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Abstract ENGLISH SUMMARY Ethyl 10H-Phenothiazine-10-yl acetate (2) [135-137] was obtained by treating of phenothiazine (1) with ethyl choloroacetate in dry acetone and anhydrous K2CO3 by refluxing in 88% yield. A solution of the respective ester 2 in absolute ethanol and hydrazine hydrate was refluxed for 3 h to afford 2-(10H-Phenothiazine-10-yl)acetohydrazide (3) as a pale yellow powder in 88% yield (Scheme 1). Amino acid ethyl ester hydrochlorides (5,10,15) were prepared by treating of the corresponding amino acid with thionyl chloride in absolute ethanol at -10oC followed by addition of absolute ethanol. The melting points of the prepared ester were agreed with these in Aldrich fine chemical catalog. Methyl [(10Hphenothiazine-10-ylacetyl)amino]acetate (6) was prepared by treating of the acid hydrazide 3 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added and the formed 10HPhenothiazine- 10-ylacetyl azide (4) was treated with glycine methyl ester Summary hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 6 in 78% yield. A mixture of the above ester 6 and hydrazine hydrate (N2H4.H2O) in ethanol was heated under reflux for 3 h to give N-(2-Hydrazinyl-2-oxoethyl)- 2-(10H-phenothiazine-10-yl)acetamide (7) in 88% yield (Scheme 2) Summary Methyl{{{[(10H-phenothiazin-10-ylacetyl)amino]acetyl}amino}} acetate (9) was prepared by treating of the acid hydrazide 7 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 8 was treated with glycine methyl ester hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 9 in 70% yield (Scheme 3). Methyl-2-[(10H-phenothiazin-10-ylacetyl)amino]-3- sulfanylpropanoate (11) was prepared by treating of the acid hydrazide 3 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 4 was treated with cystin methyl ester hydrochloride (10) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 11 in 75% yield (Scheme 4). Summary A mixture of the above ester 11 and hydrazine hydrate (N2H4.H2O) in ethanol was heated under reflux for 3 h to give N-(1-hydrazinyl-1-oxo- 3-sulfanylpropan-2-yl)-2-(10H-phenothiazin-10-yl)acetamide (12)in 85% yield (Scheme 5). The peptide (14) was prepared by treating of the acid hydrazide 12 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 13 was treated with cystine methyl ester hydrochloride (10) in ethyl acetate containing Et3N at 0oC. The Summary formed product was purified by recrystallization from ethanol to afford peptide 14 in 78% yield (Scheme 6). The peptide (16) was prepared by treating of the acid hydrazide 3 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 4 was treated with lysin methyl ester hydrochloride (15) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 16 in 79% yield (Scheme 7). Summary A mixture of the above ester 16 and hydrazine hydrate (N2H4.H2O) in ethanol was heated under reflux for 3 h to give acid hydrazide (17) in 88% yield (Scheme 8). The peptide (19) was prepared by treating of the acid hydrazide 17 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 18 was treated with glycine methyl Summary ester hydrochloride (15) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford 19 in 78% yield (Scheme 9). Ethyl [3-(10H-phenothiazin-10-ylmethyl)-1,2,4-oxadiazol-4(5 H)- yl]acetate (21) was obtained by treating of 10-(4,5-dihydro-1,2,4-oxadiazol- 3-ylmethyl)-10H-phenothiazine (20) with ethyl choloroacetate in dry acetone and anhydrous K2CO3 by refluxing in 88% yield. A solution of the respective ester 21 in absolute ethanol and hydrazine hydrate was refluxed for 3 h to afford 2-[3-(10H-phenothiazin- 10-ylmethyl)-1,2,4-oxadiazol-4(5H)-yl]acetohydrazide (22) as a pale yellow powder in 88% yield (Scheme 10). Summary Methyl {[(3-(10H-phenothiazin-10-ylmethyl)-1,2,4-oxadiazol-4(5 H)-yl)acetyl]amino}acetate (24) was prepared by treating of the acid hydrazide 22 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 23 was treated with glycin methyl ester hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 24 in 74% yield (Scheme 11). Summary A solution of the respective ester 24 in absolute ethanol and hydrazine hydrate was refluxed for 3 h to afford N-(2-hydrazinyl-2- oxoethyl)-2-[3-(10H-phenothiazin-10-ylmethyl)-1,2,4-oxadiazol-4(5 H)- yl]acetamide (25) as a pale yellow powder in 88% yield (Scheme 12). The peptide (27) was prepared by treating of the acid hydrazide 25 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 26 was treated with glycin methyl ester hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 27 in 75% yield (Scheme 13). The structure of 27 was elucidated by I.R and mass spectroscopy . Summary A solution of the respective ester 27 in absolute ethanol and hydrazine hydrate was refluxed for 3 h to afford N-(2-hydrazinyl-2- oxoethyl)-2-[3-(10H-phenothiazin-10-ylmethyl)-1,2,4-oxadiazol-4(5 H)- yl]acetamide (28) as a pale yellow powder in 88% yield (Scheme 13). The peptide (30) was prepared by treating of the acid hydrazide 28 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 29 was treated with glycin methyl ester hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford 30 in 77% yield (Scheme 14). Summary The peptide (31) was prepared by treating of the acid hydrazide 22 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 23 was treated with cystin methyl ester hydrochloride (10) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford peptide 31 in 75% yield (Scheme 15). Summary A mixture of the above ester 31 and hydrazine hydrate (N2H4.H2O) in ethanol was heated under reflux for 3 h to give acid hydrazide (32) in 85% yield (Scheme 16). Summary The peptide (34) was prepared by treating of the acid hydrazide 32 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 33 was treated with cystine methyl ester hydrochloride (10) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 34 in 78% yield (Scheme 17). The peptide (35) was prepared by treating of the acid hydrazide 22 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 23 was treated with lysin methyl ester hydrochloride (15) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford 35 in 79% yield (Scheme 18). Summary A mixture of the above ester 35 and hydrazine hydrate (N2H4.H2O) in ethanol was heated under reflux for 3 h to give acid hydrazide (36) in 88% yield (Scheme 19). The peptide (38) was prepared by treating of the acid hydrazide 36 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 37 was treated with lysin methyl Summary ester hydrochloride (15) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford 38 in 78% yield (Scheme 20). 5-(10H-phenothiazin-10-ylmethyl)pyrimidine-2,4(1 H,3H)-dione (40) was obtained by treating of phenothiazine (1) with uracile (39) in the presence of paraformaldehyde by refluxing in 68% yield (Scheme 21). Summary Ethyl[2,6-dioxo-5-(10H-phenothiazin-10-ylmethyl)-3,6- dihydropyrimidin-1(2 H)-yl]acetate (41) was obtained by treating of 5- (10H-phenothiazin-10-ylmethyl)pyrimidine-2,4(1 H,3H)-dione (40) with ethyl choloroacetate in dry acetone and anhydrous K2CO3 by refluxing in 88% yield. Asolution of the respective ester 41 in absolute ethanol and hydrazine hydrate was refluxed for 3 h to afford 2-[2,6-dioxo-5-(10Hphenothiazin- 10-ylmethyl)-3,6-dihydropyrimidin-1(2H)- yl]acetohydrazide (42) as a pale yellow powder in 88% yield(Scheme 22) The peptide (44) was prepared by treating of the acid hydrazide 42 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 43 was treated with glycin methyl ester hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford 44 in 74% yield (Scheme 23). Summary A solution of the respective ester 44 in absolute ethanol and hydrazine hydrate was refluxed for 3 h to afford acid hydrazide (45) as a pale yellow powder in 88% yield (Scheme 24). Summary The peptide (47) was prepared by treating of the acid hydrazide 45 with AcOH and 1N HCl and cooling at -5oC. Sodium nitrite (NaNO2) was added, and the formed acid azide 46 was treated with glycin methyl ester hydrochloride (5) in ethyl acetate containing Et3N at 0oC. The formed product was purified by recrystallization from ethanol to afford the corresponding peptide 47 in 75% yield (Scheme 25). - 1 - METHODS FOR THE PEPTIDE SYNTHESIS AND ANALYSIS 1. INTRODUCTION Peptides as drugs show unique characteristics (high biological activity, high specificity, and low toxicity) thereby making them particularly attractive therapeutic agents [1]. However, the role of peptides in drug discovery has suffered ups and downs during the last four decades. A first analysis of the new chemical entities (NCEs) accepted by the Food and Drug Administration (FDA) indicated that while 53 NCEs were introduced as drugs in 1996, only 17 were introduced in 2002. This number increased to 31 in 2004, but decreased again in 2005 with just 18 new drugs, 17 in 2007, and a slight increase to 21 in 2008 (Figure 1.1) [2, 3]. An analysis of these 21 drugs approved in 2008 indicated that almost 50% of the new drugs can be considered nonclassical, in the sense that they are nonclassical small molecules. Figure 1.1 Distribution by chemical structure of the newdrugs approved by the FDAin 2008. (See insert for color representation of this figure.) |