Title of Invention	"A PROCESS FOR THE DEVELOPMENT OF NOVEL LINEAR ANTIMICROBIAL PEPTIDES( AMPS)"
Abstract	The present invention deals with preparation and antimicrobial activities of hexapeptides of general formula (1), which offer an improved means for the treatment and prevention of fungal and bacterial infections. X-D-Trp-D-Phe-X 1-D-Phe-X2-NH2 1 Wherein X, Xi and Xa represents all twenty naturally occurring L-amino acids, their D-counterparts; unnatural amino acids omithine (OM), phenylglycine (Phg), aminoisobutyric acid (Aib); all three isomeric L/D-pyridylalanines; all halogenated L/D-phenylalanines, and all synthetic derivatives of D- and L-histidine such as 5-halogenated/2,5-dihalogenated-D/L-histidines where halogen is F, Cl, Br, and I groups; 1-alkyl-L/D-histidines where alkyl groups represents various primary alkyl, secondary alkyl and aromatic groups; 2-alkyl-L/D-histidines where alkyl group represents various primary, secondary and tertiary alkyl groups. The development of these peptides offers and adds one more line of antibiotics for the treatment and control of opportunistic fungal infections caused by Candida albicans, Cryptococcous neoformans, Aspergillus fumigatus, Candida glabrata, and Candida krusei and antibacterial infections caused by Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare.

Title of Invention

"A PROCESS FOR THE DEVELOPMENT OF NOVEL LINEAR ANTIMICROBIAL PEPTIDES( AMPS)"

Abstract

The present invention deals with preparation and antimicrobial activities of hexapeptides of general formula (1), which offer an improved means for the treatment and prevention of fungal and bacterial infections. X-D-Trp-D-Phe-X 1-D-Phe-X2-NH2 1 Wherein X, Xi and Xa represents all twenty naturally occurring L-amino acids, their D-counterparts; unnatural amino acids omithine (OM), phenylglycine (Phg), aminoisobutyric acid (Aib); all three isomeric L/D-pyridylalanines; all halogenated L/D-phenylalanines, and all synthetic derivatives of D- and L-histidine such as 5-halogenated/2,5-dihalogenated-D/L-histidines where halogen is F, Cl, Br, and I groups; 1-alkyl-L/D-histidines where alkyl groups represents various primary alkyl, secondary alkyl and aromatic groups; 2-alkyl-L/D-histidines where alkyl group represents various primary, secondary and tertiary alkyl groups. The development of these peptides offers and adds one more line of antibiotics for the treatment and control of opportunistic fungal infections caused by Candida albicans, Cryptococcous neoformans, Aspergillus fumigatus, Candida glabrata, and Candida krusei and antibacterial infections caused by Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare.

Full Text	FIELD OF THE INVENTION The present invention deals with preparation and antimicrobial activities of hexapeptides of general formula (1), which offer an improved means for the treatment and prevention of fungal and bacterial infections. X-D-Trp-D-Phe-X1D-Phe-X2-NH2 1 Wherein X, X] and \2 represents all twenty naturally occurring L-amino acids, their D-counterparts; unnatural amino acids ornithine (OM), phenylglycine (Phg), aminoisobutyric acid (Aib); all three isomeric L/D-pyridylalanines; all halogenated L/D-phenylalanines, and all synthetic derivatives of D- and L-histidine such as 5-halogenated/2,5-dihalogenated-D/L-histidines where halogen is F, Cl, Br, and I groups; 1-alkyl-L/D-histidines where alkyl groups represents various primary alkyl, secondary alkyl and aromatic groups; 2-alkyl-L/D-histidines where alkyl group represents various primary, secondary and tertiary alkyl groups. The development of these peptides offers and adds one more line of antibiotics for the treatment and control of opportunistic fungal infections caused by Candida albicans, Cryptococcous neoformam, Aspergillus fumigatus, Candida glabrata, and Candida krusei and antibacterial infections caused by Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare. BACKGROUND OF THE INVENTION The widespread and irrational use of antibiotics has led to the emergence of resistant strains of pathogenic microorganisms. Hence, the search for novel antimicrobial agents has gained importance over the past several years. Fungi are one of the most neglected pathogens apparent from the fact that the Amphotericin B, a polyene antibiotic, discovered way back in 1956 is still used as a 'gold standard' for antifungal therapy. Past two decades have witnessed a dramatic rise in the incidences of life threatening systemic fungal infections. This can be ascribed to the increase in the number of immune-compromised patients due to rise in HIV infected population, cancer chemotherapy and indiscriminate use of antibiotics. Majority of clinically used antifungals suffer from various drawbacks in terms of toxicity, efficacy and cost, and their frequent use has led to the emergence of resistant strains. Hence, there is a great demand for novel antifungals belonging to wide range structural classes, selectively acting on novel targets with fewer side effects. At the same time, development of resistant by all major drugs to opportunistic antibacterial infections has created a need to design, discover and develop entirely new structural classes of these agents which are not only effective but also offer an alternate means of antibacterial therapy. Nature provides the answer in the form of antimicrobial peptides that are not only lethal to a broad spectrum of pathogens but also have a unique low tendency for resistance development. A variety of antimicrobial peptides and proteins have been isolated from virtually all the kingdoms and phyla including plants, microbes, insects, animals and humans (Kitajima, S.; Sato, F. J. Biochem. 1999, 725, 1; De Lucca, A. J.; Walsh, T. J. Antimicrob. Agents Chemother. 1999, 43, 1; Fehlbaum, P.; Bulet, P.; Chernych, S.; Briand, J.-P.; Roussel, J. P.; Letellier, L.; Hetru, C.; Hoffmann, J. A. Proc. Natl. Acad. Sci. USA 1996, 93, 1221; Diamond, G.; Zasioff, M.; Eck, H.; Brasseur, M.; Maloy, W. L.; Bevins, C. L. Proc. Natl. Acad. Sci. USA 1991, 88, 3952). These naturally occurring peptides are the integral part of the host defense system and are supposed to be strategically evolved against the common infections of the host cells. Consequently, these are specifically toxic to the pathogens and are able to differentiate between host and microbial cells. This specific recognition may be governed by the difference in the membrane composition and hence charge, membrane asymmetry, transmembrane potential, and specific receptor binding (Yeaman, M. R.; Yount, N. Y. Pharmacol. Rev. 2003, 55, 27). Most of these peptides are cationic in nature with significant number of hydrophobic residues, and antimicrobial potency has been correlated with the positive charge, although not always linearly (Bessalle, R.; Haas, H.; Goria, A.; Shalit, I.; Fridkin, M. Antimicrob. Agents Chemother. 1992, 36, 313). The antimicrobial mechanism of action of these peptides includes either formation of multimeric pores in the cell membranes leading to cell lysis (Hancock, R. E. Lancet 1997, 349, 418) or interaction with the RNA or DNA after penetration into the cell (Park, C. B.; Kim, H. S.; Kim, S. C. Biochem. Biophys. Res. Commun. 1998, 244, 253). DETAILED DISCRIPTION OF THE INVENTION The widespread use of antibiotics led to the development of numerous antibiotic-resistant strains, resulting in an urgent need for new antibiotic drugs. Antibiotic Resistance and Surveillance has reported that resistance to antimicrobial agents is already a serious public health problem in developed and developing countries alike. Levels of resistance had been increasing at an alarming rate and were "expected to increase at a similar or even greater rate in future as antimicrobial agents lose their effectiveness." One of the reasons that antibiotic resistance is so widespread is the pervasive use of antibiotics in animal feeds to prevent infections and promote growth. This has encouraged spontaneous mutation of the targets of antimicrobial drugs as well as the exchange of plasmids encoding antibiotic resistant genes. There are many mechanisms by which bacteria evade antibiotic treatment, such as reduced drug uptake, active pumping of drugs out of the cell, enzymatically altering the antibiotic, modification of targets, drug sequestering by protein binding, overproduction of the target, metabolic bypass of the targeted pathway The proliferation of multi-drug resistant bacteria and fungi has necessitated the search for new antimicrobial agents and treatment strategies. The search for antibiotics with a new mode of action has increased the interest in peptides as potential therapeutic agents. Peptide-based host defense can be considered as a pervasive and evolutionary ancient mechanism of immunity. Antimicrobial peptides (AMPs) are a major component of innate self-defense system. They provide an immediate response to invading microorganisms and display a broad spectrum of bactericidal and fungicidal actions. Development of resistance by sensitive microbial strains against these antimicrobial peptides is improbable because antimicrobial peptides exert their action by forming pores in microbial membranes or disrupting membrane structure. Antimicrobial peptides possess several advantages for making them suitable candidates for anti-infective drugs. These advantages are: (i) broad-spectrum activity (antibacterial, antifungal, antiviral); (ii) rapid onset of killing; (iii) cidal activity, (iv) potentially low levels of induced resistance; (v) concomitant broad anti-inflammatory activities. Antimicrobial peptides contain both hydrophilic and hydrophobic amino which are arranged in such a fashion in the peptide that the most positively charged amino acids are localized to one side of the molecule, opposite to those that are most hydrophobic. This amphipathic organization is common to many antimicrobial peptides and allows them to bind by charge attraction to negative membranes and enter lipid phase as a result of hydrophobic amino acid cluster. Unfolded cationic peptides are proposed to associate with the negatively charged (mainly due to the presence of highly anionic lipopolysaccharide [LPS]) surface of the outer membrane. They then either neutralize the charge over a patch of outer membrane, creating cracks through which the peptide can cross the outer membrane (a) or actually bind to the divalent cation binding sites on lipopolysaccharide, and disrupt the membrane (b). Once the peptide has crossed the outer membrane, it will interact with the negatively charged surface of the cytoplasmic membrane. This is referred to as self-promoted uptake of cationic peptides across the outer membrane. Many research groups are working on antimicrobial peptides but still several important topics regarding AMPs will have to be addressed in the near future. These are: (i) Identification of novel AMPs - it is likely that the families of AMPs consist of multiple functions with different functions; (ii) Analysis of biologically relevant functions of AMPs involving both antimicrobial activity and other functions; (iii) Development of AMPs as drugs involves optimized strategies for candidate modification, for modification of pharmacodyanamic and pharmacokinetic profiles, and for production. Keeping these points in mind, this invention describes discovery of new peptides and consisting of several synthetic unnatural residues. Though naturally occurring antimicrobial peptides appear to be promising candidates against resistant pathogens due to their novel mechanism of action, specificity and rare resistance problems, their clinical utility is rather less convincing owing to problems of enzymatic degradation, availability and cost. Thus, synthetic congeners possessing similar structural features but containing unnatural amino acids may provide the solution. In fact, the availability of standard protocols for solid phase peptide synthesis and combinatorial technology has been exploited to design and synthesize large libraries of small synthetic peptides containing unnatural amino acids, which were successfully used to identify leads against various microbial pathogens (Blondelle, S. E.; Perez-Paya, E.; Houghten, R. A. Antimicrob. Agents Chemother. 1996, 40, 1067; Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84). EXPERIMENTSUsing modern peptide synthesis and combinatorial techniques we have discovered that several hexapeptides (1) of general formula given above displayed highly promising antifungal and antibacterial activities. The requisite peptides 1 were synthesized using solid phase peptide synthesis protocol developed in our laboratory (Figure 1). Accordingly, hexapeptides were assembled on 4-methylbenzhydrylamine-functionalized, 1% cross-linked polystyrene resin (0.31 mequiv g"1) in 0.31 mmol scale on CS Bio Co. (San Carlos, CA; model no. CS 136) synthesizer, using the following protocol: deblocking, 20% TFA in DCM (25 min); DCM wash cycle (two washes); neutralization, 10% DIEA in DCM (10 min); DMF wash cycle (one wash); DCM wash cycle (two washes); coupling with preformed TBTU esters (3 equiv), 120 min in DMF, with a catalytic amount of DIEA; DMF wash (one wash); DCM wash (two washes). Coupling reactions were monitored qualitatively with the Kaiser test. The peptides were cleaved from the resin support with simultaneous side-chain deprotection by acidolysis using anhydrous trifluoromethane sulfonic acid (1 mL) containing thioanisole (1 mL) and 1,2-ethanedithiol (0.5 mL) as scavengers in TFA (10 mL) for 2 h at ambient temperature, and crude peptides were then purified and analyzed (Scheme 1). The crude peptide was separated from solid support by filtration and resin was washed with TFA (3x4 mL). Combined extracts were evaporated under reduced pressure and residue was neutralized with saturated ammonium bicarbonate solution. The non-polar impurities were removed by extracting the aqueous layer with diethyl ether (3x10 mL). The aqueous layer was evaporated under reduced pressure to afford crude peptide. The crude peptides were purified by semi-preparative RP-HPLC on CIS bonded silica gel using Merck Hiber® RT 250-25 RP-18 (10 (am) column. A linear gradient elution system at a flow rate of 20 mL min"1 was employed: A, 0.1% TFA; B, 0.1% TFA in 80% MeCN; 20% B to 50% B at 1% min"1. The separations were monitored at 215 nm, by TLC on silica gel coated (Merck Kiesel 60 F254, 0.2 mm thickness) sheets and by analytical RP-HPLC. A representative example of hexapeptide synthesis HCI. H2N 1. DIEA, DCM 2. Boc-2-Pal-OH, TBTU, DMF Boc-2-Pal-HN- 3.40% TFA in DCM 4. DIEA, DCM 5. Boc-D-Phe-OH, TBTU, DMF, 90 min Boc-D-Phe-2-Pal-HN steps (3) and (4) 6. Boc-lle-OH, TBTU, DMF Boc-lle-D-Phe-2-Pal-HN steps (3) and (4) 7. Boc-D-Phe-OH, TBTU, DMF Boc-D-Phe-lle-D-Phe-2-Pal-HN- steps (3) and (4) 8. Boc- D-Trp-OH, TBTU, DMF Boc-D-Phe-lle-D-Phe-2-Pal-HN- steps (3) and (4) 9. Boc-Arg (Z)2 -OH, TBTU, DMF Boc-Arg(Z)2-D-Phe-lle-D-Phe-2-Pal-HN - 10. CF3SO3H, TFA, anisole, ehtanedithiol Arg-D-Trp-D-Phe-lle-D-Phe-2-Pal-NH2 free peptide resin The fractions containing the product were pooled, concentrated in vacuo, and lyophilized. Each peptide was obtained as a white powder by lyophilization from 80% acetic acid. The purity of the final peptides was assessed by analytical RP-HPLC. Analytical RP-HPLCs were recorded using a Merck Lichrospher® 100 RP-18 (10 urn) column with a flow rate of 1 mL/min. Column eluent was monitored at 215 nm. All peptides were found to be >95% pure and retention time and HRMS analysis of some representative peptides is given in Table 1. Table 1. Identities and analytical data of the some representative hexapeptides (TableRemoved)HPLC: In a solvent system of 10% CH3CN in 0.1% TFA and a gradient of 90% CH3CN over 45 min. An analytical Merck Lichrospher® 100 RP-18 (lO^m) column was used with a flow rate of 1 mL/min. bMolecular Weight C[M+H]+ BIOLOGICAL ACTIVITIES The antifungal activities of the target compounds against pathogenic fungi associated with opportunistic infections, C. albiccms, C. neoformans, and Aspergillusfumigatus, are summarized in Table 2. Amphotericin B was included as a standard drug for comparison. ICso, MICs, and MFCs were determined according to NCCLS methods (NCCLS, In Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard M27-A, National Committee on Clinical Laboratory Standards, 1997, 17, pp 9; NCCLS, In Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically M7-A5, National Committee on Clinical Laboratory Standards, 2000, 20, pp 2; NCCLS, In Susceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Tentative Standard—Second Edition, M24-T2, National Committee on Clinical Laboratory Standards, 2000, 20, pp 26).The was defined as the concentration that affords 50% growth, whereas MIC was defined as the lowest concentration resulting in inhibition of visible growth after incubation, and MFC was the minimum fungicidal concentration that kills 100% of the organism. Representative peptides 1-4 displayed selective antifungal activity against C. neoformcms, but they were not active against C. albicans andA.fumigatus. Peptide 1, in which Arg-1 is replaced with Orn and His-6 is replaced with 1-Bzl-His showed pronounced antifungal activity with an ICso value of 1.6 uM, MIC of 5.3 uM and MFC of 5.3 ^M and is therefore most promising antifungal compound of the series. Peptides 2-4 also displayed promising activities and exhibited IC5os in the range of 3.5-6.6 u.M, MICs between 5.0-11.0 jiM and killed 100% of C. neoformans in concentration ranging between 5.0-11.0 uM (Table 2). Table 2. In vitro antifungal activities (\M] of the representative hexapeptides (1-4) (TableRemoved)IC50, The concentration that affords 50% inhibition of bacterial growth bMIC, (Minimum inhibitory concentration) is the lowest test concentration that allows detectable growth of bacteria °MFC, Minimum fungicidal concentration (the lowest test concentration that kills 100% of the organism) NT, Not tested NA, not active at 20 ug/mL (20-22 ( The antibacterial activities of representative hexapeptides 1-4 against Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare are reported as ICso, MIC, and MBC in Table 3. Ciprofoxacin was included as positive control for comparison. Susceptibility of S. aureus and methicillin-resistant S. aureus to test compounds was determined according to the rocedure as described by the National Committee for Clinical Laboratory Standards (NCCLS) (NCCLS, In Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard M27-A, National Committee on Clinical Laboratory Standards, 1997, 17, pp 9; NCCLS, In Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically M7-A5, National Committee on Clinical Laboratory Standards, 2000, 20, pp 2; NCCLS, In Susceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Tentative Standard—Second Edition, M24-T2, National Committee on Clinical Laboratory Standards, 2000, 20, pp 26). Table 3. In vitro antibacterial activities (^M) of the hexapeptides (1-4) (TableRemoved)aIC50, The concentration that affords 50% inhibition of bacterial growth bMIC, (Minimum inhibitory concentration) is the lowest test concentration that allows detectable growth of bacteria CMBC, Minimum bactericidal concentration (the lowest test concentration that kills 100% of the organism) NT, Not tested NA, not active at 20 ug/mL (20-22 Susceptibility of M. intracellulare was done using the modified Alamar Blue procedure of Franzblau et al. (Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; G. Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.; Oilman, R. H. J. Clin. Microbiol. 1998, 36, 362). The MBC was defined as the minimum bactericidal concentration that kills 100% of the organism. None of the peptides were active against M. intracellulare. In contrast, peptide 1, 3 and 4 displayed promising antibacterial activities against S. aureus and methicillin-resistant S. aureus (MRS). Peptide 4, in which His-6 was replaced with 2-Pal exhibited promising antibacterial activities (IC50 = 3.8µM, MIC = 5.5µM, MBC = 10.9 µM) against MRS. While, 6 displayed (ICso = 3.8 uM, MIC = 10.9 fiM, MBC = 10.9 (o,M) against S. aureus and is the most promising compound of the series. Peptides 1 and 3 also exhibited encouraging antibacterial activity ranging between 3.7-6.5 uM, MICs of 10 \M and killed 100% of MRS and S. aureus at concentrations ranging between 10.0-20.0 uM. In contrast, Peptide 2, in which His-6 was replaced with D-His was found to be inactive against all microorganisms. It is important to note that lead peptide 2 was found inactive against all three microorganisms. Finally, cytotoxicity of the peptides was tested against four human cancer cell lines (SK-MEL, KB, BT-549, SK-OV-3) and two noncancerous mammalian kidney cells (VERO and LLC-PKi) up to a highest concentration of 10.0 ^g/mL (10-11 jaM) using a neutral red assay procedure as described earlier (Borenfreund, E.; Babich, H.; Martin-Alguacil, N. In Vitro Cell Dev. Biol. 1990, 26, 1030; Mustafa, J.; Khan, S. I.; Ma, G.; Walker, L. A.; Khan, I. A. Lipids 2004, 39, 167). None of the analogues showed any cytotoxic effects. DESCRIPTION OF THE DRAWING Figure 1 shows peptide synthesis cycle. SUMMARY OF THE INVENTION The present invention provides novel liner antimicrobial peptides of general formula (I) for the treatment and prevention of a panel of opportunistic fungi and bacterial pathogens. Some of these new derivatives have shown excellent in vitro antifungal against Candida albicans, Cryptococcous neoformans, Aspergillus fumigatus, Candida glabrata, and Candida krusei and excellent in vitro antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare. These promising antimicrobial peptides possess several advantages for making them suitable candidates for anti-infective drugs such as: Broad-spectrum activity of antibacterial and antifungal effects, rapid onset of pathogen killing, potentially low levels of induced resistance due to peptide nature of drug, and elimination of cyto-toxic side effects. It is expected that development of these peptides as ideal anti-infective agents will add to already existing armament for the suppression as well as radical cure of the various opportunistic fungal and bacterial infections. We claim 1. A process for the development of novel linear Antimicrobial hexapeptide composition, comprising of general formula (1); X-D-Trp-D-Phe-X1-D-Phe-X2-NH2 (1) Wherein X, XI and X2 represents all twenty naturally occurring L-amino acids, their D-counter parts; unnatural amino acids ornithine (OM), phenylglycine (Phg), aminoisobutyric acid (Aib); all three isomeric L/D- pyridyalanines, and all synthetic derivatives of D- and L- histidine such as 5-halogenated/ 2,5-dihalogenated-D/L-histidine where halogen is F, Cl, Br and I groups; 1 alkyl-L/D-histidine, 2 alkyl-histidines. 2. A process as claimed in claim 1, wherein the process is a solid phase peptide synthesis protocol. 3. The process as claimed in claim 2, wherein the said protocol is carried out in a 4-methylbenzhydrylamine-functionalised, 1% cross linked polystyrene resin (0.31 mequiv g"1) in 0.31 mmol. 4. The process as claimed in claim 2, wherein, the said protocol comprising the following, I. 4-Methylbenzhydrylamine (MBHA.HC1) resin (500 nig, 0.31 mmol) was charged into the reaction vessel II. The requisite suitably protected amino acids (0.93 mmol) were loaded sequentially into the amino acid vessels, III. The MBHA.HC1 resin was neutralized with (N,.N'-Diisopropylethylaniine) DIEA (10%inDCM)for5min. IV. After neutralization the resin is washed with DMF (2 * 10 niL) and once with DCM (lOmL). V. The coupling of the first amino acid was done in the presence of coupling reagent TBTU (2-(lH-Benztriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate) and DIE A (10% in DCM) for 90 min. VI. After coupling, the tert-Boc group was cleaved with trifluoroacetic acid (40% solution in DCM) for 20 min. VII. The amino acid linked resin was again neutralized with DIEA (10% in DCM) for 5 min and washed with DMF (2 x 10 mL) and once with DCM (10 mL) to afford dipeptide. VIII. The coupling and deprotection steps were repeated to obtain desired sequence of peptide resins. IX. The peptides were cleaved from the resin support with simultaneous side chain deprotection, then purified and analyzed. 5. The process as claimed as claimed in claim 4, wherein the said coupling reactions were monitored qualitatively with the Kaiser test. 6. The process as claimed claim 4, wherein the said peptides were cleaved from the resin support as claimed in claim 4(i), wherein cleavage of crude peptides from the resin support is carried out by acidolysis using anhydrous trifluromethane sulfonic acid (1 mL) containing thianisole (ImL) and 1, 2-ethanedithiol (0.5 mL) as scavengers in TFA (10 mL) for 2 hrs at ambient temperature 7. The process as claimed in claim 4, wherein the said purification as claimed in claim 4(1), wherein the purification is carried out as under; I. The crude peptide was separated from solid support by filtration 11. The resin was washed with TFA (3X4 mL) III. Combined extracts were evaporated under reduced pressure IV. The residue obtained from step 7 (III) was neutralized with saturated ammonium bi carbonate solution V. Non polar impurities are removed by extracting the aqueous layer with diethyl ether (3 X lOmL) VI. Crude peptide was recovered by evaporating the aqueous layer under reduced pressure VII. The crude peptides were further purified by semi-preparative RP-HPLC on CIS bonded silica gel using (l0µm) column VIII. A linear gradient elution system at a flow rate of 20 mL min"1 was employed IX. The separations were monitored at 215 nm, by TLC on silica gel coated sheets. 8. The process as claimed in claim 4, wherein the said analysis is carried out by analytical RP-HPLC and is recorded using 100 RP-18(10um) column with a flow rate of 1 mL/min. 9. The process as claimed in claim 8, wherein the said column eluent was monitored at 215 nm. 10. Peptides as claimed in claim 1, wherein the peptides were found to be > 95% pure. 11. A process for the development of novel linear Antimicrobial hexapeptide composition substantially described herewith foregoing description and diagrams.

Full Text

FIELD OF THE INVENTION
The present invention deals with preparation and antimicrobial activities of hexapeptides of general formula (1), which offer an improved means for the treatment and prevention of fungal and bacterial infections.
X-D-Trp-D-Phe-X1D-Phe-X2-NH2
1
Wherein X, X] and \2 represents all twenty naturally occurring L-amino acids, their D-counterparts; unnatural amino acids ornithine (OM), phenylglycine (Phg), aminoisobutyric acid (Aib); all three isomeric L/D-pyridylalanines; all halogenated L/D-phenylalanines, and all synthetic derivatives of D- and L-histidine such as 5-halogenated/2,5-dihalogenated-D/L-histidines where halogen is F, Cl, Br, and I groups; 1-alkyl-L/D-histidines where alkyl groups represents various primary alkyl, secondary alkyl and aromatic groups; 2-alkyl-L/D-histidines where alkyl group represents various primary, secondary and tertiary alkyl groups. The development of these peptides offers and adds one more line of antibiotics for the treatment and control of opportunistic fungal infections caused by Candida albicans, Cryptococcous neoformam, Aspergillus fumigatus, Candida glabrata, and Candida krusei and antibacterial infections caused by Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare.
BACKGROUND OF THE INVENTION
The widespread and irrational use of antibiotics has led to the emergence of resistant strains of pathogenic microorganisms. Hence, the search for novel antimicrobial agents has gained importance over the past several years. Fungi are one of the most neglected pathogens apparent from the fact that the Amphotericin B, a polyene antibiotic, discovered way back in 1956 is still used as a 'gold standard' for antifungal therapy. Past two decades have witnessed a dramatic rise
in the incidences of life threatening systemic fungal infections. This can be ascribed to the increase in the number of immune-compromised patients due to rise in HIV infected population, cancer chemotherapy and indiscriminate use of antibiotics. Majority of clinically used antifungals suffer from various drawbacks in terms of toxicity, efficacy and cost, and their frequent use has led to the emergence of resistant strains. Hence, there is a great demand for novel antifungals belonging to wide range structural classes, selectively acting on novel targets with fewer side effects. At the same time, development of resistant by all major drugs to opportunistic antibacterial infections has created a need to design, discover and develop entirely new structural classes of these agents which are not only effective but also offer an alternate means of antibacterial therapy.
Nature provides the answer in the form of antimicrobial peptides that are not only lethal to a broad spectrum of pathogens but also have a unique low tendency for resistance development. A variety of antimicrobial peptides and proteins have been isolated from virtually all the kingdoms and phyla including plants, microbes, insects, animals and humans (Kitajima, S.; Sato, F. J. Biochem. 1999, 725, 1; De Lucca, A. J.; Walsh, T. J. Antimicrob. Agents Chemother. 1999, 43, 1; Fehlbaum, P.; Bulet, P.; Chernych, S.; Briand, J.-P.; Roussel, J. P.; Letellier, L.; Hetru, C.; Hoffmann, J. A. Proc. Natl. Acad. Sci. USA 1996, 93, 1221; Diamond, G.; Zasioff, M.; Eck, H.; Brasseur, M.; Maloy, W. L.; Bevins, C. L. Proc. Natl. Acad. Sci. USA 1991, 88, 3952). These naturally occurring peptides are the integral part of the host defense system and are supposed to be strategically evolved against the common infections of the host cells. Consequently, these are specifically toxic to the pathogens and are able to differentiate between host and microbial cells. This specific recognition may be governed by the difference in the membrane composition and hence charge, membrane asymmetry, transmembrane potential, and specific receptor binding (Yeaman, M. R.; Yount, N. Y. Pharmacol. Rev. 2003, 55, 27). Most of these peptides are
cationic in nature with significant number of hydrophobic residues, and antimicrobial potency has been correlated with the positive charge, although not always linearly (Bessalle, R.; Haas, H.; Goria, A.; Shalit, I.; Fridkin, M. Antimicrob. Agents Chemother. 1992, 36, 313). The antimicrobial mechanism of action of these peptides includes either formation of multimeric pores in the cell membranes leading to cell lysis (Hancock, R. E. Lancet 1997, 349, 418) or interaction with the RNA or DNA after penetration into the cell (Park, C. B.; Kim, H. S.; Kim, S. C. Biochem. Biophys. Res. Commun. 1998, 244, 253).
DETAILED DISCRIPTION OF THE INVENTION
The widespread use of antibiotics led to the development of numerous antibiotic-resistant strains, resulting in an urgent need for new antibiotic drugs. Antibiotic Resistance and Surveillance has reported that resistance to antimicrobial agents is already a serious public health problem in developed and developing countries alike. Levels of resistance had been increasing at an alarming rate and were "expected to increase at a similar or even greater rate in future as antimicrobial agents lose their effectiveness." One of the reasons that antibiotic resistance is so widespread is the pervasive use of antibiotics in animal feeds to prevent infections and promote growth. This has encouraged spontaneous mutation of the targets of antimicrobial drugs as well as the exchange of plasmids encoding antibiotic resistant genes. There are many mechanisms by which bacteria evade antibiotic treatment, such as reduced drug uptake, active pumping of drugs out of the cell, enzymatically altering the antibiotic, modification of targets, drug sequestering by protein binding, overproduction of the target, metabolic bypass of the targeted pathway
The proliferation of multi-drug resistant bacteria and fungi has necessitated the search for new antimicrobial agents and treatment strategies. The search for antibiotics with a new mode of
action has increased the interest in peptides as potential therapeutic agents. Peptide-based host defense can be considered as a pervasive and evolutionary ancient mechanism of immunity. Antimicrobial peptides (AMPs) are a major component of innate self-defense system. They provide an immediate response to invading microorganisms and display a broad spectrum of bactericidal and fungicidal actions. Development of resistance by sensitive microbial strains against these antimicrobial peptides is improbable because antimicrobial peptides exert their action by forming pores in microbial membranes or disrupting membrane structure.
Antimicrobial peptides possess several advantages for making them suitable candidates for anti-infective drugs. These advantages are: (i) broad-spectrum activity (antibacterial, antifungal, antiviral); (ii) rapid onset of killing; (iii) cidal activity, (iv) potentially low levels of induced resistance; (v) concomitant broad anti-inflammatory activities.
Antimicrobial peptides contain both hydrophilic and hydrophobic amino which are arranged in such a fashion in the peptide that the most positively charged amino acids are localized to one side of the molecule, opposite to those that are most hydrophobic. This amphipathic organization is common to many antimicrobial peptides and allows them to bind by charge attraction to negative membranes and enter lipid phase as a result of hydrophobic amino acid cluster. Unfolded cationic peptides are proposed to associate with the negatively charged (mainly due to the presence of highly anionic lipopolysaccharide [LPS]) surface of the outer membrane. They then either neutralize the charge over a patch of outer membrane, creating cracks through which the peptide can cross the outer membrane (a) or actually bind to the divalent cation binding sites on lipopolysaccharide, and disrupt the membrane (b). Once the peptide has crossed the outer membrane, it will interact with the negatively charged surface of the cytoplasmic membrane. This is referred to as self-promoted uptake of cationic peptides across the outer membrane.
Many research groups are working on antimicrobial peptides but still several important topics regarding AMPs will have to be addressed in the near future. These are: (i) Identification of novel AMPs - it is likely that the families of AMPs consist of multiple functions with different functions; (ii) Analysis of biologically relevant functions of AMPs involving both antimicrobial activity and other functions; (iii) Development of AMPs as drugs involves optimized strategies for candidate modification, for modification of pharmacodyanamic and pharmacokinetic profiles, and for production.
Keeping these points in mind, this invention describes discovery of new peptides and consisting of several synthetic unnatural residues. Though naturally occurring antimicrobial peptides appear to be promising candidates against resistant pathogens due to their novel mechanism of action, specificity and rare resistance problems, their clinical utility is rather less convincing owing to problems of enzymatic degradation, availability and cost. Thus, synthetic congeners possessing similar structural features but containing unnatural amino acids may provide the solution. In fact, the availability of standard protocols for solid phase peptide synthesis and combinatorial technology has been exploited to design and synthesize large libraries of small synthetic peptides containing unnatural amino acids, which were successfully used to identify leads against various microbial pathogens (Blondelle, S. E.; Perez-Paya, E.; Houghten, R. A. Antimicrob. Agents Chemother. 1996, 40, 1067; Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84).
EXPERIMENTSUsing modern peptide synthesis and combinatorial techniques we have discovered that several hexapeptides (1) of general formula given above displayed highly promising antifungal and
antibacterial activities. The requisite peptides 1 were synthesized using solid phase peptide synthesis protocol developed in our laboratory (Figure 1). Accordingly, hexapeptides were assembled on 4-methylbenzhydrylamine-functionalized, 1% cross-linked polystyrene resin (0.31 mequiv g"1) in 0.31 mmol scale on CS Bio Co. (San Carlos, CA; model no. CS 136) synthesizer, using the following protocol: deblocking, 20% TFA in DCM (25 min); DCM wash cycle (two washes); neutralization, 10% DIEA in DCM (10 min); DMF wash cycle (one wash); DCM wash cycle (two washes); coupling with preformed TBTU esters (3 equiv), 120 min in DMF, with a catalytic amount of DIEA; DMF wash (one wash); DCM wash (two washes).
Coupling reactions were monitored qualitatively with the Kaiser test. The peptides were cleaved from the resin support with simultaneous side-chain deprotection by acidolysis using anhydrous trifluoromethane sulfonic acid (1 mL) containing thioanisole (1 mL) and 1,2-ethanedithiol (0.5 mL) as scavengers in TFA (10 mL) for 2 h at ambient temperature, and crude peptides were then purified and analyzed (Scheme 1).
The crude peptide was separated from solid support by filtration and resin was washed with TFA (3x4 mL). Combined extracts were evaporated under reduced pressure and residue was neutralized with saturated ammonium bicarbonate solution. The non-polar impurities were removed by extracting the aqueous layer with diethyl ether (3x10 mL). The aqueous layer was evaporated under reduced pressure to afford crude peptide. The crude peptides were purified by semi-preparative RP-HPLC on CIS bonded silica gel using Merck Hiber® RT 250-25 RP-18 (10 (am) column. A linear gradient elution system at a flow rate of 20 mL min"1 was employed: A, 0.1% TFA; B, 0.1% TFA in 80% MeCN; 20% B to 50% B at 1% min"1. The separations were monitored at 215 nm, by TLC on silica gel coated (Merck Kiesel 60 F254, 0.2 mm thickness) sheets and by analytical RP-HPLC.
A representative example of hexapeptide synthesis
HCI. H2N
1. DIEA, DCM
2. Boc-2-Pal-OH, TBTU, DMF
Boc-2-Pal-HN-
3.40% TFA in DCM
4. DIEA, DCM
5. Boc-D-Phe-OH, TBTU, DMF, 90 min
Boc-D-Phe-2-Pal-HN
steps (3) and (4)
6. Boc-lle-OH, TBTU, DMF
Boc-lle-D-Phe-2-Pal-HN
steps (3) and (4) 7. Boc-D-Phe-OH, TBTU, DMF
Boc-D-Phe-lle-D-Phe-2-Pal-HN-
steps (3) and (4)
8. Boc- D-Trp-OH, TBTU, DMF
Boc-D-Phe-lle-D-Phe-2-Pal-HN-
steps (3) and (4) 9. Boc-Arg (Z)2 -OH, TBTU, DMF
Boc-Arg(Z)2-D-Phe-lle-D-Phe-2-Pal-HN -
10. CF3SO3H, TFA, anisole, ehtanedithiol

Arg-D-Trp-D-Phe-lle-D-Phe-2-Pal-NH2 free peptide

resin

The fractions containing the product were pooled, concentrated in vacuo, and lyophilized. Each peptide was obtained as a white powder by lyophilization from 80% acetic acid. The purity of the final peptides was assessed by analytical RP-HPLC. Analytical RP-HPLCs were recorded using a Merck Lichrospher® 100 RP-18 (10 urn) column with a flow rate of 1 mL/min. Column
eluent was monitored at 215 nm. All peptides were found to be >95% pure and retention time and HRMS analysis of some representative peptides is given in Table 1.
Table 1. Identities and analytical data of the some representative hexapeptides

(TableRemoved)HPLC: In a solvent system of 10% CH3CN in 0.1% TFA and a gradient of 90% CH3CN over 45 min. An analytical Merck Lichrospher® 100 RP-18 (lO^m) column was used with a flow rate of 1 mL/min.
bMolecular Weight C[M+H]+
BIOLOGICAL ACTIVITIES
The antifungal activities of the target compounds against pathogenic fungi associated with opportunistic infections, C. albiccms, C. neoformans, and Aspergillusfumigatus, are summarized in Table 2. Amphotericin B was included as a standard drug for comparison. ICso, MICs, and MFCs were determined according to NCCLS methods (NCCLS, In Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard M27-A, National Committee on Clinical Laboratory Standards, 1997, 17, pp 9; NCCLS, In Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically M7-A5, National Committee on Clinical Laboratory Standards, 2000, 20, pp 2; NCCLS, In Susceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Tentative Standard—Second Edition, M24-T2, National Committee on Clinical Laboratory Standards, 2000, 20, pp 26).The was defined as the concentration that affords 50% growth, whereas MIC was defined as the

lowest concentration resulting in inhibition of visible growth after incubation, and MFC was the minimum fungicidal concentration that kills 100% of the organism.
Representative peptides 1-4 displayed selective antifungal activity against C. neoformcms, but they were not active against C. albicans andA.fumigatus. Peptide 1, in which Arg-1 is replaced with Orn and His-6 is replaced with 1-Bzl-His showed pronounced antifungal activity with an ICso value of 1.6 uM, MIC of 5.3 uM and MFC of 5.3 ^M and is therefore most promising antifungal compound of the series. Peptides 2-4 also displayed promising activities and exhibited IC5os in the range of 3.5-6.6 u.M, MICs between 5.0-11.0 jiM and killed 100% of C. neoformans in concentration ranging between 5.0-11.0 uM (Table 2).
Table 2. In vitro antifungal activities (\M] of the representative hexapeptides (1-4)

(TableRemoved)IC50, The concentration that affords 50% inhibition of bacterial growth
bMIC, (Minimum inhibitory concentration) is the lowest test concentration that allows detectable growth of bacteria
°MFC, Minimum fungicidal concentration (the lowest test concentration that kills 100% of the organism)
NT, Not tested
NA, not active at 20 ug/mL (20-22 (
The antibacterial activities of representative hexapeptides 1-4 against Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare are reported as ICso, MIC, and MBC in Table 3. Ciprofoxacin was included as positive control for comparison. Susceptibility of S. aureus and methicillin-resistant S. aureus to test compounds was determined according to the
rocedure as described by the National Committee for Clinical Laboratory Standards (NCCLS) (NCCLS, In Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard M27-A, National Committee on Clinical Laboratory Standards, 1997, 17, pp 9; NCCLS, In Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically M7-A5, National Committee on Clinical Laboratory Standards, 2000, 20, pp 2; NCCLS, In Susceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Tentative Standard—Second Edition, M24-T2, National Committee on Clinical Laboratory Standards, 2000, 20, pp 26).
Table 3. In vitro antibacterial activities (^M) of the hexapeptides (1-4)

(TableRemoved)aIC50, The concentration that affords 50% inhibition of bacterial growth
bMIC, (Minimum inhibitory concentration) is the lowest test concentration that allows detectable growth of bacteria
CMBC, Minimum bactericidal concentration (the lowest test concentration that kills 100% of the organism)
NT, Not tested
NA, not active at 20 ug/mL (20-22
Susceptibility of M. intracellulare was done using the modified Alamar Blue procedure of Franzblau et al. (Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; G. Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.; Oilman, R. H. J. Clin. Microbiol. 1998, 36, 362). The MBC was defined as the minimum bactericidal concentration that kills 100% of the organism.
None of the peptides were active against M. intracellulare. In contrast, peptide 1, 3 and 4 displayed promising antibacterial activities against S. aureus and methicillin-resistant S. aureus (MRS). Peptide 4, in which His-6 was replaced with 2-Pal exhibited promising antibacterial activities (IC50 = 3.8µM, MIC = 5.5µM, MBC = 10.9 µM) against MRS. While, 6 displayed (ICso = 3.8 uM, MIC = 10.9 fiM, MBC = 10.9 (o,M) against S. aureus and is the most promising compound of the series. Peptides 1 and 3 also exhibited encouraging antibacterial activity ranging between 3.7-6.5 uM, MICs of 10 \M and killed 100% of MRS and S. aureus at concentrations ranging between 10.0-20.0 uM. In contrast, Peptide 2, in which His-6 was replaced with D-His was found to be inactive against all microorganisms. It is important to note that lead peptide 2 was found inactive against all three microorganisms.
Finally, cytotoxicity of the peptides was tested against four human cancer cell lines (SK-MEL, KB, BT-549, SK-OV-3) and two noncancerous mammalian kidney cells (VERO and LLC-PKi) up to a highest concentration of 10.0 ^g/mL (10-11 jaM) using a neutral red assay procedure as described earlier (Borenfreund, E.; Babich, H.; Martin-Alguacil, N. In Vitro Cell Dev. Biol. 1990, 26, 1030; Mustafa, J.; Khan, S. I.; Ma, G.; Walker, L. A.; Khan, I. A. Lipids 2004, 39, 167). None of the analogues showed any cytotoxic effects.
DESCRIPTION OF THE DRAWING
Figure 1 shows peptide synthesis cycle.
SUMMARY OF THE INVENTION
The present invention provides novel liner antimicrobial peptides of general formula (I) for the treatment and prevention of a panel of opportunistic fungi and bacterial pathogens. Some of
these new derivatives have shown excellent in vitro antifungal against Candida albicans, Cryptococcous neoformans, Aspergillus fumigatus, Candida glabrata, and Candida krusei and excellent in vitro antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare. These promising antimicrobial peptides possess several advantages for making them suitable candidates for anti-infective drugs such as: Broad-spectrum activity of antibacterial and antifungal effects, rapid onset of pathogen killing, potentially low levels of induced resistance due to peptide nature of drug, and elimination of cyto-toxic side effects. It is expected that development of these peptides as ideal anti-infective agents will add to already existing armament for the suppression as well as radical cure of the various opportunistic fungal and bacterial infections.

We claim
1. A process for the development of novel linear Antimicrobial hexapeptide
composition, comprising of general formula (1);
X-D-Trp-D-Phe-X1-D-Phe-X2-NH2
(1)
Wherein X, XI and X2 represents all twenty naturally occurring L-amino acids, their D-counter parts; unnatural amino acids ornithine (OM), phenylglycine (Phg), aminoisobutyric acid (Aib); all three isomeric L/D- pyridyalanines, and all synthetic derivatives of D- and L- histidine such as 5-halogenated/ 2,5-dihalogenated-D/L-histidine where halogen is F, Cl, Br and I groups; 1 alkyl-L/D-histidine, 2 alkyl-histidines.
2. A process as claimed in claim 1, wherein the process is a solid phase peptide synthesis
protocol.
3. The process as claimed in claim 2, wherein the said protocol is carried out in a
4-methylbenzhydrylamine-functionalised, 1% cross linked polystyrene resin (0.31
mequiv g"1) in 0.31 mmol.
4. The process as claimed in claim 2, wherein, the said protocol comprising the
following,
I. 4-Methylbenzhydrylamine (MBHA.HC1) resin (500 nig, 0.31 mmol) was charged
into the reaction vessel
II. The requisite suitably protected amino acids (0.93 mmol) were loaded sequentially into the amino acid vessels,
III. The MBHA.HC1 resin was neutralized with (N,.N'-Diisopropylethylaniine) DIEA
(10%inDCM)for5min.
IV. After neutralization the resin is washed with DMF (2 * 10 niL) and once with DCM
(lOmL).
V. The coupling of the first amino acid was done in the presence of coupling reagent TBTU (2-(lH-Benztriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate) and DIE A (10% in DCM) for 90 min. VI. After coupling, the tert-Boc group was cleaved with trifluoroacetic acid (40%
solution in DCM) for 20 min.
VII. The amino acid linked resin was again neutralized with DIEA (10% in DCM) for 5 min and washed with DMF (2 x 10 mL) and once with DCM (10 mL) to afford dipeptide. VIII. The coupling and deprotection steps were repeated to obtain desired sequence of
peptide resins.
IX. The peptides were cleaved from the resin support with simultaneous side chain deprotection, then purified and analyzed.
5. The process as claimed as claimed in claim 4, wherein the said coupling reactions
were monitored qualitatively with the Kaiser test.
6. The process as claimed claim 4, wherein the said peptides were cleaved from the resin
support as claimed in claim 4(i), wherein cleavage of crude peptides from the resin
support is carried out by acidolysis using anhydrous trifluromethane sulfonic acid (1
mL) containing thianisole (ImL) and 1, 2-ethanedithiol (0.5 mL) as scavengers in
TFA (10 mL) for 2 hrs at ambient temperature
7. The process as claimed in claim 4, wherein the said purification as claimed in claim
4(1), wherein the purification is carried out as under;
I. The crude peptide was separated from solid support by filtration 11. The resin was washed with TFA (3X4 mL)
III. Combined extracts were evaporated under reduced pressure
IV. The residue obtained from step 7 (III) was neutralized with saturated ammonium
bi carbonate solution
V. Non polar impurities are removed by extracting the aqueous layer with diethyl
ether (3 X lOmL) VI. Crude peptide was recovered by evaporating the aqueous layer under reduced
pressure
VII. The crude peptides were further purified by semi-preparative RP-HPLC on CIS bonded silica gel using (l0µm) column
VIII. A linear gradient elution system at a flow rate of 20 mL min"1 was employed IX. The separations were monitored at 215 nm, by TLC on silica gel coated sheets.
8. The process as claimed in claim 4, wherein the said analysis is carried out by
analytical RP-HPLC and is recorded using 100 RP-18(10um) column with a flow rate
of 1 mL/min.
9. The process as claimed in claim 8, wherein the said column eluent was monitored at
215 nm.
10. Peptides as claimed in claim 1, wherein the peptides were found to be > 95% pure.
11. A process for the development of novel linear Antimicrobial hexapeptide composition
substantially described herewith foregoing description and diagrams.

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Patent Number

269454

Indian Patent Application Number

1010/DEL/2006

PG Journal Number

44/2015

Publication Date

30-Oct-2015

Grant Date

23-Oct-2015

Date of Filing

18-Apr-2006

Name of Patentee

NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH

Applicant Address

SECTOR 67, S.A.S.NAGAR,PUNJAB 160062,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ROHIT KUMAR SHARMA	DEPARTMENT OF MEDICINAL CHEMISTRY,NIPER,SECTOR 67, S.A.S.NAGER,PUNJAB-160062,INDIA.
2	SANDEEP SUNDRIYAL	DEPARTMENT OF MEDICINAL CHEMISTRY,NIPER,SECTOR 67, S.A.S.NAGER,PUNJAB-160062,INDIA.
3	RAHUL JAIN	DEPARTMENT OF MEDICINAL CHEMISTRY,NIPER,SECTOR 67, S.A.S.NAGER,PUNJAB-160062,INDIA.

PCT International Classification Number

C07D 211/86

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA