2. MATERIALS AND METHODS

Transcription

1 2. MATERIALS AND METHODS 2.1 MATERIALS Table 2.1 List of materials used & their manufacturer S.No. Name of Material Name & Address of Manufacturer 1. Agar Powder Qualigens fine chemicals, Navi Mumbai. 2. Albendazole GlaxoSmithKline Pharmaceuticals Ltd., Mumbai. 3. Beef Extract powder Qualigens fine chemicals, Navi Mumbai. 4. n-butanol Qualigens fine chemicals, Navi Mumbai bromosalocyladehyde Himedia Laboratories Pvt. Ltd., Mumbai. 6. Chloroacetic acid Ranbaxy Fine Chemicals Ltd., New Delhi 7. 4-chloro-3,5-dimethyl phenol S d fine chem Limited, Mumbai. LR 8. Chloroform Spectrochem Pvt. Ltd., Mumbai (India). 9. Ciprofloxacin Cipla (P) Ltd., Solan 10. Dextrose (anhydrous) purified Central Drug House(P) Ltd., New Delhi 11. Dicyclohexylcarbodiimide Spectrochem Pvt. Ltd., Mumbai (India). (DCC) 12. Diethyl ether Qualigens fine chemicals, Navi Mumbai. 13. Dimethya Sulphoxide Central Drug House(P) Ltd., New Delhi 14. Di-tert-butyl-pyrocarbonate Spectrochem Pvt. Ltd., Mumbai (India). (Boc 2 O) 15. Glacial acetic acid Qualigens fine chemicals, Navi Mumbai. 16. Griseofulvin GlaxoSmithKline Pharmaceuticals Ltd., Mumbai. 17. n-hexane S d fine chem Limited, Mumbai. 18. Hydrochloric Acid Qualigens fine chemicals, Navi Mumbai.

2 19. Isopropanol Qualigens fine chemicals, Navi Mumbai. 20. L-Amino Acids (Aspargenin, Spectrochem Pvt. Ltd., Mumbai (India). Alanine, Phenylalanine, Proline, Tyrosine, Leucine, Isoleucine, Serine, Tryptophan,) and Glycine 21. Lithium Hydroxide Himedia (Mumbai). 22. Methanol Qualigens fine chemicals, Navi Mumbai. 23. N,N- Dimethyl Formamide Qualigens fine chemicals, Navi Mumbai. 24. N-Methylmorpholine (NMM) Spectrochem Pvt. Ltd., Mumbai (India). 25. Para-nitrophenol Spectrochem Pvt. Ltd., Mumbai (India). 26. Petroleum ether C Rankem (New Delhi). 27. Sodium Bicarbonate Qualigens fine chemicals, Navi Mumbai. 28. Sodium Chloride Qualigens fine chemicals, Navi Mumbai. 29. Sodium Hydroxide Rankem (New Delhi). 30. Sodium Sulphate Rankem (New Delhi). 31. Sulfuric Acid Qualigens fine chemicals, Navi Mumbai. 32. Tetrahydrofuron (THF) Rankem (New Delhi). 33. Thionyl Chloride Spectrochem Pvt. Ltd., Mumbai (India). 34. Triethylamine (TEA) Qualigens fine chemicals, Navi Mumbai. 35. Trifluoroacetic acid (TFA) Spectrochem Pvt. Ltd., Mumbai (India).

3 TABLE 2.2 List of Instruments used & their manufacturer S.No Name Name & Address of Manufacturer 1. Antibiotic Zone Reader HICON, Grover Enterprises, New Delhi, India 2. Autoclave HICON, Grover Enterprises, New Delhi, India 3. B.O.D. Incubator (Super Delux Model) S. M. Industries, Delhi, India. 4. Digital Balance Parkar TH Digital melting Point Apparatus Jyoti Scientific Industries 6. Electrical Single Pan Balance K- ROY Instruments, Varanasi, India. 7. Electric Water Bath Jyoti Scientific Industries 8. Fourier Transform Infrared SHIMADZU, Japan. Spectrophotometer (FTIR-RXI) 9. 1 H-NMR Spectrometer (AVANCE II- BURKER, Japan 400) 10. Heating Mantle Labtech Sunbim. 11. Hot Air Oven Universal hot air oven 12. Laminar Air Flow Cabinet S. M. Industries, Delhi, India. 13. Magnetic Stirrer Remi 2 MLH 14. ph Meter Simtronics digital ph meter Model SE 962-P 15. Refrigerator Godrej

4 TABLE 2.3 List of glasswares used & their manufacturer S.No Name of Glassware Specification Name & Address of Manufacturer 1. Beakers 50ml, 100ml, 250ml, 500 ml Borosil Glass Works Ltd., Wori, Mumbai Condenser 250 mm Asgi 3. Desiccators 150 mm Jyoti Scientific Industries 4. Distillation Unit 3 lit. Borosil Glass Works Ltd., Wori, Mumbai Funnel 75 mm Borosil Glass Works Ltd., Wori, Mumbai Glass rod 15 cm Jyoti Scientific Industries 7. Measuring Cylinder 10ml, 50ml, 100 ml Borosil Glass Works Ltd., Wori, Mumbai Petri dish 200 mm Borosil Glass Works Ltd., Wori, Mumbai Pipette 1ml, 2ml, 5ml, 10 ml Borosil Glass Works Ltd., Wori, Mumbai RBF 500 ml Borosil Glass Works Ltd., Wori, Mumbai Separating Funnel 250 ml Borosil Glass Works Ltd., Wori, Mumbai Test tubes mm Borosil Glass Works Ltd., Wori, Mumbai

5 2.2 METHODS EXPERIMENTAL Determination of melting point Melting points of newly synthesized compounds were determined by open capillary method using the digital melting point apparatus and were uncorrected. Compounds were placed in one end sealed capillary and placed in the caves made for the capillary. Thermometer was already placed in their caves because it is digital apparatus. The temperature at which compound start melting to the temperature at which it completely melts was recorded as the melting point range Solubility of compounds in different solvents The various solvents such as water, ethanol, chloroform, carbon disulphide, ether, benzene, carbon tetrachloride, tetrahydrofuron, methanol, dimethyl formamide (DMF) and dimethyl sulphoxide (DMSO) were taken for dissolving intermediates and final products. 10 mg of each compound was weighed and added to 10 ml of each solvent individually taken in 50 ml beaker. The observation was recorded observed for different compounds Thin Layer Chromatography Thin layer chromatographic analysis of compounds was done on silica gel G coated glass plates. The adsorbent silica gel G was coated to a thickness of about 0.3 mm on previously cleaned TLC plates of 20 x 10 cm. using conventional spreader. The plates were placed in hot air oven at 105 C for 30 min. The solution of compounds was applied as a spot on the activated plate about 2 cm above from the lower edge. The mobile phases were selected according to the polarity of the products. CHCl 3 : CH 3 OH (99.5:0.5) is used as mobile phase. The spots were visualized by exposure to iodine vapor Spectral Analysis UV spectral analysis 10 mg of compounds dissolved in Chloroform was diluted to 20 ml. 2 ml of above solution was further diluted to 10 ml with chloroform, again 1 ml from the solution was further more diluted to 50 ml with chloroform and UV spectra were recorded on a UV- Visible spectrophotometer ELICO BL 198 Bio spectrophotometer FTIR spectral analysis

6 IR spectra of the compound were obtained from FTIR spectrophotometer using KBr pellets, recorded on a 417 Jasco FTIR. Preparation of KBr pellet of compounds 100 mg of dehydrated KBr was accurately weighed. To this added 1 mg of compound and mixed well. The mixture was placed in an evacuable die and subjected to a pressure of 5-6 tones for 5 min. A transparent disc was produced which was then placed in a pellet holder and IR spectra were taken H-NMR spectral analysis 1 H NMR spectra of compounds was recorded on Bruker NMR spectrophotometer in deuterium- substituted chloroform using TMS as internal standard (Chemical shift in δ ppm) Mass spectral analysis Mass spectra were recorded on 3200 Qtrap Mass spectrophotometer and the result are observed in 72 ev (M+H) + molecule in peak Physical Characteristic Physical character identified by - Elemental analysis Elemental analysis of compounds was done on Vario micro elemental analyser (Elementar Germany).

7 2.2.2 SYNTHETIC PROCEDURE SYNTHESIS OF BOC- AMINO ACIDS tert-butyloxycarbonyl-proline-oh (1) Molecular Formula C 10 H 17 NO 4 Molecular Weigh L-proline (1.15gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml of 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was recrystallized from methanol and ether at 0 o C to give white semisolid mass of compound (1) (1.79 gm, 83.4%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): Rf 0.26 tert-butyloxycarbonyl--alanine-oh (2) Molecular Structure: Molecular Formula: C 9 H 17 NO 4 Formula Weight: L-alanine (.89gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml of 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was recrystallized from methanol and ether at 0 o C to give white semisolid mass of compound (2) (1.76gm, 79.0%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.58

8 tert-butyloxycarbonyl--tyrosine-oh (3) Molecular Formula: C 14 H 19 NO 5 Formula Weight: L-tyrosiine (1.81gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml of 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was recrystallized from methanol and ether at 0 o C to give white semisolid mass of compound (3) (2.04gm, 72.6 %). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.29 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (C-OH), (Ar-C=C), (Ar-CH), (- COOH acidic), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend). tert-butyloxycarbonyl--isoleucine-oh (4) Molecular Formula: C 11 H 21 NO 4 Formula Weight:

9 L-isoleucine (1.31gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to the resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 10 ml). The lower organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure on electronic water bath to give crude product. The crude product was crystallized from chloroform and petroleum ether (b.p o C) to get pure Boc-L- Isoleucine-OH white semisolid mass of compound (4) (1.58gm, 68.4%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.38 Analysis IR (KBr) Vmax (cm -1 ): (-COOH acidic), (-C=O amide), (OH carboxylic), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend). tert-butyloxycarbonyl-glycine-oh (5) Molecular Formula: C7H13NO4 Formula Weight: Glycine (0.75gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml of 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was recrystallized from methanol and ether at 0 o C to give white semisolid mass of compound (5) (1.47gm, 84.1%).

10 TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.30 tert-butyloxycarbonyl-tryptophen-oh (6) L-tryptophen (2.04gm, 10mmol) was dissolved in 20 ml of sodium hydroxide (1 mol L -1 ) and 20 ml of isopropanol. Di-tert-Butylpyrocarbonate (4.55 gm, mol) in 10 ml of isopropanol was added followed by 20 ml of sodium hydroxide (1 mol L -1 ) to the resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 20 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The lower organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure on electronic water bath to give crude product. The crude product was crystallized from chloroform and petroleum ether (b.p o C) to get pure Boc-L-Tryptophen-OH white semisolid mass of compound (6) (1.92gm, 73%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.38 tert-butyloxycarbonyl-l-serine-oh (7) L-serine (1.05 gm, 10 mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to the resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The lower organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure on electronic water bath to give crude product. The crude product was crystallized from methanol and ether at 0 o C to give white needle type crystals of compound (7) (1.70gm, 77.0%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.56

11 tert-butyloxycarbonyl-l-phenylalanine-oh (8) Molecular Formula: C 14 H 19 NO 4 Monoisotopic mass: L-phenylalanine (1.65 gm, 10 mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to the resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The lower organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure on electronic water bath to give crude product. The crude product was crystallized from methanol and ether at 0 o C to give white needle type crystals of compound (8) (2.30gm, 87.0%), M.p C. TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.40 tert-butyloxycarbonyl-l-asparagine-oh (9) L-asparagine (1.32 gm, 10 mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L - 1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to the resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The lower organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure on electronic water bath to give crude product. The crude product was crystallized from methanol and ether at 0 o C to give white needle type crystals of compound (9) (1.69 gm, 77.0%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.64

12 tert-butyloxycarbonyl-leucine-oh (10) Molecular Formula: C 11 H 21 NO 4 Formula Weight: L-leucine (1.31 gm, 10 mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L -1 ) and 10 ml of isopropanol. Di-tert-Butylpyrocarbonate (3 ml of 13 mmol) in 5 ml of isopropanol was added followed by 10 ml of sodium hydroxide (1 mol L -1 ) to the resulting solution. The solution was stirred at room temperature for 2 hrs, washed with 10 ml of light petroleum ether (b.p o C), acidified to ph 3.0 with 1 mol L -1 sulphuric acid and finally extracted with chloroform (3 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was recrystallized from methanol and ether at 0 o C to give white semisolid mass of compound..(1.90 gm, 82.6%). TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f 0.31

13 SYNTHESIS OF L-AMINO ACID METHYL ESTER HYDROCHLORIDES L-Isoleucin methyl ester hydrochloride (11) Thionyl chloride (0.73 ml, 10 mmol) was slowly added to 50 ml of methanol at 0 o C and 1.31 gm of L- isoleucin (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 o C. Methanol was evaporated and the residue was triturate with ether at 0 o C until excess dimethyl sulphite was removed. The crude product was recrystallized from methanol and ether at 0 o C to give white needle type crystals of comound (11) (65%). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.42 L-Alanine methyl ester hydrochloride (12) Thionyl chloride (1.4 ml, 0.02 mol) was slowly added to methanol (100 ml) at 0 C and L-Alanine (1.6 g, 0.02 mol) was added to the above solution. The resulting mixture was refluxed for 8-10 hr at ambient temperature. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude solid was crystallized from methanol and ether at 0 C to get L- Alanine methyl ester hydrochloride (12). Percentage Yield = 100 % TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.35 Analysis IR (KBr) Vmax (cm -1 ): (-C=O ester), (-C-O ether), (1 amide str), (1 amide bend), (CH in CH 3 ), (-CH bend). L-Leucine methyl ester hydrochloride (13) Thionyl chloride (1.4 ml, 0.02 mol) was slowly added to methanol (100 ml) at 0 C and L-Leucine (2.6 g, 0.02 mol) was added to the above solution. The resulting mixture was refluxed for 8-10 hr at ambient temperature. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude solid was crystallized from methanol and ether at 0 C to get L-Leucine methyl ester hydrochloride (13). Percentage Yield = 91 % M.P o C. TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.47 Analysis: IR (KBr) Vmax (cm -1 ):

14 (-C=O ester), (-C-O ether), (1 amide str), (1 amide bend), (CH in CH 3 ), (-CH bend). L-Phenylalanine methyl ester hydrochloride (14) Thionyl chloride (1.4 ml, 0.02 mol) was slowly added to methanol (100 ml) at 0 C and L-Phenylalanine (3.3 g, 0.02 mol) was added to the above solution. The resulting mixture was refluxed for 8-10 hr at ambient temperature. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude solid was crystallized from methanol and ether at 0 C to get L-Phenylalanine methyl ester hydrochloride (14). (2.3 gm, 69.3%). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.73 Analysis IR (KBr) Vmax (cm -1 ): (-C=O ester), (-C-O ether), (1 amide str), (1 amide bend), (Ar-CH ), (Ar-C=C), (CH in CH 3 ), (-CH bend). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.50 L-Proline methyl ester hydrochloride (15) Thionyl chloride (0.73 ml, 10 mmol) was slowly added to 50 ml of methanol at 0 o C and 1.15 gm of L-proline (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 o C. Methanol was evaporated and the residue was triturate with ether at 0 o C until excess dimethyl sulphite was removed. The crude product was recrystallized from methanol and ether at 0 o C to give pure dark broun semisolid mass of comound (15) (1.15 gm, 69.3%). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.43 Glycine methyl ester hydrochloride (16) Thionyl chloride (1.4 ml, 0.02 mol) was slowly added to methanol (100 ml) at 0 C and Glycine (1.5 g, 0.02 mol) was added to the above solution. The resulting mixture was refluxed for 8-10 hr at ambient temperature. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude solid was crystallized from methanol and ether at 0 C to get Glycine methyl ester hydrochloride (16). Percentage Yield = 95.3 % Analysis

15 IR (KBr) Vmax (cm -1 ): (-C=O ester), (-C-O ether), (1 amide str), (1 amide bend), (CH in CH 3 ), (-CH bend). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.46 L-Tyrosine methyl ester hydrochloride (17) Thionyl chloride (0.73 ml, 10 mmol) was slowly added to 50 ml of methanol at 0 o C and 1.81 gm of L- tyrosine (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 o C. Methanol was evaporated and the residue was triturate with ether at 0 o C until excess dimethyl sulphite was removed. The crude product was recrystallized from methanol and ether at 0 o C to give white needle type crystals of comound (17) (85%). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.38 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (C-OH), (Ar-C=C), (Ar-CH), (- COOH acidic), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend). L-Serine methyl ester hydrochloride (18) Thionyl chloride (0.73 ml, 10 mmol) was slowly added to 50 ml of methanol at 0 o C and 1.05 gm of L- serine (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 o C. Methanol was evaporated and the residue was triturate with ether at 0 o C until excess dimethyl sulphite was removed. The crude product was recrystallized from methanol and ether at 0 o C to give white needle type crystals of comound (18) (79%). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.54 L-Isoleucine methyl ester hydrochloride (19) Thionyl chloride (0.73 ml, 10 mmol) was slowly added to 50 ml of methanol at 0 o C and 1.05 gm of L- isoleucine (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 o C. Methanol was evaporated and the residue was triturate with ether at 0 o C until excess dimethyl sulphite was removed. The crude product was recrystallized from methanol and ether at 0 o C to give white needle type crystals of comound (19) (89%). TLC (n-butanol: Acetic acid: Water, 4:1:1, v/v): R f 0.70 General Procedure for synthesis of N-Methyl Amino acid methyl ester hydrochloride

16 N-methyl amino acid methyl ester hydrochloride or dipeptide/tripeptide methyl ester (0.01 mol) was dissolved in DMF (25 ml). To this, NMM (0.021 mol) was added at 0 C and the reaction mixture was stirred for 15 min. Boc-dipeptide (0.01 mol) in DMF (25 ml) and DIPC (0.01 mol) were added with stirring. Stirring was first don for 1 h at 0-5 C and then further for 24 h at room temperature (RT). After the completion of the reaction, the reaction mixture was diluted with an equal amount of water. The precipitated solid was then, filtered and washed with water, and finally recrystallized from mixture of chloroform and petroleum ether (b.p C) followed by cooling at 0 C to get title compounds (20) N-Methyl-Phenylalanine methyl ester hydrochloride Percentage Yield: 77.5%, Rf: 0.81 IR (CHCl3): 2625, 2851 (m, -CH str, asym and sym, CH2), 2869, 2866 (m, -CH str, sym, CH3), 1742 (s, -C=O str, ester), 1586, 1473 (m, skeletal bands, arom. ring), 1271 (s, C-O str, ester), 728, 687 (s, -CH bend, oop, arom. ring) cm-1; 1 H NMR (300 MHz, CDCl3): (2H, m, NH2+), (1H, t, J = 6.3 Hz, p-h, Phe), (2H, dd, J = 8.75, 4.15 Hz, o-h s, Phe), (2H, tt, J = 6.7, 4.45 Hz, m-h s, Phe), 3.85 (3H, s, OCH3), (1H, m, a-h, Phe), (2H, d, J = 5.55 Hz, b-h s, Phe), 2.52 (3H, s, NCH3) ppm. (21) N-Methyl-Glycine methyl ester hydrochloride Percentage Yield: 67.8%, R f : 0.65 (22) N-Methyl-Alanine methyl ester hydrochloride Percentage Yield: 87.8%, R f : 0.85

17 SYNTHESIS OF BOC-DIPEPTIDE METHYL ESTERS General procedure for the preparation of linear dipeptide fragments L-Amino acid methyl ester hydrochloride (0.01 mol) was dissolved in CH 2 Cl 2 (20 ml), NMM (2.3ml, mol) was added at 0ºC. The reaction mixture was stirred for 15 min. Boc-L- Pro-OH Compound (0.01 mol) in CH 2 Cl 2 (20 ml) followed by addition of EDC.HCl (1.92 g, 0.01 mol) and HOBt (1.34 g, 0.01 mol). The resulting mixture was added to above solution with constant shaking and stirring was continued for 24 h. The reaction mixture was filtered and the residue was washed with CH 2 Cl 2 (30 ml) and added to the filtrate. The filtrate was washed with 5% NaHCO 3 and saturated NaCl solutions. The organic layer was dried over anhydrous Na 2 SO 4, filtered and evaporated in vacuum. The crude product was recrystallized from a mixture of chloroform and petroleum ether (b.p C) followed by cooling at 0 C to get the title compounds. (23) tert-butyloxycarbonyl-l-proline-l-leucine methyl ester Percentage Yield: 87% Anal: TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : IR (CHCl 3 /KBr, v cm -1 ): 3128, 3123 (N H str, amide), (C H str, CH 2, Pro), 2966, 2928 (C H str, asym, CH 3 and CH 2 ), 1748 (C=O str, ester), 1675, 1643 (C=O str, tert and sec amide), 1539 (N H def, sec amide), 1391, 1377 (C H def, tert-butyl), 1384, 1369 (C H def, iso-propyl), 1267 (C O str, ester). 1 H/ NMR (CDCl 3, δ ppm): 6.65 (1H, br. s, NH), 4.40 (1H, q, H-, Leu), 4.05 (1H, t, J = 7.2 Hz, H-, Pro), 3.62 (3H, s, OCH 3 ), 3.22 (2H, t, J = 7.2 Hz, H-, Pro), 2.58 (2H, q, H-, Pro), (2H, m, H-, Pro), 1.47 (9H, s, tert-butyl), (3H, m, H- and H-, Leu), 0.94 (6H, d, J = 6.35 Hz, H-, Leu). (24) tert-butyloxycarbonyl-l-alanine-l-isoleucine methyl ester Percentage Yield: - 93% Anal: TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : IR (CHCl 3 /KBr, v cm -1 ):

18 3129, 3125 (N H str, amide), 2969, 2926 (C H str, asym, CH 3 and CH 2 ), 2876, 2872 (C H str, sym, CH 3 ), 1744 (C=O str, ester), 1645, 1641 (C=O str, sec amide), 1538, 1533 (N H def, sec amide), 1392, 1377 (C H def, tert-butyl), 1269 (C O str, ester). 1 H/ NMR (CDCl 3, δ ppm): 6.68 (1H, br. s, NH), 6.55 (1H, br. s, NH), (1H, m, H-, Ala), 4.22 (1H, t, J = 6.4 Hz, H-, Ile), 3.53 (3H, s, OCH 3 ), (1H, m, H-, Ile), (2H, m, H-, Ile), 1.58 (3H, d, J = 7.3 Hz, H-, Ala), 1.55 (9H, s, tert-butyl), 0.92 (3H, t, J = 7.15 Hz, H-, Ile), 0.88 (3H, d, J = 6.5 Hz, H-, Ile). (25) tert-butyloxycarbonyl-l-proline-l-phenylanine methyl ester Percentage Yield: - 83% Anal: TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : IR (CHCl 3 /KBr, v cm -1 ): 3124 (N H str ), (C H str ), 2966, 2928, 2874 (C H str ), 1744 (C=O str ), 1673, 1644 (C=O str ), 1546, 1417 (C=C), 1536 (N H def ), 1390, 1375 (C H def ), 1271 (C O str ), 725, 689 (C H def ). 1 H/ NMR (CDCl 3, δ ppm): 7.18 (br. s, 1H, NH), 7.11 (t, J=5.7 Hz, 1H, H-p, Phe), (m, 2H, H-m, Phe), 6.85 (dd, J = 7.2 Hz, 2H, H-o, Phe), 4.22 (t, J=5.7 Hz, 1H, H-, Phe), 4.05 (t, J=6.5 Hz, 1H, H-, Pro), 3.56 (s, 3H, OCH 3 ), 3.23 (t, J=7.2 Hz, 2H, H-, Pro), 2.98 (d, J=4.9 Hz, 2H, H-, Phe), 2.58 (q, 2H, H-, Pro), (m, 2H, H-, Pro), 1.48 (s, 9H, tert-butyl). (26) tert-butyloxycarbonyl-l-alanine-l- Phenylanine methyl ester Percentage Yield = 79 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : 0.65 (27) tert-butyloxycarbonyl-l-alanine-glycine methyl ester Percentage Yield = 89 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : 0.74 (28) tert-butyloxycarbonyl-l-isoleucine-l- Phenylanine methyl ester

19 Percentage Yield = 74 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : 0.83 Analysis IR (KBr) Vmax (cm -1 ): (Ar-C=C), (Ar-CH), (-COOH acidic), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend). (29) tert-butyloxycarbonyl-l- Tyrosine -L-Alanine methyl ester Percentage Yield = 73.4 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : 0.81 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (-C=O ester), (Ar-C=C), (Ar-CH), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (CH in CH 3 str), (CH in CH 3 bend). (30) tert-butyloxycarbonyl-l- Tyrosine -L-Phenylanine methyl Percentage Yield = 83.2 % (31) tert-butyloxycarbonyl-glysine-l-leucine methyl ester Percentage Yield = 2.39 gm, 79.2 % Analysis TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : 0.76 UV ʎmax(CHCl 3 ): nm IR (KBr) Vmax (cm -1 ): (N-H stretching, amide), (C-H stretching, asymmetric, CH 3 ), (C-H stretching, isopropyl group), (C-H stretching, symmetric, CH 3 ), (C-H stretching, OCH 3 ), (C=O stretching, 2º amide), (N-H bend,2º amide)3, , (Ch bend, t butyl), cm -1 (C-O stretching, OCH 3 ). 1 H NMR (300 MHz, CDCl3) : 7.93 (br. S, 2H, NH, amide), (m, ih, α-h, Leu), 3.83 (s, 2H, Gly), 3.61 (s, 3H, OCH 3 ), (s, 9H, t butyl), (m, 3H,β-H s and γ-h, Leu), and ppm (d, 6H,

20 δ- H s, Leu). Calcd for C 14 H 26 N 2 O 5 : C, 55.62; H, 8.60; N, Found: C, 55.65; H, 8.63; N, (32) tert-butyloxycarbonyl-l- Tryptophen -L-Proline methyl Percentage Yield = 67.5 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : 0.66 (33) tert-butyloxycarbonyl-glysine-l-serine methyl ester Percentage Yield = 87.9 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : (34) Tert-Butyloxycarbonyl-L-Serine-L-Leucine methyl ester Percentage Yield = 77.1 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.38 (35) tert-butyloxycarbonyl-l-proline-l-isoleucine methyl ester Percentage Yield = 59 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.79 (36) tert-butyloxycarbonyl -L-Phenylanine-L-Proline methyl ester Percentage Yield = 72.1 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.58 (37) tert-butyloxycarbonyl-l-proline-l-tyrosine methyl ester Percentage Yield = 74.1 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.55 (38) tert-butyloxycarbonyl-l-leucine-l-isoleucine methyl ester Percentage Yield = 69.9 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.58 IR (CHCl3): 3139, 3136 (m, -NH str, amide), 2926, 2851 (m, -CH str, asym and sym, CH2), 2965, 2961, (m, -CH str, asym and sym, CH3), 1743 (s, -C=O str, ester), 1645, 1641 (s, - C=O str, 2 amide), 1536, 1532 (m, -NH bend, 2 amide), 1462 (m, -CH bend(scissoring), CH2), 1391, 1374 (s, -CH bend, tert-butyl group), 1380, 1362 (s, -CH bend, iso-propyl group), 1269 (s, CO str, ester), 934, 921 (w, CH3 rocking, tert-butyl and iso-propyl groups) cm-1; 1 H NMR (300 MHz, CDCl3):

21 6.45 (1H, br. s, -NH, Ile), 6.02 (1H, br. s, -NH, Leu), (1H, q, J = 6.75 Hz, a-h, Leu), (1H, t, J = 8.6 Hz, a-h, Ile), 3.52 (3H, s, OCH3), (3H, m, b-h s, Leu and Ile), (2H, m, g-h s, Ile), (1H, m, g-h s, Leu), 1.54 (9H, s, tert-butyl group), (6H, d, J = 6.25 Hz, δ-h s, Leu), (3H, d, J = 7.75 Hz, δ-h s, Ile), (3H, d, J = 5.9 Hz, g -H s, Ile) ppm. (39) tert-butyloxycarbonyl-l-asperganine-l-proline methyl ester Percentage Yield = 89.7 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.75 (40) tert-butyloxycarbonyl -L-Leucine-L-Proline methyl ester Percentage Yield = 88.1 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.81 (I) tert-butyloxycarbonyl -L-Proline-Glycine methyl ester Percentage Yield = 78.7 % TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f: 0.70 General method for the synthesis of dipeptide fragments (41-42) N-methyl amino acid methyl ester hydrochloride (0.01 mol) was dissolved in CHCl3 (20 ml). To this, NMM (0.021 mol, 2.23 ml) was added at 0 C38 and the reaction mixture was stirred for 15 min. Boc-amino acid (0.01 mol) in 20 ml of CHCl3 and DIPC (0.01 mol) were added with stirring. After 24 h, the reaction mixture was filtered and the residue was washed with CHCl3 (25 ml) and added to the filtrate. The filtrate was washed with 5% NaHCO3 and saturated NaCl solutions. The organic layer was dried over anhydrous Na 2 SO 4, filtered and evaporated in vacuum. The crude product was recrystallized from a mixture of chloroform and petroleum ether (b.p C) followed by cooling at 0 C. (41) tert-butyloxycarbonyl-phenylalanyl-n-methyl-glycine methyl ester Percentage Yield = 73.4 % IR (CHCl3): 3138 (m, -NH str, amide), 2929, 2624 (m, -CH str, asym, CH2), 2854 (m, -CH str, sym, CH2), 2869, 2865 (m, -CH str, sym, CH3), 1746 (s, -C=O str, ester), 1644 (s, -C=O str, 2 amide), 1589, 1478 (m, skeletal bands, arom. ring), 1533 (m, -NH bend, 2 amide), 1465 (m, -

22 CH bend(scissoring), CH2), 1393, 1377 (s, -CH bend, tert-butyl group), 1272 (s, C-O str, ester), 932 (w, CH3 rocking, tert-butyl group), 725, 689 (s, -CH bend, out-of-plane, arom. ring) cm-1; 1 H NMR (300 MHz, CDCl3): (2H, tt, J = 6.75, 4.45 Hz, m-h s, Phe), 6.96 (1H, br. s, -NH, Phe), (1H, t, J = 6.25 Hz, p-h, Phe), (2H, dd, J = 8.8, 4.15 Hz, o-h s, Phe), (1H, q, J = 5.5 Hz, a-h, Phe), 4.24 (2H, s, a-h, Gly), 3.72 (3H, s, OCH3), (2H, d, J = 5.6 Hz, b-h s, Phe), 3.05 (3H, s, NCH3, Gly), 1.53 (9H, s, tert-butyl group) ppm. (42) tert-butyloxycarbonyl-prolyl-n-methyl-phenylalanine methyl ester IR (CHCl3): 2998, 2995 (m, -CH str, cyclic CH2, Pro), 2925 (m, -CH str, asym, CH2), 2872 (m, -CH str, sym, CH3), 1744 (s, -C=O str, ester), 1662, 1659 (s, -C=O str, 3 amide), 1588, 1475 (m, skeletal bands, arom. ring), 1392, 1379 (s, -CH bend, tert-butyl group), 1269 (s, C-O str, ester), 930 (w, CH3 rocking, tert-butyl group), 722, 686 (s, -CH bend, oop, arom. ring) cm-1; 1 H NMR (300 MHz, CDCl3): (1H, t, J = 6.2 Hz, p-h, Phe), (2H, tt, J = 6.7, 4.5 Hz, m-h s, Phe), (2H, dd, J = 8.75, 4.2 Hz, o-h s, Phe), (1H, t, J = 5.15 Hz, a-h, Phe), (1H, t, J = 6.85 Hz, a-h, Pro), 3.52 (3H, s, OCH3), (2H, t, J = 7.2 Hz, δ-h s, Pro), (2H, d, J = 5.55 Hz, b-h s, Phe), 3.02 (3H, s, NCH3, Phe), (2H, q, b-h s, Pro), (2H, m, γ-h s, Pro), 1.48 (9H, s, tert- Butyl group) ppm.

23 DEPROTECTION OF DIPEPTIDE AT CARBOXYL END General Procedure: To a solution of compound (10 mmol) in 36 ml THF/H 2 O (1:1), 0.36 gm of lithium hydroxide (15 mmol) was added at 0ºC. The mixture was stirred at room temperature for 1 hr, and acidified to ph 3.5 with 0.5 mol L -1 sulphuric acid. The aqueous layer was extracted with diethyl ether (3 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from methanol and ether to give deprotected product. (43) (tert-butyloxycarbonyl -L-Isoleucine-L-Phenylalanine-OH) Percentage Yield = 86.3 % R f 0.72 Analysis IR (KBr) Vmax (cm -1 ): (C-OH), (-C=O ester), (Ar-C=C), (Ar-CH), (- COOH acidic), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend). (44) tert-butyloxycarbonyl -L-Tyrosine-L-Alanine-OH To a solution of Boc-L-Tyr-L-Ala-OMe compound (29) (3.7 g, 0.01 mol) in THF/H 2 O (1:1, 36 ml), LiOH (0.36 g,0.015 mol) was added at 0 C. The mixture was stirred at r.t. for 1 hr and then acidified to ph 3.5 with 0.5 mol L 1 H 2 SO 4. The aqueous layer was extracted with Et 2 O (3 x 25 ml). Combined organic extracts were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was crystallized from methanol and ether to get Boc- L-Tyr-L-Ala-OH (44). Percentage Yield = 86.6 % R f: 0.70 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (C-OH), (-C=O ester), (Ar-C=C), (Ar-CH), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend). (45) tert-butyloxycarbonyl -L-Alanine-L-Phenylalanine-OH Percentage Yield = 77 % R f 0.85 (46) tert-butyloxycarbonyl -L-Alanine-Glycine -OH

24 Percentage Yield = 72 % R f 0.83 (47) tert-butyloxycarbonyl -L-Proline-L-Leucine-OH Percentage Yield = 84 % R f 0.55 (48) tert-butyloxycarbonyl -L-Tyrosine-L-Phenylalanine-OH Percentage Yield = 90 % R f 0.81 (49) tert-butyloxycarbonyl -Glycine-L-Leucine-OH Percentage Yield = 91 % R f 0.80 (50) tert-butyloxycarbonyl -L-Tryptophen-L-Proline-OH Percentage Yield = 94% R f 0.66 (51) tert-butyloxycarbonyl -L-Proline-L-Isoleucine-OH Percentage Yield = 74% Rf 0.73 (52) tert-butyloxycarbonyl -L-Isoleucine-L-Proline -OH Percentage Yield = 77% R f 0.78 (53) tert-butyloxycarbonyl -L-Leucine-L-Proline-OH Percentage Yield = 84% R f 0.70 (54) tert-butyloxycarbonyl -L-PhenylalanineN(Me)-Glycine-OH (II) tert-butyloxycarbonyl -L-Proline-Glycine-OH Percentage Yield = 84% R f 0.65

25 DEPROTECTION OF DIPEPTIDE AT AMINO END Boc-dipeptide methyl ester (10 mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of TFA (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from mixture of chloroform and light petroleum ether (b.p ºC) to give product. (54) L-Alanine-L-Isoleucine methyl ester hydrochloride Percentage Yield = 74% R f 0.55 (55) L-Proline-L-Phenylalanine methyl ester hydrochloride Percentage Yield = 84% R f 0.48 (56) L-Serine-L-Leucine methyl ester hydrochloride Percentage Yield = 94% R f 0.77 (57) L-Proline-L-Tyrosine methyl ester hydrochloride Percentage Yield = 79% R f 0.66 (58) L-Proline-N(Me)-L-Phenylalanine methyl ester hydrochloride Percentage Yield = 90% R f 0.80 (59) L-Tyrosine-L-Alanine methyl ester hydrochloride Percentage Yield = 87% R f 0.70

26 SYNTHESIS OF BOC-TRIPTIDE METHYL ESTERS General method for tripeptide methyl ester L-Amino acid dipeptide methyl ester (10 mmol) was dissolved in dichloromethane (DCM, 20 ml). To this, NMM (2.21 ml, 20 mmol) was added at 0 C and the reaction mixture was stirred for 15 min. Boc-l-amino acid (10 mmol) in DCM (20 ml) and DIPC/DCC (1.26 g/2.1 g, 10 mmol) were added with stirring. After 24 h, the reaction mixture was filtered and the residue was washed with DCM (30 ml) and added to the filtrate. The filtrate was washed with 5% NaHCO 3 and saturated NaCl solutions. The organic layer was dried over anhydrous Na 2 SO 4, filtered and evaporated in vacuum. The crude product was recrystallized from a mixture of chloroform and petroleum ether (b.p C) followed by cooling at 0 C. (60) tert-butyloxycarbonyl-l-alanine-glycine-l-tyrosine methyl ester Percentage Yield = 82 %. R f 0.76 (61) tert-butyloxycarbonyl-l-isoleucine-l-phenylalanine-l-leunice methyl ester Percentage Yield = 82 %. R f 0.76 Analysis IR (KBr) Vmax (cm -1 ): (-C=O ester), (ar-c=c), (Ar-CH), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether), (CH in CH 3 str), (CH in CH 3 bend), (-C=O acidic). (62) tert-butyloxycarbonyl-l- Tyrosine -L- Phenylalanine - L-Proline methyl ester Percentage Yield 89%; R f 0.71 IR (CHCl 3 ), v cm 1 : 3372 (m/br, -OH str, Tyr), (m, -NH str, amide), (w, -CH str, ring), (m, -CH str, Pro), 2875 (m, -CH str, sym, CH 3 ), 2927 (m, -CH str, asym, CH 2 ), 2852 (m, -CH str, sym, CH 2 ), 1747 (s, -C=O str, ester), 1667, 1649, 1636 (s, -C=O str, 3 and 2 amide), , (m, skeletal bands, rings), , 1528, 1523 (m, -NH bend, 2 amide), 1390, 1369 (s, -CH bend, tert-butyl), 1266 (s, C O str, ester), 1230 (s, C O str, phenolic), 929 (w, CH 3 rocking, tert-butyl), 827, 725, 689 (m, -CH def, oop, rings). 1 H-NMR (DMSO-d 6 ) : 8.64 (1H, br. s, NH), 7.30 (2H, dd, J=8.6, 4.9 Hz, H-m, Tyr), 7.18 (2H, tt, J=6.75, 4.5 Hz, H-m, Phe), 6.98 (1H, t, J=6.25 Hz, H-p, Phe), 6.92 (2H, dd, J=8.55, 5.3 Hz, H-o, Tyr), 6.81 (2H,

27 dd, J=8.8, 4.15 Hz, H-o, Phe), 6.57 (1H, br. s, NH), 5.95 (1H, br. s, OH), 4.59 (1H, q, J=5.5 Hz, H-α, Phe), 4.52 (1H, q, J=7.9 Hz, H-α, Tyr), 3.94 (1H, t, J=6.9 Hz, H-α, Pro), 3.62 (3H, s, OCH 3 ), 3.40 (2H, t, J=7.25 Hz, H-δ, Pro), (4H, m, H-, Phe and Tyr), (4H, m, H- and H-γ, Pro), 1.53 (9H, s, tert-butyl). Anal. Calcd for C 29 H 37 N 3 O 7 : C, 64.55; H, 6.91; N, Found: C, 64.58; H, 6.89; N, (63) tert-butyloxycarbonyl-glycine-l-leucine-l-proline methyl ester Percentage Yield 82%; [ ] D 73.6 (c, 0.25 in MeOH); R f 0.59; IR (CHCl 3 ), v cm 1 : (m, -NH str, amide), (m, -CH str, Pro), 2878 (m, -CH str, sym, CH 3 ), 2929, 2926 (m, -CH str, asym, CH 2 ), 2856 (m, -CH str, sym, CH 2 ), 1746 (s, -C=O str, ester), 1668, 1645, 1637 (s, -C=O str, 3 and 2 amide), 1538, (m, -NH bend, 2 amide), 1392, 1368 (s, -CH bend, tert-butyl), 1385, 1363 (s, -CH bend, iso-propyl), 1268 (s, C O str, ester), 926 (w, CH 3 rocking, tert-butyl). 1 H-NMR (DMSO-d 6 ) : 6.92 (1H, br. s, NH), 6.24 (1H, br. s, NH), 4.47 (1H, q, J=6.7 Hz, H-α, Leu), 3.92 (1H, t, J=6.95 Hz, H-α, Pro), 3.74 (2H, d, J=5.15 Hz, H-α, Gly), 3.65 (3H, s, OCH 3 ), 3.38 (2H, t, J=7.3 Hz, H-δ, Pro), (4H, m, H- and H-γ, Pro), 1.76 (2H, t, J=7.95 Hz, H-, Leu), 1.55 (9H, s, tert-butyl), (1H, m, H-γ, Leu), (6H, d, J=6.25 Hz, H-δ, Leu). Anal. Calcd for C 19 H 33 N 3 O 6 : C, 57.13; H, 8.33; N, Found: C, 57.15; H, 8.32; N, (64) tert-butyloxycarbonyl-l-asparaginyl(bzh)-l-proline-glycine methyl ester Semisolid mass; Percentage yield, 88%; R f 0.61; IR (CHCl3) v cm -1 : 3135, 3122 (m, N-H str, 2 amide), 3075, (w, C-H str, rings), (m, C-H str, Pro), 2877 (m, C-H str, sym, CH3), , 2854 (m, C-H str, asym and sym, CH2), 1754 (s, C=O str, ester), 1659, 1644, 1635 (s, C=O str, 3 and 2 amide), 1577, 1574, 1475, 1472 (m, skeletal bands, rings), 1538, 1522 (m, N-H bend, 2 amide), 1392, 1374 (s, C-H bend, butylt), 1268 (s, C O str, ester), 726, 721, 689, 684 (s, C-H bend, oop, rings); 1 H NMR (CDCl3) d (ppm): 8.62 (br. s, 1H, NH, Gly), 8.39 (br. s, 1H, NH, Asn), (m, 6H, H-m and H-p, rings, bzh), (m, 4H, H-o, rings, bzh), 6.89 (d, 1H, J = 5.6 Hz, CONH, Asn), 5.94 (d, 1H, J = 5.4 Hz, H-a, bzh), 5.94 (t, 1H, J = 6.9 Hz, H-a, Pro), 4.34 (q, 1H, H-a, Asn), 4.02 (d, 2H,

28 J = 8.2 Hz, H-a, Gly), 3.67 (t, 2H, J = 7.2 Hz, H-d, Pro), 3.59 (s, 3H, OCH3), 3.07 (d, 2H, J = 3.8 Hz, H-b, Asn), 2.65 (q, 2H, H-b, Pro), (m, 2H, H-g, Pro), 1.57 (s, 9H, butyl-t); Anal. Calc. for C 30 H 38 N 4 O 7 (566): C, 63.59; H, 6.76; N, Found: C, 63.56; H, 6.74; N, 9.90%. (65) tert-butyloxycarbonyl-l-tryptophane-l-proline-glycine methyl ester Yield 92%; [ ] D (c, 0.25 in MeOH); R f 0.66; IR (CHCl 3 ), v cm 1 : 3478 (m, -NH str, ring), (m, -NH str, amide), 3066 (w, -CH str, ring), (m, -CH str, Pro), 2877 (m, -CH str, sym, CH 3 ), 2924 (m, -CH str, asym, CH 2 ), 2853 (m, - CH str, sym, CH 2 ), 1745 (s, -C=O str, ester), 1668, 1644, 1636 (s, -C=O str, 3 and 2 amide), 1554, 1475 (m, skeletal bands, ring), , 1529 (m, -NH bend, 2 amide), 1389, 1367 (s, - CH bend, tert-butyl), 1269 (s, C O str, ester), 928 (w, CH 3 rocking, tert-butyl), 732, 675 (m, - CH def, oop, ring). 1 H-NMR (DMSO-d 6 ) : 8.96 (1H, br. s, NH, ring), 8.63 (1H, br. s, NH), (4H, m, H-, ring), 6.43 (1H, br. s, NH), 6.35 (1H, d, J=7.8 Hz, H-, ring), (1H, m, H-α, Trp), 4.39 (1H, t, J=6.9 Hz, H-α, Pro), 4.02 (2H, d, J=5.15 Hz, H-α, Gly), 3.68 (2H, t, J=7.25 Hz, H-δ, Pro), 3.62 (3H, s, OCH 3 ), 3.16 (2H, d, J=5.7 Hz, H-, Trp), (2H, m, H-, Pro), (2H, m, H-γ, Pro), 1.54 (9H, s, tert-butyl). Anal. Calcd for C 24 H 32 N 4 O 6 : C, 61.00; H, 6.83; N, Found: C, 60.99; H, 6.80; N, (66) tert-butyloxycarbonyl-l-proline -L-Isoleucine-L-Proline methyl ester Semisolid mass; Yield 87%; [α]d 41.4 ; R f 0.78; IR (CHCl3): 3,122 (N H str, amide), 2,997 2,989 (C H str, CH2, Pro), 2,965, 2,927 (C H str, asym, CH3 and CH2), 2,869 (C H str, sym, CH3), 1,752 (C=O str, ester), 1,678, 1,673, 1,644 (C=O str, 3 and 2 amide), 1,536 (N H bend, 2 amide), 1,388, 1,375 (C H bend, tert-butyl), 1,269 (C O str, ester) cm 1; 1 H-NMR (CDCl 3 ): 6.25 (1H, br. s, NH), 4.52 (1H, t, J = 8.6 Hz, H-α, Ile), 4.11 (1H, t, J = 6.9 Hz, H-α, Pro- 1), 3.92 (1H, t, J = 6.9 Hz, H-α, Pro-2), 3.62 (3H, s, OCH3), 3.41 (2H, t, J = 7.2 Hz, H-δ, Pro-2),

29 3.21 (2H, t, J = 7.1 Hz, H-δ, Pro-1), 2.55 (2H, q, H-β, Pro-1), 2.04 (3H, m, H-β, Pro-2 and H-β, Ile), 1.97 (2H, m, H-γ, Pro-2), 1.90 (2H, m, H-γ, Pro-1), 1.64 (2H, q, H-γ, Ile), 1.48 (9H, s, tertbutyl), 1.03 (3H, d, J = 5.9 Hz, H-γ, Ile), 0.97 (3H, t, J = 7.8 Hz, H-δ, Ile); Anal. Calcd. for C32H37N3O6: C, 60.12; H, 8.48; N, Found: C, 60.15; H, 8.49; N, 9.54%. (67) tert-butyloxycarbonyl-l-tyrosine -L-Alanine-Glycine methyl ester Semisolid mass; Yield 77%; [α]d 41.4 ; R f 0.88 (68) tert-butyloxycarbonyl-l-leucine-l-isoleucine-n (Me)-Phenylalanine methyl ester Percentage Yield 73%: R f : 0.77 Analysis: IR (CHCl 3 ): v 3136, 3132 (m, -NH str, amide), 2929, 2854 (m, -CH str, asym and sym, CH2), , 2869, 2863 (m, -CH str, asym and sym, CH3), 1745 (s, -C=O str, ester), 1667, (s, -C=O str, 3 and 2 amide), 1588, 1471 (m, skeletal bands, arom. ring), 1539, 1535 (m, - NH bend, 2 amide), 1466 (m, -CH bend (scissoring), CH2), 1393, 1375 (s, -CH bend, tert- Butyl group), 1379, 1364 (s, -CH bend, iso-propyl group), 1274 (s, C-O str, ester), 933, 920 (w, CH3 rocking, tert-butyl and iso-propyl groups), 729, 685 (s, -CH bend, oop, arom. ring) cm-1. 1 H-NMR (CDCl3): (300 MHz, CDCl3): d (1H, t, J = 6.25 Hz, p-h, Phe), 7.10 (1H, br. s, -NH, Ile), (2H, tt, J = 6.75, 4.45 Hz, m-h s, Phe), (2H, dd, J = 8.75, 4.2 Hz, o-h s, Phe), 6.04 (1H, br. s, - NH, Leu), (1H, t, J = 5.15 Hz, a-h, Phe), (1H, t, J = 8.55 Hz, a-h, Ile), (1H, q, J = 6.8 Hz, a-h, Leu), 3.55 (3H, s, OCH3), (2H, d, J = 5.6 Hz, b-h s, Phe), 3.02 (3H, s, NCH3), (3H, m, b-h s, Leu and Ile), (2H, m, g-h s, Ile), (1H, m, g- H s, Leu), 1.51 (9H, s, tert-butyl group), (3H, d, J = 5.85 Hz, g -H s, Ile), (6H, d, J = 6.3 Hz, δ-h s, Leu), (3H, d, J = 7.8 Hz, δ-h s, Ile) ppm.

30 DEPROTECTION OF TRIPTIDE AT AMINO END General method for deprotection of tripeptide at amino end Boc-tipeptide compound (0.01 mol) was dissolved in CHCl 3 (15 ml) and treated with trifluoro acetic acid (2.28 g, 0.02 mol). The resulting solution was stirred at r.t. for 1 hr and washed with saturated NaHCO 3 solution (25 ml). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified by crystallization from CHCl 3 and petroleum ether (b.p C) to give pure tripeptide methyl ester. (75) L-Tyr-L-Ala-Gly-OMe Percentage Yield = 90 % R f 0.83 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (Ar-C=C), , (Ar-CH), (-C=O amide), , (-NH 2 amide str), (-NH 1 amide str), (-NH 2 amide bend), (-C-O ether, str), (CH in CH 3 str), (CH in CH 3 bend). (76) L-Ala-Gly-L-Tyr-OMe Percentage Yield = 80 % R f 0.44 (77) Glycine-L-Leucine-L-Proline-OMe Percentage Yield = 88 % R f 0.66 (78) L-Leucine-L-Isoleucine-N(Me)-Phenylalaine-OMe Percentage Yield = 76 % R f 0.55

31 SYNTHESIS OF BOC-TETRAPEPTIDE METHYL ESTERS General method for tetrapeptide methyl ester Dipeptide methyl ester (0.01 mol) were dissolved in DMF (25 ml) and pyridine (0.021 mol) was added to the above solution in proportions. Finally the reaction mixture was stirred for 30 minutes, while maintaining the temperature between 0 5 C. Boc-dipeptides (0.01 mol) were dissolved in DMF (35 ml) and EDC.HCl (1.92 g, 0.01 mol) and HOBt (1.34 g, 0.01 mol) were added in proportions while stirring. Stirring was first done for 1 h at 0 5 C and then further for 24 h at room temperature (RT). After the completion of the reaction, the reaction mixture was diluted with an equal amount of water. The precipitated solid was filtered, washed with water and recrystallized from a mixture of chloroform and petroleum ether (b.p C), followed by cooling at 0 C to get Boc-tetrapeptide methyl ester. (79) Boc-L-prolyl-L-leucyl-L-alanyl-L-isoleucine methyl ester The linear tetrapeptide unit (2.63 g, mol) was deprotected at the carboxyl terminal using lithium hydroxide (LiOH, 0.18 g, mol) to obtain Boc-l-prolyl-l-leucyl-l-alanyl-lisoleucine-OH. Percentage Yield 91%; [ ] D 55.1 (c, 0.25 in MeOH); Anal: TLC (Chloroform: Methanol, 9.5:0.5, v/v): R f : IR (CHCl 3 /KBr, v cm -1 ): (N H str, amide), (C H str, CH 2, Pro), 2968, 2929, 2925 (C H str, asym, CH 3 and CH 2 ), (C H str, sym, CH 3 ), 1745 (C=O str, ester), 1676, (C=O str, tert and sec amide), 1538, 1535 (N H def, sec amide), 1392, 1375 (C H def, tert- Butyl), 1386, 1368 (C H def, iso-propyl), 1272 (C O str, ester). 1 H/ NMR (CDCl 3, δ ppm): 8.48 (1H, br. s, NH), 7.45 (1H, br. s, NH), 5.14 (1H, br. s, NH), 4.52 (1H, q, H-, Leu), 4.25 (1H, q, H-, Ala), 4.14 (1H, t, J = 7.2 Hz, H-, Pro), 3.73 (1H, t, J = 6.35 Hz, H-, Ile), 3.49 (3H, s, OCH 3 ), 3.43 (2H, t, J = 7.25 Hz, H-, Pro), 2.49 (2H, q, H-, Pro), (1H, m, H-, Ile), (2H, m, H-, Pro), (4H, m, H-, Leu and H-, Ile), 1.48 (9H, s, tert-butyl), (1H, m, H-, Leu), 1.29 (3H, d, J = 7.2 Hz, H-, Ala), 0.99 (6H, d, J = 6.4 Hz, H-, Leu), 0.93 (3H, t, J = 7.15 Hz, H-, Ile), 0.89 (3H, d, J = 6.45 Hz, H-, Ile).

32 13 C NMR (CDCl 3, δ ppm): (C=O, Leu), 172.9, (2C, C=O, Pro and Ile), (C=O, Ala), (C=O, Boc), 79.8 (C-, tert-butyl), 59.7 (C-, Pro), 58.5 (C-, Ile), 54.7 (OCH 3 ), 51.3 (C-, Ala), 49.7 (C-, Leu), 46.9 (C-, Pro), 37.6, 37.1 (2C, C-, Ile and Leu), 28.9 (C-, Pro), 28.2 (3C, C-, tert-butyl), 24.2, 23.9 (2C, C-, Ile and Leu), 23.7 (C-, Pro), 22.3 (2C, C-, Leu), 17.9 (C-, Ala), 15.1 (C-, Ile), 9.5 (C-, Ile). (80) tert-butyloxycarbonyl-l- Proline-L-Leucine-L-Proline -L-Phenylalanine methyl ester Percentage Yield = 94 % R f 0.79 Analysis IR (KBr) Vmax (cm -1 ): 3128, (N H str ), (C H str ), 2966, 2926, 2922, 2872 (C H str ), 1745 (C=O str ), 1679, (C=O str ), 1545, 1411 (C=C), (N H def ), 1397, 1376 (C H def ), 1385, 1364 (C H def ), 1276 (C O str ), 727, 686 (C H def ). 1 H NMR (CDCl3) d (ppm): 8.92 (br. s, 1H, NH), 8.89 (br. s, 1H, NH), 7.23 (t, J=5.7 Hz, 1H, H-p, Phe), (m, 2H, H-m, Phe), 6.88 (dd, J=7.1 Hz, 2H, H-o, Phe), 5.03 (t, J=5.6 Hz, 1H, H-, Phe), 4.59 (q, 1H, H-, Leu), 4.05 (t, J=7.2 Hz, 1H, H-, Pro-2), 3.89 (t, J=7.1 Hz, 1H, H-, Pro-1), 3.57 (s, 3H, OCH 3 ), 3.35 (t, J=7.2 Hz, 2H, H-, Pro-2), 3.22 (t, J=7.2 Hz, 2H, H-, Pro-1), 2.99 (d, J=4.8 Hz, 2H, H-, Phe), 2.69 (q, 2H, H-, Pro-2), 2.48 (q, 2H, H-, Pro-1), (m, 4H, H-, Pro-2 and Pro-1), (m, 3H, H- and H-, Leu), 1.49 (s, 9H, tert-butyl), 0.98 (d, J=6.4 Hz, 6H, H-, Leu). 13 C NMR (CDCl 3, δ ppm): 173.9, (2C, C=O, Pro-1 and Pro-2), 169.9, (2C, C=O, Phe and Leu), (C=O, Boc), (C-, Phe), (2C, C-o, Phe), (2C, C-m, Phe), (C-p, Phe), 79.9 (C-, tert-butyl), 59.9 (C-, Pro-1), 55.7 (C-, Pro-2), 53.8 (C-, Phe), 52.5 (OCH 3 ), 49.3 (C-, Leu), 47.2, 46.8 (2C, C-, Pro-2 and Pro-1), 37.9, 37.5 (2C, C-, Phe and Leu), 30.7 (3C, C-, tert-butyl), 28.7, 26.5 (2C, C-, Pro-1 and Pro-2), 24.2, 23.9, 23.5 (3C, C-, Pro-2, Leu and Pro-1), 22.1 (2C, C-, Leu). (81) Boc -L-Leucyl-L-Prolyl-L-Tyrosinyl-L-Alanine methyl ester Semisolid mass; yield, 84%; R f 0.87; [D] (c 0.25 in MeOH);

33 IR (CHCl3) v cm-1: 3369 (m, O-H str, Tyr), 3139, 3133 (m, N-H str, 2 amide), 3064 (w, C-H str, ring), (m, C-H str, Pro), 2956, 2870 (m, C-H str, asym and sym, CH3), 2922, 2856 (m, C-H str, asym and sym, CH2), 1753 (s, C=O str, ester), 1658, (s, C=O str, 3 and 2 amide), 1572, 1479 (m, skeletal bands, ring), (m, N-H bend, 2 amide), 1395, 1373 (s, C-H bend, butyl-t), 1383, 1362 (s, C-H def, propyl-i), 1272 (s, C-O str, ester), 1234 (s, C-O str, phenolic), 933, 921 (w, CH3 rock, butyl-t and propyl-i), 829 (s, C-H bend, oop, ring); 1 H NMR (CDCl3) d (ppm): (m, 2H, H-m, Tyr), 6.94 (br. s, 1H, NH, Ala), 6.92 (dd, 2H, J = 7.3 Hz, H-o, Tyr), 6.54 (br. s, 1H, NH, Tyr), 5.97 (br. s, 1H, OH, Tyr), 5.89 (br. s, 1H, NH, Leu), 4.97 (q, 1H, H-a, Tyr), 4.47 (t, 1H, J = 6.8 Hz, H-a, Pro), 4.21 (q, 1H, H-a, Leu), (m, 1H, H-a, Ala), 3.69 (t, 2H, J = 7.3 Hz, H-d, Pro), 3.60 (s, 3H, OCH3), 2.89 (d, 2H, J = 4.0 Hz, H-b, Tyr), 2.67 (q, 2H, H-b, Pro), (m, 4H, H-g, Pro and H-b, Leu), (m, 1H, H-g, Leu), 1.52 (s, 9H, butyl-t), 1.28 (d, 3H, J = 7.8 Hz, H-b, Ala), 1.18 (d, 6H, J = 6.2 Hz, H-d, Leu); Anal. Calc. for C29H44N4O8 (576): C, 60.40; H, 7.69; N, Found: C, 60.38; H, 7.72; N, 9.70%. (82) tert-butyloxycarbonyl-glycine-l-serine-l-serine-l-leucine methyl ester Yield 88%; [ ] D 78.4 (c, 0.25 in MeOH); R f 0.79; IR (CHCl 3 ), v cm 1 : 3334 (m/br, -OH str, Ser), (m, -NH str, amide), 2876 (m, -CH str, sym, CH 3 ), 2927, 2922 (m, -CH str, asym, CH 2 ), 2857 (m, -CH str, sym, CH 2 ), 1744 (s, -C=O str, ester), (s, -C=O str, 2 amide), , (m, -NH bend, 2 amide), 1393, 1369 (s, -CH bend, tert-butyl), 1386, 1362 (s, -CH bend, iso-propyl), 1267 (s, C O str, ester), 922 (w, CH 3 rocking, tert-butyl), 666 (m/br, -OH def, oop, Ser). 1 H-NMR (DMSO-d 6 ) : 8.42 (1H, br. s, NH), 8.04 (1H, br. s, NH), 6.98 (1H, br. s, NH), 6.84 (1H, br. s, NH), 6.72 (2H, br. s, OH), (1H, m, H-α, Ser-1), (1H, m, H-α, Ser-2), (4H, m, H-, Ser-1 & Ser-2), 3.62 (3H, s, OCH 3 ), 3.54 (2H, d, J=5.2 Hz, H-α, Gly), (1H, m, H-α, Leu), 1.52 (9H, s, tert-butyl), (3H, m, H- & H-γ, Leu), (6H, d, J=6.2 Hz, H-δ, Leu). Anal. Calcd for C 20 H 36 N 4 O 9 : C, 50.41; H, 7.61; N, Found: C, 50.40; H, 7.64 N, (83) tert-butyloxycarbonyl-l-phenylalanyl-l-prolyl-l-prolyl-l-tyrosine methyl ester: Semisolid mass; Yield 79%; [α]d 69.2 ; R f 0.59;

34 IR (CHCl3): v 3,372 (O H str, Tyr), 3,123, 3,119 (N H str, amide), 3,069, 3,055 (C H str, rings), 2,999 2,987 (C H str, CH2, Pro), 2,929, 2,925 (C H str, asym, CH2), 2,875, 2,852 (C H str, sym, CH3 and CH2), 1,748 (C=O str, ester), 1,682 1,678, 1,645, 1,639 (C=O str, 3 and 2 amide), 1,588, 1,483 (skeletal bands), 1,539, 1,532 (N H bend, 2 amide), 1,389, 1,374 (C H bend, tert-butyl), 1,266 (C O str, ester), 1,230 (C O str, phenolic), 828, 725, 689 (C H bend, out-of-plane (oop), rings) cm 1; 1H-NMR (CDCl3): δ 8.65 (1H, br. s, NH), 7.50 (2H, t, J = 7.2 Hz, 4.4 Hz, H at C-3 and C-5, Phe), 6.94 (1H, t, J = 6.2 Hz, H at C-4, Phe), 6.90 (2H, d, J = 8.5 Hz, H at C-2 and C-6, Tyr), 6.85 (2H, d, J = 8.8 Hz, 5.4 Hz, H at C-2 and C-6, Phe), 6.79 (2H, d, J = 8.6 Hz, H at C-3 and C-5, Tyr), 6.44 (1H, br. s, NH), 5.95 (1H, br. s, OH), 5.06 (1H, q, J = 7.9 Hz, H-α, Tyr), 4.60 (1H, q, J = 5.5 Hz, H-α, Phe), 4.55 (1H, t, J = 6.9 Hz, H-α, Pro-1), 4.39 (1H, t, J = 6.9 Hz, H-α, Pro-2), 3.68 (4H, m, H-δ, Pro-1 and Pro-2), 3.53 (3H, s, OCH3), 2.99 (4H, m, H-β, Phe and Tyr), 2.67 (4H, m, H-β, Pro-1 and Pro-2), 1.92 (4H, m, H-γ, Pro-1 and Pro-2), 1.55 (9H, s, tert-butyl); Anal. Calcd. for C34H44N4O8: C, 64.14; H, 6.96; N, Found: C, 64.15; H, 6.99; N, 8.79%. (84)tert-Butyloxycarbonyl-phenylalanyl-N-methylglycyl-prolyl-N-methyl- phenylalanine methyl ester Percentage Yield: 79.2%, R f : 0.48 IR (CHCl3): 3136 (m, -NH str, amide), 2999, 2996 (m, -CH str, cyclic CH2, Pro), 2927, 2622, (m, -CH str, asym and sym, CH2), 2870, 2866 (m, -CH str, sym, CH3), 1742 (s, -C=O str, ester), , 1642 (s, -C=O str, 3 and 2 amide), 1587, 1479 (m, skeletal bands, arom. rings), 1532 (m, -NH bend, 2 amide), 1391, 1376 (s, -CH bend, tert-butyl group), 1270 (s, CO str, ester), 933 (w, CH3 rocking, tert-butyl group), , 690, 687 (s, -CH bend, out-ofplane, arom. ring) cm-1; 1 H NMR (300 MHz, CDCl3): (2H, tt, J = 6.8, 4.45 Hz, m-h s, Phe-1), (1H, t, J = 6.2 Hz, p-h, Phe- 2), (2H, tt, J = 6.75, 4.5 Hz, m-h s, Phe-2), (1H, t, J = 6.25 Hz, p-h, Phe-1), (2H, dd, J = 8.8, 4.15 Hz, o-h s, Phe-1), (2H, dd, J = 8.75, 4.2 Hz, o-h s, Phe-2), 6.51 (1H, br. s, -NH, Phe-1), (1H, t, J = 5.2 Hz, a-h, Phe-2), (1H, q,

35 J = 5.55 Hz, a- H, Phe-1), (1H, t, J = 6.9 Hz, a-h, Pro), 3.89 (2H, s, a-h, Gly), (2H, t, J = 7.15 Hz, δ-h s, Pro), 3.54 (3H, s, OCH3), (4H, m, b-h s, Phe-1 and Phe-2), 2.99 (3H, s, NCH3, Phe), 2.94 (3H, s, NCH3, Gly), (2H, q, b- H s, Pro), (2H, m, γ-h s, Pro), 1.52 (9H, s, tert-butyl group) ppm.

36 DEPROTECTION OF TETRAPEPTIDE AT CARBOXYL END General Procedure for synthesis of deprotected tetrapeptides To a solution of Boc-tetrapeptide ( 0.01 mol) dissolved in THF/H 2 O (1:1, 36 ml), LiOH (0.36 g, mol) was added at 0 C. The mixture was stirred at r.t. for 1 hr and then acidified to ph 3.5 with 0.5 mol L 1 H 2 SO 4. The aqueous layer was extracted with Et 2 O (3 x 25 ml). Combined organic extracts were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was crystallized from methanol and ether to get deprotected Boctetrapeptide. (85) tert-butyloxycarbonyl-phenylalanyl-n-methylglycyl-prolyl-n-methyl-phenylalanine- OH Percentage Yield:84%, R f : 0.88

37 DEPROTECTION OF TETRAPTIDE AT AMINO END Tetrapeptide compounds (10 mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of TFA(20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from mixture of chloroform and light petroleum ether(b.p 40-60ºC) to give deprotected compound. (86)Glycine-L-Serine-L-Serine-L-Leucine methyl ester: Percentage Yield: 90.2%, R f : 0.50 (87)L-phenylalanyl-L-prolyl-L-prolyl-L-tyrosine methyl ester: Percentage Yield: 89.2%, R f : 0.69 (88) L-leucyl-L-prolyl-L-tyrosinyl-L-alanine methyl ester Percentage Yield: 94%, R f : 0.75

38 SYNTHESIS OF CYCLOTETRAPEPTIDE METHYL ESTERS Procedure for the synthesis of cyclotetrapeptide: (89) Cyclo(-L-isoleucyl-L-prolyl-L-leucyl-L-lalanyl-) To a solution of the deprotected tetrapeptide (2.56 g, mol) in CHCl 3 (50 ml), pentafluorophenol (1.23 g, mol) and EDC.HCl (0.96 g, mol) were added followed by stirring at RT for 12 h. Filtrate of the above reaction mixture was washed with 10% NaHCO 3 (3 20 ml) and 5% HCl (2 20 ml) solutions to obtain corresponding pentafluorophenyl ester Boc-L-prolyl-L-leucyl-L-alanyl-L-isoleucine-Opfp. Boc-group of resulting unit (2.71 g, mol) was removed using TFA (0.91 g, mol) and deprotected product was dissolved in CHCl 3 (25 ml) and TEA/NMM/pyridine (2.8 ml or 2.21 ml or 1.61 ml, mol) was added. Whole contents were then kept at 0 C for 7 days. The reaction mixture was washed with 10% NaHCO 3 (3 25 ml) and 5% HCl (2 25 ml) solutions. The organic layer was dried over anhydrous Na 2 SO 4 and the crude cyclized compound was recrystallized from CHCl 3 /n-hexane to obtain the pure cyclic product. Percentage Yield: 74%, R f : 0.62 Analysis: IR (CHCl 3 /KBr, v cm -1 ): 3128, 3123, 3119 (N H str, amide), 2998, (C H str, CH2, Pro), 2969, (C H str, asym, CH3 and CH2), 2879, 2875 (C H str, sym, CH3), 1675, (C=O str, tert and sec amide), (N H def, sec amide), 1385, 1369 (C H def, isopropyl) 1 H NMR (CDCl 3, δ ppm): 9.37 (1H, br. s, NH), 9.31 (1H, br. s, NH), 7.76 (1H, br. s, NH), 4.26 (1H, t, J = 7.15 Hz, H-, Pro), 3.99 (1H, q, H-, Ala), 3.95 (1H, q, H-, Leu), 3.61 (1H, t, J = 6.4 Hz, H-, Ile), 3.52 (2H, t, J = 7.3 Hz, H-, Pro), 2.35 (2H, q, H-, Pro), (2H, m, H-, Pro), (1H, m, H-, Leu), (1H, m, H-, Ile), (2H, m, H-, Leu), (2H, m, H-, Ile), 1.45 (3H, d, J = 7.25 Hz, H-, Ala), 0.98 (6H, d, J = 6.35 Hz, H-, Leu), 0.95 (3H, d, J = 6.45 Hz, H-, Ile), 0.89 (3H, t, J = 7.2 Hz, H-, Ile) 13 C NMR (CDCl 3, δ ppm): (C=O, Leu), (C=O, Ala), 170.5, (2C, C=O, Pro and Ile), 63.3 (C-, Ile), 57.5 (C-, Pro), 54.1 (C-, Leu), 49.9 (C-, Ala), 46.5 (C-, Pro), 43.8, 39.9 (2C, C-,

39 Leu and Ile), 29.9 (C-, Pro), 24.8, 24.2 (2C, C-, Ile and Leu), 22.0 (2C, C-, Leu), 21.3 (C-, Pro), 19.2 (C-, Ala), 15.4 (C-, Ile), 9.8 (C-, Ile). ESIMS/MS (rel. int., m/z): [(M + H)+, 100], [(395.4 CO)+, 11], [(H-Ile-Pro-Leu)+, 32], [(H-Leu-Ala-Ile)+, 39], [(324.4 CO)+, 10], [(H-Ala-Ile-Pro)+, 29], [(298.4 CO)+, 17], [(282.3 CO)+, 15], [(H-Ile-Pro)+, 19], [(H-Leu-Ala)+, 76], [(185.2 CO)+, 12], [(H-Leu)+, 19], 86.1 [Ile/Leu (C5H12N)+, 16], 70.1 [Pro (C4H8N)+, 10], 57.1 [(C4H9)+, 15], 44.1 [Ala (C2H6N)+, 12], 43.1 [(C3H7)+, 11], 29.1 [(C2H5)+, 9], 15.0 [(CH3)+, 13] (90) Cyclo(L-prolyl-L-leucyl-L-prolyl-L-phenylalanine-) To a solution of the deprotected tetrapeptide (0.005 mol) in CHCl 3 (50 ml), pentafluorophenol (1.23 g, mol) and EDC.HCl (0.96 g, mol) were added followed by stirring at RT for 12 h. Filtrate of the above reaction mixture was washed with 10% NaHCO 3 (3 20 ml) and 5% HCl (2 20 ml) solutions to obtain corresponding pentafluorophenyl ester Boc-L-prolyl-L-leucyl-L-alanyl-L-isoleucine-)Opfp. Boc-group of resulting unit (0.004 mol) was removed using TFA (0.91 g, mol) and deprotected product was dissolved in CHCl 3 (25 ml) and TEA/NMM/pyridine (2.8 ml or 2.21 ml or 1.61 ml, mol) was added. Whole contents were then kept at 0 C for 7 days. The reaction mixture was washed with 10% NaHCO 3 (3 25 ml) and 5% HCl (2 25 ml) solutions. The organic layer was dried over anhydrous Na 2 SO 4 and the crude cyclized compound was recrystallized from CHCl 3 /n-hexane to obtain the pure cyclic product. Percentage Yield: 79%, R f : 0.58 Analysis: IR (CHCl 3 /KBr, v cm -1 ): , 3122 (N H str ), (C H str ), 2968, 2927, 2921, 2876 (C H str ), 1677, (C=O str ), 1541, 1414 (C=C), (N H def ), 1388, 1367 (C H def ), 729, 685 (C H def ). 1 H NMR (CDCl 3, δ ppm): 9.72 (br. s, 1H, NH), 9.21 (br. s, 1H, NH), 7.39 (tt, J=5.6 Hz, 7.2 Hz, 2H, H-m, Phe), 7.32 (t, J=5.6 Hz, 1H, H-p, Phe), 7.18 (dd, J=7.2 Hz, 2H, H-o, Phe), 4.13 (q, 1H, H-, Leu), 4.06 (t,

40 J=5.7 Hz, 1H, H-, Phe), 4.28 (t, J=7.2 Hz, 1H, H-, Pro-1), 2.62 (t, J=6.5 Hz, 1H, H-, Pro-2), 3.49 (t, J=7.2 Hz, 2H, H-, Pro-2), 3.33 (t, J=7.1 Hz, 2H, H-, Pro-1), 3.17 (d, J=4.9 Hz, 2H, H-, Phe), 2.29 (q, 2H, H-, Pro-2), 1.95 (q, 2H, H-, Pro-1), 1.87 (t, 2H, H-, Leu), (m, 2H, H-, Pro-1), (m, 1H, H-, Leu), (m, 2H, H-, Pro-2), 0.95 (d, J=6.5 Hz, 6H, H-, Leu). 13 C NMR (CDCl 3, δ ppm): 171.1, (2C, C=O, Pro-1 and Pro-2), 168.0, (2C, C=O, Leu and Phe), (C-, Phe), (2C, C-m, Phe), (2C, C-o, Phe), (C-p, Phe), 58.6 (C-, Pro-1), 58.2 (C-, Phe), 57.1 (C-, Pro-2), 52.7 (C-, Leu), 46.1, 44.9 (2C, C-, Pro-2 and Pro-1), 39.2, 37.6 (2C, C-, Phe and Leu), 28.8, 27.1 (2C, C-, Pro-2 and Pro-1), 23.9 (C-, Leu), 21.8 (2C, C-, Leu), 21.4, 20.8 (2C, C-, Pro-1 and Pro-2). ESIMS/MS (rel. int., m/z): 455 [(M+H) +, 100], 427 [(455 CO) +, 16], 358 [(H-Leu-Pro-Phe) +, 71], 342 [(H-Pro-Phe- Pro) +, 38], 330 [(358 CO) +, 11], 314 [(342 CO) +, 14], 308 [(H-Pro-Leu-Pro) +, 28], 280 [(308 CO) +, 11], 245 [(H-Phe-Pro) +, 44], 217 [(245 CO) +, 9], 211 [(H-Leu-Pro) +, 49], 183 [(211 CO) +, 14], 148 [(H-Phe) +, 19], 120 [Phe immonium ion (C 8 H 10 N) +, 13], 114 [(H-Leu) +, 18], 98 [(H-Pro) +, 16], 91 [(C 7 H 7 ) +, 9], 86 [Leu immonium ion (C 5 H 12 N) +, 15], 77 [(C 6 H 5 ) +, 10], 70 [Pro immonium ion (C 4 H 8 N) +, 19], 57 [(C 4 H 9 ) +, 8], 43 [(C 3 H 7 ) +, 11], 15 [(CH 3 ) +, 12].

41 SYNTHESIS OF BOC-PENTAPEPTIDE METHYL ESTERS (91) Boc-L-Alanine-L-Phenylalanine-L-Alanine-Glycine-L-Tyrosine methyl ester The dipeptide Boc-L-Alanine-L-Phenylalanine-OH(45), after deprotection at carboxyl terminal, was coupled with deprotected tripeptide unit (76) using DIPC and NMM to get linear pentapeptide unit Boc-L-Ala-L-Phe-L-Ala-Gly-L-Tyr-OMe. Percentage Yield: 88%: R f: 0.66

42 SYNTHESIS OF CYCLOPENTAPEPTIDE (92) Cyclo(-L-Alanine-L-Phenylalanine-L-Alanine-Glycine-L-Tyrosine-) Analysis: IR (KBr), v (cm 1 ): 3372 (m/br, -OH str, Tyr), 3309, (m, -NH str, amide), (w, -CH str, arom. rings), 2955, 2952, 2876 (m, -CH str, asym and sym, CH 3 ), 2926, 2851, 2844 (m, -CH str, asym and sym, CH 2 ), (s, -C=O str, 2 amide), 1577, 1424 (m, skeletal bands, arom. rings), (m, -NH bend, 2 amide), 1230 (s, C O str, phenolic), 832, 724, 688 (s, -CH bend, oop, arom. rings); 1 H/ 13 C NMR (300 MHz, pyridine-d 5 ), δ (10 6 ): Ala-1 ( 1 H): 9.44 (1H, br. s, -NH, Ala-1), 4.61 (1H, m, α-h, Ala-1), 1.53 (3H, d, J=7.2 Hz, β-h s, Ala-1), Ala-1 ( 13 C): (C=O, Ala-1), 51.9 (α-c, Ala-1), 17.7 (β-c, Ala-1), Phe ( 1 H): 9.26 (1H, br. s, -NH, Phe), 4.84 (1H, m, α-h, Phe), 3.48 (2H, d, J=5.6 Hz, β-h s, Phe), 7.35 (2H, d, J=7.3 Hz, o-h s, Phe), 7.26 (2H, t, J=7.4 Hz, m-h s, Phe), 7.23 (1H, t, J=6.3 Hz, p- H, Phe) Phe ( 13 C): (C=O, Phe), 57.9 (α-c, Phe), 37.3 (β-c, Phe), (γ-c, Phe), (o-c s, Phe), (m-c s, Phe), (p-c, Phe), Ala-2 ( 1 H): 9.49 (1H, br. s, -NH, Ala-2), 4.55 (1H, m, α-h, Ala-2), 1.59 (3H, d, J=7.1 Hz, β-h s, Ala-2), Ala-2 ( 13 C): (C=O, Ala-2), 50.7 (α-c, Ala-2), 17.1 (β-c, Ala-2), Gly ( 1 H): 9.71 (1H, br. s, -NH, Gly), 3.85 (2H, d, J=5.1 Hz, α-h s, Gly), Gly ( 13 C): (C=O, Gly), 44.8 (α-c, Gly) Tyr ( 1 H): 8.98 (1H, br. s, -NH, Tyr), 5.12 (1H, m, α-h, Tyr), 3.47 (2H, d, J=6.6 Hz, β-h s, Tyr), 7.25 (2H, d, J=8.4 Hz, o-h s, Tyr), 7.11 (2H, d, J=8.5 Hz, m-h s, Tyr), Tyr ( 13 C): (C=O, Tyr), 55.9 (α-c, Tyr), 37.1 (β-c, Tyr), (γ-c, Tyr), (o-c s, Tyr), (m-c s, Tyr), (p-c, Tyr), ESI MS/MS: FRAGMENTATION PATTERN OF SYNTHESIZED CYCLOPENTAPEPTIDE (6) 1) Breakage at Ala-Phe amide bond level

43 510 [(M+H) +, 100], 482 [(510-CO) +, 11], 439 [(Phe-Ala-Gly-Tyr) +, 29], 411 [(439-CO) +, 16], 276 [(Phe-Ala-Gly) +, 73], 248 [(276-CO) +, 10], 219 [(Phe-Ala) +, 58], 191 [(219-CO) +, 15], 148 [(Phe) +, 19], 2) Breakage at Gly-Tyr amide bond level 453 [(Tyr-Ala-Phe-Ala) +, 33], 425 [(453-CO) +, 14], 382 [(Tyr-Ala-Phe) +, 34], 235 [(Tyr-Ala) +, 28], 3) Breakage at Tyr-Ala amide bond level 347 [(Ala-Phe-Ala-Gly) +, 22], 290 [(Ala-Phe-Ala) +, 28], 262 [(290-CO) +, 14], 4) Immonium ions 136 [Tyr:C 8 H 10 NO +, 13], 120 [Tyr:C 8 H 10 N +, 10], 44 [Ala:C 2 H 6 N) +, 19], 30 [(Gly:CH 4 N) +, 11], 5) Other fragment ions Tyr: 107 [C 7 H 7 O +, 14], 93 [C 6 H 5 O +, 10], Phe: 91 [C 7 H + 7, 19], 77 [C 6 H + 5, 13], Ala: 15 [CH + 3, 11],

44 SYNTHESIS OF LINEAR HEXAPEPTIDE METHYL ESTERS Procedure for synthesis of linear hexapeptide 5.25 g (10 mmol) of Boc-L-Tyr-L-Phe-L-Pro-OH was dissolved in DMF (25 ml) and solution was neutralized with 2.8 ml (21 mmol) of TEA at 0 C and the resulting mixture was stirred for 15 min. 3.0 g (10 mmol) of Gly-L-Leu-L-Pro-OMe was dissolved in DMF (25 ml) and resulting solution along with 2.1 g/1.26 g (0.01 mol) of DCC / DIPC were added to above mixture. Stirring was first done for 1 h at 0 to 5 C and then further for 35 h at room temperature (RT). After the completion of reaction, the reaction mixture was diluted with equal amount of water. The precipitated solid was filtered, washed with water and recrystallized from a mixture of chloroform and petroleum ether (b.p C) followed by cooling at 0 C to get Boc-L-Tyr- L-Phe-L-Pro-Gly-L-Leu-L-Pro-OMe 6 as pale-yellow semisolid mass. (93) tert-butyloxycarbonyl-l-tyrosinyl-l-phenylalanyl-l-prolyl-glycyl-lleucyl- L-proline methyl ester : Yield 76%; [a]d 55.8 (c, 0.15 in MeOH); R f 0.88; IR (CHCl3), v cm 1: 3378 (m/br, -OH str, Tyr), (m, -NH str, amide), 3067, 3059 (w, -CH str, ring), (m, -CH str, Pro), 2962 (m, -CH str, asym, CH3), 2877 (m, -CH str, sym, CH3), 2928, 2925 (m, -CH str, asym, CH2), 2854 (m, -CH str, sym, CH2), 1749 (s, -C=O str, ester), 1666, (s, -C=O str, 3 and 2 amide), , (m, skeletal bands, rings), , 1529, 1523 (m, -NH bend, 2 amide), 1392, 1368 (s, -CH bend, tert-butyl), 1386, 1362 (s, -CH bend, iso-propyl), 268 (s, C O str, ester), 1233 (s, C O str, phenolic), 927 (w, CH3 rocking, tert-butyl), 829, 722, 687 (m, -CH def, oop, rings). 1 H-NMR (DMSO-d6) d: 9.45 (1H, br. s, NH), 8.65 (1H, br. s, NH), 7.78 (1H, br. s, NH), 7.32 (2H, dd, J=8.65, 4.9 Hz, H-m, Tyr), 7.19 (2H, tt, J=6.8, 4.45 Hz, H-m, Phe), 7.02 (1H, t, J=6.3 Hz, H-p, Phe), 6.94 (2H, dd, J=8.6, 5.25 Hz, H-o, Tyr), 6.83 (2H, dd, J=8.75, 4.2 Hz, Ho, Phe), 6.59 (1H, br. s, NH), 5.97 (1H, br. s, OH), 4.55 (1H, q, J=7.85 Hz, H-_, Tyr), 4.42 (1H, q, J=5.5 Hz, H-_, Phe), 4.12 (1H, q, J=6.65 Hz, H-_, Leu), 4.05 (1H, t, J=6.9 Hz, H-_, Pro-1), 4.01 (2H, d, J=5.2 Hz, H-_, Gly), 3.89 (1H, t, J=6.85 Hz, H-_, Pro-2), 3.66 (3H, s, OCH3), 3.42 (2H, t, J=7.3 Hz, H-_, Pro- 2), 3.35 (2H, t, J=7.25 Hz, H-_, Pro-1), (4H, m, H-b, Phe and Tyr), (2H, m, H-b, Pro-1), (4H, m, H-b and H-_, Pro-2), (2H, m, H-_, Pro-1), 1.78 (2H, t,

45 J=7.9 Hz, H-b, Leu), 1.56 (9H, s, tert-butyl), (1H, m, H-_, Leu), (6H, d, J=6.2 Hz, H-_, Leu). Anal. Calcd for C42H58N6O10: C, 62.51; H, 7.24; N, Found: C, 62.49; H, 7.25; N, (94) Boc-L-Ile-L-Phe-L-Leu-L-Tyr-L-Ala-Gly-OMe To synthesize Boc-L-Ile-L-Phe-L-Leu-L-Tyr-L-Ala-Gly-OMe (94), Boc-L-Ile-L-Phe-L- Leu-OMe tripeptide unit (61) (4.8 g, 0.01 mol) was deprotected at carboxyl end to get Boc-L-Ile- L-Phe-L-Leu-OH (69). Boc-L-Tyr-L-Ala-Gly-OMe tripeptide unit (67) (4.2 g, 0.01 mol) was deprotected at amino end to get L-Tyr-L-Ala-Gly-OMe (75). The deprotected L-Tyr-L-Ala-Gly- OMe tripeptide unit (75) (3.2 g, 0.01 mol) and Boc-L-Ile-L-Phe-L-Leu-OH tripeptide unit (69) (4.6 g, 0.01 mol) were coupled in the presence of DCC and NMM to get linear hexapeptide unit (94) under same the experimental conditions as adopted for the synthesis of Boc-dipeptide methyl esters. Percentage Yield = 79 % R f 0.85 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (Ar-C=C), (Ar-CH), (-C=O amide), , (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether, str), , (CH in CH 3 str), (CH in CH 3 bend).

46 SYNTHESIS OF CYCLOHEXAPEPTIDE METHYL ESTERS Cyclization of linear hexapeptide To synthesize linear hexapeptide unit (4.0 g, 5 mmol) was deprotected at carboxyl end using lithium hydroxide (0.18 g, 7.5 mmol) to get Boc-L-Tyr-L-Phe-L-Pro-Gly-L-Leu-L-Pro-OH (93). The deprotected hexapeptide unit (3.97 g, 5 mmol) was now dissolved in chloroform (35 ml) at 0 C. To the above solution, DIPC (0.63 g, 5 mmol) and p-nitrophenol / pentafluorophenol (0.94 g/1.23 g, 6.7 mmol) were added and stirring was done at RT (12 h). The reaction mixture was filtered and the filtrate was washed with 10% NaHCO3 (3 15 ml) and 5% HCl (2 10 ml) to get the corresponding pnitrophenyl/ pentafluorophenyl ester Boc-L-Tyr-L-Phe-L-Pro-Gly-L- Leu-L-Pro-Opnp/pfp. To this compound (3.66 g/3.84 g, 4 mmol) dissolved in chloroform (25 ml), TFA (0.91 g, 8 mmol) was added, stirred at RT (1 h) and washed with 10 % NaHCO3 solution (2 25 ml). The organic layer was dried over anhydrous Na2SO4 to get L-Tyr-L-Phe-L- Pro-Gly-L-Leu-L-Pro-Opnp/pfp which was dissolved in chloroform (25 ml) and TEA/NMM/pyridine (2.8 ml/2.21 ml/1.61 ml, 21 mmol) was added. Then, whole contents were kept at 0 C (7 days). The reaction mixture was washed with sufficient quantity of 10% NaHCO3 solution until the by-product p-nitrophenol/ pentafluorophenol was removed completely and finally washed with 5 % HCl (3 25 ml). The organic layer was dried over anhydrous Na 2 SO 4. Finally, chloroform was distilled off to get the crude cyclized product. Purification of crude compound was done by dissolving it in chloroform, filtering repeatedly and finally adding nhexane dropwise in filtered cooled solution to get pure cyclic product 7 as white solid. (95) Cyclo(-L-tyrosinyl-L-phenylalanyl-L-prolyl-glycyl-L-leucyl-L-prolyl-): Yield 86% (NMM), 71% (TEA), 63% (C5H5N); [a]d 10.1 (c, 0.1 in MeOH); R f 0.62; m.p. 202 C (d); IR (KBr), v cm 1: 3377 (m/br, - OH str, Tyr), 3139, 3135, 3128 (m, -NH str, amide), 3069, 3052 (w, -CH str, rings), (m, -CH str, Pro), 2964, 2875 (m, -CH str, asym and sym, CH3), 2929, 2922 (m, -CH str, asym, CH2), 2857 (m, -CH str, sym, CH2), 1668, (s, -C=O str, 3 and 2 amide), , (m, skeletal bands, rings), 1539, 1534, (m, - NH bend, 2 amide), 1384, 1359 (s, -CH bend, iso-propyl), 1236 (s, C O str, phenolic), 825, 727, 684 (m, - CH def, oop, rings). 1 H-NMR (DMSO-d6):

47 9.89 (1H, br. s, NH), 9.15 (1H, br. s, NH), 7.59 (1H, br. s, NH), 7.38 (1H, br. s, NH), 7.17 (2H, tt, J=6.85, 4.5 Hz, H-m, Phe), 7.04 (1H, t, J=6.25 Hz, H-p, Phe), 6.97 (2H, dd, J=8.7, 4.9 Hz, H-m, Tyr), 6.91 (2H, dd, J=8.65, 5.3 Hz, H-o, Tyr), 6.84 (2H, dd, J=8.8, 4.2 Hz, H-o, Phe), 6.30 (1H, q, J=6.7 Hz, H-, Leu), 5.95 (1H, br. s, OH), 5.74 (1H, q, J=5.45 Hz, H-, Phe), 4.22 (1H, q, J=7.9 Hz, H-, Tyr), 3.95 (2H, d, J=5.15 Hz, H-, Gly), 3.92 (1H, t, J=6.9 Hz, H-, Pro-2), 3.88 (1H, t, J=6.85 Hz, H-, Pro-1), (4H, m, H-, Pro-2 and Pro-1), (4H, m, H- b, Pro-1 and Pro-2), (4H, m, H-b, Phe and Tyr), 1.88 (2H, t, J=7.85 Hz, H-b, Leu), (4H, m, H-, Pro-1 and Pro-2), (6H, d, J=6.15 Hz, H-, Leu), (1H, m, H-, Leu); 13C NMR (DMSO-d6): (C=O, Leu), (C=O, Pro-1), (C=O, Pro-2), (C=O, Tyr), (C=O, Phe), (C=O, Gly), (C-p, Tyr), 137.2, (2C, C-g, Phe and Tyr), (2C, C-o, Phe), (2C, C-o, Tyr), (2C, C-m, Phe), (2C, C-m, Tyr), (C-p, phe-1), 62.2, 58.3 (2C, C-a, Pro-1 and Pro-2), 57.1, 53.9 (2C, C-a, Tyr and Phe), 49.7 (C-a, leu), 48.2, 47.7 (2C, C-d, Pro-1 and Pro-2), 44.5 (C-b, Leu), 41.9 (C-a, Gly), 39.7, 36.2 (2C, C-b, Tyr and Phe), 32.4, 29.6 (2C, C-b, Pro-2 and Pro-1), 27.3 (C-g, Leu), 24.8 (C-g, Pro-1), 22.5 (2C, C-d, Leu), 20.3 (C-g, Pro-2). ESI-MS/MS: (m/z, rel. int.) [Leu-Gly amide bond level]: [(M + H)+, 100], [(675.7 CO)+, 17], [(Leu-Pro-Tyr-Phe-Pro)+, 59], [(618.7 CO)+, 11], [(Leu-Pro-Tyr-Phe)+, 73], [(521.6 CO)+, 19], [(Leu-Pro-Tyr)+, 29], [(374.4 CO)+, 10], [(Leu-Pro)+, 17], [(211.3 CO)+, 9], [(Leu)+, 11]; [Breakage at Pro-Phe amide bond level]: [(Pro-Gly-Leu-Pro- Tyr)+, 36], [(528.6 CO)+, 9], [(Pro-Gly-Leu)+, 54], [(268.3 CO)+, 11]; [Breakage at Pro-Leu amide bond level]: [(Pro-Tyr-Phe- Pro-Gly)+, 22], [(562.6 CO)+, 17], [(Pro-Tyr-Phe-Pro)+, 14]; [Immonium ions]: Tyr: [(C8H10NO)+, 11]; Phe: [(C8H10N)+, 13]; Pro: 70.1 [(C4H8N)+, 9]; Leu: 86.1 [(C5H12N)+, 16]; Gly: 30.1 [(CH4N)+, 10]. Anal. Calcd for C36H46N6O7: C, 64.08; H, 6.87; N, Found: C, 64.06; H, 6.89; N, (96) Cyclo(-L-isoleucine-L-phenylalanyl-L-leucine-glycyl-L-leucyl-L-prolyl-): To synthesize cyclopeptide (L-Ile-L-Phe-L-Leu-L-Tyr-L-Ala-Gly) (94c), Boc-L-Ile-L- Phe-L-Leu-L-Tyr-L-Ala-Gly-OMe linear hexapeptide unit (94) (3.8 g, mol) was

48 deprotected at carboxyl end using LiOH (0.18 g, mol) to get Boc-L-Ile-L-Phe-L-Leu-L- Tyr-L-Ala-Gly-OH (94a). The deprotected hexapeptide unit (94a) (3.7 g, mol) was dissolved in CHCl 3 (50 ml) at 0 C. To the above solution, pnp (0.94 g, mol) was added and stirred at r.t. for 12 hr. The reaction mixture was filtered and the filtrate was washed with 10% NaHCO 3 solution (3 x 15 ml) until excess of pnp was removed and finally washed with 5% HCl (2 x 10 ml) to get the corresponding p-nitrophenyl ester Boc-L-Ile-L-Phe-L-Leu-L-Tyr-L- Ala-Gly-Opnp (94b). To compound (94b) (4.37 g, mol) dissolved in CHCl 3 (35 ml), TFA (0.91 g, mol) was added, stirred at r.t. for 1 hr and washed with 10% NaHCO 3 solution (2 x 25ml). The organic layer was dried over anhydrous Na 2 SO 4 to get L-Ile-L-Phe-L-Leu-L-Tyr-L- Ala-Gly-Opnp (94c), which was dissolved in CHCl 3 (25 ml) and NMM (2.3 ml, 0.021mol) was added. After that all contents were kept at 0 C for 7 days. The reaction mixture was washed with 10% NaHCO 3 solution until the byproduct p-nitrophenol was removed completely and finally washed with 5% HCl (3 x 15 ml). The organic layer was driedover anhydrous Na 2 SO 4. Finally, chloroform was distilled off and the crude cyclized product was crystallized from CHCl 3 and hexane to get the pure compound (96). Percentage Yield = 80 % Rf 0.90 Analysis IR (KBr) Vmax (cm -1 ): (-OH str), (Ar-C=C), (Ar-CH), (-C=O amide), (-NH 2 amide str), (-NH 2 amide bend), (-C-O ether, str), (CH in CH 3 str), (CH in CH 3 bend). UV λ max (CHCl 3 ) = H-NMR (CDCl 3 ) δ (ppm): Ile, Phe, Leu, Tyr, Ala, Gly(6H, br. s, NH , 8.121, 9.511, 9.514, 7.988, 9.515) Ile ( 3H: αh , βh-2.228,γh-1.500, Meδ ) Phe (5H: αh , βh-3.690, δh-7.422, εh , ζh-7.219) Leu (3H: αh , βh-1.901,γh-1.937, Meδ- 0.7) Tyr (3H: αh , βh ,δH-7.422) Ala (2H: αh , βh-1.901), Gly (1H: αh ). 13 C NMR (CDCl 3 ) δ (ppm): Ile, Phe, Leu (3C, C=O: , 172.5, ), Tyr, Ala, Gly (3C, C=O: , , ), Ile (3C: αc- 59.1, βc- 33.9, γc-25.6, Meγ-45.6, Meδ-11.7), Phe (6C: αc- 56.8, βc- 37.1, γc , δc-129.4, εc , ζc ), Leu (3C: αc- 54.9, βc , γc-25.0,

49 Meδ-22.18), Tyr (6C: αc- 57.5, βc- 36.5, γc-127.8, δc-131.9, εc , ζc ), Ala (2C: αc- 49.4, βc- 17.5), Gly (1C: αc- 45.7) SPECIFIC ROTATION [α] D , +60 (96) FAB MS m/z [RI]-Mass, Anal. Calcd. For: C 34 H 48 N 6 O 8 : [(M+1)+, 100], [(665.6 CO)+, 18], [(gly-ala-tyr-leu-phe)+, 29], [(tyr-leu-phe-ile)+, 25], [(537.7 CO)+, 13], [(gly-ala-tyr-leu)+, 32], [(phe-ilegly-ala)+, 17], [(405.4 CO)+, 11], [(gly-ala-tyr)+, 39], [(tyr-leu)+, 19], [(292.3 CO)+, 15], [(phe)+, 9], [(gly-ala)+, 24], [(129.1 CO)+, 8], 58.0 [(gly)+, 14], [(C 8 H 10 NO)+, 16], [(C 8 H 10 N)+, 13], 94.1 [(C 6 H 6 O)+, 19], 93.1 [(C 6 H 5 O)+, 11], 91.1 [(C 7 H 7 )+, 15], 86.1 [(C 5 H 12 N)+, 18], 65.1 [(C 5 H 5 )+, 11], 57.1 [(C 4 H 9 )+, 6], 43.1 [(C 3 H 7 )+, 7], 44.1 [(C 2 H 6 N)+, 5], 30.0 [(CH 4 N)+, 5], 29.0 [(C 2 H 5 )+, 9], 15.0 [(CH 3 )+, 13]. Elemental Analysis: C, 30.34; H, 4.83; N,

50 SYNTHESIS OF BOC-HEPTAPEPTIDE METHYL ESTERS Procedure for Synthesis of Linear Heptapeptide (97) 4.6 g (10 mmol) of Boc-L-Trp-L-Pro-Gly-OH was dissolved in dimethylformamide (DMF, 25 ml) and solution was neutralized with 2.21 ml (21 mmol) of NMM at 0 C and the resulting mixture was stirred for 15 min. 3.8 g (10 mmol) of Gly-L-Ser-L-Ser-L-Leu-OMe was dissolved in DMF (25 ml) and resulting solution along with 2.1 g (0.01 mol) of DCC were added to above mixture. Stirring was first done for 1 h at 0-5 C and then further for 35 h at RT. After the completion of reaction, the reaction mixture was diluted with equal amount of water. The precipitated solid was filtered, washed with water and recrystallized from a mixture of chloroform and petroleum ether (b.p C) followed by cooling at 0 C to get Boc-L-Trp-L- Pro-Gly-Gly-L-Ser-L-Ser-L-Leu-OMe 7 as pale-yellow semisolid mass. (97) tert-butyloxycarbonyl-tryptophanyl-prolyl-glycyl-glycyl-seryl-seryl-leucine methyl ester: Yield 71%; [ ] D 53.9 (c, 0.15 in MeOH); R f 0.52; IR (CHCl 3 ), v cm 1 : 3475 (m, -NH str, ring), 3336 (m/br, -OH str, Ser), (m, -NH str, amide), 3068 (w, -CH str, ring), (m, -CH str, Pro), 2877 (m, -CH str, sym, CH 3 ), 2928, 2923 (m, - CH str, asym, CH 2 ), 2854 (m, -CH str, sym, CH 2 ), 1747 (s, -C=O str, ester), 1667, (s, -C=O str, 3 and 2 amide), 1556, 1473 (m, skeletal bands, ring), , (m, - NH bend, 2 amide), 1387, 1369 (s, -CH bend, tert-butyl), 1382, 1361 (s, -CH bend, iso-propyl), 1269 (s, C O str, ester), 928 (w, CH 3 rocking, tert-butyl), 733, 678 (m, -CH def, oop, ring), 669 (m/br, -OH def, oop, Ser). 1 H-NMR (DMSO-d 6 ) : 9.46 (1H, br. s, NH), 8.75 (1H, br. s, NH), 8.52 (1H, br. s, NH), 8.39 (1H, br. s, NH), 7.48 (3H, br. s, NH, ring & OH, Ser-1 & Ser-2), (4H, m, H-, ring), 6.82 (1H, br. s, NH), 6.46 (1H, br. s, NH), 6.33 (1H, d, J=7.75 Hz, H-, ring), (1H, m, H-α, Trp), 4.45 (1H, t, J=6.85 Hz, H-α, Pro), (1H, m, H-α, Ser-2), (1H, m, H-α, Ser-1), 4.04 (2H, d, J=5.2 Hz, H-α, Gly-1), 3.84 (2H, d, J=5.15 Hz, H-α, Gly-2), (4H, m, H-, Ser-1 & Ser-2), 3.65 (2H, t, J=7.3 Hz, H-δ, Pro), 3.60 (3H, s, OCH 3 ), (1H, m, H-α, Leu), 3.38 (2H, d, J=5.65 Hz, H-, Trp), (2H, m, H-, Pro), (2H, m, H-γ, Pro), 1.57

51 (9H, s, tert-butyl), (3H, m, H- and H-γ, Leu), (6H, d, J=6.15 Hz, H-δ, Leu). Anal. Calcd for C 38 H 56 N 8 O 12 : C, 55.87; H, 6.91; N, Found: C, 55.85; H, 6.90; N, Procedure for the synthesis of the linear heptapeptide (98) Deprotected tetrapeptide (5.36 g, 0.01 mol) was dissolved in tetrahydrofuran (THF, 35 ml). To this solution, TEA (2.8 ml, mol) was added in portions at 0 C and the resulting mixture was stirred for 15 min. Deprotected tripeptide (4.25 g, 0.01 mol) was dissolved in THF (35 ml) and DIPC (1.26 g, 0.01 mol) was added in portions to the above mixture with stirring, maintaining the temperature between 0 10 C. Stirring was continued at room temperature for 36 h, after which the reaction mixture was filtered and the filtrate was washed with 5% NaHCO3 and saturated NaCl solutions (30 ml each). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated in vacuum. The crude product was recrystallized from a mixture of chloroform and petroleum ether (bp C) followed by cooling at 0 C. (98) tert-butyloxycarbonyl-l-prolyl-l-isoleucyl-l-prolyl-l-phenylalanyl-l-prolyl-lprolyl-l-tyrosine methyl ester. Semisolid mass; Yield 83%; [α]d 52.7 ; R f 0.85; IR (CHCl3): 3,375 (O H str, Tyr), 3,128 3,123 (N H str, amide), 3,065 (C H str, ring), 2,999 2,986 (C H str, CH2, Pro), 2,968 2,963, 2,927 2,922 (C H str, asym, CH3 and CH2), 2,876 2,872, 2,858 2,853 (C H str, sym, CH3 and CH2), 1,750 (C=O str, ester), 1,679 1,673, 1,645 (C=O str, 3 and 2 amide), 1,589, 1,482 (skeletal bands), 1539, 1,534 (N H bend, 2 amide), 1,389, 1,373 (C H bend, tert-butyl), 1,271 (C O str, ester), 1,233 (C O str, phenolic), 829, 722, 687 (C H bend, out-of-plane (oop), rings) cm 1; 1 H-NMR (CDCl3): 9.85 (1H, br. s, NH), 8.62 (1H, br. s, NH), 7.17 (2H, dd, J = 7.2 Hz, 4.4 Hz, H at C-3 and C-5, Phe), 7.01 (1H, t, J = 6.2 Hz, H at C-4, Phe), 6.89, 6.81 (2H each, A2B2, J = 8.5 Hz, H at C-2, 6, 3 and 5, Tyr), 6.84 (2H, dd, J = 8.7 Hz, 5.3 Hz, H at C-2 and C-6, Phe), 6.25 (1H, br. s, NH), 5.97 (1H, br. s, OH), 5.11 (1H, m, H-α, Phe), 5.04 (1H, q, H-α, Tyr), 4.38 (1H, t, J = 6.9 Hz, H-α, Pro-2), 4.33 (1H, t, J = 8.6 Hz, H-α, Ile), 4.16 (1H, t, J = 6.9 Hz, H-α, Pro-1), 4.09 (2H, m, H-α, Pro-1 and Pro-2), 3.66 (2H, t, J = 7.1 Hz, H-δ, Pro-4), 3.54 (3H, s, OCH3), 3.33 (4H, m,

52 H-δ, Pro-2 and Pro-3), 3.21 (2H, t, J = 7.2 Hz, H-δ, Pro-1), 3.01 (4H, m, H-β, Phe and Tyr), 2.68 (6H, m, H-β, Pro-2, Pro-3 and Pro-4), 2.54 (2H, q, H-β, Pro-1), 2.04 (1H, m, H-β, Ile), 1.92 (8H, m, H-δ, Pro-1-4), 1.64 (2H, m, H-γ, Ile), 1.49 (9H, s, tert-butyl), 1.00 (3H, d, J = 5.9 Hz, H-γ, Ile), 0.96 (3H, t, J = 7.8 Hz, H-δ, Ile); Anal. Calcd. for C50H69N7O11: C, 63.61; H, 7.37; N, Found: C, 63.60; H, 7.39; N, 10.40%. (99) tbutyloxycarbonyl-l-asparaginyl(bzh)-l-prolyl-glycyl-l-leucyl-l-prolyl-l-tyrosinyl- L-alanine methyl ester Semisolid mass; yield, 77%; R f 0.79; _[D] (c 0.35 in MeOH); IR (CHCl3) v cm-1: 3369 (m, O-H str, Tyr), (m, N-H str, 2 amide), , 3047 (w, C-H str, rings), (m, C-Hstr, Pro), 2959, (m, C-H str, asym and sym, CH3), , (m, C-H str, asym and sym, CH2), 1756 (s, C=O str, ester), , (s, C=O str, 3 and 2 amide), , (m, skeletal bands, rings), 1539, 1533, 1529 (m, N-H bend, 2 amide), 1390, 1376 (s, C-H bend, butyl-t), 1381, 1366 (s, C-H def, propyl-i), 1271 (s, C O str, ester), 1236 (s, C-O str, phenolic), 932, 925 (w, CH3 rock, butylt and propyl-i), 827, , (s, C-H bend, oop, rings). 1 H NMR (pyridine-d6) d (ppm): (br. s, 1H, NH, Gly), 8.39 (br. s, 1H, NH, Tyr), 8.34 (br. s, 1H, NH, Asn), 8.09 (br. s, 1H, NH, Leu), (m, 6H, H-m and H-p, rings, bzh), (m, 4H, H-o, rings, bzh), (m, 2H, H-m, Tyr), 6.92 (d, 1H, J = 5.7 Hz, CONH, Asn), 6.88 (br. s, 1H, NH, Ala), 6.83 (dd, 2H, J = 7.3 Hz, H-o, Tyr), 5.99 (d, 1H, J = 5.3 Hz, H-a, bzh), 5.94 (br. s, 1H, OH, Tyr), 5.17 (q, 1H, H-a, Leu), 5.05 (q, 1H, H-a, Tyr), 4.47 (t, 1H, J = 6.8 Hz, H-a, Pro-1), 4.30 (q, 1H, H-a, Asn), 4.14 (t, 1H, J = 6.9 Hz, H-a, Pro-2), 3.98 (d, 2H, J = 8.2 Hz, H-a, Gly), (m, 1H, H-a, Ala), 3.73 (t, 2H, J = 7.3 Hz, H-d, Pro-1), 3.61 (s, 3H, OCH3), 3.39 (t, 2H, J = 7.2 Hz, H-d, Pro-2), 2.96 (d, 2H, J = 4.2 Hz, H-b, Tyr), 2.80 (d, 2H, J = 4.0 Hz, Hb, Asn), (m, 4H, H-d, Pro-1 and Pro-2), (m, 2H, H-b, Leu), (m, 1H, H-g, Leu), (m, 4H, H-b, Pro-1 and Pro-2), 1.56 (d, 3H, J = 7.8 Hz, H-b, Ala), 1.51 (s, 9H, butyl-t), 1.13 (d,

53 6H, J = 6.1 Hz, H-d, leu); Anal. Calc. for C53H70N8O12 (1011): C, 62.95; H, 6.98; N, Found: C, 62.92; H, 6.99; N, 11.10%. (100) tert-butyloxycarbonyl-phenylalanyl-n-methyl -glycyl-prolyl-n-methyl-phenylalanylleucylisoleucyl-n-methyl-phenylalanine methyl ester. Semisolid mass; yield, 77%; Rf 0.79; [D] (c 0.35 in MeOH); IR (CHCl3): (m, -NH str, amide), 2998, 2995 (m, -CH str, cyclic CH2, Pro), , (m, -CH str, asym and sym, CH2), 2966, 2869, 2865 (m, -CH str, asym and sym, CH3), 1747 (s, -C=O str, ester), , 1645, 1641 (s, -C=O str, 3 and 2 amide), , (m, skeletal bands, arom.rings), 1536, (m, -NH bend, 2 amide), 1468 (m, -CH bend(scissoring), CH2), 1395, 1374 (s, -CH bend, tert-butyl group), 1378, 1365 (s, -CH bend, iso-propyl group), 1268 (s, CO str, ester), 935, 919 (w, CH3 rocking, tert-butyl and iso-propyl groups), , (s, -CH bend, oop, arom. ring) cm-1; 1 H NMR (300 MHz,CDCl3): 8.78 (1H, br. s, -NH, Leu), 7.59 (1H, br. s, -NH, Ile), (2H, tt, J = 6.75, 4.5 Hz, m- H s, Phe-1), (2H, tt, J = 6.8, 4.45 Hz, m- H s, Phe-2), (1H, t, J = 6.3 Hz, p-h, Phe- 3), (3H, m, p-h, Phe-2 and m-h s, Phe- 3), (1H, t, J = 6.25 Hz, p- H, Phe-1), (2H, dd, J = 8.75, 4.2 Hz, o-h s, Phe-1), (4H, m, o-h s, Phe-2 and Phe-3), 6.45 (1H, br. s, -NH, Phe-1), (1H, t, J = 5.15 Hz, a- H, Phe-2), (1H, q, J = 5.6 Hz, a-h, Phe-1), (1H, t, J = 6.85 Hz, a-h, Pro), (1H, t, J = 5.2 Hz, a-h, Phe-3), (1H, q, J = 6.75 Hz, a-h, Leu), (1H, t, J = 8.6 Hz, a-h, Ile), 3.87 (2H, s, a-h, Gly), (2H, t, J = 7.2 Hz, δ-h s, Pro), 3.53 (3H, s, OCH3), 3.20 (3H, s, NCH3, Gly), (4H, m, b-h s, Phe-1 and Phe-3), 3.05 (3H, s, NCH3, Phe-3), 3.02 (3H, s, NCH3, Phe-2), (2H, d, J = 5.6 Hz, b-h s, Phe-2), (2H, q, b-h s, Pro), (1H, m, b-h, Ile), (2H, m, g-h s, Pro), (2H, t, b-h s, Leu), (2H, m, g- H s, Ile), 1.54 (9H, s, tert-butyl group), (1H, m, g-h s, Leu), (3H, d, J = 5.9 Hz, g - H s, Ile), (6H, d, J = 6.25 Hz, δ-h s, Leu), (3H, d, J = 7.75 Hz, δ-h s, Ile) ppm.

54 SYNTHESIS OF CYCLIC HEPTA PEPTIDES Procedure for cyclization of linear heptapeptide To synthesize (101), linear heptapeptide unit (99) (5.06 g, mol) was deprotected at carboxyl end using lithium hydroxide (0.18 g, mol) to get Boc-L-Asn(bzh)-L-Pro-Gly-L- Leu-L-Pro-L-Tyr-L-Ala-OH. The deprotected linear peptide unit (4.98 g, mmol) was now dissolved in chloroform (50 ml) at 0 C. To the above solution, pentafluorophenol (1.23 g, mol) was added and stirred at room temperature (12 h). The reaction mixture was filtered and the filtrate was washed with 10% sodium bicarbonate solution (3 x 25 ml) until excess of pentafluorophenol was removed and finally washed with 5% HCl (2 x 20 ml) to get the corresponding pentafluorophenyl ester Boc-L-Asn(bzh)-L-Pro-Gly-L-Leu-L-Pro-LTyr- L-Ala- Opfp. To this compound (4.65 g, mol) dissolved in chloroform (35 ml), TFA (0.91 g, mol) was added, stirred at room temperature (1 h) and washed with 10% sodium bicarbonate solution (2 x 25 ml). The organic layer was dried over anhydrous Na2SO4 to get L- Asn(bzh)-L-Pro-Gly-L-Leu-L-Pro-L-Tyr-L-Ala-Opfp which was dissolved in chloroform (25 ml) and TEA/NMM (2.21 ml/2.8 ml, mol) was added. Then, whole contents were kept at 0 C (7 days). The reaction mixture was washed with 10% sodium carbonate and 5% HCl (4 x 25 ml) solutions. The organic layer was dried over anhydrous Na2SO4. Finally, chloroform was distilled off and crude product was crystallized from chloroform/n-hexane to get pure cyclic product (101). (101) Cyclo (-asparaginyl-prolyl-glycyl-leucyl-prolyl-tyrosinyl-alanyl-) White solid; M.p. 202 C (d); yield, (89%, NMM), (68%, TEA); R f 0.66; [D] (-41.2 for natural peptide, c in MeOH); Yield IR (KBr) v cm-1: 3372 (m, O-H str, Tyr), 3349, 3175 (m, N-H str, asym and sym, 1 amide), , (m, N-H str, 2 amide), 3069 (w, C-H str, ring), (m, C-H str, Pro), , 2872 (m, C-H str, asym and sym, CH3), 2928, 2922, 2858 (m, C-H str, asym and sym, CH2), 1660, 1655 (s, C=O str, 3 and 1 amide), , 1636, 1632 (s, C=O str, 2 amide), 1623 (m, - NH2 bend, 1 amide), 1575, 1478 (m, skeletal bands, ring), , 1523 (m, N- H bend, 2 amide), 1468 (m, C-H bend(scissoring), CH2), 1409 (m, C N str, 1 amide), 1381, 1364 (s, C-H def, propyl-i), 1231 (s, C-O str, phenolic), 923 (w, CH3 rocking, propyl-i), 827 (s, C-H bend, oop, ring);

55 1 H NMR (pyridine-d6) d (ppm): 9.34 (br. s, 1H, NH, Gly), 9.27 (br. s, 1H, NH, Tyr), 8.91 (br. s, 1H, NH, Ala), 8.63 (br. s, 1H, NH, Asn), 8.05 (br. s, 1H, NH, Leu), (m, 2H, H-m, Tyr), 7.14 (dd, 2H, J = 7.4 Hz, H-o, Tyr), 6.73 (s, 2H, CONH2, Asn), 5.94 (br. s, 1H, OH, Tyr), 5.41 (q, 1H, Ha, Tyr), 5.29 (q, 1H, H-a, Leu), 4.94 (q, 1H, H-a, Asn), (m, 1H, H-a, Ala), 4.04 (d, 2H, J = 8.3 Hz, H-a, Gly), (m, 2H, H-a, Pro-1 and Pro-2), (m, 4H, H-d, Pro-1 and Pro-2), 3.36 (d, 2H, J = 4.1 Hz, H-b, Tyr), 3.07 (d, 2H, J = 3.9 Hz, H-b, Asn), (m, 2H, H-b, Leu), (m, 1H, H-g, Leu), (m, 4H, H-b, Pro-1 and Pro-2), 1.85 (d, 3H, J = 7.9 Hz, Hb, Ala), (m, 4H, H-g, Pro-1 and Pro-2), 1.18 (d, 6H, J = 6.1 Hz, H-d, leu); 13 C NMR (pyridine-d6) d (ppm): (C=O, C-g, Asn), (C=O, Asn), (C=O, Tyr), 172.7, (2C, C=O, Pro-1 and Pro-2), (C=O, Ala), (C=O, Leu), (C=O, Gly), (C-p, Tyr), (2C, C-o, Tyr), (C-g, Tyr), (2C, C-m, Tyr), 65.2, 63.2 (2C, C-a, Pro-1 and Pro- 2), 59.3 (C-a, Tyr), 56.8, 54.4, 52.2 (3C, C-a, Asn, Leu and Ala), 48.9, 48.0 (2C, C-d, Pro-1 and Pro-2), 43.9 (C-a, Gly), 39.7, 36.8 (2C, C-b, Tyr and Asn), 33.5 (C-g, Leu), 31.6 (C-b, Leu), 30.6, 29.9 (2C, C-b, Pro-2 and Pro-1), 26.8, 25.1 (2C, C-g, Pro-1 and Pro-2), 19.8 (2C, C-d, Leu), 17.7 (C-b, Ala); ESIMS/MS (m/z, rel. int.): 713 [(M + H)+, 100], 642 [(Asn-Pro-Gly-Leu-Pro-Tyr)+, 29], 614 [(642 CO)+, 16], 685 [(713 CO)+, 13], 599 [(Pro-Gly-Leu-Pro-Tyr-Ala)+, 21], 571 [(599 CO)+, 10], 528 [(Pro-Gly- Leu-Pro-Tyr)+, 39], 500 [(528 CO)+, 17], 446 [(Pro-Tyr-Ala-Asn)+, 55], 365 [(Pro-Gly-Leu- Pro)+, 51], 332 [(Pro-Tyr-Ala)+, 18], 304 [(332 CO)+, 11], 283 [(Ala-Asn-Pro)+, 34], 269 [(Asn-Pro-Gly)+, 18], 268 [(Pro-Gly-Leu)+, 72], 240 [(268 CO)+, 15], 212 [(Asn-Pro)+, 10], 186 [(Ala-Asn)+, 25], 184 [(212 CO)+, 21], 158 [(186 CO)+, 9], 155 [(Pro-Gly)+, 42], 136 [Tyr immonium ion (C8H10NO)+, 13], 127 [(155 CO)+, 14], 116 [(Asn)+, 16], 107 [(C7H7O)+, 10], 87 [Asn immonium ion (C3H7N2O)+, 15], 86 [Leu immonium ion (C5H12N)+, 19], 70 [Pro immonium ion (C4H8N)+, 23], 58 [(C2H4NO)+, 9], 57 [(C4H9)+, 11], 44 [Ala immonium ion (C2H6N)+, 9], 43 [(C3H7)+, 8], 30 [Gly immonium ion (CH4N)+, 11], 15 [(CH3)+, 10]; Anal. Calc. for C34H48N8O9 (712): C, 57.29; H, 6.79; N, Found: C, 57.32; H, 6.80; N, 15.69%.

56 Procedure for Cyclization of Linear Heptapeptide To synthesize (102), linear heptapeptide unit (97) (4.1 g, 5 mmol) was deprotected at carboxyl end using lithium hydroxide (0.18 g, 7.5 mmol) to get Boc-L-Trp-L-Pro-Gly-Gly-L- Ser-L-Ser-L-Leu-OH. The deprotected heptapeptide unit (4.0 g, 5 mmol) was now dissolved in chloroform (35 ml) at 0 C. To the above solution, DCC (1.1 g, 5 mmol) and p- nitrophenol/pentafluorophenol (0.94 g/1.23 g, 6.7 mmol) were added and stirred was done at RT (12 h). The reaction mixture was filtered and the filtrate was washed with 10% NaHCO 3 (3 15 ml) and 5% HCl (2 10 ml) to get the corresponding p-nitrophenyl/pentafluorophenyl ester Boc- L-Trp-L-Pro-Gly-Gly-L-Ser-L-Ser-L-Leu-Opnp/pfp. To this compound (3.7 g/4.84 g, 4 mmol) dissolved in chloroform (25 ml), TFA (0.91 g, 8 mmol) was added, stirred at RT (1 h) and washed with 10% NaHCO 3 solution (2 25 ml). The organic layer was dried over anhydrous Na 2 SO 4 to get L-Trp-L-Pro-Gly-Gly-L-Ser-L-Ser-L-Leu-Opnp/pfp which was dissolved in chloroform (25 ml) and TEA/NMM/pyridine (21 mmol) was added. Then, whole contents were kept at 0 C (7 days). The reaction mixture was washed with sufficient quantity of 10% NaHCO 3 solution and finally washed with 5% HCl (3 25 ml). The organic layer was dried over anhydrous Na 2 SO 4. Finally, chloroform was distilled off and crude product was crystallized from chloroform/n-hexane to get pure cyclic product (102). (102) Cyclo (-tryptophanyl-prolyl-glycyl-glycyl-seryl-seryl-leucyl-) Yield 65% (TEA), 71% (NMM), 83% (C 5 H 5 N); [ ] D 17.9 ( 18.0 for natural peptide; c, 0.1 in MeOH); R f 0.76; m.p C (d); IR (KBr), v cm 1 : 3477 (m, -NH str, ring), 3332 (m/br, -OH str, Ser), (m, -NH str, amide), 3064 (w, -CH str, ring), (m, -CH str, Pro), 2874 (m, -CH str, sym, CH 3 ), 2929 (m, -CH str, asym, CH 2 ), 2857, 2952 (m, -CH str, sym, CH 2 ), 1668, (s, -C=O str, 3 and 2 amide), 1559, 1475 (m, skeletal bands, ring), , 1529, 1525 (m, -NH bend, 2 amide), 1384, 1363 (s, -CH bend, iso-propyl), 730, 679 (m, -CH def, oop, ring), 664 (m/br, -OH def, oop, Ser). 1 H-NMR (DMSO-d 6 ) : 9.14 (1H, br. s, NH), 8.79 (1H, br. s, NH), 8.67 (1H, br. s, NH), 7.96 (3H, br. s, NH, ring & OH, Ser-1 & Ser-2), 7.67 (1H, br. s, NH), 7.64 (1H, br. s, NH), 7.39 (1H, d, J=7.8 Hz, H-,

57 ring), (4H, m, H-, ring), 6.42 (1H, br. s, NH), (1H, m, H-α, Leu), (1H, m, H-α, Trp), (2H, m, H-α, Ser-1 & Ser-2), 5.10 (2H, d, J=5.2 Hz, H-α, Gly- 2), (4H, m, H-, Ser-1 & Ser-2), 3.94 (2H, d, J=5.15 Hz, H-α, Gly-1), 3.90 (1H, t, J=6.9 Hz, H-α, Pro), 3.26 (2H, t, J=7.25 Hz, H-δ, Pro), 2.88 (2H, d, J=5.7 Hz, H-, Trp), (2H, m, H-, Pro), (2H, m, H-γ, Pro), (2H, t, J=5.4 Hz, H-, Leu), (6H, d, J=6.2 Hz, H-δ, Leu), (1H, m, H-γ, Leu). 13 C NMR (DMSO-d 6 ) : (C=O, Leu), (C=O, Pro), 171.4, (2C, C=O, Ser-2 & Ser-1), (C=O, Trp), (2C, C=O, Gly-2 & Gly-1), 135.4, (2C, C- & C-, ring), (C-, ring), 120.7, 119.8, 118.5, (4C, C-δ, ring), (C-, ring), 63.9 (C-, Ser-1), 62.2 (C-, Pro), 60.6 (C-, Ser-2), 56.4 (C-, Ser-2), 56.1 (C-, Trp), 54.8 (C-, Ser-1), 54.2 (C-, Leu), 48.8 (C-δ, Pro), 43.7 (C-, Leu), 42.5, 39.9 (2C, C-, Gly-2 & Gly-1), 29.5 (C-, Pro), 27.2 (C-, Leu), 26.6 (C-, Trp), 24.9 (C-, Pro), 22.5 (2C, C-δ, Leu). ESIMS/MS: (m/z, rel. int.) [(M + H)+, 100], [(685.7 CO)+, 11], [(Leu-Trp-Pro-Gly-Gly-Ser)+, 48], [(Gly-Gly-Ser-Ser-Leu-Trp)+, 29], [(Trp-Pro-Gly-Gly-Ser-Ser)+, 23], [(598.6 CO)+, 18], [(Gly-Ser-Ser-Leu-Trp)+, 15], [(Leu-Trp-Pro-Gly-Gly)+, 36], [(Pro-Gly-Gly-Ser-Ser-Leu)+, 27], [(511.5 CO)+, 10], [(Ser-Ser-Leu-Trp)+, 27], [(Leu-Trp-Pro-Gly)+, 67], [(454.5 CO)+, 17], [(Leu-Trp-Pro)+, 34], [(Pro-Gly-Gly-Ser-Ser)+, 41], [(Pro-Gly-Gly-Ser)+, 19], [(Ser-Ser)+, 12], (Trp immonium ion), [(C 9 H 8 N)+, 9], [(C 8 H 6 N)+, 11], [(Gly-Gly)+, 19], 86.1 (Leu immonium ion), 70.1 (Pro immonium ion), 60.1 (Ser immonium ion), 57.1 [(C 4 H 9 )+, 12], 42.0 [(C 3 H 6 )+, 8], 31.0 [(CH 3 O)+, 11], 30.0 (Gly immonium ion). Anal. Calcd for C 32 H 44 N 8 O 9 : C, 56.13; H, 6.48; N, Found: C, 56.15; H, 6.47; N,

58 Synthesis of the cyclic heptapeptide (103) To synthesize stylisin 2 (103), linear heptapeptide unit (98) (4.72 g, mol) was deprotected at carboxyl end using LiOH (0.18 g, mol) to get Boc-L-prolyl-L-isoleucyl-Lprolyl-Lphenylalanyl- L-prolyl-L-prolyl-L-tyrosine-OH. The deprotected heptapeptide unit (4.65 g, mol) was now dissolved in CHCl3 (50 ml) at 0 C. To this solution, p-nitrophenol or pentafluorophenol (0.94 g or 1.23 g, mol) and DIPC (0.63 g, mol) were added and the mixture was stirred at room temperature for 12 h. The reaction mixture was filtered and the filtrate was washed with 10% NaHCO3 solution (3 25 ml) and finally washed with 5% HCl (2 30 ml) to get the corresponding p-nitro-phenyl or pentafluorophenyl ester Boc-L-prolyl-Lisoleucyl-L-prolyl-L-phenylalanyl-L-prolyl-Lprolyl- L-tyrosine-Opnp or Boc-L-prolyl-Lisoleucyl-L-prolyl-L-phenylalanyl-L-prolyl-L-prolyl-L-tyrosine- Opfp. To this compound (4.2 g or 4.38 g, mol) dissolved in CHCl3 (35 ml), CF3COOH (0.91 g, mol) was added, stirred at room temperature for 1 h and washed with 10% NaHCO3 solution (2 25 ml). The organic layer was dried over anhydrous Na2SO4 to get L-prolyl-L-isoleucyl-L-prolyl- Lphenylalanyl-L-prolyl-L-prolyl-L-tyrosine-Opnp or L-prolyl-L-isoleucyl-L-prolyl-Lphenylalanyl-L-prolyl- L-prolyl-L-tyrosine-Opfp, which was dissolved in CHCl3 (25 ml) and TEA/NMM/pyridine (2.8 ml or 2.21 ml or 1.61 ml, mol) was added. Then, above alkaline solution was kept at 0 C for 7 days. The reaction mixture was washed with 10% NaHCO3 (3 25 ml) and 5% HCl (2 30 ml) solutions. The organic layer was dried over anhydrous Na2SO4 and crude cyclized product was crystallized from CHCl3/n-hexane to get pure cyclic product (103). (103) Cyclo(L-prolyl-L-isoleucyl-L-prolyl-L-phenylalanyl-L-prolyl-L-prolyl-L-tyrosinyl) White solid; Yield 89% (3.61 g, C5H5N), 82% (3.33 g, NMM), 79% (3.21 g, TEA); mp 216 C (dec); [α]d 39.8 (c 0.2, MeOH) ( 39.9 for natural stylisin 2); R f 0.68 (CHCl3:AcOH:H2O, 3:2:5); IR (KBr): 3,377 (O H str, Tyr), 3,129, 3,125-3,121 (N H str, amide), 3,069 3,066 (C H str, ring), 2,998 2,985 (C H str, CH2, Pro), 2,969 2,965, 2,929, 2,925 (C H str, asym, CH3 and CH2), 2,875 2,872, 2,859 2,855 (C H str, sym, CH3 and CH2), 1,676 1,672, 1,645 1,640 (C=O str, 3 and 2 amide), 1,587, 1,480 (skeletal bands), 1,537, 1,533 1,530 (N H bend, 2 amide), 1,236 (C O str, phenolic), 828, 720, 689 (C H bend, out-of-plane (oop), rings) cm 1;

59 1 H-NMR (CDCl3): 9.75 (1H, br. s, NH), 9.72 (1H, br. s, NH), 8.52 (1H, br. s, NH), 7.18 (2H, dd, J = 7.2 Hz, 4.4 Hz, H at C-3 and C-5, Phe), 7.03 (1H, t, J = 6.2 Hz, H at C-4, Phe), 6.97, 6.90 (2H each, A2B2, J = 8.5 Hz, H at C-2, 6, 3 and 5, Tyr), 6.85 (2H, d, J = 8.6 Hz, 5.4 Hz, H at C-2 and C-6, Phe), 5.95 (1H, br. s, OH), 4.39 (2H, m, H-α, Phe and Tyr), 4.25 (1H, t, J = 7.0 Hz, H-α, Pro-4), 4.21 (1H, t, J = 6.9 Hz, H-α, Pro-3), 3.91 (2H, m, H-α, Pro-1 and Pro-2), 3.85 (1H, t, J = 8.6 Hz, H-α, Ile), 3.60 (2H, t, J = 7.2 Hz, H-δ, Pro-4), 3.26 (6H, m, H-δ, Pro-1 3), 2.67 (8H, m, H-β, Pro-1 4), 2.61 (4H, m, H-β, Phe and Tyr), 1.83 (8H, m, H-δ, Pro-1 4), 1.61 (2H, m, H-γ, Ile), 1.55 (1H, m, H-β, Ile), 1.00 (3H, d, J = 5.9 Hz, H-γ, Ile), 0.95 (3H, t, J = 7.8 Hz, H-δ, Ile); 13 C-NMR (CDCl3): (C=O, Ile), 172.8, (2C, C=O, Pro-2 and Pro-4), (C=O, Pro-1), 170.3, (2C, C=O, Phe and Tyr), (C=O, Pro-3), (C-p, Tyr), 138.5, (2C, C-γ, Phe and Tyr), (2C, C-o, Phe), (2C, C-o, Tyr), (2C, C-m, Phe), (2C, C-m, Tyr), (C-p, Phe), 58.7 (2C, C-α, Pro-1 and Pro-2), 57.3, 55.7 (2C, C-α, Pro-4 and Pro-3), 54.3 (C-α, Ile), 53.6, 52.8 (2C, C-α, Phe and Tyr), 48.9, 48.2 (2C, C-δ, Pro-1 and Pro-3), 46.6, 45.8 (2C, C-δ, Pro-2 and Pro-4), 43.2 (C-β, Tyr), 37.8 (C-β, Phe), 35.6 (C-β, Ile), 34.4, 33.9 (2C, C-β, Pro-1 and Pro-4), 32.2, 30.5 (2C, C-β, Pro-2 and Pro-3), 24.8, 23.6 (2C, C-γ, Ile and Pro-3), 23.2, 22.6, 21.7 (3C, C-γ, Pro-1, Pro-2 and Pro-4), 16.2 (C-γ, Ile), 10.3 (C-δ, Ile); ESI-MS/MS m/z [RI]: [(M + 1)+, 100], [(812.9 CO)+, 14], [(H-Pro-Phe-Pro-Pro-Tyr-Pro)+, 37], [(699.8 CO)+, 11], [(H-Pro-Pro-Tyr-Pro-Ile-Pro)+, 29], [(H-Pro-Phe- Pro-Pro-Tyr)+, 64], [(602.7 CO)+, 19], [(H-Pro-Pro-Tyr-Pro)+, 38], [(H- Pro-Phe-Pro-Pro)+, 49], [(439.5 CO)+, 21], [(H-Pro-Pro-Tyr)+, 66], [(H- Pro-Phe-Pro)+, 32], [(358.4 CO)+, 11], [(H-Pro-Phe)+, 25], [(245.3 CO)+, 11], [(H-Pro-Pro)+, 17], [Tyr (C8H10NO)+, 14], [Phe (C8H10N)+, 10], [(C7H7O)+, 12], 98.1 [(Pro)+, 15], 93.1 [(C6H5O)+, 9], 91.1 [(C7H7)+, 11], 86.1 [Ile (C5H12N)+, 12], 77.1 [(C6H5)+, 7], 70.1 [H-Pro (C4H8N)+, 23], Ile: 57.1 [(C4H9)+, 6], 29.1 [(C2H5)+, 9], 15.0 [(CH3)+, 13]; Anal. Calcd. for C44H57N7O8: C, 65.09; H, 7.08; N, Found: C, 65.11; H, 7.05; N, 12.10%. PROCEDURE FOR SYNTHESIS OF CYCLOHEPTAPEPTIDE

60 To synthesize cycloheptapeptide (104), linear peptide unit (100) (0.005 mol, 4.98 g) was deprotected at carboxyl end using lithium hydroxide ( mol, 0.18 g) to get Boc-Phe- N(Me)Gly-Pro-N-(Me)Phe-Leu-Ile-N-Me Phe-OH. The deprotected heptapeptide unit (0.005 mol, 4.9 g) was now dissolved in 50 ml of chloroform at 0 C. To the above solution, pentafluorophenol (pfp, mol) was added and stirred at room temperature for 12 h. The reaction mixture was filtered and the filtrate was washed with 10% sodium bicarbonate solution (3 15 ml) until excess of pentafluorophenol was removed and finally washed with 5% HCl (2 10 ml) to get the corresponding pentafluorophenyl ester Boc-Phe-N(Me)Gly-Pro-N-(Me)Phe- Leu-Ile-N- (Me)Phe-Opfp. To this compound (0.004 mol, 4.6 g) dissolved in 35 ml of chloroform, trifluoroacetic acid (0.008 mol, 0.91 g) was added, stirred at room temperature for 1 h and washed with 10% sodium bicarbonate solution (2 25 ml). The organic layer was dried over anhydrous sodium sulphate to get the Phe-N-(Me) Gly-Pro-N-(Me) Phe-Leu-Ile-N-(Me) Phe-Opfp which was dissolved in 25 ml of chloroform and triethylamine/n-methyl morpholine/pyridine (0.021 mol, 2.21 ml / 2.8 ml /1.61 ml) was added. Then, whole contents were kept in a refrigerator at 0 C for 7 days. The reaction mixture was washed with 10% sodium carbonate solution until the byproduct pentafluorophenol (pfp) was removed completely and finally washing was done with 5% HCl (3 15 ml). The organic layer was dried over anhydrous sodium sulphate. Finally, chloroform was distilled off and the crude cyclized product was crystallized using a mixture of chloroform and n-hexane to get the pure cyclo(-phe-n(me)gly- Pro-N-(Me)Phe-Leu- Ile -N-(Me) Phe-) (104). (104)Cyclo(-phenylalanyl-N-methyl-glycyl-prolyl-Nmethyl-phenylalanyl-leucyl-isoleucyl-Nmethylphenylalanyl-) Yield 87.3% (3.77 g, pyridine), 74.1% (3.2 g, NMM), 66.0% (2.85 g, TEA); IR (KBr): 3139, (m, -NH str, amide), (m, - CH str, cyclic CH2, Pro), 2926, 2622, (m, -CH str, asym and sym, CH2), 2969, (m, -CH str, asym and sym, CH3), 1669, 1665, (s, -C=O str, 3 and 2 amide), 1588, 1585, 1477, 1473 (m, skeletal bands, arom. rings), 1538, (m, -NH bend, 2 amide), 1381, 1366 (s, -CH bend, iso-propyl group), 921 (w, CH3 rocking, iso-propyl groups), 728, 722, (s, -CH bend, oop, arom. ring) cm-1; 1 H NMR (300 MHz, CDCl3):

61 9.58 (1H, br. s, -NH, Phe-1), 9.12 (1H, br. s, -NH, Leu), 9.09 (1H, br. s, -NH, Ile), (4H, m, m-h s, Phe-2 and Phe-3), (2H, tt, J = 6.85, 4.5 Hz, m- H s, Phe-1), (2H, m, p-h, Phe-2 and Phe-3), (1H, t, J = 6.25 Hz, p-h, Phe-1), (2H, dd, J = 8.75, 4.15 Hz, o-h s, Phe-1), (4H, m, o-h s, Phe-2 and Phe-3), (1H, t, J = 5.15 Hz, a-h, Phe-3), (1H, t, J = 8.55 Hz, a-h, Ile), 5.28 (2H, s, a-h, Gly), (1H, q, J = 6.8 Hz, a-h, Leu), (1H, t, J = 6.9 Hz, a-h, Pro), (2H, m, a- H s, Phe-1 & Phe-2), (2H, t, J = 7.15 Hz, δ-h s, Pro), 3.05 (3H, s, NCH3, Gly), 2.90 (3H, s, NCH3, Phe-3), 2.86 (3H, s, NCH3, Phe-2), (2H, q, b-h s, Pro), (4H, m, b-h s, Phe-2 and Phe-3), (2H, d, J = 5.55 Hz, b-h s, Phe-1), (2H, t, b- H s, Leu), (2H, m, g-h s, Pro), (2H, m, g-h s, Ile), (1H, m, b-h, Ile), (6H, d, J = 6.3 Hz, δ-h s, Leu), (3H, d, J = 5.85 Hz, g -H s, Ile), (3H, d, J = 7.75 Hz, δ-h s, Ile), (1H, m, g-h s, Leu) ppm. 13 C NMR (300 MHz, CDCl3): 174.1, 172.8, (3C, C=O, Ile, Phe-1 and Phe-2), 171.0, 170.7, (3C, C=O, Phe- 3, Leu and Pro), (C=O, Gly), (g-c, Phe-2), 139.0, (2C, g-c s, Phe-1 & Phe- 3), (2C, m-c s, Phe-2), (2C, m-c s, Phe-3), (2C, o-c s, Phe-1), (2C, o- C s, Phe-3), (2C, o-c s, Phe-2), (2C, m-c s, Phe-1), 127.3, (2C, p-c s, Phe-2 and Phe-3), (p-c, Phe-1), 61.2, 59.8 (2C, a-c s, Phe-3 and Phe-2), 54.9, 54.4 (2C, a-c s, Leu and Ile), 52.7 (a-c, Pro), 47.7, 47.3 (2C, a-c s, Phe-1 and Gly), 45.6 (δ-c, Pro), 44.0 (b-c, Leu), 41.5 (b-c, Phe-1), 38.8, 38.4, 35.6 (3C, b-c s, Phe-2, Phe-3 and Ile), 35.1, 33.9, 33.4 (3C, NCH3, Phe-2, Gly and Phe-3), 30.7 (b-c, Pro), 26.9 (g-c, Leu), 24.4, 23.6 (2C, g-c s, Ile and Pro), 22.7 (2C, δ-c s, Leu), 16.4 (g -C, Ile), 10.1 (δ-c, Ile) ppm; FAB MS (m/z, relative intensity): 865 [(M + H)+, 100], 837 [(865 CO)+, 12.7], 794 [(Pro-N(Me)Phe-Leu-Ile- N(Me)Phe- Phe)+, 38.5], 766 [(794 CO)+, 15.3], 751 [(Ile-N(Me)Phe-Phe-N(Me)Gly-Pro-N(Me) Phe)+, 33.2], 723 [(751 CO)+, 11.6], 703 [(Phe- N(Me)Gly-Pro-N(Me)Phe-Leu-Ile)+, 22.9], 675 [(703 CO)+, 13.7], 646 [(Pro-N(Me)Phe-Leu-Ile- N(Me)Phe)+, 76.8], 618 [(646 CO)+, 21.5], 590 [(Ile-N(Me)Phe-Phe-N(Me)Gly-Pro)+, 48.6], 562 [(590 CO)+, 16.5], 493 [(Ile-N(Me)Phe-Phe-N (Me)Gly)+, 27.4], 485 [(Pro-N(Me)Phe-Leu-Ile)+, 52.4], 477 [(Phe-N(Me)Gly-Pro-N(Me)Phe)+, 37.2], 465 [(493 CO)+, 10.2], 457 [(485 CO)+, 11.9], 449 [(477 CO)+, 11.2], 422 [(Ile- N(Me)Phe-Phe)+, 22.5], 394 [(422 CO)+, 14.8], 372 [(Pro-N(Me)Phe-Leu)+, 60.2], 344 [(372

62 CO)+, 14.8], 316 [(Phe-N(Me)Gly-Pro)+, 19.9], 288 [(316 CO)+, 9.6], 275 [(Ile-N(Me)Phe)+, 40.3], 259 [(Pro-N(Me)Phe)+, 29.6], 247 [(275 CO)+, 15.1], 231 [(259 CO)+, 10.2], 219 [(Phe- N(Me)Gly)+, 31.3], 191 [(219 CO)+, 10.9], 148 [(Phe)+, 14.7], 120 [(C8H10N)+, 8.9], 114 [(Ile)+, 19.6], 98 [(Pro)+, 13.3], 91 [(C7H7)+, 17.3], 86 [(C5H12N)+, 11.4], 77 [(C6H5)+, 12.7], 57 [(C4H9)+, 14.2], 43 [(C3H7)+, 9.8], 42 [(C3H6)+, 8.7], 29 [(C2H5)+, 7.9], 15 [(CH3)+, 13.9].

63 2.2.3 BIOLOGICAL EVALUATION ANTIMICROBIAL ACTIVITY The synthesized cyclic peptides were screened for antibacterial activity against Escherichia coli (Gram- negative bacteria) and Staphylococcus aureus, Bacillus subtills (Grampositive bacteria) using modified Kirby-Bauer disc diffusion method. All synthesized compounds were dissolved separately to prepare a stock solution of 1 mg ml -1 using DMF. A spore suspension in sterile distilled water was prepared from five days old culture of the test bacteria growing on nutrient broth media. About 20 ml of the growth media was transferred into sterilized petri discs of 6 mm diameter and 2 mm thickness were sterilized by autoclaving at C for 15 min. Each petri plate was divided into five equal portions along the diameter to place one disc. Three discs of test sample were placed on three portions together with one disc with reference drug ciprofloxacin (50 µg ml -1 ) and a disc impregnated with the solvent (DMF) as negative control. The test samples were tested at the concentration 25, 50, 100 µg ml -1. The Petri plates inoculated with bacterial cultures were incubated at 37 C for24 hrs. Diameters of the inhibition zones (mm) were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced the standard drugs of ciprofloxacin and Gatifloxacin. The synthesized cyclic peptides were screened for antifungal activity against fungal strain Aspergillus niger and Candida albicans. A spore suspension in normal saline was prepared form culture of the test fungi on sabourud s broth media. After transferring growth media, petri plates were inoculated with spore suspension. After drying, wells were made using agar punch and the test sample; reference drug and negative control (DMSO) were placed in labelled wells in each petri plate. Test sample were tested at the concentration of 25, 50, 100 µg ml -1. The standard drug griseofulvin was tested at the concentration of 50 µg ml -1. The petri plates inoculated with fungal cultures were incubated at 25 0 C for 48 hrs. Antifungal activity was determined by measuring the diameter of the inhibition zone triplicate sets. Activity of the each compound was compared griseofulvin as standard drug. The results of antibacterial and antifungal studies are listed in following Tables. Table 2.4. Antimicrobial screening data for compound79 and 90.

64 Compound Diameter of zone of inhibition (mm) Bacterial strains Fungal strains B. sub. S. aur. P. aeru. K. pneu. C. alb. M. audo. A. niger T. menta (6) 16(6) 12(6) 18(6) 19(6) 90 18(6) 20(6) 15(6) 23(6) 25(6) Control * Gatifloxacin 18(12.5) 28(6) 22(6) 25(6) Griseofulvin 20(6) 17(6) 18(12.5) 20(6) B. sub.: Bacillus subtilis; S. aur.: Staphylococcus aureus; P. aeru.: Pseudomonas aeruginosa; K. pneu.: Klebsiella pneumoniae; C. alb.: Candida albicans; M. audo.: Microsporum audouinii; A. niger: Aspergillus niger; T. menta.: Trichophyton mentagrophytes. Values in bracket are MIC values ( g/ml); * DMF/DMSO. FIGURE:1 Antimicrobial screening data for compound79 and 90

65 Table 2.5 Antimicrobial screening data for 80 and 89. Compound Diameter of zone of inhibition (mm) Bacterial strains Fungal strains B. sub. S. aur. P. aeru. K. pneu. C. alb. M. audo. A. niger T. menta (25) 16(25) 9(12.5) 18(6) 19(6) 89 15(25) 19(25) 12(12.5) 25(6) 27(6) Control* Gatifloxacin 18(12.5) 28(6) 22(6) 25(6) Griseofulvin 20(6) 17(6) 18(12.5) 20(6) B. sub.: Bacillus subtilus, S. aur.: Staphylococcus aureus, P. aeru.: Pseudomonas aeruginosa, K. pneu.: Klebsiella pneumonia, C. alb.: Candida albicans, M. audo.: Microsporum audouinii, A. niger: Aspergillus niger, T. menta.: Trichophyon mentagrophytes Values in bracket are MIC values ( g/ml) * DMF / DMSO FIGURE:2 Antimicrobial screening data for 80 and 89.

66 Table 2.6. Antimicrobial activity data for compound 92 Compd. Diameter of zone of inhibition (mm) Bacterial strains Fungal strains B. S. P. E. C. M. A. T. Subtilis aureus aeruginosa Coli albicans audouinii niger Mentagrophytes 92 28(6)* 23(12.5) 24(6) 11(12.5) 10(12.5) Control Ciprofloxacin 20(6) 20(12.5) 25(6) 19(12.5) Griseofulvin 20(6) 17(6) 18(12.5) 20(6) * Values in bracket are MIC values ( g/ml).

67 FIGURE:3 Antimicrobial activity data for compound 92 Table 2.7. Antibacterial activity data of Compound 96 Zone of inhibition (mm) Compd. Bacteria Bacillus Escherichia Staphylococcus Pseudomonas subtilis coli aureus aeruginosa DMF Ciprofloxacin a a c = 50 g ml -1

68 FIGURE:4 Antibacterial activity data of Compound 96 Table 2.8. Antifungal activity data of compound 96 Zone of inhibition (mm) Compd. Fungus Candida Ganoderma Microsporum Trichophyton albicans species audouinii mentagrophytes DMF Griseofulvin a

69 a c = 50 g ml -1 Table 2.9. Antibacterial activity data of 95. Compound Diameter of zone of inhibition (ZI) in mm Bacterial strains B. subtilis S. epidermidis P. aeruginosa K. Pneumonia 95 Control b Gatifloxacin 9(25) 20(12.5) 11(12.5) 28(6.25) a Values in bracket are MIC values ( g/ml); b DMF; 27(6.25) 26(6.25) 24(6.25) 25(6.25) indicates no activity Table Antifungal activity data of 95. Compound Diameter of zone of inhibition (ZI) in mm Fungal strains C. albicans M. audouinii A. niger T. mentagrophytes 95 Control b Griseofulvin 19(6.25) 20(6.25) 24(6.25) 17(6.25) 9(25) 18(12.5) 28(6.25) 22(6.25) a Values in bracket are MIC values ( g/ml); b DMSO; indicates no activity

70 FIGURE:5 Antifungal activity data of 95.

71 Table 2.11: Response of Pathogenic Microorganisms to Synthesized Cyclopeptide 102 Compd. B. Subtilis Diameter of zone of inhibition (in mm) Bacterial strains Fungal strains P. K. C. M. A. aeruginosa pneumoniae albicans audouinii niger S. epidermidis T. Mentag rophyt es 102 9(25) 20(6.25) 19(6.25) 22(6.25) 20(6.25) 26(6.2 5) Control Gatifloxacin 20(12.5) 28(6.25) 24(6.25) 25(6.25) Griseofulvin 20(6.25) 17(6.25) 18(12.5) 22(6.2 5) # Values in bracket are MIC values ( g/ml). FIGURE:6 Response of Pathogenic Microorganisms to Synthesized Cyclopeptide 102

72

73 Table2.12. Antimicrobial activity data for compounds 98 and 103. Compound B. sub. Bacterial strains S. aur. Diameter of zone of inhibition (mm) P. aeru. K. pneu. C. alb. Fungal strains M. audo (25) 14(12.5) 22(6) 23(6) 21(6) 11(6) 14(6) (25) 16(12.5) 26(6) 28(6) 27(6) 15(6) 18(6) A. niger Control* Gatifloxacin 18(12.5) 28(6) 22(6) 25(6) Griseofulvin 20(6) 17(6) 18(12.5) 20(6) Values in bracket are MIC values ( g/ml) * DMF / DMSO T. menta. FIGURE:7 Antimicrobial activity data for compounds 98 and 103.

74 Table Antimicrobial activity data for synthesized cyclopeptide 104 Diameter of zone of inhibition (mm) Bacterial strains Fungal strains Compd. B. S. P. K. C. M. A. T. subtilis aureus aeruginosa pneumoniae albicans audouini Niger Mentagrop i hytes (6) a 19(6) 9(12.5) 20(6) 22(6) Control Gatifloxaci n 19(12.5) 28(6) 24(6) 25(6) Griseofulvi n 20(6) 17(6) 18(12.5) 20(6) a Values in bracket are MIC values (μg/ml)

75 FIGURE:8 Antimicrobial activity data for synthesized cyclopeptide 104

76 Table Antibacterial activity data for compound 99 and 101 Compd. Diameter of zone of inhibition (mm) Bacterial strains K. pneumonia P.aeruginosa B. subtilis S. aureus 99 17(12.5) 24(6) 9(12.5) (12.5) 28(6) 9(6) 10(12.5) Control Ciprofloxacin 19(6) 25(6) 20(6) 20(12.5) * Values in bracket are MIC values ( g/ml).

77

78 Anthelmintic activity An anthelmintic studies were carried out against three different species of earthworms according to Garg and Atal method at 4 mg -1 concentration. Suspension of the samples were prepared by triturating synthesized compounds (200 mg) with Tween 80 (0.5%) and distilled water. The resulting mixture were stirred using a mechanical stirrer for 30 min. The suspensions were diluted to contain 0.4% w/v of the test samples. Suspensions of standard drugs albendazole, piperazine citrate and mebendazole were prepared in a similar way. Three sets of five earthworms of similar sizes (5.1 cm in length) were placed in Petri plates of 10.2 cm diameter containing 50 ml of the suspension of the distilled water and Tween 80 (0.5%). The paralyzing and death times were noted and their mean was calculated for triplicate sets. The death time was ascertained by placing the earth worms in warm water (50 C), which stimulated movement if the worm was alive. Results of the anthelmintic activity of the test compounds and reference drugs are listed below.

79 Compoun d Control # Mebendazol e Mean paralyzing time (min) ± ± ± 0.64 Table Anthelmintic screening data for 79 and 90. Earthworm species M. konk. P. core. E. euge. Mean death time (min) ± ± ± 0.53 Mean paralyzing time (min) ± ± ± 0.43 Mean death time (min) ± ± ± 0.22 Mean paralyzing time (min) ± ± ± 0.45 Mean death time (min) ± ± ± 0.62 M. konk.: Megascoplex konkanensis; P. core.: Pontoscotex corethruses; E. euge.: Eudrilus eugeniea; Conc. = 2 mg/ml; Data are given as mean ± S.D. (n = 3); # Tween 80 (0.5%) in distilled water. Table Anthelmintic screening data for 80 and 89. Compound Earthworm species M. konk. P. core. E. euge. Mean paralyzing time (min) Mean death time (min) Mean paralyzing time (min) Mean death time (min) Mean paralyzing time (min) Mean death time (min) ± ± ± ± ± ± ± ± ± ± ± ± 0.23 Control # Mebendazole ± ± ± ± ± ± 0.62 M. konk.: Megascoplex konkanensis, P. core.: Pontoscotex corethruses, E. euge.: Eudrilus eugeniea Data are given as mean ± S.D. (n = 3) # Tween 80 (0.5%) in distilled water

80 Table Anthelmintic activity data for compound 92 Eudrilus species Megascoplex konkanensis Compd. Mean paralyzing Mean death Mean paralysing Mean death time(min) S.E. time(min) S.E. time(min) S.E. time(min) S.E. 0.5% tween 80 in distilled water Piperazine citrate a Mebendazole a a c = 2 mg ml -1 Table Anthelmintic activity data of compound (96) Eudrilus species Megascoplex konkanensis Compd. Mean paralyzing Mean death Mean paralysing Mean death time(min) S.E. time(min) S.E. time(min) S.E. time(min) S.E. 0.5% tween 80 in distilled water Piperazine citrate a Mebendazole a a c = 2 mg ml -1

81 Table Antihelmintic activity data of 95. Compound Earthworm species M. konkanensis P. corethruses Eudrilus sp. Mean Mean Mean Mean Mean Mean paralyzing death time paralyzing death time paralyzing death time time (min) a (min) time (min) (min) time (min) (min) ± ± ± ± ± ± 0.11 Control c Mebendazo ± ± ± ± ± ± 0.62 le b a Data are given as mean ± S.D. (n = 3); b c = 2 mg/ml; c 0.5% tween 80 in distilled water. Table 2.20: Anthelmintic activity data for Synthesized Cyclopeptide 102 Compd. Mean paralyzing time (min) a Earthworm species M. konkanensis P. corethruses Eudrilus sp. Mean Mean Mean Mean death time paralyzing death time paralyzing (min) time (min) (min) time (min) Mean death time (min) 8 b Control c Mebendaz ole b 8.28 ± ± ± ± ± ± ± ± ± ± ± ± 0.62 a Data are given as mean ± S.D. (n = 3); b c = 2 mg/ml; c 0.5% tween 80 in distilled water.

82

83 Compd Control Standard (5-FU) Cytotoxicity screening Synthesized cyclopeptide were subjected to short term in vitro cytotoxicity study at μg/ml using 5-fluorouracil (5-FU) as reference compound (Kuttan et al 1985). Activity was assessed by determining the percentage inhibition of DLA and EAC cells. Both cells were cultured in the peritoneal cavity of healthy albino mice by injecting the suspension of cells ( cells/ml) intra peritoneally. After days, cells were withdrawn from the peritoneal cavity of the mice with help of sterile syringe and counted using haemocytometer and adjusted to cells/ml. Different dilution of all selected compounds ranging from μg/ml were prepared in dulbecoccs minimum essential medium and 0.1 ml of each diluted test compound were added to 0.1 ml of DLA cells ( cells/ml) and EAC cells ( cells/ml). Resulted suspensions were incubated at 37 C for 3 h. After 3 h, tryphan blue dye exclusion test was performed and percentage growth inhibition was calculated. CTC 50 values were determined by graphical extrapolation method. Controls were also tested at μg/ml against both cell lines. The results of cytotoxicity studies are listed in the Tables. Table Cytotoxic activity data for synthesized cyclopeptide 104 Conc. (μg/ml) Live cells counted No. of dead cells DLA cells % CTC growth 50 inhibition a (μm) b Live cells counted EAC cells No. of dead cells % growth inhibition CTC 50 (μm)

84 a % growth inhibition = 100 [{(Cell total Cell dead ) 100} / Cell total ]; b CTC 50 = conc. inhibiting 50% of percentage growth FIGURE:13 Cytotoxic activity data for synthesized cyclopeptide 104 Table Cytotoxic activity data for synthesized cyclopeptide 101 Compd. 104 Control Standard (5-FU) Conc. (μg/ml) Live cells counte d DLA cells No. of dead cells % growth inhibitio n a CTC 50 b (μm) Live cells counte d EAC cells No. of dead cells % growth inhibitio n CTC 50 (μm)

85 a % growth inhibition = 100 [{(Cell total Cell dead ) 100} / Cell total ]; b CTC 50 = conc. inhibiting 50% of percentage growth FIGURE:14 Cytotoxic activity data for synthesized cyclopeptide 101