T inact
Scheme 3.1. Synthetic procedure of triazolyl decorated donor/acceptor tripeptides
N- H (Ala)
3.9. Experimental Section
3.9.2. Synthesis and Characterization
Synthesis of L-Alanine methyl ester (3.41): To a degassed and cold solution of L- Alanine in dry methanol, thionyl chloride (1.5 equivalent) was added at 0 oC. After 15
minutes the reaction mixture was refluxed at 75-80 oC overnight.
The solvent was evaporated in vacuum. Material was washed
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with toluene three times to remove excess thionyl chloride and the amine salt of methyl ester -L-alanine was isolated, washed with ether twice, dried and used for the next step.
Synthesis of methyl 2-(2-(tert-butoxycarbonyl)-3 hydroxypropanamido) propanoate (3.42): To a solution N-protected L-serine 3.40, (378 mg, 1.84 mmol) in 3:1 mixture of dry CH2Cl2 and DMF , 1-[3-dimethyl amino propyl]-3-ehtylcarbo- diimide hydrochloride (EDC.HCl) (422 mg, 2.21 mmol) and HOBT (373 mg, 2.21mmol) were added and the reaction mixture was stirred for 1h at 0 oC. Then the amine salt of methyl ester -L-alanine (257 mg, 1.84 mmol) was added followed by diisopropyl-ethylamine (DIPEA) (0.8 ml, 4.41 mmol). The reaction mixture was stirred for another 18-20 h at 0 oC to room temperature. Then solvent was dried by rotary evaporator, after which it was partitioned between EtOAc and aqueous NaHCO3 solution (50 ml each). The organic layer was washed with brine solution.
Pure product 3.42 (300 mg, 1.03 mmol) was isolated in pure form by column chromatography (Si-gel, PE : EtOAc = 1:1) as colourless oil. Yield 62%; 1H NMR (CDCl3; 400 MHz) δ 1.38 (3H, d, J = 7.2 Hz); 1.42 (9H, s); 2.303 (1H, m); 3.62 (1H, q, J = 5.6, 10.8 Hz); 3.72 (3H, s); 4.02 (1H, dd, J = 3.6, 12.4 Hz); 4.16 (1H, m); 4.52 (1H, m); 5.5 (1H, d, J = 8.0 Hz); 7.02 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3; 100 MHz); δ 17.1, 28.1, 48.3, 52.6, 55.7, 62.7, 80.34, 156.0, 171.4, 173.6.
Synthesis of methyl 2-(2-(tert-butoxycarbonyl)-3(methylsulfonyloxy) propanamido) propanoate (3.43): Dipeptide 3.42 (190 mg, 0.61 mmol) was taken in dry DCM and was cooled to 0 oC. Methane sulphonyl chloride (61 µl, 0.72 mmol) and triethyl amine (109 µl, 0 .72 mmol) were added to the reaction mixture and stirred for an hour till the total reactant was consumed. After that reaction mixture was poured into water and CH2Cl2 (20 ml each).The organic layer was washed with water two times, dried over Na2SO4 and then evaporated. The pure compound 3.43, was then isolated by column chromatography (si-gel, PE:EA = 2:1) as brown oil (yield, 89%). 1H NMR (CDCl3; 400 MHz) δ 1.40 (3H, d, J
= 8 Hz); 1.44 (9H, s); 3.30 (3H, s); 3.73 (3H, s); 4.31 (1H, dd, J = 4.8 Hz, 10.4 Hz);
4.53 (2H, m); 4.61 (1H, dd, J = 4 Hz, 10.4 Hz); 5.37 (1H, d, J = 8 Hz); 6.94 (1H, d, 5.6 Hz).
Synthesis of methyl 2-(3-azido-2-(tert-butoxycarbonyl) propanamido) propanoate (3.44) : To a solution of the mesylate 3.43 (180 mg, 0.49 mmol) in dry DMF (6 ml), NaN3 (48.75 mg, 0.75 mmol) was added and stirrer for 18 h at 40 oC. The reaction mixture was partitioned between EtOAc and water (20 ml each). The organic layer was washed with brine solution, dried, filtered and then evaporated. The title compound 3.44 was isolated by column chromatography (Si-gel, PE:EA = 2:1), as light brown oil (yield, 78 %). IR (KBr) 3425, 2981, 2107, 1661, 1515, 1368, 1251, 1163, 1062, 859. 1H NMR (CDCl3; 400 MHz) δ 1.35 (3H, d, J = 7.2); 1.39 (1H,
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s); 3.49 (2H, dd, J = 2.8, 11.6 Hz); 3.69 (3H, s); 4.31(1H, m); 4.52 (1H, m); 5.52(1H, d, J = 5.2 Hz); 7.09 (1H, s); 13C NMR (CDCl3; 100 MHz); δ 18.2, 28.3, 48.4, 52.4, 52.6, 53.8, 80.8, 155.3, 169.2, 173.
General procedure for the synthesis triazolyl unnatural dipeptide: Using the azido derivative of dipeptide (Boc-NH-Ser.Ala-OMe) 3.44, we have prepared three different unnatural tri-peptide using Donor/Acceptor terminal alkynes.
General procedure for the synthesis triazolyl unnatural dipeptide (Boc-NH- Ser.Ala-OMe) by [3+2] cycloaddition reaction: The azido derivative of dipeptide (Boc-NH-Ser.Ala-OMe) 3.44 (1.0 equiv) was taken in 5:1 dry THF and water and degassed for 5 min with N2. Then, alkyne (1.1 equiv) was added and both the stirring and degassing were continued for the next 5 min. After that a 6 mol % sodium ascorbate and 1 mol% powderd CuSO4 were added. Then the reaction mixture was degassed and allowed to proceed for 18-20 h about 65 to 70 oC. After total consumption of the starting azide, the reaction mixture was evaporated completely and work up was done by EtOAc and NH4Cl solution. The organic layer was washed with brine, dried over Na2SO4.The title trizolyl derivative of dipeptides 3.47 and 3.48 were separated by column chromatography and characterized.
Synthesis of methyl 2-(2-(tert-butoxycarbonyl)-3-(4-(phenanthren-9-yl)-1H- 1,2,3-triazol-1-yl) propanamido) propanoate (3.47): Using general procedure, starting from 155 mg (0.49 mmol) of azido derivative of dipeptide 3.44 and 109 g (0.54 mmol) of 9- ethynylphenanthrene (3.45), 210 mg (0.41 mmol) of the title compound 3.47 was isolated as a light brown gummy material.
Yield 83%. 1H NMR (CDCl3; 400 MHz) δ =1.27 (3H, d, J = 6.8 Hz); 1.46 (9H, s); 3.69 (3H, s); 4.48 (1H, t, J = 7.6 Hz);
4.82 (2H, m); 5.03 (1H, m); 5.79 (1H, d, J = 7.60 Hz); 7.11 (1H, d, J = 6 Hz); 7.62 (4H, dd, J = 8.4, 24 Hz); 7.95 (1H, d, J
= 5.2 Hz); 8.26 (2H, d, J = 6.8 Hz); 8.32 (1H, d, J = 7.6 Hz); 8.71 (2H, dd, J = 8 Hz, 25.6 Hz); 13C NMR (CDCl3; 100 MHz); δ 17.7, 28.2, 48.4, 51.4, 52.4, 54.3, 80.7, 122.5, 122.9, 124.9, 126.1, 126.8, 128.4, 128.8, 129.9, 130.3, 130.6, 131.2, 146.5, 155.53, 169.0, 172.7.
Synthesis of methyl 2-(2-(tert-butoxycarbonyl)-3-(4-(4-cyanophenyl)-1H-1,2,3- triazol-1yl)propanamido) propanoate (3.48): Using general procedure, starting from 162 mg (0.51 mmol) of azido derivative of dipeptide 3.44 and 71 mg (0.56 mmol) of 4-ethynyl benzonitrile (3.46), 165 mg (0.37 mmol) of the title compound 3.48 was isolated as a colourless gummy material. Yield 73%. 1H NMR (CDCl3; 400 MHz) δ 1.24 (3H, d, J=7.2 Hz); 3.69 (3H, s); 4.43 (1H, m); 4.73 (2H, d, J
= 10.4 Hz); 4.93 (1H, m); 5.66 (1H, d, J = 5.2 Hz); 7.02 (1H, d, J = 6.4 Hz); 7.68 (2H, d, J = 7.6 Hz); 7.89 (2H, d, J = 7.2 Hz); 7.98 (1H, s);
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13C NMR (CDCl3; 100 MHz); δ 18.0, 28.3, 48.5, 51.4, 52.7, 54.3, 81.3, 111.7, 118.8, 123.1, 126.1, 132.9, 134.8, 146.0, 155.5, 168.6, 172.8.
General procedure for the deprotection of the methyl ester. To a solution of the respective methyl ester protected peptide in Water : THF = 1 : 10, lithium hydroxide (1.5 equivalent) was added at 0 oC. Then the reaction mixture was stirred about 2-3 hour until starting material was consumed. Reaction was monitored by TLC. After completion of the reaction, solvent dried by rotary evaporator. Then water (4-5 ml) was added to the reaction mixture and cooled to 0 oC. The dilute acetic acid was added to the reaction mixture to adjust pH ~ 3 to 4. The reaction mixture was extracted with CH2Cl2. The combined organic layers were dried over anhydrous Na2SO4. The hydrolysed compound was isolated by column chromatography (Si-gel, CHCl3:MeOH = 10:1). Yield was 96-97%.
Synthesis of 2-(2-(tert-butoxycarbonyl)-3-(4-(phenanthren-9-yl)-1H-1,2,3- triazol-1-yl)propanamido) propanamido) propanoic acid (3.49): Using general procedure of deprotection of the methyl ester, starting from 205 mg (0.40 mmol) of 3.47, 193 mg (0.38 mmol) of the title compound 3.49 was isolated as a white solid material and used for the next step without characterization. Yield 97%.
Synthesis of 2-(2-(tert-butoxycarbonyl)-3-(4-(4-cyanophenyl)-1H-1,2,3-triazol-1- yl) propanamido) propanoic acid (3.50): Using general procedure of deprotection of
the methyl ester, starting from 159 mg (0.36 mmol) of 3.44, 148 mg (0.35 mmol) of the title compound 3.50 was isolated as a white solid material and used for the next step without characterization. Yield 96%.
General procedure for the deprotection of the Boc-group. The respective protected amino acid or peptide was dissolved in CH2Cl2 and cooled to 0 oC. TFA (equal amount as the solvent) was added and the solution was allowed to warm to room temperature. After stirring at room temperature until starting material was consumed (TLC monitoring), the reaction mixture was evaporated in vacuo. The residual TFA was evaporated by titurating the mixture with dry toluene thrice, evaporated thrice and dried to afford the product in quantitative yield.
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Synthesis of 3.51 and 3.52 : Using the general procedure of Boc-deprotection, compound 2.74 ( 170 mg, 0.42 mmol) and 2.79 (200 mg, 0.42 mmol) were reacted.
The products 3.51 and 3.52 were obtained in quantitative yield and were used without further purification and characterization.
Synthesis of triazolyl donor/acceptor tripeptides: The target tripeptides 3.37-3.39 were synthesized following the general procedure of peptide coupling protocol as was discussed for the synthesis of dipeptides. The pure product was isolated by column chromatography (Si-gel, CHCl3:MeOH = 30:1).
Synthesis of tert-butyl 3-(4-(4-cyanophenyl)-1H-1,2,3-triazol-1-yl)-1-(1-(1- (methoxy(methyl)amino)-1-oxo-3-(4-(phenanthren-9-yl)-1H-1,2,3-triazol-1-
yl)propan-2-ylamino)-1-oxopropan-2-ylamino)-1- oxopropan-2-ylcarbamate (3.37): Using general procedure, starting from 144 mg (0.28 mmol) of 3.49 and 126 mg (0.31 mmol) of 3.52, 95 mg (0.12 mmol) of the title compound 3.37 was isolated as a gummy material, after washing with ether it converted to white solid. Yield 43%. IR (KBr) 3320, 2973, 2931, 2226, 1660, 1516, 1452, 1367, 1248, 1164, 1053, 849, 729, 556 cm-1. 1H NMR (d6-DMSO; 400 MHz) δ 1.1 (3H, d, J = 6.9 Hz); 1.17 (9H, s); 3.16 (3H, s); 3.75 (3H, s); 4.35 (1H, bs);4.46 (2H, d, J = 13.2 Hz); 4.7 (2H, d, J = 13.5 Hz); 4.81 (1H, bs); 5.33 (1H, bs); 6.99 (1H, d, J = 7.5 Hz); 7.65 (2H, d, J = 6.4 Hz); 7.72 (2H, d, J = 6.0 Hz); 7.87 (2H, m); 7.79 (1H, t, J = 4.5 Hz); 7.95 (1H, s); 8.06 (1H, t, J = 3.9 Hz); 8.25 (1H, d, J = 9.0 Hz);
8.44 (1H, d, J = 9.0 Hz); 8.36 (1H, d, J = 5.7 Hz); 8.44(1H, d, J = 9.0 Hz); 8.50 (1H, s); 8.56 (1H, s); 8.58 (1H, m); 8.84 (1H, d, J = 8.1 Hz); 8.93 (1H, d, J = 7.8 Hz); 13C NMR (CDCl3; 100MHz); δ 17.7, 28.1, 32.4, 36.3, 49.2, 50.3, 60.3, 61.8, 66.9, 80.4, 110.8, 118.7, 122.4, 122.9, 124.8, 125.5, 126.0, 126.4, 126.8, 128.2, 128.7, 129.8, 130.1, 130.5, 131.0, 132.3, 134.4, 145.2, 146.3, 156.1, 168.2, 169.0, 172.2. HRMS calcd for C41H44N11O6 ([M + H]+) 786.3471, found 786.3476. C41H43N11O6Na([M + Na]+) 809.3376, found 809.3374.
Synthesis of tert-butyl 1-(1-(1-(methoxy(methyl)amino)-1-oxo-3-(4- (phenanthren-9-yl)-1H-1,2,3-triazol-1-yl)propan-2-ylamino)-1-oxopropan-2-
ylamino)-1-oxo-3-(4-(phenanthren-9-yl)-1H-1,2,3- triazol-1-yl)propan-2-ylcarbamate (3.38) : Using general procedure, starting from 193 mg (0.37 mmol) of 3.49 and 182 mg (0.37 mmol) of 3.51, 147 mg (0.17 mmol) of the title compound 3.38 was isolated as a brown solid compound after washing with ether.
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Yield 45%. IR (KBr) 3327, 2979, 1655, 1519, 1451, 1367, 1248, 1165, 1054, 728 cm-
1. 1H NMR 1H NMR (d6-DMSO; 300 MHz) δ 1.13 (3H, d, J = 6.9 Hz); 1.26 (9H, s);
3.15 (3H, s); 3.71 (3H, s); 4.39 (1H, bs);4.69- 4.53 (4H, m); 4.78 (1H, bs); 5.33 (1H, bs); 7.24-7.13 (1H, m); 7.72-7.66 (8H, m); 8.07-8.01 (4H, m); 8.34 (1H, d, J = 7.2 Hz); 8.45 (3H, d, J = 8.1 Hz); 8.52 (1H, s); 8.57 (1H, s); 8.82 (2H, d, J = 7.8 Hz); 8.85 (2H, d, J = 8.1 Hz); 13C NMR (CDCl3; 100 MHz); δ 17.3, 28.3, 32.5, 49.5, 50.4, 51.0, 51.3, 54.9, 61.9, 80.8, 122.5, 122.9, 124.8, 126.8, 126.9, 128.4, 128.9, 130.0, 130.4, 130.7, 131.3, 146.7, 155.9, 168.2, 169.6, 172.1. HRMS calcd for C48H49N10O6 ([M + H]+) 861.3831, found 861.3837. C48H48N10O6Na ([M + Na]+) 884.3734, found 884.3731.
Synthesis of tert-butyl 3-(4-(4-cyanophenyl)-1H-1,2,3-triazol-1-yl)-1-(1-(3-(4-(4- cyanophenyl)-1H-1,2,3-triazol-1-yl)-1-(methoxy(methyl)amino)-1-oxopropan-2- ylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-ylcarbamate (3.39): Using general procedure, starting from 148 mg (0.35 mmol) of 3.50 and 159 mg (0.39 mmol) of 3.52, 100 mg (0.14 mmol) of the title compound 3.39 was isolated as a transparent gummy material which became solid particle after ether wash.
Yield 44%. IR (KBr) 3325, 2979, 2931, 2227, 1664, 1517, 1368, 1237, 1164, 1049, 848, 556 cm-1. 1H NMR (CDCl3; 400 MHz) δ = 1.18 (3H, d, J = 7.2 Hz); 1.34 (9H, s); 3.2 (3H, s); 3. 77 (3H, s); 4.41 (1H, m); 4.78 (2H, m); 4.89 (2H, m); 5.27 (1H, m); 5.80 (1H, m); 6.43 (1H, d, J = 8 Hz); 6.65 (1H, d, J = 6.8 Hz); 6.83 (1H, d, J = 7.6 Hz); 7.65 (4H, d, J = 7.2 Hz); 7.89 (6H, m); 13C NMR (CDCl3; 100 MHz); δ 17.9, 28.1, 32.4, 49.4, 50.0, 51.7, 53.4, 54.2, 61.9, 80.8, 111.1, 111.2, 118.7, 122.8, 126.0, 132.6, 134.7, 135.0, 145.5, 145.7, 155.3, 167.8, 169.2, 172.2. HRMS calcd for C34H39N12O6 ([M + H]+) 711.3116, found 711.3110. C34H38N12O6Na([M + Na]+) 734.3013, found 734.301.