Experimental section
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Esperimental Section.
Matherials and Methods.
Melting points were determined using a Reichert Kofler hot-stage apparatus and are uncorrected. Rutine nuclear magnetic resonance spectra were recorded in DMSOd6 solution on a Vrian Gemini 200 spectrometer operating at 200 MHz. Optcital rotatory powers (α) were determined using a Perkin Elmer model 343 polarimeter. Evaporation was performed in vacuo (rotary evaporator). Analytical TLC was carried out on Merck 0.2 mm precoated silica gel aluminium sheets (60 F-254). Combustion analyses on target compounds were performed by our Analytical Laboratory in Pisa. All compounds showed > 95% purity.
All reagent used were obtained from commercial sources. All solvents were of an analytical grade.
Chemestry.
Procedure for the synthesis of 2-substituted-(1H-indol-3-yl)glyoxylyl chloride derivatives (4-6, 11, 22).
Oxalyl chloride (0.31 mL, 3.6 mmol) was added dropwise, at 0°C to a well-stirred mixture of the appropriate indole 1, 2, 3, 10 or 21, commercially avaiable (indole 1, methylindole 2, 2-phenylindole 3, 5-nitroindole 14) or obtained following experimental protocols reported in
Scheme 3 (2-(4-chlorophenyl)-1H-indole) 21 (3.0 mmol) in freshly distilled diethyl ether (10
mL). The mixture was mainteined at room temperature for 2-4 h (TLC analysis). The generated precipitates were collected by vacuum filtration and were directly used in the subsequent reaction.
Procedure for the synthesis of N-(4-chlorophenyl)-substituted-(1H-indol-3yl) 2-oxoacetamide (7, 8, 9)
A solution of 4-chloroaniline (0.130 g, 2.24 mmol) in 5 mL of dry toluene was added dropwise to a stirred suspension, cooled at 0°C, of the 2-substituted-(indol-3-yl)glyoxylyl chloride derivatives 4-6 (2.0 mmol), in 50 mL of the same solvent, followed by the addition of a solution of triethylamine (0.34 mL, 2.4 mmol). The reaction mixtures were left under stirring for 12-24 h (TLC analysis) at room temperature and then filtered. The collected precipitates were washed with a NaHCO3 5% aqueous solution and collected again to give a first portion of crude
products. The toluene solution was instead removed under reduced pressure, and the residues obtained were extracted with CH2Cl2 and purified by washing with 1) a solution of NaHCO3 dil.
5%; 2) H2O; 3) HCl dil. 10%; and finally 4) H2O. After drying with MgSO4, the dichloromethane
solution was evaporated to dryness to yield an additional amount of crude products. Products 7-9 were isolated directly in a pure state.
mp not reported).
N-(4-chlorophenyl)-2-(2-methyl-1H-indol-3-yl)-2-oxoacetamide 8. Yield 78%, mp 167-169 °C
1H NMR (200 MHz, DMSO-d6, ppm): 2.56 (s, 1H, CH3); 7.09-7.16 (m, 2H, Ar-H); 7.40 (t, 3H,
Ar-H, J = 7.7 Hz); 7.76 (d, 2H, Ar-H, J = 8.6 Hz); 7.81-7.86 (m, 1H, Ar-H); 10.01 (bs, exch. D2O, 1H, NH); 12.55 (bs, exch. D2O, 1H, NH). Anal. Calcd. for C17H13ClN2O2: C, 65.29; H, 4.19;
Cl, 11.34; N, 8.96; O, 10. Found: C, 64.96; H, 4.02; Cl, 11.22; N, 8.67; O, 9,88.
N-(4-chlorophenyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 9. Yield 78%, mp 233-235 °C
1H NMR (200 MHz, DMSO-d6, ppm): 7.15-7.30 (m, 9H, Ar-H); 7.50-7.54 (m, 3H, Ar-H); 7.60
(bs, exch. D2O, 1H, NH); 8.07-8.11 (m, 1H, Ar-H); 10.64 (bs, exch. D2O, 1H, NH). Anal. Calcd.
for C22H15ClN2O2: C, 70.50; H, 4.03; Cl, 9.46; N, 7.47; O, 8.54. Found: C, 70.31; H, 3.70; Cl,
9.30; N, 7.37; O, 8.38.
General Procedure for the Synthesis of N-L-Glycine-L-Phenylalanine ethylester-2-indol-glyoxylamide derivatives (13-14).
A solution of L-Glycine-L-Phenylalanine-ethylester 10 (0.685 g, 2.24 mmol) in 5 mL of dry toluene was added dropwise to a stirred suspension, cooled at 0°C, of the 5-substituted indolylglyoxylyl chloride derivatives 4,12 (2.0 mmol), in 15 mL of the same solvent, followed by the addition of a solution of triethylamine (0.34 mL, 2.4 mmol). The reaction mixtures were left under stirring for 12-24 h at room temperature and then filtered (TLC analysis). The collected precipitates were washed with a NaHCO3 5% aqueous solution and collected again to
give a first portion of crude products. The toluene solution was instead removed under reduced pressure, and the residues obtained were extracted with CH2Cl2 and purified by washing with 1)
a solution of NaHCO3 dil. 5%; 2) H2O; 3) HCl dil. 10%; and finally 4) H2O. After drying with
MgSO4, the dichloromethane solution was evaporated to dryness to yield an additional amount of
crude products. Products 13 and 14 were purified by flash-chromatography (AcOEt/Ether petrol 60°-80° C= 4/6 as eluent).
N-[(1H-indol-3-yl)glyoxylyl]-glycylphenylalanine ethyl ester 13. Yield 70%, mp = 145-147 °C,
lit ref n36 mp = 137-139 °C
N-[(5-nitro-1H-indol-3-yl)glyoxylyl]-glycylphenylalanine ethyl ester 14. Yield 60%, mp =
200-207 °C, lif ref n36 mp = 211-212 °C
Procedure for the synthesis of N-Boc-L-Leucine-L-Phenylalanine-ethylester (17).
N-Boc-L-Leu 15 (0.208 g, 0.9 mmol) was solubilized in dry DMF (10 mL) in a nitrogen atmosphere; N,N’-carbonyldiimidazole (0.146 g, 0.9 mmol) was added and the solution was left to stir for 2h at room temperature. Then L-Phenylalanine ethyl ester hydrochloride (16) (0.413 g, 1.8 mmol) in 5 mL of the same solvent was added dropwise. The product formed quantitatively within 4-5 h. The solvent was evaporated under reduce pressure and the residue was triturated with saturated aqueous NaHCO3 solution, washed with water and filtered. The product was
isolated directly in a pure state.
N-Boc-L-Leucine-L-Phenylalanine-ethylester 17. Yield 70%, mp 78-80 °C,= -19 1H NMR (200
MHz, DMSO-d6, ppm): 0.79-0.84 (m, 6H, 2CH3); 1.07 ( t, 3H, CH3, J = 6.3 Hz); 1.34-1.55 (m,
12H, 3CH3, CH, CH2); 2.89-2.98 (m, 2H,CH2); 3.97-4.03 (m, 3H, CH, CH2); 4.40-4.46 (m, 1H,
CH); 6.81 (d, exch. D2O, 1H, NH, J = 7 Hz); 7.21 (s, 5H, Ar-H); 8.13 (d, exch. D2O, 1H, NH, J =
7.4 Hz). Anal. Calcd. for C22H34N2O5: C, 65.00; H, 8.43; N, 6.89; O, 19.68. Found: C, 64.82; H,
8.22; N, 6.67; O, 19.43.
Procedure for the synthesis of L-Leucine-L-Phenylalanine-ethylester (18).
Trifluoroacetic acid (2 mL, 20 mmol) was added to a stirred solution of derivative 17 (0.122 g, 0.3 mmol) in 10 mL of CH2Cl2. The mixture was stirred at room temperature for 6 h. Then the
reaction mixture was concentrated under reduce pressure. The resulting solid residue was taken up with water, and cooled in an ice bath. Then K2CO3 was added until pH = 10. The reaction
eliminated under vacuum and the product was isolated directly in a pure state.
L-Leucine-L-Phenylalanine-ethylester 18. Yield 50%, = -7; mp 268-270 °C(lit.ref. n37: mp not
reported).
Procedure for the Synthesis of 2-(4-chlorophenyl)-1H-indole (21).
Compound 21 was prepared in accordance with reported procedures33.
2-(4-chlorophenyl)-1H-indole 21. Yield 70%, mp 168-170 °C (toluene) 1H NMR (200 MHz, DMSO-d6, ppm): 6.93-7.11 (m, 3H, Ar-H); 7.40 (d, 1H, Ar-H, J = 7.6 Hz); 7.51 (d, 3H, Ar-H, J =
6 Hz); 7.86 (d, 2H, Ar-H, J = 6.6 Hz); 11.6 (bs, exch. D2O, 1H, NH). Anal. Calcd. for C14H10ClN:
C, 73.85; H, 4.43; Cl, 15.57; N, 6.15. Found: C, 73.66; H, 4.57; Cl, 15.71; N, 6.38.
General procedure for the Synthesis of N-[(2-substituted indol-3-yl)glyoxylyl]-leucinephenylalanine ethyl ester (23-24).
A solution of L-Leucine-L-Phenylalanine-ethylester 18 (0.685 g, 2.24 mmol) in 5 mL of dry toluene was added dropwise to a stirred suspension, cooled at 0°C, of the 2-substituted-(indol-3-yl)glyoxylyl chloride derivatives 6 and 22 (2.0 mmol), in 15 mL of the same solvent, followed by the addition of a solution of triethylamine (0.34 mL, 2.4 mmol). The reaction mixtures were left under stirring for 12-24 h (TLC analysis) at room temperature and then filtered. The collected precipitates were washed with a NaHCO3 5% aqueous solution and collected again to give a first
portion of crude products. The toluene solution was instead removed under reduced pressure, and the residues obtained were extracted with CH2Cl2 and purified by washing with 1) a solution of NaHCO3 dil. 5%; 2) H2O; 3) HCl dil. 10%; and finally 4) H2O. After drying with MgSO4, the
products. Products 23 and 24 were purified by flash-chromatography (AcOEt/Ether petrol 60-80° C = 4/6 as eluent).
N-[(2-phenylindol-3-yl)glyoxylyl]-leucinephenylalanine ethyl ester 23. Yield 64%, mp 86-88 °C,
= -20 1H NMR (200 MHz, DMSO-d6, ppm): 0.75-0.82 (m, 6H, 2CH3); 1.01-1.32 (m, 6H, CH,
CH2, CH3); 2.91-2.96 (m, 2H, CH2); 3.92-4.03 (m, 3H, CH2, CH); 4.41-4.47 (m, 1H, CH);
7.18-7.23 (m, 5H, Ar-H); 7.41-7.57 (m, 7H, Ar-H); 7.99 (d, 1H, Ar-H, J = 7.4 Hz); 8.39 (d, exch. D2O,
1H, NH, J = 7.4 Hz); 8.57 (d, exch. D2O, 1H, NH, J = 8.6 Hz); 12.34 (bs, exch. D2O, 1H, NH).
Anal. Calcd. for C33H35N3O5: C, 71.59; H, 6.37; N, 7.59; O, 14.45. Found: C, 71.81; H, 6.11; N,
7.42; O, 14.52.
N-[2-(4’-chlorophenylindol-3-yl)glyoxyly]-leucinephenylalanine ethyl ester 24. Yield 62%, mp
77-80°C, = -40 1H NMR (200 MHz, DMSO-d6, ppm): 0.78-0.86 (m, 6H, 2CH3); 1.01-1.13 (m,
3H, CH3); 1.23-1.42 (m, 3H, CH, CH2); 2.85-2.99 (m, 2H, CH2); 3.93-4.47 (m, 1H, CH);
4.33-4.47 ( m, 1H, CH); 7.18-7.24 (m, 8H, Ar-H); 7.34 (d, 2H, Ar-H, J = 8.6 Hz); 7.58-7.64 (m, 3H, Ar-H); 8.02 (d, 1H, Ar-H, J = 7.2 Hz); 8.44 (d, exch. D2O, 1H, NH, J = 6.8 Hz); 8.61 (d, exch
D2O, 1H, NH, J = 8.6 Hz); 12.42 (bs, exch. D2O, 1H, NH). Anal. Calcd. for C33H34ClN3O5: C,
67.40; H, 5.83; Cl, 6.03; N, 7.15; O, 13.60. Found: C, 67.30; H, 6.03; Cl, 6.31; N, 6.95; O, 13.30.
Procedure for the Synthesis of N-[(2-phenylindol-3-yl)glyoxylyl]-leucinephenylalanine (25).
Lithium hydroxide monohydrate (0.007 g, 0.3 mmol) was added to a suspension of N-[(2-phenylindol-3-yl)glyoxylyl]-leucinephenylalanine ethyl ester 23 (0.277 g, 0.5 mmol) in 20 mL of
a MeOH/H2O (3:1) solution. The mixture was stirred under reflux at 80 °C overnight (TLC
analysis). Subsequently, the solid precipitate was eliminate through vacuum filtration, and the solution was acidified with HCl dil. 10% to pH 5. The acid precipitated in the solution was collected by filtration. Any further purification did not needed.
1H NMR (200 MHz, DMSO-d6, ppm): 0.71-0.83 (m, 6H, 2CH3); 1.23-1.45 (m, 3H, CH, CH2);
2.89-3.03 (m, 2H, CH2); 4.12-4.14 (m, 1H, CH); 4.43-4.48 (m, 1H, CH); 7.19-7.59 (m, 13H,
Ar-H); 8.01 (d, 1H, Ar-H, J = 7.4 Hz); 8.20 (d, exch. D2O, 1H, NH, J = 7.8 Hz); 8.59 (d, exch. D2O,
1H, NH, J = 8.6 Hz); 12.36 (bs, exch. D2O, 1H, NH); 12.78 (bs, exch. D2O, 1H, COOH). Anal.
Calcd. for C31H31N3O5: C, 70.84; H, 5.94; N, 7.99; O, 15.22. Found: C, 70.71; H, 6.05; N, 7.53;
