m,2H), 1.16(t,J=7 Hz,2 H), 1.09(t,J=7 Hz,1H). C,H,N analysis calculated for C.sub.27 H.sub.28 N.sub.2 O.sub.6 0.25 H.sub.2 O: C 67.42, H 5.97, N 5.82; found C 67.43, H 5.88, N 5.77.
EXAMPLE 82
Ethyl N-(3'-Quinolylcarbonyl)-(.gamma.-benzylester)-R-Glutamyl (N-benzyl)glycinate
The hydrochloride of example 79 (325 mg, 0.67 mmol) was coupled to quinoline-3-carboxylic acid in a similar manner to that in example 70 to provide product (331 mg, 87.7% yield) after purification by chromatography. [.alpha.].sub.D =+5.5.degree. (c=0.86, MeOH). MS(CI) m/e 568(m+H).sup.+ 375, 342. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 9.34(d,J=2 Hz,1H), 8.60(d,J=1.8 Hz,1H), 8.17(d,J=7.5 Hz,1H), 7.92(m,1H), 7.82(m,1H), 7.63(m,1H), 7.50(m,1H), 7.20-7.40(m,9H), 5.50(dt,J=1,4.5 Hz,0.67H), 5.00-5.12(m,3H), 4.69(s,0.67H), 4.62(d,J=16 Hz,0.67H), 4.10-4.45(m,3H), 3.74(d,J=16 Hz,1H), 2.50-2.70(m,2H), 2.30-2.50(m,1H), 2.03- 2.11(m,1H), 1.60(bs,H.sub.2 O), 1.26(m,3H). C,H,N analysis calculated for C.sub.33 H.sub.33 N.sub.3 O.sub.6 0.7 H.sub.2 O: C 8.31, H 5.98, N 7.24; found C 68.28, H 5.88, N 7.13.
EXAMPLE 83
Ethyl N-(3'-Quinolylcarbonyl)-R-Glutamyl(N-benzyl)glycinate
The product of example 82 (100 mg) was subjected to hydrogenolysis as in example 21 utilizing cyclohexadiene as the reductant. Product was purified by chromatography over silica with 0.5% acetic acid in ethyl acetate solution and recovered by lyophilization to provide 55.3 mg (66% yield) of product. mp=70.degree.-79.degree. C. [.alpha.].sub.D =-30.4.degree. (c=0.23, acetone). MS(CI m/e 478(m+H).sup.+ 287, 257, 194, 156. .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 9.29(d,J=2 Hz,0.33), 9.25(d,J=2 Hz,0.67H), 9.15(bd,J=7.5 Hz,0.33), 9.05(bd,J=7.5 Hz,0.67H), 8.91(d,J=2 Hz,0.33H), 8.85(d,J=2 Hz,0.67H), 8.10(m,2H), 7.88(m,1H), 7.72(m,1H), 7.45(bd,J=7 Hz,1H), 7.22-7.37(m,4H), 5.13(m,l.33H), 4.96(m, 0.67H), 4.58(m,2H), 4.22(m,0.67H), 4.08(q,J=7 Hz,l.33H), 3.96(m,0.33H), 3.65(bd,J=17 Hz,0.67H), 3.35(bs,H.sub.2 O), 2.33-2.48(m,2H), 1.92-2.12(m,2H), 1.17(t,J=7 Hz(2H), 1.09(t,J=7 Hz,1H). C,H,N analysis calculated for C.sub.26 H.sub.27 N.sub.3 O.sub.6 0.33 H.sub.2 O: C 64.59, H 5.77, N 8.69; found C 64.47, H 5,69, N 8.71.
EXAMPLE 84
Ethyl N-(4', 8'-Dihydoxyquinolyl-2-carbonyl)-(.gamma.-benzylester) -R-Glutamyl(N-benzyl)glycinate
The hydrochloride of example 79 (288 mg, 0.64 mmol) was coupled to 4,8-dihydroxyquinoline-2-carboxylic acid in a similar manner to that in example 20 to provide product (234 mg, 60% yield) after purification by chromatography. mp=71.degree.-74.degree. C. [.alpha.].sub.D =-10.7.degree. (c=0.56, acetone). MS(CI) m/e 600(m+H).sup.+ 407, 379. .sup.1 H NMR(CDCl.sub.3,300 MHz) .delta. 7.76(m,1H), 7.55(m,1H), 7.20-7.45(m,l1H), 6.95(m,1H), 5.25(m,1H), 5.18(bs,2H), 5.07(bd,J=16 Hz,1H), 4.70-4.90(m,1H), 4.62(dd,J=4.5,16 Hz,1H), 4.53(bd,J=16 Hz,1H), 4.18(m,2H), 3.69(bd,J=16 Hz,1H), 2.55-2.85(m,2H), 2.50-2.68(m,1H), 2.44(m,2H), 1.23(bt,J=7.5 Hz,3H).
EXAMPLE 85
Ethyl N (4',8'-Dihdroxyquinolyl-2-carbonyl)-R-Glutamyl (N-benzyl)glycinate
The product of example 84 (57 mg, 0.095 mmol) was subjected to hydrogenolysis as in example 21 utilizing cyclohexadiene as the reductant and DMF (1 mL) to aid in solubilization of the substrate. Product was purified by chromatography over silica with 0.5% acetic acid in ethyl acetate solution and recovered by lyophilization to provide 30.3 mg (60% yield) of product. mp=103.degree.-109.degree. C. [.alpha.].sub.D =-10.0.degree. (c=0.13, MeOH). [.alpha.].sub.D =-10.0.degree. (c=0.13, MeOH). MS(CI m/e 510(m+H).sup.+ 482, 464, 305. 1H NMR(DMSO.sub.d6,300 MHz) .delta. 10.12(bs,1H), 9.72(m,1H), 7.96(bd,J=7 Hz,0.67H), 7.69(bd,J=7.5 Hz,0.33H), 7.54(m,1H), 7.37-7.50(m,3H), 7.19-7.33(m,4H), 7.12(bd,J=7.5 Hz,1H), 5.18(m,0.33H), 4.95(m,0.67 H), 4.55(bd,J=7.5 Hz,1H), 4.50(m,1H), 4.19(m,0.67H), 4.07(bq,J=7 Hz,l.33H), 3.90(m,0.33H), 3.70(bd,J=16 Hz,0.67H), 3.34bs,H.sub.2 O), 2.25-2.37(m,2H), 2.15(m,1H), 2.00(m,1H), 1.14(t,J=7 Hz,2H), 1.07(t,J=7 Hz,1H).
EXAMPLE 86
N-(2'-Methylphenylaminocarbonyl)-(.gamma.-benzyl ester)-S-Glu-di-n-pentylamide
To a solution of the hydrochloride salt of (.gamma.-benzyl ester)-S-Glu-di-n-pentylamide (0.1 g, 0.24 mmol) in THF (7 mL) was added TEA (40 .mu.L, 0.3 mmol) and a slight excess of 1-methylphenylisocyanate (0.055 g, 0.42 mmol). The reaction mixture was stirred overnight at ambient temperature. The solvents were evaporated and residue purified by chromatography using EtOAc and hexane as the elutant mixture. Pure fractions were pooled and the volatiles removed in vacuo to yield an oily product (0.075 g) in 62% yield. MS(CI) m/e 510(m+H).sup.+ .sup.1 H NMRCDCl ,300 MHz) .delta. 0.8-0.95(m,6H), 1.1-1.35(m,8H), 1.36-1.5(m,4H), 1.72-1.83(m,1H), 2.0-2.12(m,1H), 2.21(s,3H), 2.38-2.60(m,2H), 2.97-3.08(m,1H), 3.15-3.3(m 1H), 3.4-3.55(m,2H), 4.9(m,1H), 5.1(s,2H), 6.02(d,J=9 Hz,1H), 6.47(s,1H), 7.05(t,J=7 Hz,1H), 7.08-7.19(m,2H), 7.35(m,5H), 7.48(d,J=9 Hz,1H). C,H,N analysis calculated for C.sub.30 H.sub.43 N.sub.3 O.sub.4 0.25 H.sub.2 O: C 70.07, H 8.53, N 8.17; found C 69.79, H 8.30, N 8.18.
EXAMPLE 87
Methyl (.gamma.-benzyl ester)-R-Glutamyl-S-phenylglycinate hydrochloride
The product of example 35 was deprotected in a similar manner to that in example 79. The product was concentrated under vacuum to an oil which was used without further purification.
EXAMPLE 88
Methyl N-(2'-Naphthoyl)-(.gamma.-benzyl ester)-R-Glutamyl -S-phenylglycinate
The hydrochloride of example 87 (157 mg, 0.37 mmol) was coupled with 2-naphthoic acid as in example 80 to provide product (156.2 mg, 78% yield) after chromatography. mp=154.degree.-6.degree. C. [.alpha.].sub.D =+38.4.degree. (c=0.24, acetone). MS(CI) m/e 539(m+H).sup.+ 374, 347, 155. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 8.36(s,1H), 7.89(m,4H), 7.56(m,3H), 7.43(d,J=7 Hz,1H), 7.32-7.40(m,10H), 5.55(d,J=7 Hz,1H), 5.12(s,2H), 4.84(dt,J=4.8,7.5 Hz,1H), 3.70(s,3H), 2.72(m,1H), 2.45(m,1H), 2.10-2.33(m,2H). C,H,N analysis calculated for C.sub.32 H.sub.30 N.sub.2 O.sub.6 C 69.05, H 5.79, N 5.03; found C 69.26, H 5.74, N 4.90.
EXAMPLE 89
Methyl N-(2'-Naphthoyl)-R-Glutamyl-S-phenylglycinate
The product of example 88 (73 mg, 0.136 mmol) was subjected to hydrogenolysis as in example 21 using cyclohexadiene as the reductant and DMF (1 mL) to solubilize the substrate for transfer to the reaction vessel. Yield 48 mg (79%). mp=56.degree.-63.degree. C. D =+32.9.degree. (c=0.34, MeOH). MS(CI) m/e 449(m+H).sup.+ 329, 284, 273, 257, 155. .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 8.90(d,J=7 Hz,1H), 8.64(d,J=8 Hz,1H), 8.52(bs,1H), 7.95-8.06(m,4H), 7.58-7.67(m,2H), 7.35-7.42(m,6H), 5.48(d,J=7 Hz,1H), 4.68(m,1H), 3.63(s,3H), 2.32(m,2H), 1.98(m,2H).
EXAMPLE 90
Methyl N-(3'-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-GlutamylS-phenylglycinate
The hydrochloride of example 87 (221 mg, 0.528 mmol) was coupled with quinoline-3-carboxylic acid as in example 80 to provide product (170 mg, 63% yield) after chromatography. mp =136-137.5.degree. C. [.alpha.].sub.D =+37.9.degree. (c=0.24, acetone). MS(CI) m/e 40(m+H).sup.+ 480, 432, 375, 156. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 9.34(d,J=2 Hz,1H), 8.62(d,J=2 Hz,1H), 8.18(d,J=7.5 Hz,1H), 7.90(dd,J=1,7.5 Hz,1H), 7.83(m,1H), 7.70(bd,J=7 Hz,1H), 7.6(m,1H), 7.54(bd,J=7 Hz,1H), 7.28-7.40(m,10H), 5.55(d,J=7 Hz,1H), 5.16(d,J=12 Hz,1H), 5.12(d,J=12 Hz,1H), 4.85(dt,J=5,7 Hz,1H), 3.71 (s,3H), 2.75(m,1H), 2.48(m,1H), 2.15-2.33(m,2H). C,H,N analysis calculated for C.sub.31 H.sub.29 N.sub.3 O.sub.6 C 69.01, H 5.42, N 7.79; found C 68.63, H 5.46, N 7.76.
EXAMPLE 91
Methyl N-(3'-Quinolylcarbonyl) R-Glutamyl-S-phenylglycinate
The product of example 90 (67 mg, 0.124 mmol) was subjected to hydrogenolysis as in example 21 using cyclohexadiene as the reductant and DMF (1 mL) to solubilize the substrate for transfer to the reaction vessel. Yield 28 mg (50%). mp=182.degree.-185.degree. C. [.alpha.].sub.D =+46.6.degree. (c=0.075, MeOH). MS(CI) m/e 450(m+H).sup.+.285 256, 156. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 9.30(d,J=2 Hz,1H), 8.96(d,J=7 Hz,1H), 8.90(m,2H), 8.11(m,2H), 7.88(m,1H), 7.71(m,1H), 7.39(m,5H), 5.49(d,J=7 Hz,1H), 4.72(m,1H), 3.64(s,3H), 3.35(bs,H.sub.2 O), 2.32(m,2H), 1.98(m,2H). C,H,N analysis calculated for C.sub.24 H.sub.23 N.sub.3 O.sub.6 0.67 H.sub.2 O: C 62.48, H 5.31, N 9.11; found C 62.23, H 5.29, N 8.93.
EXAMPLE 92
Methyl N-(4',8'-Dihydroxyquinolyl-2-carbonyl)-(.gamma.-benzyl ester)- R-Glutamyl-S-phenylglycinate
The hydrochloride of example 87 (146 mg, 0.35 mmol) was coupled with 4,8-dihydroxyquinoline-2-carboxylic acid as in example 80.to provide product 65.6 mg, (33% yield) after chromatography. mp=234.degree.-5.degree. C. MS(CI) m/e 572(m+H).sup.+ 385, 294, 277, 211, 192, 166. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 10.08(bs,1H); 9.56(d,J=7 Hz,1H), 9.00(d,J=7 Hz,1H), 7.55(m,2H), 7.25-7.45(m,10H), 7.12(d,J=7 Hz,1H), 5.48(d,J=7 Hz,1H), 5.05(s,2H), 4.75(m,1H), 3.61(s,3H), 3.30(bs,H.sub.2 O), 2.43(m,2H), 2.10(m,2H). C,H,N analysis calculated for C.sub.31 H.sub.29 N.sub.3 O.sub.8 0.5 H.sub.2 O: C 64.13, H 5.21, N 7.24; found C 64.43, H 5.07, N 7.25.
EXAMPLE 93
Methyl N-(4',8'-Dihydroxyquinolyl-2-carbonyl)-R-Glutamyl -S-phenylglycinate
The product of example 92 (49.7 mg, 0.087 mmol) was subjected to hydrogenolysis as in example 21 using cyclohexadiene as the reductant and DMF (2 mL) to solubilize the substrate for transfer to the reaction vessel. Yield 31.4 mg (75%). MS(CI) m/e 482(m+H).sup.+ 464, 317, 289, 277, 205. .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 9.56(m,1H), 8.96(bd,J=7 Hz,1H), 7.57(bd,J=8 Hz,1H), 7.50(bs,1H), 7.33-7.45(m,7H), 7.12(bd,J=8 Hz,1H), 5.47(d,J=7 Hz,1H), 4.71(m,1H), 3.62(s,3H), 3.3(bs,H.sub.2 O), 2.28(m,2H),. 2.00(m,2H). C,H,N analysis calculated for C.sub.24 H.sub.23 N.sub.3 O.sub.8 0.33H.sub.2 O: C 59.14, H 4.89, N 8.62; found C 59.07, H 4.95, N 8.39.
EXAMPLE 94
Methyl N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-Glutamyl -S-leucinate
N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-glutamic acid (1.077 grams, 3.0 mmol) and methyl-S-leucinate hydrochloride were coupled in a manner similar to that in example 21 to provide 1.28 grams (86% yield) of product after chromatography. mp=69.degree.-71.degree. C. [.alpha.].sub.D =-1.1.degree. (c=0.62, acetone). MS(CI) m/e 482(m+NH.sub.4)+, 465(m+H).sup.+, 409, 365, 309, 292, 274, 257. .sup.1 H, NMR(CDCl.sub.3, 300 MHz) .delta. 7.36(m,5H), 6.55(m,1H), 5.30(m,1H), 5.14(s,2H), 4.56(m,1H), 4.19(m,1H), 3.72(s,3H), 2.56(dt,J=7,17 Hz,1H), 2.47(dt,J=7,17 Hz,1H), 2.17(m,1H), 1.97(m,1H), 1.50-1.67(m,3H), 1.41(s,9H), 0.92(d,J=6 Hz,6H). C,H,N analysis calculated for C.sub.24 H.sub.36 N.sub.2 O.sub.7 C 62.05, H 7.81, N 6.03; found C 61.98, H 7.80, N 5.96.
EXAMPLE 95
Methyl (.gamma.-benzyl ester)-R-Glutamyl-S-leucinate hydrochloride
The product of example 94 (1.07 grams, 2.30 mmol) was treated with 6.0 mL of 4.5 N hydrochloric acid in dioxane. The hydrochloride was isolated as an oil as in example 87, yield 914 mg (99%). MS(CI) m/e 365(m+H).sup.+ 192. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 9.05(d,J=7 Hz,1H), 8.34(bs,3H), 7.32-7.42(m,5H), 5.11(s,2H), 4.28-4.36(m,1H), 3.91(t,J=6 Hz,1H), 3.64(s,3H), 2.36-2.58(m,2H), 2.02-2.09(m,2H), 1.49-1.65(m,3H), 0.86(d,J=6 Hz,3H), 0.82(d,J=6 Hz,3H).
EXAMPLE 96
Methyl N-(2'-naphthoyl)-(.gamma.-benzyl ester)-R-Glutamyl-S-leucinate
Naphthalene-2-carboxylic acid i121 mg, 0.70 mmol) and the product of example 95 (200 mg, 0.50 mmol) were coupled and extracted as in example 80. The resulting residue was chromatographed on silica gel eluted with a step gradient of 9:1 to 2:1 hexanes-EtOAc to give 149 mg, 0.29 mmol (44% yield). mp.TM.95.degree.-98.degree. C. [.alpha.].sub.D =+17.3.degree. (c=0.55, CHCl.sub.3). MS(CI) m/e 519(m+H).sup.+ 428, 411. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.94(dd,J=2,6 Hz,6H), 1.55-1.71(m,2H), 2.13-2.38(m,3H), 2.55(dt,J=7,17 Hz,1H), 2.72-2.82(m,1H), 3.67(s,3H), 4.54-4.65(m,1H), 4.78-4.84(m,1H), 5.13(s,2H), 6.91(d,J=8 Hz,1H), 7.28-7.33(m,5H), 7.50- 7.61(m,3H), 7.86-7.94(m,4H), 8.36(s,1H). C,H,N analysis calculated for C.sub.30 H.sub.34 N.sub.2 O.sub.6 : C 69.48, H 6.61, N 5.40; found C 69.21, H 6.71, N 5.33.
EXAMPLE 97
Methyl N-(2'-Naphthoyl)-R-Glutamyl-S-leucinate
The product of example 96 (74 mg, 0.14 mmol) was dissolved in 10 mL MeOH and treated with 20 mg of 10% Pd/C (prewetted with solvent under N.sub.2) and 1 atmosphere of hydrogen gas. After 3 hours, the catalyst was filtered and rinsed with fresh solvent. The residue was dissolved in MeOH, filtered through a 0.5 .mu. filter and then diluted with water. The aqueous mixture was frozen and lyophilized to give 31 mg, 0.072 mmol (52% yield). R.sub.f =0.22 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH) mp=58.degree.-65.degree. C. [.alpha.].sub.D =-31.8.degree. (c=1.0, MeOH). MS(CI) m/e 429(m+H).sup.+ 284. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 0.83(d,J=6 Hz,3H), 0.86(d,J=6 Hz,3H), 1.43-1.63(m,3H); 1.94-2.03(m,2H), 2.33(bt,J=7 Hz,2H), 3.62(s,3H), 4.28-4.36(m,1H), 4.56(q,J=7 Hz,1H), 7.56-7.65(m,2H), 7.96-8.06(m,4H), 8.42(d,J=8 Hz,1H), 8.52(s,1H), 8.83(d,7 Hz,1H). C,H,N analysis calculated for C.sub.23 H.sub.28 N.sub.2 O.sub.6 0.6 H.sub.2 O: C 62.89, H 6.70, N 6.38; found C 62.75, H 6.51, N 6.44.
EXAMPLE 98
Methyl N-(3'-Quinolylcarbonyl)-(.gamma.-benzyl ester)-R-GlutamylS-leucinate
Quinoline-3-carboxylic acid (139 mg, 0.8 mmol) and the product of example 95 (200 mg, 0.50 mmol) were coupled as in example 82. Silica gel chromatography of the residue eluted with a step gradient of 9:1 to 1:1 hexane-EtOAc gave 99 mg, 0.19 mmol (39% yield). R.sub.f =0.22 (1:1 hexane-EtOAc). mp=105.degree.-107.degree. C. [.alpha.].sub.D =+16.9.degree. (c=1.5, CHCl.sub.3) MS(CI) m/e 520(m+H).sup.+ 429, 412. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.93(d,J=2 Hz,3H), 0.96(d,J=2 Hz,3H), 1.55-1.71(m,3H), 2.17-2.37(m,2H), 2.57(ddd,J=5,7,17 Hz,1H), 2.80(ddd,J=5,8,17 Hz,1H), 3.69(s,3H), 4.58-4.65(m,1H), 4.76-4.82(m,1H), 5.13(d,J=12 Hz,1H), 5.18(d,J=12 Hz,1H), 6.90(d,J=8 Hz,1H), 7.28-7.35(m,5H), 7.63(dt,J=1,8 Hz,1H), 7.76(d,J=7 Hz,1H), 7.82(dt,J=1,7 Hz,1H), 7.91(d,J=8 Hz,1H), 8.16(d,J=8 Hz,1H), 8.62(d,J=2 Hz,1H), 9.36(d,J=2 Hz,1H). C,H,N analysis calculated for C.sub.29 H.sub.33 N.sub.3 O.sub.6 0.3 H.sub.2 O: C 66.35, H 6.45, N 8.00; found C 66.34, H 6.43, N 7.96.
EXAMPLE 99
Methyl N-(3'-Quinolylcarbonyl)-R-Glutamyl-S-leucinate
The product of example 98 (100 mg, 0.192 mmol) was treated with 25 mg of 10% Pd/C (prewetted with EtOH under N.sub.2) and 1 atmosphere of hydrogen gas in 25 mL of EtOH. Another 20 mg of catalyst was carefully added after 4 hours and the reaction was allowed to continue overnight, After filtration of the catalyst and evaporation of the solvent, the residue was chromatographed on silica gel eluted with a 9:1 CH.sub.2 Cl.sub.2 -EtOH to 80:20:1 CHCl.sub.3 -MeOH-NH-40H step gradient. After evaporation of the product fractions, the residue was mixed with aqueous EtOH, frozen and lyophilized to give 39 mg, 0.092 mmol (48% yield). mp=194.degree.-6.degree. C. [.alpha.].sub.D =-26.6.degree. (c=1.64, MeOH). MS(CI) m/e 430(m+H).sup.+ 412, 285, 257, 199, 174, 156. .sup.1 H NMR(CDCl.sub.3 300 MHz) .delta. 0.95(dd,J=5,6 Hz,6H), 1.62-1.72(m,3H), 2.08-2.27(m,2H), 2.49-2.53(m,2H), 3.70(s,3H), 3.98-4.02(m,1H), 4.22(dd,J=6,8 Hz,1H), 7.21(dt,J=1,8 Hz,1H), 7.39(dt,J=1,7 Hz,1H), 8.06-8.12(m,2H) 8.86(d,J= 2 Hz,1H), 9.28(d,J=2 Hz,1H).
EXAMPLE 100
Methyl N-(4',8'-Dihydroxyquinolyl-2-carbonyl)-(.gamma.-benzyl ester)-R-Glutamyl-S-leucinate
4,8-Dihydroxyquinoline-2-carboxylic acid (62 mg, 0.3 mmol), the product of example 95 (100 mg, 0.25 mmol), HOBt (41 mg, 0.3 mmol), and TEA (42 .mu.L, 0.3 mmol) were dissolved in 5 mL DMF and treated with EDCI (57 mg, 0.3 mmol). After 3 days, the solvent was evaporated and the residue in EtOAc was extracted as in example 82. The residue from evaporation was chromatographed in silica gel eluted with a 1% to 5% EtOH in CH.sub.2 Cl.sub.2 to give 108 mg, 0.196 mmol (78% yield) as a light yellow solid. mp=208.degree.-10.degree. C. [.alpha.].sub.D =-28.0.degree. (c=0.46, MeOH). MS(FAB+) m/e 552(m+H).sup.+ 374. .sup.1 H NMR(CDCl.sub.3 --CD.sub.3 OD, 300 MHz) .delta. 0.92(bd,J=5 Hz,6H), 1.55-1.68 (m,3H), 2.15-2.37(m,2H), 2.48-2.66(m,2H), 3.70(s,3H), 4.52-4.57(m,1H), 4.73(dd,J=5,12 Hz,1H), 5.13(s,2H), 7.16(bd,J=7 Hz,1H), 7.28-7.33(m,6H), 7.40(s,1H), 7.72(dd,J=1,8 Hz,1H). C,H,N analysis calculated for C.sub.29 H.sub.33 N.sub.3 O.sub.8 0.25 H.sub.2 O: C 2.64, H 6.07, N 7.56; found C 62.41, H 5.91, N 7.39.
EXAMPLE 101
Methyl N-(4', 8'-Dihydroxyquinolyl-2-carbonyl)-R -Glutamyl-S-leucinate
The product of example 100 (50 mg, 0.091 mmol) was treated with 10 mg of 10% Pd/C in 5 mL MeOH as in example 97. After 2 hours, the catalyst was filtered and the solvent was evaporated. mp=228.degree.-30.degree. C. [.alpha.].sub.D =-42.0.degree. (c=1.07, DMF). MS(CI) m/e 462(m+H).sup.+. .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 0.83(d,J=6Hz, 3H), 0.88(d,J=6Hz, 3H), 1.43-1.66(m,3H), 1.42-2.15(m,2H), 2.32(bt,J=7Hz,2H), 3.61(s,3H), 4.29-4.36(m,1H), 4.56-4.63(m,1H), 7.10(dd,J=1,7Hz, 1H), 7.40(t,J=8Hz, 1H), 7.47(bs,1H), 7.56(dd,J=1,8Hz,1H), 8.45(d,J=7Hz,1H), 9.55(bd,J=7Hz,1 H). C,H,N analysis calculated for C.sub.22 H.sub.27 N.sub.3 O.sub.8 1.2 H.sub.2 O: C 54.70, H 6.13, N 8.70; found C 54.63, H 5.79, N 8.30.
EXAMPLE 102
N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-Glutamylbis(carboethoxymethyl)amide
N-(t-Butyloxycarbonyl)-(.gamma.-benzyl ester)-R-glutamic acid (1.98 gram, 5.9 mmol) and diethyl imidodiacetate (5.32 gram, 28.15 mol) were coupled in a manner similar to that in example 78 to provide 2.62 grams 88% yield) of product after chromatography. [.alpha.].sub.D =+16.5.degree. (c=1.16, MeOH). MS(CI) m/e 526(m+NH.sub.4).sup.+, 470, 453, 436, 423, 409, 380. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 7.35(m,5H), 5.31(bd,J=8Hz,1H), 5.17(d,J=16Hz,1H), 5.12(d,J=16Hz, 1H), 4.70(dt,J=4,9Hz,1H), 4.38(bs,2H), 4.05-4.27(m,6H), 2.40-2.60(m,2H), 2.13(m,1H), 1.73(m,1H), 1.43(bs,9H), 1.28(t,J=7Hz, 3H), 1.26(t,J=7Hz, 3H).
EXAMPLE 103
(.gamma.-Benzyl ester)-R-Glutamyl-bis(carboethoxymethyl)amide hydrochloride
The product of example 102 (2.16 gram, 4.25 mmol) was deprotected as in example 95 to provide 1.88 grams as an oil which was utilized as is. MS(CI) m/e 409(m+H).sup.+ 391, 380, 363. .sup.1 H NMR(DMSO.sub.d6 ,300 MHz) .delta. 8.46(bs,3H), 7.30-7.41(m,5H), 5.12(s,2H), 4.36-4.52(m,3H), 4.02-4.23(m,6H), 2.63-2.73(m,1H), 2.56(dd,J=5,10 Hz,1H), 1.85-2.12(m,2H), 1.15-1.23(m,6H).
EXAMPLE 104
N-(2'-Naphthoyl)-(.gamma.-Benzyl ester)-R-Glutamyl-bis(carboethoxymethyl)amide
EDCI (96 mg, 0.50 mmol) was added to a solution of 2-naphthoic acid (87 mg, 0.50 mmol), the product of example 103 (200 mg, 0.45 mmol) and TEA (70 .mu.L, 0.5 mmol) in 5 mL CH.sub.2 Cl.sub.2 at room temperature overnight. After evaporation of the solvent, the residue was dissolved in EtOAc and extracted with 0.1 M citric acid(3x) and water(3x). The EtOAc was then dried over MgSO.sub.4, filtered and the solvent evaporated in vacuo. The residue was chromatographed on silica gel eluted with 4:1 to 2:1 step gradient to give 193 mg, 0.34 mmol (76% yield) as an oil. [.alpha.].sub.D =+27.7.degree. (c=0.73, MeOH). MS(CI) m/e 563 (m+H).sup.+ 374. .sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta. 1.27(dt,J=1,7 Hz,6H), 1.92-2.03(m,1 H), 2.27-2.38(m,1H), 2.50(dt,J=6,17 Hz,1H), 2.59-2.70(m,1H), 4.10(d,J=17 Hz,1H), 4.15-4.23(m,4H), 4.30(d,J=17 Hz,1H), 4.45(d,J=l1Hz,1H), 4.50(d,J=17 Hz,1H), 5.11(d,J=l1Hz,1H), 5.15(d,J=l1Hz,1H), 5.25-5.32(m,1H), 7.22(d,J=8 Hz,1H), 7.29-7.35(m,5H), 7.51-7.60(m,2H), 7.82-7.93(m,4H), 8.32(s,1H). C,H,N analysis calculated for C.sub.31 H.sub.34 N.sub.2 O.sub.8 0.3 H.sub.2 O: C 65.55, H 6.14, N 4.93; found C 65.52, H 6.17, N 4.90.
EXAMPLE 105
N-(2'-Naphthoyl)-R-Glutamyl-bis(carboethoxymethyl)amide
The product of example 104 (100 mg, 0.18 mmol) was dissolved in 10 mL-EtOH. and then treated with 25 mg of 10% Pd/C (prewetted with solvent under N.sub.2) under 1 atmosphere H.sub.2 (balloon) overnight. The catalyst was removed by filtration and the solvent was evaporated in vacuo. The residue was redissolved in EtOH and filtered through a 5 .mu. filter. The filtrate was then mixed with -water, frozen and lyophilized to yield an oil 67 mg, (0.14 mmol, 79% yield). R.sub.f =0.35 (80:20:1 CHCl.sub.3 -MeOH-NH.sub.4 OH). [.alpha.].sub.D =+20.4.degree. (c=1.41, MeOH). MS(FAB+) m/e 473(m+H).sup.+, 495(m+Na).sup.+, 284, 256. .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.10(t,J.TM.7 Hz,3H), 1.17(t,J=7 Hz,3H), 1.83-2.08{m,2H), 2 32(bt,J=8 Hz,2H), 3.86-4.11(m,5H), 4.19(d,J=17 Hz,1H), 4.48(d.J=18 Hz.1H). 4.55(d,J=i8 Hz,1H), 4.43-5.01(m,1H), 7.57-7.65(m,ZH). 7.93-8.04(m,4H), 8.5Z(s,1H), 8.89(d,J=8 Hz,1H). C,H,N analysis calculated for C.sub.24 H28N2O.sub.8 0.6 H.sub.2 O: C 59.65, H 6.09, N 5.80; found C 59.69, H 5.70, N 5.70.
EXAMPLE 106
N-(3'-Quinolylcarbonyl)-(.gamma.-Benzyl ester)-R-Glutamyl-bis(carboethoxymethyl)amide
Quinoline-3-carboxylic acid (87 mg, 0.5 mmol) was coupled to the product of example 103 (200 mg, 0.45 mmol) as in example 104. Column chromatography on silica gel eluted with a 9:1 to 2:1 hexane-EtOAc step gradient yielded an oil 131 mg (0.23 mmol, 52% yield). [.alpha.].sub.D =+28.0.degree. (c=0.55, MeOH). MS(CI) m/e 564(m+H).sup.+ .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 1.23-1.32(m,6H), 1.92-2.03(m,1H), 2.28-2.38(m,1H), 2.51(dt,J=6,17 Hz,1H), 2.60-2.71(m,1H), 4.07-4.27(m,5H), 4.31(d,J=17 Hz,1H), 4.49(s,2H), 5.13(d,J=l1Hz,1H), 5.18(d,J=11Hz,1H), 5.25-5.32(m,1H), 7.28-7.38(m,6H), 7.62(dt,J=1,8 Hz,1H), 7.82(dt,J= 1,8 Hz,1H), 7.90(dd,J=1,8 Hz,1H), 8.15(d,J=8 Hz,1H), 8.58(d,J=2 Hz,1H), 9.32(d,J=2 Hz,1H). C,H,N analysis calculated for C.sub.30 H.sub.33 N.sub.3 O.sub.8 0.5 H.sub.2 O: C 62.93, H 5.99, N 7.94; found C 62.95, H 6.09, N 7.22.
EXAMPLE 107
N-(3'-Quinolylcarbonyl)-R-Glutamyl-bis(carboethoxymethyl)amide
The product of example 106 (70mg, 0.124 mmol) was treated with 20 mg of 10% Pd/C-and 1 atmosphere hydrogen gas in 7 mL EtOH. After 6 hours, the catalyst was filtered and the solvent concentrated, mixed with water and then frozen and lyophilized to yield 49 mg (0.10 mmol, 84% yield). mp=48.degree.-52.degree. C. [.alpha.].sub.D =+17.1.degree. (c=1.37, MeOH). MS(FAB+) m/e 474(m+H).sup.+ 496(m+Na)+.sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 1.12(t,J=7 Hz,3H), 1.17(t,J=7 Hz,3H), 1.83-2.10(m,2H), 2.34(bt,J=7 Hz,2H), 3.42-4.12(m,5H), 4.19(d,J=17 Hz,1H), 4.51(d,J=17 Hz,1H), 4.55(d,J=17 Hz,1H), 4.95-5.02(m,1H), 7.71(dt,J=1;8 Hz, 1H), 7.88(dt,J=1,8 Hz,1H), 8.10(d,J=8 Hz,2H), 8.88(d,J=2 Hz,1H), 9.18(d,J=7 Hz,1H), 9.28(d,J=2 Hz,1H). C,H,N analysis calculated for C.sub.23 H.sub.27 N.sub.3 O.sub.8 0.7 H.sub.2 O: C 56.83, H 5.89, N 8.65; found C 56.75, H 5.53, N 8.52.
EXAMPLE 108
N-(4',8'-Dihydroxyquinolyl-2-carbonyl)-(.gamma.-Benzyl ester)-R-Glutamyl-bis(carboethoxymethyl)amide
4,8-Dihydroxyquinoline-2-carboxylic acid (103 mg, 0.50 mmol), the product of example 103 (202 mg, 0.45 mmol), HOBt (68 mg, 0.5 mmol) and TEA (70 .mu.L, 0.5 mmol) were dissolved in 5 mL DMF and treated with EDCI (96 mg, 0.50 mmol) overnight. After evaporation of the solvent, the residue was extracted as in example 82. The residue was chromatographed on silica gel eluted with 1% to 6% EtOH in methylene choride step gradient to yield 193 mg (0.32 mmol, 72% yield) of nearly pure product. A second silica gel column eluted with 4:1 to 1:3 hexane-EtOAc step gradient yielded pure product. mp=79.degree.-82.degree. C. [.alpha.].sub.D =+23.9.degree. (c=1.6, MeOH). MS(CI) m/e 596(m+H).sup.+ 4.07, 379. .sup.1 H NMR(DMSO.sub.d6, 500 MHz) .delta. 1 18(t J=6 Hz 3H) 1.23(t,J=6 Hz 3H) 1.98-2.05(m,1H), 2.13-2.20(m,1H), 2.37-2.47(m,2H), 3.82-3.94(m,2H), 4.00(d,J=16 Hz,1H), 4.04(d,J=7 Hz,1H), 4.07(d,J=7 Hz,1H), 4.18(d,J=16 Hz,1H), 4.38(d,J=17 Hz,1H), 4.53(d,J=17 Hz,1H), 4.98-5.03(m,1H), 5.05(d,J=l1Hz,1H), 5.08(d,J=11HZ,1H), 7.08(d,J=8 Hz,1H), 7.28-7.36(m,4H), 7.42(t,J=7 Hz,1H), 7.52(s,1H), 7.54(d,J=7 Hz,1H), 7.71(d,J=8 Hz,1H), 10.04(s,1H), 11.75(bs,1H). C,H,N analysis calculated for C.sub.30 H.sub.33 N.sub.3 O.sub.10 0.5 H.sub.2 O: C 59.60, H 5.67, N 6.97; found C 59.69, H 5.56, N 6.88.
EXAMPLE 109
N-(4',8'-Dihydroxyquinolyl-2-carbonyl)-R-Glutamyl-bis(carboethoxymethyl)ami de
The product of example 108 (22 mg, 0.37 mmol) was treated with 5 mg of 10% Pd/C in 5 mL MeOH as in example 97. After 2 hours, the catalyst was filtered and the solvent was evaporated. The crude product was chromatographed on silica gel eluted with 70:30:1 EtOAc-hexanes-HOAc. [.alpha.].sub.D =+10.0.degree. (c=0.21, MeOH). MS(CI) m/e 506(m+H).sup.+ .sup.1 H NMR(DMSO.sub.d6, 300 MHz) .delta. 1.07-1.17(m,6H), 1.86-1.97(m,1H), 2.04-2.14(m,1H), 2.18-2.36(m,2H),.3 82-3.96(m,2H), 4.02-4.09(m,3H), 4.18(d,J=16 Hz,1H), 4.42(d,J=18 Hz,1H), 4.56(d,J=18 Hz,1H), 4.92-5.00(m,1H), 7.08(dd,J=1,7 Hz,1H), 7.39(t,J=8 Hz,1H), 7.46(s,1H), 7.55(dd,J= 1,8 Hz,1H), 9.68(d,J=8 Hz,1H), 12.0(bs,1H). C,H,N analysis calculated for C.sub.23 H.sub.27 N.sub.3 O.sub.10 1.5 H.sub.2 O: C 1.88, H 5.68, N 7.89; found C 52.32, H 5.51, N 7.33.
EXAMPLE 110
N-(2'-Indolylcarbonyl)-(N.sup..gamma. -n-pentylamide)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentyl amide
The product of example 4 (50 mg, 0.116 mmol) and n-amylamine (35 .mu.L, 0.30 mmol) were dissolved in 5 mL CH.sub.2 Cl.sub.2 and treated with EDCI (23 mg, 0.12 mmol). TEA (17 .mu.L, 0.12 mmol) and additional EDCI (23 mg) were added after 1 day. After 2 days, the solvent was evaporated and the residue in EtOAc was washed with 0.1% citric acid, water, dried and evaporated. The crude residue was chromatographed on silica gel eluted with a 4:1 to 1:1 hexane-EtOAc stepped gradient to yield 26.7 mg, 0.054 mmol (46%). mp=103.degree.-5.degree. C. R.sub.f =0.56(1:1 hexane-EtOAc). MS(CI) m/e 499(m+H).sup.+ 341. .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.92(t,J=7 Hz,3H), 1.23-1.38(m,12H), 1.49-1.60(m,6H), 1.83-1.95 (m,1H), 2.13-2.23(m,1H), 2.29-2.33(m,2H), 3.03(dt,J=7,14 Hz,1H), 3.12-3.47(m,4H), 3.63(dt,J=7,14 Hz,1H), 4.99(dt,J=2,9 Hz,1H), 6.73(bt,J=5 Hz,1H), 7.01(dd,J=1,2 Hz,1H), 7.15(dt,J=1,7 Hz,1H), 7.29(dt,J=1,7 Hz,1H), 7.42(dd,J=1,8 Hz,1H), 7.47(d,J=8 Hz,1H), 7.66(dd,J=1,7 Hz,1H), 9.12(s,1H). C,H,N analysis calculated for C.sub.29 H.sub.46 N.sub.4 O.sub.3 C 69.84, H 9.30, N 11.24; .found C 69.78, H 9.28, N 11.13.
EXAMPLE 111
N-(2'-Indolylcarbonyl)-[N.sup..alpha.-( 2'-phenylethylamide)]-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentyl amide
The product of example 4 (50 mg, 0.116 mmol) was reacted with .beta.-phenethylamine (38 .mu.L, 0.30 mmol) in the same manner as in example 110. The reaction yielded 36 mg, 0.068 mmol (58%). MS(CI) m/e 533(m+H).sup.+ 376, 348. .sup.1 H NMRCDCl.sub.3, 300 MHz) .delta. 0.85-0.92(m,6H), 1.25-1.36(m,8H), 1.48-1.65(m,4H), 1.85-1.96(m,1H), 2.13-2.32(m,3H), 2.82(t,J=8 Hz,2H), 3.02-3.22(m,2H), 3.37-3.65(m,4H), 5.02(dt,J=2,9 Hz,1H), 6.54(t,J=5 Hz,1H), 6.99(dd,J=1,2 Hz,1H), 7.12-7.32(m,8H), 7.40-7.46(m,2H), 7.68(dd,J=1,7 Hz,1H), 9.11(s,1H). C,H,N analysis calculated for C.sub.32 H.sub.44 N.sub.4 O.sub.3 0.3 H.sub.2 O: C 71.42, H 8.35, N 10.41; found C 71.46, H 8.32, N 10.43.
EXAMPLE 112
N-(4', 8'-Dihydroxyquinolyl-2'-carbonyl)-(.gamma.-benzyl ester)-R-Glu-di-n-pentylamide
To a solution of the hydrochloride of example 2 (1.0 grams, 2.42 mmol) in DMF (10mL) at 0.degree. C. were added NMM (0.55 mL, 5 mmol), HOBt (0.33 gram, 2.42 mmol), 4,8-dihydroxyquinoline-2-carboxylic acid (0.5 gram, 2.44 mmol) and DCC (0.55 grams, 2.6 mmol). The reaction was stirred overnight with warming to ambient temperature. The solvents were evaporated in vacuo and the resulting residue was dissolved in EtOAc. The dicyclohexylurea was filtered away, the filtrate was concentrated and the residue chromatographed on silica gel using CHCl.sub.3 and MeOH (18:1). The solvents were removed and the semisolid yellow residue dried under vacuum at ambient temperature. Product was isolated in 55% yield 0.75 grams. MS(CI) m/e 564(m+H).sup.+ .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.85-0.95(m,6H), 1.22-1.42(m,8H), 1.55-1.75(m,4H), 1.75-2.0(m,2H), 2.5-2.62(m,1 H), 2.65-2.8(m,1H), 3.05-3.20(m,1H), 3.30-3.52(m,2H), 3.64-3.78(m,1H), 5.0(m,1H), 5.2(s,2H), 6.95(m,2H), 7.15(m,7H), 7.6(d,J=9 Hz,1H), 8.1(bs,1H), 9:2(bs,1H). C,H,N analysis calculated for C.sub.32 H.sub.41 N.sub.3 O.sub.6 0.25 H.sub.2 O: C 67.64, H 7.36, N 7.40; found C 67.62, H 7.03, N 7.66.
EXAMPLE 113
N-(4',8'-Dihydroxyquinolyl-2'-carbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-penty amide
To a suspension of 0.3 grams 10% Pd/C in MeOH (15 mL) and cyclohexadiene (2 mL) under inert atmosphere (N.sub.2) was added via cannula solution of the benzyl ester of example 112 (0.4 grams, 0.71 mmol) in MeOH. The reaction mixture was stirred overnight at ambient temperature. The mixture was filtered through celite and washed several times with MeOH. The filtrate and washings were combined and concentrated in vacuo. The product was purified by flash chromatography using EtOAc/hexane and HOAc (70:30:1) as the elutant mixture. Removal of the volatiles in vacuo provided pure product in 60% yield (0.2 grams). MS(CI) m/e 474(m+H).sup.+ .sup.1 H NMR(DMSO.sub.d6,300 MHz) .delta. 0.75-0.88(m,6H), 1.1-1.35(m,8H), 1.4-1.65(m,4H), 1.95-2.10(m,2H), 2.35-2.42(m,2H), 3.1(m,1H), 3.2-3.4(m,3H), 5 0(m,1H), 7.10(d,J=9 Hz,1H), 7.42(t,J=10 Hz,1H), 7.49(bs,1H), 7.55(d,J=9 Hz,1H), 9.6(bd,J=9 Hz,1H), 10.1(bs,1H), 12.00(bs,1H). C,H,N analysis calculated for C.sub.25 H.sub.35 N.sub.3 O.sub.6 H.sub.2 O HOAc: C 58.79, H 7.49, N 7.62; found C 58.78, H 7.04, N 7.87.
EXAMPLE 114
N-(2'-Indolylcarbonyl)-R-Glu-N.sup..gamma. -methoxyamide -N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
The product of example 4 (50 mg, 0.116 mmol) was reacted with methoxyamine hydrochloride (24 mg, 0.29 mmol) in the same manner as example 110. The reaction yielded 35 mg, 0.076 mmol (66%). .sup.1 H NMR(CDCl.sub.3, 300 MHz) .delta. 0.89(t,J=7 Hz,6H), 1.23-1.37(m,8H), 1.48-1.62(m,4H), 1.85-1.94(m,1H), 2.20(bs,1H), 2.24(bs,2H), 3.00-3.08(m,1H), 3.10-3.21(m,1H), 3.32-3.43(m,1H), 3.62(dt,J=8,13 Hz,1H), 3.81(s,3H), 4.94(bt,J=8 Hz,1H), 7.02(s,1H), 7.15(dt,J=1,8 Hz,1H), 7.30(dt,J=1,7 Hz,1H), 7.42(d,J=8 Hz,1H), 7.62(bd,J=7 Hz,1H), 7.67(d,J=8 Hz,1H), 9.28(s,1H), 9.94(s, 1H).
EXAMPLE 115
N-Benzyl-(.gamma.-benzyl ester)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
The product of example 2 (413 mg, 1.0 mmol) was dissolved in 5 mL of dry ethanol and treated consecutively with TEA (139 .mu.L, 1.0 mmol), benzaldehyde (127 .mu.L, 1.25 mmol) and acetic acid (57 .mu.L, 1.0 mmol) each at 10 minute intervals. Then sodium cyanoborohydride (126 mg, 2.0 mmol) was added in 4 portions over 30 minutes. Bubbling commenced immediately and the reaction became opaque. The reaction was allowed to stir overnight. The reaction was then poured into ethyl acetate, extracted with 0.5 M sodium bicarbonate and then dried over MgSO.sub.4, filtered and the ethyl acetate evaporated in vacuo. The resultant oil was chromatographed on silica gel eluted with a 9:1 to 4:1 step gradient.
EXAMPLE 116
N-Benzyl-N-(3'-quinolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide
The product of example 115, quinoline-3-carboxylic acid and TEA were dissolved in methylene chloride and treated with BOPCl. The residue after evaporation of the solvent was extracted as in example 70 and then purified by column chromatography.
EXAMPLE 117
N-Methyl-N-(3'-quinolylcarbonyl)-(.gamma.-benzyl ester)-R-Glu-N.sup..alpha., N.sup..alpha. -di-n-pentylamide
The product of example 5 (100 mg, 0.176 mmol) was dissolved in 5 mL of DMF and then treated with lithium bis(trimethylsilyl) amide (170 .mu.L, 0.17 mmol, 1.0 M in THF) followed by methyl iodide (33 .mu.L, 0.035 mmol). After 2 hours, the solvents were evaporated and the residue was extracted with 0.1 M citric acid and 0.5 M sodium bicarbonate. Column chromatography on silica gel eluted with 9:1 to 2:1 hexane-ethyl acetate step gradient yielded 48 mg of product (0.089 mmol, 52%).
EXAMPLE 118
N-(3'-Quinolylcarbonyl)-R-Glu-N.sup..alpha.,N.sup..alpha. -di-n-pentylamide Dicyclohexylamine salt
The product of example 15 (880 mg, 2 mmol) was dissolved in 10 mL of methylene chloride and then treated with dicyclohexylamine (4 mL, 20 mmol- After complete mixing, hexane was added (200 mL) and a solid formed. The solid was collected and rinsed with fresh hexanes and then dried in vacuo. mp=129.degree.-131.degree. C. MS(CI m/e 442(m+H).sup.+ 398 1H NMR(DMSO.sub.d6 ,300 MHz) .delta. 0.83(d,J=7 Hz,3H), 0.88(d,J=5 Hz,3H), 1.12-1.32(m,20H), 1.42-1.70(m,8H), 1.82-1.92(m,6H), 2.26-2.32(m,2H), 2.67-2.74(m,2H), 3.02(quintet,J=7 Hz,1H), 3.23-3.35(m,1H), 3.42-3.60(m,2H), 4.82-4.90(m,1H), 7.70(dt,J=1,7 Hz,1H), 7.86(dt,J=1,7 Hz,1H), 8.09(d,J=8 Hz,2H), 8.90{ d,J=2 Hz,1H), 9.31(d,J=2 Hz,1H), 9.50(d,J=7 Hz,1H). C,H,N analysis calculated for C.sub.37 H.sub.58 N.sub.4 O.sub.4 C 71.34, H 9.39, N 9.00; found C 71.50, H 9.40, N 8.91.
The compounds of Formula I antagonize CCK which makes the compounds useful in the treatment and prevention of disease states wherein CCK or gastrin may be involved, for example, gastrointestinal disorders such as irritable bowel syndrome, ulcers, excess pancreatic or gastric secretion, hyperinsulinemia, acute pancreatitis, cancers of the gall bladder and pancreas, motility disorders, pain (potentiation of opiate analgesia), central nervous system disorders caused by CCK's interaction with dopamine such as neuroleptic disorders, tardive dyskinesia, Parkinson's disease, psychosis or Gilles de la Tourette Syndrome, disorders of appetite regulatory systems, Zollinger Ellison syndrome, and central G cell hyperplasia.
The ability of the compounds of Formula I to interact with CCK receptors and to antagonize CCK can be demonstrated in vitro using the following protocols.
CCK.sub.8 [Asp Tyr(SO.sub.3 H) Met-Gly Trp Met-Asp-Phe-NH.sub.2 ]was purchased from Beckman Instruments (Palo Alto, Calif.) and Peptide International (Louisville, Ky.). Chymostatin, L Try Gly, puromycin, bestatin, EGTA, HEPES and BSA were. purchased from Sigma Chemical Co. (St. Louis, Mo.). [.sup.125 I]BH-CCK.sub.8 (specific activity, 2200 Ci/mmol) and Aquasol 2 scintillation cocktail were obtained from New England Nuclear (Boston, Mass.). Male guinea pigs, 250 to 325 q, were obtained from Scientific Small Animal Laboratory and Farm (Arlington Heights, Ill.). Collagenase, 300 units per mg, was purchased from Worthington (Frehold, N.J.)
Protocol For Radioligand Binding Experiments
1. Guinea Cerebral Cortical and Pancreatic Membrane Preparations
Cortical and pancreatic membrances were prepared as described (Lin and Miller; J. Pharmacol. Exp. Ther. 232, 775-780, 1985). In brief, cortex and pancreas were removed and rinsed with ice cold saline. Visible fat and connective tissues were removed from the pancreas. Tissues were weighed and homogenized separately in approximately 25 mL of ice-cold 50 mM Tris HCl buffer, pH 7.4 at 4.degree. C., with a Brinkman Polytron for 30 sec, setting 7. The homogenates were centrifuged for 10 min at 1075 x g and pellets were discarded. The supernatants were saved and centrifuged at 38,730 x g for 20 min. The resultant pellets were rehomogenized in 25 mL of 50 mM Tris HCl buffer with a Teflon glass homogenizer, 5 up and down strokes. The homogenates were centrifuged again at 38,730 x g for 20 min. Pellets were then resuspended in 20 mM HEPES, containing 1 mM EGTA, 118 mM NaCl, 4.7 mM KCl, 5 mM MgCl.sub.2, 100 microM bestatin, 3 microM phosphoramidon, pH 7.4 at 22.degree. C, with a Teflon-glass homogenizer, 15 up and down strokes. Resuspension volume for the cortex was 15-18 mL per gm of original wet weight and 60 mL per gm for the pancreas.
2. Incubation Conditions
[.sup.125 I]Bolton Hunter CCK , and test compounds were diluted with HEPES-EGTA-salt buffer (see above) containing 0.5% bovine serum albumin (BSA). To 1 mL Skatron polystyrene tubes were added 25 microliters of test compounds, 25 microliters of [.sup.125 I]BH-CCK.sub.8, and 200 microliters of membrane suspension. the final BSA concentration was 0.1%. The cortical tissues were incubated at 30.degree. C. for 150 min and pancreatic tissues were incubated at 37.degree. C. for 30 min. Incubations were terminated by filtration using Skatron Cell Harvester and SS32 microfiber filter mats. The specific binding of [125I]BH-CCK.sub.8, defined as the difference between binding in the absence and presence of 1 microM CCK.sub.8, was 85-90% of total binding in cortex and 90-95% in pancreas. IC.sub.50 's were determined from the Hill analysis. The results of these binding assays are shown in Table I.
Protocol for Amylase
This assay was performed using the modified protocol of Lin et al., J. Pharmacol. Exp. Ther. 236, 729-734, 1986.
1. Guinea Pig Acini Preparation
Guinea pig acini were prepared by the method of Bruzzone et al. (Biochem. J. 226, 621-624, 1985) as follows. Pancreas was dissected out and connective tissues and blood vessels were removed. The pancreas was cut into small pieces (2mm) by a scissor and placed in a 15 mL conical plastic tube containing 2.5 mL of Krebs-Ringer HEPES (KRH) buffer plus 400 units per mL of collagenase. The composition of the KRH buffer was: HEPES, 12.5 mM: NaCI, 118 mM; KCl, 4.8 mM; CaCl.sub.2, mM; KH.sub.2 PO.sub.4, 1.2 mM; MgSO.sub.4, 1.2 mM; NaHCO.sub.3, 5 mM; glucose, 10 mM, pH 7.4. The buffer was supplemented with 1% MEM vitamins, 1% MEM amino acids and 0.001% aprotinin. The tube was shaken by hand until the suspension appeared homogeneous, usually 5 to 6 min. 5 mL of the KRH, without collagenase and with 0.1% BSA, were added and the tube was centrifuged at 50 x g for 35 sec. The supernatant was discarded and 6 mL of the KRH were added to the cell pellet. Cells were triturated by a glass pipet and centrifuged at 50 x g for 35 sec. This wash procedure was repeated once. The cell pellet from the last centrifugation step was then resuspended in 15 mL of KRH containing 0.1% BSA. The contents were filtered through a dual nylon mesh, size 275 and 75 micrometers. The filtrate, containing the acini, were centrifuged at 50 x g for 3 min. The acini were then resuspended in 5 mL of KRH BSA buffer for 30 min at 37.degree. C., under 100% 0.sub.2, with a change of fresh buffer at 15 min.
2. Amylase Assay
After the 30 min incubation time, the acini were resuspended in 100 volumes of KRH-BSA buffer, containing 3 microM phosphoramidon and 100 microM bestatin. While stirring, 400 microliters of acini were added to 1.5 mL microcentrifuge tubes containing 50 microliters of CCK.sub.8, buffer, or test compounds. The final assay volume was 500 microliters. Tubes were vortexed and placed in a 37.degree. C water bath, under 100% O.sub.2, for 30 min. Afterward, tubes were centrifuged at 10,000 g for 1 min. Amylase activity in the supernatant and the cell pellet were separately determined after appropriate dilutions in 0.1% Triton X 100, 10 mM NaH.sub.2 PO.sub.4, pH 7.4 by Abbott Amylase A gent test using the Abbott Bichromatic Analyzer 200. The reference concentration for CCK.sub.8 in determining the IC.sub.50 's of the compounds of Formula I was 3.times.10.sup.-10 M. The results of this assay are shown in Table 2.
The results of the assays indicate that the compounds of the invention inhibit specific [.sup.125 I]-BH-CCK-8 receptor binding in the concentration range of 10.sup.-9 to 10.sup.-6 M.
The ability of the compounds of Formula I to interact with CCK receptors and to antagonize CCK can be demonstrated in vivo using the following protocol.
Inhibition Of CCK Induced Gastric Emptying
Three fasted mice were dosed (p.o.) with the test compound. CCK.sub.8 (8micrograms/kg s.c.) was administered within 60minutes and charcoal meal (0.1 mL of 10% suspension) was given orally 5 minutes later. The animals were sacrificed within an additional 5 minutes.
Gastric emptying, defined as the presence of charcoal within the intestine beyond the pyloric sphincter, is inhibited by CCK.sub.8 Gastric emptying observed in more than 1 or 3 mice (greater than 1) indicates antagonism of CCK.sub.8.
Gastric emptying was observed with a dose of 100 mg/kg of the compound of example 118 in three out of three mice.
The ability of the compounds of Formula I to antagonize CCK induced hyperinsulinemia can be demonstrated in vivo using the following protocol.
Measurement of Plasma Insulin Level Following Treatment with CCK.sub.8 and a Compound of Formula I
Male mice, 20-30 g, were used in all experiments. The animals were fed with laboratory lab chow and water ad libitum. The compound of Formula I (1 to 100 mg/kg in 0.2 mL of 0.9% saline) was administered i.p. Ten minutes later CCK.sub.8 (0.2 to 200 nmole/kg in 0.2 mL of 0.9% saline) or saline was injected into the tail vein. Two minutes later the animals were sacrificed and blood was collected into 1.5 mL heparinized polypropylene tubes. The tubes were centrifuged at 10,000 x g for 2 min. Insulin levels were determined in the supernatant (plasma) by an RIA method using kits from Radioassay Systems Laboratory (Carson, Calif.) or Novo Biolabs (Mass.).
The compounds of the present invention can be used in the form of salts derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Also, the basic nitrogen-containing groups can be guaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.
The pharmaceutically acceptable salts of the present invention can be synthesized from the compounds of Formula I which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt forming inorganic or organic acid or base in a suitable solvent or various combinations of solvents.
Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium or with organic bases.
The pharmaceutically acceptable salts of the acid of Formula I are also readily prepared by conventional procedures such as treating an acid of Formula I with an appropriate amount of a base, such as an alkali or alkaline earth metal hydroxide e.g. sodium, potassium, lithium, calcium, or magnesium, or an organic base such as an amine, e.g., dibenzylethylenediamine, cyclohexylamine, dicyclohexylamine, trimethylamine, piperidine, pyrrolidine, benzylamine and the like, or a guaternary ammonium hydroxide such as tetramethylammonium hydroxide and the like.
When a compound of Formula I is used as an antagonist of CCK or gastrin in a human subject, the total daily dose administered in single or divided doses may be in amounts, for example, from 0.001 to 1000 mg a day and more usually 1 to 1000 mg. Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dose.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular treatment and the particular mode of administration.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
The compounds of the present invention may be administered orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
Injectable preparations, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable prepartion may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In additon, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or, diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsion, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
The foregoing is merely illustrative of the invention and is not intended to limit the invention to the disclosed compounds. Variations and changes which are obvious to one skilled in the art are intended to be within the scope and nature of the invention which are defined in the appended claims.
Claims
What is claimed is:
1. A compound of the formula: ##STR7## wherein R.sub.1 is --(CH.sub.2).sub.m C(O)OR.sub.4 wherein m is 1 to 4 and R.sub.4 is loweralkyl, loweralkenyl, loweralkynyl, aryl, substituted aryl, arylalkyl or (substituted aryl)alkyl; R.sub.2 is loweralkyl, lowralkenyl, aryl, substituted aryl, arylalkyl, (substituted aryl)alkyl or --(CH.sub.2).sub.m cycloalkyl wherein m is independently selected from 0,1,2,3 and 4; R.sub.3 is --OR.sub.4 wherein R.sub.4 is independently selected from loweralkyl, loweralkenyl, loweralkynyl, aryl, substituted aryl, arylalkyl and (substituted aryl)alkyl; wherein R.sub.5 is hydrogen, loweralkyl, cycloalkyl, loweralkenyl, loweralkynyl, hydroxy, halogen, trihalomethyl, nitro, --CN, --NH.sub.2, thioalkoxy, --NHOH, carboxy, carboalkoxy or carboxamide; and n is 1 to 3; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1 wherein R.sub.1 is --(CH.sub.2).sub.m C(O)OR.sub.4 wherein m is 1 to 4 and R.sub.4 is loweralkyl; R.sub.2 is arylaklyl; R.sub.3 is --OR.sub.4 wherein R.sub.4 is arylalkyl; R.sub.5 is hydroxy and n is 1 to 3.
3. The compound of claim 2 wherein m is 1; R.sub.1 is --CH.sub.2 C(O)OEt; R.sub.4 is --O(benzyl); R.sub.2 is benzyl and n is 2.
4. The compound having the formula: ##STR8##
5. A pharmaceutical composition useful for antagonizing CCK comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of claim 1.
6. The composition of claim 5 wherein the compound is ##STR9##
7. A method for antagonizing CCK comprising administering to a human or animal in need of such treatment a therapeutically effective amount of a compound of claim 1.
8. The method of claim 7 wherein the compound is: ##STR10##
Patent Citations (9)
Non-Patent Literature (4)
- F. Makovec et al., Arzneim.-Forsch./Drug Res. 35(II), 1048 (1985) "New Glutaramic Acid Derivatives with Potent Competitive and Specific Cholecystokinin-antagonistic Activity".
- F. Makovec et al., Arzneim-Forsch./Drug Res. 36(I), 98 (1986) "Differentiation of Central & Peripheral Cholecystokinin Receptors by New Glutaramic Acid Derivatives with Cholecystokinin-antagonistic Activity".
- J. Regan et al., Eur. J. Pharmacol. 144, 241 (1987) "Fasting and L-364,718 Prevent Cholecystokinin-induced Elevators of Plasma Insulin Levels".
- L. Rossetti et al., Diabetes 36, 1212 (1987) "Physiological Role of Cholecystokinin in Meal.varies.Induced Insulin Secretion in Conscious Rats-Studies with L364,718, A Specific Inhibitor of CCK-Receptor Binding".