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TABLE 2: SAMPLE CALCULATION FOR WEIGHT (wt) TABLE 3: SIMILARITY FACTORS FOR DIFFERENT TEST FOR TEST FORMULATION1 FORMULATIONS

Time R T SDR SDT CVR CVT wt Test formulations f2-m f2-m3 f2 (min) 1 51.34 54.68 60.04 30 34.92 40.34 2.26 4.10 6.47 10.16 1.83 2 45.01 46.88 51.08 60 59.50 67.15 3.85 6.34 6.47 9.44 2.59 3 41.86 48.30 51.19 90 79.27 87.01 5.12 4.76 6.45 5.47 2.19 4 37.38 46.46 50.07 180 95.08 97.73 6.14 1.48 6.45 1.51 1.79 5 42.05 44.98 48.05

R = reference, T = Test , SDR = standard deviation of Rt, SDT = standard deviation f2-m = Similarity factor calculated using new approach, f2-m3 = Similarity factor of Tt, CVR = percentage coefficient of variation of Rt, CVT = percentage calculated using approach 3, f2 = Similarity factor calculated using conventional coefficient variation of tT , wt = 1 + (%CV of Rt/MCVE/L) + (% CV of Tt/MCVE/L) method (wt = 1) average value of reference and test at all the time point in Abbreviated New Drug Application (ANDA) point is similar then it is irrational to compute weight submission. If the value of f2-m is greater than 50 for calculating similarity factor because the product than we may safely conclude that products show similar dissolution. The positive and negative points of weight (wt) and (Rt-Tt) will be zero. Therefore f2 is equal to 100. of the new approach will emerge out when various researchers will try the approach with their data sets.

In the new scheme of weight (wt) calculation, no parameter was decided on an arbitrary ground. REFERENCES The new approach appears to be more realistic as compared to approach 3. Another advantage of the 1. Moore JW, Flanner HH. Mathematical comparison of dissolution profiles. Pharma Tech 1996;20:64-74. new method is simple calculation steps than approach 2. Gohel MC, Sarvaiya KG, Mehta NR, Soni CD, Vyas VU, Dave RK. 3. Equal stress is given to variability in reference and Assessment of similarity factor using different weighting approaches. test formulation in the new approach. The maximum Diss Tech 2005;12:22-37. 3. Guidance for industry: Dissolution testing of immediate release solid allowable %CV is also considered in the proposed oral dosage forms. US Food and Drug Administration, Rockville, MD, method. It considers within samples (12 units of USA, 1997. reference and 12 units of test) as well as between 4. Shah VP, Tsong Y, Sathe P, Liu JP. In vitro dissolution profile comparison statistics and analysis of the similarity factor, f2. Pharma samples (reference and test formulations) variability Res 1998;15:889-95. because of utilization of standard deviation and averages of reference and test formulations at each time points for calculating weight. The use of new method is recommended in deciding equivalence of Accepted 5 April 2009 test product with innovators product. The approach Revised 9 January 2009 may also find application in selection of a bio-batch. Received 11 September 2006 The use of new approach may become a strong Indian J. Pharm. Sci., 2009, 71 (2): 142-144

Synthesis and Biological Evaluation of Fatty Hydrazides of By-products of Oil Processing Industry

S. Toliwal*, K. Jadav and K. Patel Department of Industrial Chemistry, Institute of Science and Technology for Advanced Studies and Research (ISTAR), Vallabh Vidyanagar-388 120, India

Toliwal, et al.: Fatty Hydrazides of By-products of Oil Processing Industry

Some new 2-alkyl-5-mercapto-1,3,4-Oxadiazoles and 3-alkyl-5-mercapto-1,2,3-4H triazoles were synthesized from

*Address for correspondence E-mail: [email protected]

144 Indian Journal of Pharmaceutical Sciences March - April 2009 www.ijpsonline.com hydrazides of acid oil and oil recovered from spent bleaching earth. These newly synthesized compounds were characterized on the basis of elemental analysis and evaluated for biological properties. Certain derivatives exhibited fairly high antibacterial and antifungal activities when compared with streptomycin and immidil used as standard antibacterial and antifungal agents respectively.

Key words: Acid oil, oil recovered from spent bleaching earth (ORSBE), oxadiazoles, triazoles, hydrazides, antibacterial, antifungal, streptomycin, immidil

Hydrazides, the acylated derivatives[1] of hydrazine gas at flow rate 10 ml/min using FID at an injector are usually encountered as the simple or temperature of 2500 was found to be: palmitic, 12.03 monosubsituted (RCONHNH2), or as sym-disubsituted for acid oil and 10.54 for ORSBE; stearic, 10.26 for (RCONHNHOR) compounds. The latter have been acid oil and 4.62 for ORSBE; oleic, 36.08 for acid oil referred to as sec-hydrazides. Besides being useful and 21.39 for ORSBE; linoleic, 40.01 for acid oil and for a number of biological properties, hydrazides 52.53 for ORSBE and linolenic, 1.2 for acid oil and are important starting materials for a wide range 6.09% for ORSBE. All other chemicals used in the of derivatives utilizable in pharmaceutical products study were of laboratory grade and were used without and as surfactants. Hydrazides have been known any modification. to be associated with antibacterial[2], antifungal[3], anthelmintic[4] and anticonvulsant[5] activities. Methyl esters of acids from oil were prepared by acid Various thiosemicarbazide derivatives are reported catalyzed esterification using standard method[14]. The to possess useful pharmacological properties like, esters were purified by distillation under 4-5 mm Hg antidepressant[6], antiinflammatory[7] and analgesic[8] pressure. activities. In addition to the antibacterial[9] activities exhibited by several triazole derivatives, they are Preparation of fatty hydrazides and their derivatives is also known for their fungicidal, analgesic and schematically represented in Fig 1. For the preparation [14] antiinflammatory[10] activities. Oxadiazoles and of fatty acid hydrazides a solution of fatty acid their derivatives are well known chemotherapeutic esters (0.1 M) in ethanol (150 ml) was mixed with agents and their utility has muscle relaxant, and hydrazine hydrate (95%, 0.2 M) was added. The bacteriostatic[11] are well known. Biological assessment reaction mixture was refluxed for 3-4 h. It was cooled, of fatty hydrazide and their derivatives has been the and the solid separated was collected, washed and focus of earlier investigative studies[12,13]. recrystallised from ethanol.

The present work is carried out with a view to impart For the preparation of 2-alkyl-5-mercapto-1,3,4- [15] value addition targets by-products, acid oil and oil oxadiazole a solution of a fatty acid hydrazide recovered from spent bleaching earth (ORSBE) used (0.01 M) in 10 ml of ethanol, a solution of carbon for soya bean oil. The fatty hydrazides are further derivatized to obtain new antibacterial and antifungal N N N NH NH2NH2 CS2/KOH RCOOC2H5 RCONHNH2 agents. (A) R C C-SH R C C=S O O (B)

The by-products of oil processing industries- acid oil KSCN RCONHNH2 RCONHNHCSNH2 and ORSBE were procured from Ashwin Vanaspti (A) (C) Ltd, Samlaya. Analysis of oils for physicochemical characteristics was tested by standard BIS methods NaOH/I /KI KOH/C H OH and it gave: sp.gr at 30°, 0.915 and 0.921; acid value, 2 2 5 130.38 and 18.82; iodine value, 115.51 and 123.24; N N N N N NH R C R C R C C=S saponification value, 182.75 and 186.01 for acid oil C NH2 C SH and ORSBE, respectively. The fatty acid composition O N N (E) H H of oils was determined by gas liquid chromatography (D) (GLC) of methyl esters using capillary column (2 Fig. 1: Preparation of fatty hydrazides and their alkyl derivatives. A- Fatty hydrazide, B- 2-alkyl-5-mercapto-1,3,4-oxadiazole, C- m×0.32 mm) packed with 50% cynopropyl phenyl thiosemicarbazide, D- 3-alkyl-5-mercapto-1,2,4-4H-triazoles, E- polysiloxane (BP225) at 2200 with nitrogen as carrier 2-alkyl-5-amino-1,3,4-oxadiazole

March - April 2009 Indian Journal of Pharmaceutical Sciences 145 www.ijpsonline.com disulfide (2 g) in 3 ml of water and 1 g of potassium TABLE 1: Physico-chemical properties of hydroxide were refluxed for 7-8 h until the H S is hydrazides and their derivatives 2 Sample Melting Yield Nitrogen ceased. The contents were cooled and acidified with code point0 % Content (%) dilute hydrochloric acid. The separated solid was Calculated Found filtered, collected, washed with water, dried and II 81.0 82.1 9.52 8.68 recrystallised from ethanol. A IIB 76.8 74.6 11.90 10.34 II 71.8 72.0 8.33 9.24 [16] F For the preparation of fatty acid thiosemicarbazide IIG 86.9 76.3 12.54 12.55 II 74.5 84.2 13.17 11.78 a solution of a fatty acid hydrazide (0.02 M) in H V 79.3 69.3 9.52 9.89 methanol (50 ml), potassium thiocynate (0.03 M) A VB 76.1 72.5 11.90 10.86 and hydrochloric acid 3 ml were mixed added with VF 73.2 81.2 8.33 8.94 VG 84.7 78.3 12.54 10.58 constant stirring. The mixture was immediately V 78.1 72.6 13.17 12.18 evaporated to dryness on a steam bath and heated for H IIA- Fatty hydrazide of acid oil, IIB-thiosemicarbazide of acid oil, IIF-2-alkyl-­ 5-mercapto-1,3,4-oxadiazole of acid oil, II - 3-alkyl-5-mercapto-1,2,4-4H- an additional hour with another 50 ml ethanol. The G triazoles of acid oil, IIH- 2-alkyl-5-amino- 1,3,4-oxadiazole of acid oil, VA- fatty resulting solid was treated with water, little ethanol hydrazide of ORSBE, VB -thiosemicarbazide of ORSBE, VF- 2-alkyl-5-mercapto-

1,3,4-oxadiazole of ORSBE, VG- 3-alkyl-5-mercapto-1,2,4-4H-triazoles, VH and recrystallised from ethanol. - 2-alkyl-5-amino-1,3,4-oxadiazole of ORSBE.

For the preparation of 3-alkyl-5-mercapto-1,2,4,-4H- for anti bacterial activity against Bacillus subtilis [17] triazoles a solution of thiosemicarbazide (0.01 M) and Escherichia coli and antifungal activity against in 15 ml of ethanol, and potassium hydroxide (20 Aspergillus niger by agar-agar cup method[18]. ml, 10%) was refluxed for 7-8 h on a steam bath. It Streptomycin and immidil were used as standard was cooled and acidified with dilute hydrochloric acid antibacterial and antifungal agents respectively. to adjust the pH between 5-6. Resulting solid was filtered, dried and recrystallised from ethanol. It can be observed that (Table 1) upon cyclization to thiosemicarbazides, 2-alkyl 5-mercapto 1,3,4 For the preparation of 2-alkayl-5-amino-1,3,4- oxadiazoles and 2-alkyl-5-amino-1,3,4-oxadiazoles, oxadiaazole[14] a solution of thiosemicarbazide (0.01 the melting points are decreased and are lower than M) in 15 ml of ethanol was added to a solution that of the original hydrazide from which they are of sodium hydroxide (5 ml, 5N) with cooling and derived. However, the melting point increase is stirring. To this clear solution, a solution of I /KI 2 observed when the fatty hydrazides were cyclised was added till permanent tinge of iodine persisted at room temperature. The mixture was immediately to 3-alkyl-5-mercapto-1,2,4-4H triazoles with latter showing higher melting point than that of the former. refluxed and more I2/KI was added till permanent tinge was obtained. The mixture was then cooled and This is in agreement with results of the synthesis poured into ice-cold water; the solid that separated work of some fatty hydrazide derivatives conducted [19] [20] was collected by filtration, washed with water and by Badami et al. and by Daulatabad et al. . They with dilute thiosulfate solution and again with water. also reported similar trend of increase in melting point The solid was dried and recrystallised from absolute for 3-alkyl 5 mercapto-1,3,4-4H triazoles of different ethanol. The melting point, nitrogen content and fatty hydrazides and increasing melting points for percent yield of the compound is given in Table 1. oxadiazoles of the two fatty hydrazides as compared to respective fatty (oleic and linoleic) hydrazides. Characterization of synthesized compounds was conformed by IR. Mercaptotriazoles and The melting point increase for various hydrazide aminooxadiazoles showed band at 3100 cm-1 for NH derivatives was observed in the following order: group. The C=N stretching as observed at 1600 cm-1. 3-alkyl-5-mercapto-1,2,4-(4H)-triazoles>2-alkyl-5- The mercaptooxadiazoles showed strong band at 1160 amino-1,3,4-oxadiazoles>2-alkyl-5-mercapto-1,3,4- cm-1 for C=S and absence of band around 2550-2600 oxadiazoles. This result is also in agreement with the cm-1. This showed that this compound existed in results obtained by Badami et al.[19]and Daulatabad et thione form rather than thiol. al.[20] for corresponding fatty derivatives.

The hydrazides and their derivatives were tested The results of antibacterial activities of hydrazides

146 Indian Journal of Pharmaceutical Sciences March - April 2009 www.ijpsonline.com

TABLE 2: Antibacterial activities of Acid oil and ORSBE hydrazides and their derivatives Bacillus subtilis Escherichia coli Sample Zone standard Zone sample Control growth Zone standard Zone sample Control growth code 200 µg (mm) 200 µg (mm) % 200 µg (mm) 200 µg (mm) %

IIA --- -Ve ------23 -Ve ------

IIB 22 4.0 --- + 23 1.2 --- +

IIF 23 4.4 --- + 22 1.2 17 +

IIG 22 3.2 --- + 22 0.8 17 +

IIH 24 2.4 --- + 26 1.2 16 +

VA -Ve -Ve ------24 0.8 16 +

VB 20 7.2 --- ++ 23 -Ve ------

VF 23 6.4 --- ++ 26 0.4 18 +

VG 21 4.8 --- + 21 -Ve 16 ---

VH -Ve -Ve ------20 -Ve 17 ---

IIA- Fatty hydrazide of acid oil, IIB- thiosemicarbazide of acid oil, IIF-­ 2-alkyl-5-mercapto-1,3,4-oxadiazole of acid oil, IIG- 3-alkyl-5-mercapto-1,2,4-4H triazoles of acid oil, IIH- 2-alkyl-5-amino-1,3,4-oxadiazole of acid oil, VA- fatty hydrazide of ORSBE, VB- thiosemicarbazide of ORSBE, VF -2-alkyl-5-mercapto-1,3,4-oxadiazole of ORSBE, VG- 3-alkyl-5-mercapto-1,2,4-4H-triazoles, VH- 2-alkyl-5-amino-1,3,4-oxadiazole of ORSBE. + = 0-25%, ++ = 25-50%, +++ = 50-75%, ++++ = 75-100%, +++++ = More than 100%. and their derivatives (Table 2) highlights following Table 3: Antifungal activities of Acid oil and points: When tested for antibacterial activity against ORSBE hydrazides and their derivatives Sample Zone standard Zone sample Control growth Bacillus subtilis and Escherichia colid acid oil-based code 100ppm (mm) 100ppm (mm) % derivatives namely thiosemicarbazide, 2-alkyl-5- IIA ------II --- -Ve ------mercapto-1,3,4-oxadiazole, 3-alkyl-5-mercapto-1,2,4- B II --- -Ve ------4H-triazoles, 2-alkyl-5-amino-1,3,4–oxadiazole, F IIG 13 7.0 --- +++ exhibited mild (0–25%) activities. ORSBE-based IIH 13 4.0 --- + V 13 2.0 --- + derivatives namely thiosemicarbazide, 2-alkyl-5- A VB --- -Ve ------mercapto-1,3,4-oxadiazole exhibited fair (25-50%), VF 13 8.0 --- +++ V --- -Ve ------while hydrazides, 2-alkyl-5-amino-1,3,4-oxadiazole of G V 13 2.0 --- + ORSBE a showed poor and 3-alkyl-5-mercapto-1,2,4- H IIA- Fatty hydrazide of acid oil, IIB- thiosemicarbazide of acid oil, IIF-­ 2-alkyl- 4H-triazoles demonstrated mild (0-25%) acvtivities 5-mercapto-1,3,4-oxadiazole of acid oil, IIG- 3-alkyl-5-mercapto-1,2,4-4H triazoles of acid oil, II - 2-alkyl-5-amino-1,3,4-oxadiazole of acid oil, V - fatty against Bacillus subtilis relative to streptomycin H A hydrazide of ORSBE, VB- thiosemicarbazide of ORSBE, VF -2-alkyl-5-mercapto- 1,3,4-oxadiazole of ORSBE, V - 3-alkyl-5-mercapto-1,2,4-4H-triazoles, V - used as standard. ORSBE based derivatives namely G H 2-alkyl-5-amino-1,3,4-oxadiazole of ORSBE. + = 0-25%, ++ = 25-50%, +++ = hydrazide, 2-alkyl-5-mercapto-1,3,4–oxadiazole 50-75%, ++++ = 75-100%, +++++ = More than 100%. exhibited mild (0-25%), while thiosemicarbazide, 3-alkyl-5-mercapto-1,2,4-4H-triazoles, 2-alkyl-5- amino-1,3,4-oxadiazole demonstrated poor bacterial It can be concluded that thiosemicarbazide and 2-alkyl-5-mercapto-1,3,4-oxadiazoles of ORSBE growth retardation against Escherichia coli relative to can be used as antibacterial agent against Bacillus streptomycin used as standard. subtilis due to their fair antibacterial activities (25- 50%) whereas 3-alkyl-5-mercapto-1,2,4-4H-triazoles The results of antifungal activities of acid oil and of acid oil, and 2-alkyl-5 mercapto-1,3,4-oxadiazole ORSBE hydrazides and their derivatives (Table 3) of ORSBE exhibited good (50-75%) fungal growth highlight following points: when tested for antifungal retardation against Aspergillus niger due to their fairly activity against Aspergillus niger acid oil based high antifungal activities. 2-alkyl-5-amino-1,3,4-oxadiazole, ORSBE-based hydrazide and 2-alkyl-5-amino- 1,3,4-oxadiazole REFERENCES showed mild (0-25%) antifungal activity, while 3-alkyl- 5-mercapto-1,2,4-4H triazoles of acid oil, 2-alkyl-5- 1. Markley KS. Fatty acids, their chemistry, properties and uses. New mercapto-1,3,4-oxadiazole of ORSBE exhibited good York: Interscience Publishers; 1964. p. 1604-8. (50-75%) fungal growth retardation. The hydrazide, 2. Mir I, Siddiqui MT, Comrie A. 6-Benzylidene-2-(2-chlorophenyl) thiazolo[3,2-b]-1,2,4-triazol-5(6H)-one. Tetrahedron 1970;26:5235-7. thiosemicarbazide and 2-alkyl-5-mercapto-1,3,4- 3. Degener E, Scheinplug H, Schemelzer HG. Brit Patent No, 1035,474, oxadiazole of acid oil, thiosemicarbazide and 3-alkyl- 1967. 5-mercapto-1,2,4-4H triazoles of ORSBE, showed poor 4. Cavier R, Rips R. Antianthelminate effect of thioamides. J Med Chem 1965;8:706-9. antifungal growth against Aspergillus niger relative to 5. Trepanier DL,Wagner ER, Harris G, Rudzik AD. 1,4,5,6-Tetrahydro- immidil used as a standard. as-triazines. I. Sulfuric Acid Catalyzed Condensation of Nitriles and

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Hydrazino Alcohols. J Med Chem 1966;9:881–5. Standards; 1976. p. 6–7. 6. Schilling K. Laballenic Acid: A New Allenic Acid from Leonotis 15. Daulatabad CD, Mirajkar AM. Epoxy oleic acid. J Oil Tech Assn nepetaefolia Seed Oil. Fette Seifen Antrichmittel 1961;67:421-5. (India) 1988;20:9-11. 7. Takagi T, Craig BM. Hydrogenation of conjugated fatty acids with 16. Hoggarth E. Synthesis of triazoles and thione derivatives. J Chem Soc hydrazine. J Am Oil Chem Soc 1964;41:660-1. 1961;48:111. 8. Lieberman D, Rist N, Grumbach F, Moyewx M, Gautheir V, Rouqix 17. Boots SG, Cheng CC. Sydnone derivatives as synthons for novel A, et al. Antituberculosis activity of isonicotinic thioamides. Bull Soc bismesoionic compounds. J Heteroycl Chem 1967;4:272-7. Chim; France, 1954;1440. 18. Hoggarth E. Synthesis and biological activity of N-aryl-N-subsituted 9. Sengupta AK, Garg M, Chandra U. antibacterial activity of thiourea derivatives. J Chem Soc 1949;1163-5. 1,3,4-oxadiazoles. J Indian Chem Soc 1974;56:1230-2. 19. Badami RC, Hendi SB, Patil KB. Synthesis of some hydrazide 10. George T, Mehta DV, Ahiramani R, David J, Talwalker PK. Synthesis derivatives. J Oil Tech Assoc India 1979;11:78-9. of some 5-triazoles with potential analgesic and inflammatory activities. 20. Daulatabad CD, Mirajkar AM, Hosamani KM. Oleochemicals J Med Chem 1971;14:335-7. II: Synthesis and biological evalution of some substituted 1,3,4– 11. Sherman WR. 5-Nitro-2-furyl substituted 1,3,4-oxadiazoles oxadiazoles and 1,2,4, 4H - triazoles. J Oil Tech Assn (India) 1,3,4-thiadizoles and 1,3,5-triazoles. J Org Chem 1961;26:88-91. 1989;21:27-9. 12. Raval DA, Toliwal SD. New Oleochemicals from non-traditional oils. J Oil Tech Assn (India) 1994;1:26. 13. Yousef EA, Zaki ME, Megahed MG. Fatty acid hydrazides in organic synthesis: Novel synthesis of 6-alkyl-3-aryl-5-imino-7-oxo-2,5,6,7- tetrahydro-1H-1,2-diazepine-4-carbonitrile and 6-alkyl-3-aryl-5,7-dioxo- Accepted 10 April 2009 2,5,6,7-tetrahydro-1H-1,2-diazepine-4-carbonitrile. Heterocycl Comm Revised 8 January 2009 2003;9:293-8. Received 2 April 2008 14. BIS: 548 (Part–III–Analysis by liquid chromatography), Methods of sampling and test for oils and fats. New Delhi: Bureau of Indian Indian J. Pharm. Sci., 2009, 71 (2): 144-148

RP-HPLC Estimation of Ramipril and Telmisartan in Tablets

V. P. KURADE*, M. G. PAI AND R. GUDE Department of Pharmaceutical Analysis, Goa College of Pharmacy, Panaji-403 001, India

Kurade, et al.: RP-HPLC Estimation of Ramipril and Telmisartan

A rapid high performance liquid chromatographic method has been developed and validated for the estimation of ramipril and telmisartan simultaneously in combined dosage form. A Genesis C18 column having dimensions of 4.6×250 mm and particle size of 5 µm in isocratic mode, with mobile phase containing a mixture of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid): methanol:acetonitrile (15:15:70 v/v/v) was used. The mobile phase was pumped at a flow rate of 1.0 ml/min and the eluents were

monitored at 210 nm. The selected chromatographic conditions were found to effectively separate ramipril (Rt: 3.68 min) and telmisartan (Rt: 4.98 min) having a resolution of 3.84. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation. Linearity for ramipril and telmisartan were found in the range of 3.5-6.5 µg/ml and 28.0-52.0 µg/ml, respectively. The percentage recoveries for ramipril and telmisartan ranged from 99.09-101.64% and 99.45-100.99%, respectively. The limit of detection and the limit of quantitation for ramipril was found to be 0.5 µg/ml and 1.5 µg/ml respectively and for telmisartan was found to be 1.5 µg/ml and 3.0 µg/ml, respectively. The method was found to be robust and can be successfully used to determine the drug content of marketed formulations.

Key words: Simultaneous estimation, RP-HPLC, ramipril, telmisartan, validation

Telmisartan is a new angiotensin II receptor antagonist given in combination with ramipril. Telmisartan for the treatment of essential hypertension usually (TEL) is chemically 4’-((1,4’-dimethyl-2’-propyl(2,6’- bi-1H-benzimidazol)-1’-yl)methyl)-(1,1’-biphenyl)- *Address for correspondence E-mail: [email protected] 2-carboxylic acid. Ramipril (RAM) is chemically “Indira”, PMC,W-5,166/16, Shantinagar, Ponda, Goa, India (1S,5S,7S)-8-((2S)-2-(((1S)-1-ethoxycarbonyl-3-

148 Indian Journal of Pharmaceutical Sciences March - April 2009