DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME CYCLIC PENTAPEPTIDES

Transcription

1 UIQUE JURAL F PARMACEUTICAL AD BILGICAL SCIECES Researh Artile DESIG, SYTESIS AD BILGICAL EVALUATI F SME CYCLIC PETAPEPTIDES M. imaja 1*, Arhana 2, Karigar Asif 3 1 Pharmaeutial Chemistry Division, Shool of Advaned Sienes, VIT University, Vellore , India 2 Dept of Pharmaeutial Chemistry, GSM Institute of Pharmaeutial Sienes, Mangalore, KTK, India 3 Dept of Pharmaeutial Analysis, Maratha Mandal College of Pharmay, Belgaum, Karnataka, India *Corresponding Author: Dr. (Mrs). M. imaja, Professor, Pharmaeutial Chemistry Division. Shool of Advaned Sienes, VIT University, Vellore Tamil adu, India. dr_himaja@yahoo.om Moile: Reeived ; Revised ; Aepted Cyli peptides exhiiting antimiroial ativity showed that almost all yli peptides ontain proline, valine, phenylalanine in their strutures. Three yli pentapeptides have een designed ontaining nitro-arginine, two proline units, phenylalanine and a valine unit, with varied sequenes in order to arry out a study on variation of ativity with variation in the sequene of amino aids. Strutures of all the newly synthesized ompounds were onfirmed y FTIR, 1 MR and Mass spetral data. All the synthesized analogs were sreened for their ateriidal ativity and fungiidal ativity. Bateriidal ativity against two Gram-positive ateria Staphyloous aureus, Baillus sutilis and two Gram-negative ateria Esheridhia oli, Pseudomonas aeruginosa and fungiidal ativity against two fungi Aspergillus niger and Candida alians and found to e ative against these miroorganisms. Keywords: Cyli Pentapeptides, Amino aids, Diylohexylarodiimide, Antiaterial ativity, Antifungal ativity. ITRDUCTI Peptides are the important lass of organi ompounds with potent iologial ativities 1-7. A review of the strutures of yli peptides exhiiting antimiroial ativity showed that almost all yli peptides ontain proline, valine, phenylalanine in their strutures. Moreover, ompounds ontaining proline units showed good antimiroial ativity. Peptide antiiotis are atagorised as homomeri (uilt up of only amino aids) and heteromeri (ontaining amino aids along with other moieties). Further division leads to homodeti and heterodeti peptides (rings ontaining other linkages) suh as depsipeptides, whih ontain hydroxy and amino aid residues joined y amide and ester onds. Antiiotis effetive against all miroorganisms are not likely to e found, and a reasonale effort should e onentrated on the more speialised antiiotis. The following have een linially used effetively: aitrains, apreomyin, yloserine, atinomyin D, gramiidin S, polymixin, tyroidine and viomyin. The inherent mediinal properties of yli peptides prompted sientists to isolate these ompounds from natural soures. Sine minute quantities are otained from natural soures, many of these ompounds are attempted to synthesise in the ABSTRACT laoratories of many sientists. Antiaterial ativity studies performed on these syntheti peptides proved to give good results. MATERIALS AD METDS All the three yli pentapeptides ontain two proline units, one phenylalanine unit, one valine unit and a nitro-arginine unit with varied sequene. Synthesis of the yli pentapeptides was arried out y the solution phase peptide synthesis. In order to arry out the synthesis, all the three yli pentapeptides were disonneted into two dipeptide units and a single amino aid -(nitro)arginine. The dipeptides were prepared from the respetive proteted amino aids. The amino group was proteted with tert. Butyloxyaronyl (-) group and the aroxyl group was proteted y onverting into the methyl ester. The -amino aids were oupled with the amino aid methyl ester hydrohlorides y diylohexylarodiimide (DCC) as the oupling agent and - methylmorpholine (MM) as the ase to get the proteted dipeptides. These are oupled, after appropriate deprotetion, with -(nitro)arginine to get the pentapeptides, whih were finally ylised y p-nitrophenyl ester method using highdilution tehnique to get the yli pentapeptides. Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug

2 Synthesis of Cyli Pentapeptide 1: Cyli pentapeptide: ylo[nitroarg-phe-pro-pro-val] (1) was disonneted into -Phe-Pro-Me (2), -Pro-Val-Me (3) and -(nitro)arginine (4). The synthesis of the pentapeptide 1 (1) was arried out as follows. The ester group of -Phe-Pro-Me (2) was removed with Li and the -group of -Pro-Val-Me (3) was removed with trifluoroaeti aid (TFA). The deproteted dipeptides were oupled using DCC and MM to get the tetrapeptide -Phe-Pro-Pro-Val-Me (5). The -group of the tetrapeptide was removed using TFA. The deproteted unit was oupled with -(nitro)arg (4) to otain the pentapeptide -(nitro)arg-phe-pro-pro-val-me (6). The ester group of the pentapeptide was removed y Li and p-nitrophenyl group (pnp-) was introdued. The - group was removed y TFA and the linear fragment was ylised y adding MM and keeping the whole ontents at 0 0 C for seven days to get the yli pentapeptide-1 (1). The struture of the moleule was onfirmed y IR, 1 MR, FABMS and elemental analysis. The aove syntheti steps are shown in Sheme-1. MeC MeC Sheme-1 (3) (2) MeC C a CMe CMe 2 ( 2 ) (5) ( 2 ) Me (6) (4) ( 2 ) a,, d, e a = Li, TF: 2 (1:1), RT, 1 hr = TFA, CCl 3, RT, 1 hr = DCC, MM, CCl 3, RT, 36 hr d = pnp, CCl 3, RT, 12 hr e = MM, CCl 3, 7 days, 0 0 C (1) Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug

3 Synthesis of Cyli Pentapeptide 2: Cyli pentapeptide: ylo[nitroarg-pro-phe-pro-val] (7) was disonneted into -Pro-Phe-Me (8), -Pro-Val-Me (3) and -(nitro)arginine (4). The synthesis of the pentapeptide 2 (7) was arried out as follows. The ester group of -Pro-Phe-Me (8) was removed with Li and the -group of -Pro-Val-Me (3) was removed with trifluoroaeti aid (TFA). The deproteted dipeptides were oupled using DCC and MM to get the tetrapeptide -Pro-Phe-Pro-Val-Me (9). The -group of the tetrapeptide was removed using TFA. The deproteted unit was oupled with - (nitro)arg (4) to otain the pentapeptide -(nitro)arg- Pro-Phe-Pro-Val-Me (10). The ester group of the pentapeptide was removed y Li and p-nitrophenyl group (pnp-) was introdued. The -group was removed y TFA and the linear fragment was ylised y adding MM and keeping the whole ontents at 0 0 C for seven days to get the yli pentapeptide-2 (7). The struture of the moleule was onfirmed y IR, 1 MR, FABMS and elemental analysis. The aove syntheti steps are shown in Sheme-2. MeC Sheme- 2 MeC (3) (8) Me a C 3 CMe CMe ( 2 ) (9) ( 2 ) Me a,, d, e a = Li, TF: 2 (1:1), RT, 1 hr = TFA, CCl 3, RT, 1 hr = DCC, MM, CCl 3,RT, 36 hr d = pnp, CCl 3, RT, 12 hr e = MM, CCl 3, 7 days, 0 0 C (10) (4) ( 2 ) (7) Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug (7)

4 Synthesis of Cyli Pentapeptide 3: Cyli pentapeptide: ylo[nitroarg-pro-val-pro-phe] (11) was disonneted into -Pro-Val-Me (3), -Pro-Phe- Me (8) and -(nitro)arginine (4). The synthesis of the pentapeptide 3 (11) was arried out as follows. The ester group of -Pro-Val-Me (3) was removed with Li and the -group of -Pro-Phe-Me (8) was removed with trifluoroaeti aid (TFA). The deproteted dipeptides were oupled using DCC and MM to get the tetrapeptide -Pro-Val-Pro-Phe-Me (12). The -group of the tetrapeptide was removed using TFA. The deproteted unit was oupled with - (nitro)arg (4) to otain the pentapeptide -(nitro)arg- Pro-Val-Pro-Phe-Me (13). The ester group of the pentapeptide was removed y Li and p-nitrophenyl group (pnp-) was introdued. The - group was removed y TFA and the linear fragment was ylised y adding MM and keeping the whole ontents at 0 0 C for seven days to get the yli pentapeptide-3 (11). The struture of the moleule was onfirmed y IR, 1 MR, FABMS and elemental analysis. The aove syntheti steps are shown in Sheme-3. MeC MeC Sheme- 3 (8) a (3) Me Me Me ( 2 ) (12) ( 2 ) Me a,, d, e (13) ( 2 ) (4) a = Li, TF: 2 (1:1), RT, 1 hr = TFA, CCl 3, = DCC, MM, CCl 3, RT, 36 hr d = pnp, CCl 3, RT, 12 hr e = MM, CCl 3, 7 days, 0 0 C (11) Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug

5 General Method of Peptide Synthesis: Preparation of the Dipeptides: ut of the various reported methods for peptide oupling, we adopted the methods proposed y Bodanszky 8 method, whih was onvenient and useful. From these proedures we derived our own more onvenient method for the peptide oupling reations. The dipeptides were prepared y using -amino aid and amino aid methyl ester hydrohloride, TEA and DCC (Sheme 4). Sheme 4 R 1 C DCC, DCM, MM, RT, 24h. R 2 Me 2 R 1 R 2 Me The strutures of all the dipeptides were onfirmed y spetral analysis. Preparation of the tetrapeptides: The tetrapeptides were prepared from the dipeptide units after appropriate deprotetion at the desired funtional groups. The - group was deproteted y trifluoroaeti aid (TFA) and the ester group was removed y hydrolysing with Li. The following tetrapeptides were prepared y oupling the deproteted dipeptide units using the proedure similar to that of the dipeptide oupling (Tale-2). Preparation of the Pentapeptides (Linear Fragments) The -group of the tetrapeptides was removed y TFA/CCl 3 and the deproteted units were oupled with -(nitro)arginine the proedure similar to that of the dipeptide oupling to get the pentapeptide 1,2 and 3. Struture of ompounds was onfirmed y spetral analysis (Tale 3). Cylisation of the Linear Pentapeptide 1, 2 & 3: Cylisation of the linear pentapeptide unit was arried out y the p-nitrophenyl ester method of Bodanszky 8 with ertain modifiations. The ester group of the linear fragment was removed with Li and the p-nitrophenyl eater group was introdued y stirring the deproteted pentapeptide in CCl 3 with p- nitrophenol. The reation mixture was washed several times with saturated ac 3 until the unreated p-nitrophenol was removed ompletely. The - group was removed y TFA using the standard proedure. To the deproteted linear fragment a atalyti amount of -methylmorpholine was added and the reation mixture was kept at 0 0 C for 10 days. Washed several times with saturated ac 3 until the yprodut, p-nitrophenol was ompletely removed, finally washed with 5% Cl and water dried over anhydrous a 2 S 4 and evaporated in vauum to get the ylised produt whih was rystallised from CCl 3. The strutures yli pentapeptide 1, 2 and 3 were onfirmed y IR, 1 MR, FABMS and elemental analysis. Evaluation of Antimiroial Ativity All the plates were kept in the refrigerator for 30 minutes to allow the diffusion of the sample into the surrounding agar medium. Then the plates inoulated with aterial ultures were inuated at 37 0 C for 18 hours and those with fungi were inuated at 25 0 C for 48 hours. Diameter of the zones of inhiition wherever produed were measured and the average diameter for eah sample was alulated. The diameters otained for the test samples were ompared with that produed y the standard antiiotis, enzylpeniillin for antiaterial ativity and griseofulvin for antifungal ativity 9. The results are given in Tale 5. RESULTS AD DISCUSSI Evaluation of antiaterial and antifungal ativity was arried out for all the synthesised yli pentapeptides. All the ompounds showed moderate antiaterial and antifungal ativities. Cyli Peptide 1: ylo[nitro-arginyl-phenylalanyl-prolyl- Prolyl-Valine] (1) 1 MR (300Mz, CDCl 3 ) : δ (6, m), 6.9(2, d, J = 7.2z), (1, r. S), (2, r. S), (2, m), (1, m), (2, m), (4, m), (4, m), (8, m), (1, m), 0.9(6, d, J = 7.0z). IR (CCl 3 ): 3290(r. s), 2910 (s), 2885(s), 1740(s), 1690(s), 1675(s), 1670(s), 1660(s), 1495(s), 1410(m), 1380(s), 1330(s), 1135(m), 1050 (r. s), 910(s)m 1. FABMS: m/z (M 1) : 643 Cyli Peptide-2: ylo[nitro-arginyl-prolyl- Phenylalanyl-Prolyl-Valine](7) 1 MR (300Mz, CDCl 3 ) : δ (4, m), (2, m), 7.0(2, d, J = 7.2z), (1, r. S), (2, r. S), (2, m), (1, m), (2, m), (4, m), (4, m), (8, m), (1, m), 0.9(6, d, J = 7.0z). IR (CCl 3 ): 3300(r. s), 2950(s), 2880(s), 1745(s), 1700(s), 1685(s), 1670(s), 1660(s), 1495(s), 1380(s), 1330(s), 1135(m), 1050 (r. s), 910(s)m 1. FABMS: m/z (M 1) : 643 Cyli Peptide-3: ylo[nitro-arginyl-prolyl-valyl-prolyl- Phenylalanine] (11) 1 MR (300Mz, CDCl 3 ) : δ (6, m), 6.9(2, d, J = 7.2z), (1, r. s), (2, r. s), (2, m), (1, m), (2, m), (4, m), (4, m), (8, m), (1, m), 0.9(6, d, J = 7.0z). IR (CCl 3 ): 3320(r. s), 2990 (s), 1735(s), 1695(s), 1670(s), 1655(s), 1640(s), 1495(s), 1415(m), 1390(s), 1330(s), 1135(m), 1050 (r. s), 915(s)m 1. FABMS: m/z (M 1) : 643 Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug

6 Tale 1: Physial data of the synthesised dipeptides Sl. o -dipeptide-me Physial state % Yield 1 -Pro-Val-Me Visous liquid Pro-Phe-Me Semisolid mass Phe-Pro-Me Semisolid mass Tale 2: Physial Data of the Tetrapeptides Sl. o -tetrapeptide-me Physial state % Yield 1 -Phe-Pro-Pro-Val-Me Semisolid mass Pro-Phe-Pro-Val-Me Semisolid mass Pro-Val-Pro-Phe-Me Semisolid mass Tale 3: Physial Data of the Pentapeptides Sl. no -tetrapeptide-me Physial state % Yield 1 -(nitro)arg-phe-pro-pro-val-me Semisolid mass (nitro)arg-pro-phe-pro-val-me Semisolid mass (nitro)arg-pro-val-pro-phe-me Semisolid mass Tale 4: Physial Data of the Cyli Pentapeptides Sl no Cylised Produt Physial state % Yield 1 Cylo-(nitro)Arg-Phe-Pro-Pro-Val-Me Semisolid mass Cylo-(nitro)Arg-Pro-Phe-Pro-Val-Me Semisolid mass Cylo-(nitro)Arg-Pro-Val-Pro-Phe-Me Semisolid mass 89.3 Tale 5: Data of Antimiroial Ativity Sl. o. Compound o. Diameter of Zone of Inhiition (in mm) B. su. S. aur. E. oli P. aer. C. al. A. nigar Benzyl peniillin Griseofulvin CCLUSI Three yli pentapeptides have een synthesized y presried Sheme with good yields y solution phase peptide synthesis. Strutures of all the newly synthesized ompounds were onfirmed y FTIR, 1 MR and Mass spetral data. All the synthesized analogs were sreened for their ateriidal ativity and fungiidal ativity. Bateriidal ativity against two Gram-positive ateria Staphyloous aureus, Baillus sutilis and two Gram-negative ateria Esheridhia oli, Pseudomonas aeruginosa and fungiidal ativity against two fungi Aspergillus niger and Candida alians, All the synthesized ompounds found to e ative against test miroorganisms. REFERECES 1. Beverly AT, Ara G, Roy, Palomella VJ, Julian A, The Proteasome Inhiitor PS-341 in Caner Therapy, Clinial Caner Researh., 1999; 5: Boger DL, Jiaheng Zhou, Brian Writer and Kitos PA. Key analogs of the Tetrapeptide Suunit of RA- VII and Deoxyouvardin, Bioorgani and Mediinal Chemistry, 1995; 3: Katsuhirokudo S, Yasuyuki T, Benkelia, Shiomi, Antioxidative ativities of some peptides isolated from hydrolyzed potato protein extrat, Journal of funtional foods., 2009; 1: Luke Simmons KL. MPhail B, Eduardo B, Susan L, William G, Belamide A, A new antimitoti tetrapeptide from a Panamanian marine yanoaterium, Tetrahedron Letters., 2006; 47: Gunnur Dikmen Z, Gellert GC., Pakize Dogan, eejeong Yoon, Lee YB, Chang A, Shay JW. In vivo and Invitro Effets of a IF-1α Inhiitor, RX- 0047, Journal of Cellular Biohemistry, 2008; 104: Lee MD, Yuhong MJ, Soskis CP, Borella M. umanmitohondrial peptide deformylase, a new Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug

7 antianer target of atinonin-ased antiiotis, J. Clinial Investigation., 2004; 114: Alejandro MS. Mayera, Kirk R. Gustafson. Marine pharmaology in : Antitumor and ytotoxi ompounds, European journal of Caner, 2008; 44: Bodanszky M, and Bodanszky A. Pratie of Peptide Synthesis. (Springer-Verlag, ew York); p Clinial miroiology proedures handook. enry D Isenerg. Volume 1. Amerian soiety for miroiology/ Washington, D.C, Soure of support: il, Conflit of interest: one Delared Unique Journal of Pharmaeutial and Biologial Sienes 01(01), Jul-Aug