|Title of Invention||
"VASOACTIVE INTESTINAL PEPTIDE ANALOGS"
|Abstract||The present invention provides a A vasoactive intestinal peptide analog of the formula (I) His-Ser-Asp-R 1 - Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu- Arg-Lys-Gin-R3 - Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NHi wherein Rl is Aib, Deg or Ac5c, R2 is Phe or 4-Cl-D-Phe, R3 is Met, Leu or Dpg wherein Aib is a-amino-isobutyric acid, Deg is diethylglycine, Ac5c is 1-amnocyclopentane carboxylic acid and Dpg is di-n-propylglycine, and a method of preparation thereof.|
|Full Text||FIELD OF THE INVENTION;
The present invention encompasses novel analogs of vasoactive intestinal peptide (VIP), containing substitutions at appropriately selected amino acids. The invention particularly relates to the design and synthesis of novel biologically active VIP analogs containing α,α-dialkylated amino acids in a site-specific manner. Specifically, the invention relates to the synthesis of VIP peptide derivatives, which bind selectively to VIP receptors on target cells. The invention encompasses methods for the generation of these peptide reagents and the pharmacological applications of these aforesaid derivatives in neoplasia.
BACKGROUND OF THE INVENTION;
Vasoactive intestinal peptide is known to play critical roles in modulating the intracellular and extracellular events involved in homeostasis, and are intimately involved in all major cognitive and non-cognitive homeostatic systems (Schofl et al, 1994). The multiple biological activities of peptides has led to extensive research focused on the exploitation of these peptide hormones as therapeutic drugs. Multiple replacements have been used to avoid the susceptibility of the amide bond to proteolytic cleavage. These include the use of unnatural amino acids like D-amino acids, N-alkyl and N-hydroxy- amino acids, α-aza amino acids, thioamide linkage, design of peptide mimetics and prodrugs as well as amide bond modifications under the pseudopeptide linkage rubric (Dutta, 1993 ; Pasternak et al, 1999). Another approach has been the blockage of N-terminus or C-terminus of the peptide by acylation or amidation.
The design of conformationally constrained bioactive peptide derivatives has been one of the widely used approaches for the development of pepticle-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of α, α- dialkylated amino acids have been well studied. The incorporation of these amino acids restricts the rotation of , ψ angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of α, α-
dialkylated aminoacids, α-aminoisobutyric acid (Aib) or α, α- dimethylglycine has been shown to induce p-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, Karle and Balaram, 1990) The conformational properties of the higher homologs of α, α- dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg), di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of a, a- dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), as 1-aminocycloheptane carboxylic acid (Ac7c) and as 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., 1995 ; Karle et a/., 1995). a, a- dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad et a/., 1996) However, the applicants are not aware of any prior art for the -synthesis of novel peptide analogs, encompassed in the present invention, particulary the synthesis of such VIP peptide analogs, alalogs, containing α, α-dialkylated amino acids, by solid phase peptide synthesis methodology. Moreover, the use of such constrained amino acids for the design of peptides possessing anti-neoplastic activity is also unknown in any previous prior art. The present invention exploits the conformational properties of such a, a-dialkylated amino acids for the design of biologically active peptide derivatives, taking VIP as the model system under consideration. Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said and Mutt, 1970). It bears extensive homology to secretin, peptide histidine isoleucine (PHI) and glucagon. The amino acid sequence for VIP is : His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 VIP is known to exhibit a wide variety of biological activities in the autocrine, endocrine and paracrine functions in living organisms (Said, 1984). In the gastrointestinal tract, it has been known to stimulate pancreatic and biliary secretions, hepatic glycogenesis as well as the secretion of insulin and glucagon (Kerrins and Said, 1972 ; Domschke et al, 1977). In the nervous system it acts as a neurotransmitter and neuromodulator, regulating the release and secretion of several key hormones (Said, 1984). In recent years, attention has been focussed on the function of VIP in certain .areas of the CNS as well its role in the progression and control of neoplastic disease (Reubi, 1995). The importance of peptide growth factors and regulatory hormones in the etiology and pathogenesis in several carcinomas has long been recognized Data from epidemiological and endocrinological studies suggest that neuropeptides like VIP which are responsible for the normal growth of tissues like the pancreas can also cause conditions for their neoplastic transformation (Sporn et al., 1980). Several lines of evidence indicate that VIP acts as a growth factor and plays a dominant autocrine and paracrine role in the sustained proliferation of cancer cells (Said, 1984). The stimulatory effect of VIP on tumor growth can be mediated directly by its receptors on cell membranes or indirectly by potentiation of the activities of other growth factors in tumor cells (Scholar el al., 1991). The synergistic effect of VIP and related pituitary adenylate cyclase activating polypeptide (PACAP) in glioblastomas is an illustration to the above fact (Moody et al., 1996) The multiple physiological and pharmacological activities of VIP are mediated by high affinity G-protein coupled transmembrane receptors on target cells (Reubi et al., 1996). VIP receptors are coupled to cellular effector systems via adenyl cyclase activity (Xia et al., 1996). The VIP receptor, found to be highly over expressed in neoplastic cells, is thought to be one of the biomarkers in human cancers (Reubi et al., 1996). High affinity VIP receptors have been localized and characterized in neoplastic cells of most breast carcinomas, breast and prostate cancer metastases, ovarian, colonic and pancreatic adenocarcinomas, endometrial and squamous cell carcinomas, non small cell lung cancer, lymphomas, glioblastomas, astrocytomas, meningiomas and tumors of mesenchymal origin. Amongst, neuroendocrine tumors all differentiated and non-differntiated gastroenteropancreatic tumors, pheochromocytomas, small-cell lung cancers, neuroblastomas, pituitary adenomas as well tumors associated with hypersecretory states like Verner-Morrison syndrome were found to overexpress receptors for vasoactive intestinal peptide (Tang et al, 1997a & b; Moody et al, 1998a &b; Oka et al, 1998)). These findings suggest that new approaches for the diagnosis and treatment of these cancers may be based on functional manipulation of VTP activity, by designing suitable peptide derivatives of the same. The present invention relates to the anti-neoplastic activity of novel VIP peptide analogs using selected constrained amino acids. These novel VIP analogs were found to bind to VIP receptor on cell membranes. The anti-neoplastic activity of the aforesaid peptides was also determined. REFERENCES: Domschke, S. etal (1977) Gastroenterology, 73, 478-480. Dutta, A.S. (1993) Small Peptides: Chemistry, Biology and Clinical Studies, Elsevier, Pharmacochemistry Library, 19, pp 293-350. Karle, I.L. etal. (1995) J. Amer. Chem. Soc. 117, 9632-9637. Karle, I.L. and Balaram, P. (1990) Biochemistry 29, 6747-6756.
Kerrins, C. and Said, S.I. (1972) Proc. Soc. Exp. Biol. Med. 142, 1014-1017.
Oka, H. et al. (1998) Am. J. Pathol. 753, 1787-1796.
Pasternak, A. etal. (1999)Biorg. Med. Chem. 9, 491-496.
Prasad, B.V.V and Balaram, P. (1984) CRC Crit. Rev. Biochem. 16, 307-347.
Prasad, S etal. (1995) Biopolymers 35, 11-20
Prasad, S et al. (1996) Int. J. Peptide Protein Res. 48, 312-318.
Reubi, J.C. etal Cancer Res., 56 (8), 1922-1931,1996.
Said, S. I. and Mutt, V. (1970) Science, 169, 1217-1218.
Said, S.I. (1984) Peptides, 5,143-150.
Scofl, C. etal. (1994) Trends. Endocrinol. Metab. 5, 53-59.
Sporn, M.B., and Todaro, G.J. (1980) N. Engl. J. Med., 303, 378-379.
Stewart, J. and Young , Y.D. (1969) Solid Phase Peptide Synthesis, W.H. Freeman &
Tang, C. etal., (1997a) Gut, 40, 267-271.
Tang, C. etal., (1997b) Br. J. Cancer, 75,1467-1473.
Xia, M. etal., J. Clin. Immunol., 16 (1), 21-30, 1996
SUMMARY OF THE INVENTION
The present invention comprises of vasoactive intestinal peptide antagonists of the following general formula, wherein appropriate amino acids in VIP have been replaced by α, α-dialkylated amino acids in a specific manner, and all pharmaceutically acceptable salts thereof:
wherein Rl is Aib, Deg or Ac5c,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg
wherein Aib is α-amino-isobutyric acid, Deg is diethylglycine, Ac5c is 1-amnocyclopentane carboxylic acid and Dpg is di-n-propylglycine. The present invention also provides a solid phase synthesis process for the preparation of a peptide analog described above which comprises sequentially loading the corresponding protected α-α-dialkylated amino acids in sequential cycles to the amino terminus of a solid phase resin, coupling the amino acids in the presence of conventional solvents such as herein described and reagents in a manner herein described to assemble a peptide-resin assembly, removing in a manner herein described the protecting groups and cleaving the peptide from the resin to obtain a crude peptide analog. The preferred VIP antagonists of the present invention are as follows: (Rl=Aib, R2=4-D-Cl-Phe, and R3=Leu)
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lyr-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:1) (Rl=Deg, R2=4-D-Cl-Phe, and R3=Leu)
His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lyr-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:2) (Rl=Ac5c, R2=4-D-Cl-Phe, and R3=Leu)
His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lyr-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:3) (Rl=Aib, R2=Phe, and R3=Met and the )
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Tnr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:4) (Rl=Aib, R2=Phe, and R3=Leu):
His-SetAsp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr- Leu -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO: 5);
(R1=Ac5c, R2=Phe, and R3 =Leu):
His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr- Le.u -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO:6);
(Rl=Deg, R2=Phe, and R3 -Leu):
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr- Leu -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO:7);
(Rl=Aib, R2=Phe, and R3 =Dpg):
His-Ser-Asp-Aib -Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg -Ala-Val-Lys-Lys-
Tyr- Leu -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO:8);
(Rl=Aib, R2=4-Cl-D-Phe, and R3 =Dpg):
His-Ser-Asp-Aib -Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg -Ala-Val-Lys-Lys-
Tyr- Leu -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO:9);
(Rl=Deg, R2=Phe, and R3 =Dpg):
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr- Leu -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO: 10);
(Rl=Ac5c, R2=Phe, and R3 =Dpg):
His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr- Leu -Asn-Ser-Ile-Leu -Asn-NH2 (SEQID NO: 11);
The novel compounds of the present invention have important pharmacological applications. They are potent anti-neoplastic agents and thereby possess therapeutic potential in a number of human cancers.
DETAILED DESCRIPTION OF THE INVENTION;
The novel peptide analogs embodied in the present invention contain amino acids, namely a, a-dialkylated amino acids, which have been known to induce highly specific constraints in the peptide backbone.
The α, α-dialkylated amino acids, used in the present invention are synthesized from the corresponding ketones In a preferred embodiment of the invention, the ketones are first converted into the corresponding hydantoins, which are hydrolyzed to yield the aforesaid amino acids. In a preferred embodiment of the present invention, 60% sulphuric acid has been employed as the hydrolyzing agent. The conversion of the ketones to their appropriate a, a-dialkylated amino acids is shown in Example 1.
The novel peptides in the present invention have been generated by using solid phase techniques or by a combination of solution phase procedures and solid phase techniques or by fragment condensation. Although these methods for the chemical synthesis of polypeptides are well known in the art (Stewart and Young, 1969), the use of solid phase methodology for the synthesis of peptides incorporating a, a-dialkylated amino acids is not known in the prior art. The applicants are also not aware of any prior art describing the synthesis of the novel VIP analogs encompassed in the present invention.
In a preferred embodiment of the present invention the peptides were synthesized using the Fmoc strategy, on a semi automatic peptide synthesizer (CS Bio, Model 536), using optimum side chain protection. The peptides were assembled from C-terminus to N-terminus. Peptides amidated at the carboxy-terminus were synthesized using the Rink Amide resin. The loading of the first Fmoc protected amino acid was achieved via an amide bond formation with the solid support, mediated by Diisopropylcarbodiimide (DIPCDI) and HOBt. Substitution levels for automated synthesis were preferably between 0.2 and 0.6 mmole amino acid per gram resin. The steps involved in the synthesis of the VIP analogs employed the following protocol:
The resin employed for the synthesis of carboxy-terminal amidated peptide analogs was 4-(2', 4'-
Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl-derivatized polystyrene 1 %
divinylbenzene (Rink Amide) resin (100-200 mesh), procured from Calbioichem-Novabiochem Corp., La Jolla, U.S.A., (0.47 milliequivalent NH. sub. 2 /g resin).
Accordingly, the present invention provides A vasoactive intestinal peptide analog of the
following general formula (I)
His-Ser-Asp-R 1 - Val-R2-Thr- Asp- Asn-Tyr-Thr-Arg-Leu- Arg-Ly s-Gln-R3 - Ala-Val-Lys-Lys-Tyr-
Leu -Asn-Ser-Ile-Leu -Asn-NHi
Wherein Rl = Aib or Deg or Ac5c
R2 = Pheor4-Cl-D-Phe
R3 = Met or Leu or Dpg
or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salts thereof.
The present invention also provides a solid phase synthesis process for the preparation of a peptide
analog of general formula (I):
Leu -Asn-Ser-Ile-Leu -Asn-NHz
Wherein Rl = Aib or Deg or Ac5c
R2 = Phe or 4-Cl-D-Phe
R3 = Met or Leu or Dpg
which comprises sequentially loading the corresponding protected a- a-dialkylated amino acids in sequential cycles to the amino terminus of a solid phase resin, coupling the amino acids in the presence of conventional solvents and reagents to assemble a peptide-resin assembly, removing the protecting groups and cleaving the peptide from the resin to obtain a crude peptide analog.
In a particularly preferred embodiment of the present invention the following chemical moieties were used to protect reactive side chains of the peptides during the synthesis procedure: The N-terminal amino group was protected by 9-flourenylmethoxycarbonyl (Fmoc) group. Trityl (trt) or t-butyloxycarbonyl (Boc) were the preferred protecting groups for imadazole group of Histidine residue. The hydroxyl groups of Serine, Threonine and Tyrosine were preferably protected by t-butyl group (tBu) 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl (Pmc) or 2,2,4,7,-pentamethyl-dihydrobenzenofuran-5-sufonyl (Pbf) were the preferred protecting groups for the guandino group of Arginine. Trityl was the preferred protecting group for Asparagine and Glutamine and tertiary butyl group (tBu) was the preferred protecting group for Aspartic acid and Glutamic acid. The tryptophan residue was either left unprotected or used with Boc protection. The side chain amino group of Lysine was protected using preferably Boc group.
In a preferred embodiment of the invention, 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent were used. The activating reagents used for coupling amino acids to the resin, in solid phase peptide synthesis, are well known in the art. These include BOP, PyBOP, HBTU, TBTU, PyBOP, HOBt. Preferably, DCC or DIPCDI / HOBt or HBTU/HOBT and DIEA were used as activating reagents in the coupling reactions.
The protected amino acids were either activated in situ or added in the form of preactivated esters known in the art such as NHS esters, Opfp esters etc.
The coupling reaction was carried out in DMF, DCM or NMP or a mixture of these solvents and was monitored by Kaiser test [Kaiser et al. Anal. Biochem., 34, 595-598 (1970)]. In case of a positive Kaiser test, the appropriate amino acid was re-coupled using freshly prepared activated reagents.
After the assembly of the peptide analog was completed, the amino-terminal Fmoc group was removed using steps 1-6 of the above protocol and then the peptide-resin was washed with methanol and dried. The analogs were then deprotected and cleaved from the resin support by treatment with trifluoroacetic acid, crystalline phenol, ethanedithiol, thioanisole and de-ionized water for 1.5 to 5 hours at room temperature. The crude peptide was obtained by precipitation with cold dry ether, filtered , dissolved, and lyophilized.
The resulting crude peptide was purified by preperative high performance liquid chromatography (HPLC) using a LiChroCART® C18 (250. Times. 10) reverse phase column (Merck, Darmstadt, Germany) on a Preparative HPLC system (Shimadzu Corporation, Japan) using a gradient of 0.1% TFA in acetonitrile and water . The eluted fractions were reanalyzed on Analytical HPLC system (Shimadzu Corporation, Japan) using a C18 LiChrospher®, WP-300 (300 X 4) reverse- phase column. Acetonitrile was evaporated and the fractions were lyophilized to obtain the pure peptide. The identity of each peptide was confirmed by electron-spray mass spectroscopy.
An analog of the present invention can be made by exclusively solid phase techniques, by partial solid phase / solution phase techniques and fragment condensation. Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples discussed in the subsequent section of this document.
The present invention will be further described in detail with reference to the following examples, as will be appreciated by a person skilled in the art is merely illustrative and should not be
construed as limiting. Various other modifications of the invention will be possible without departing from the spirit and scope of the present invention
Synthesis of Amino Acids
a,a- Dialkylated amino acids were synthesized from the appropriate ketones These ketones were first converted into their corresponding hydantoins which on hydrolysis with strong acid or alkali gave the respective amino acids
Cyclopentanone (0.1 mol), KCN (0.3mol) and (NH4)2CO3 were dissolved in 300ml of 50% aqueous methanol and the mixture was refluxed for 4-6 hrs on water bath. Subsequently, the solution was concentrated to half of its volume and chilled in an ice bath. The chilled solution was acidified with 2N HC1. The precipitate thus obtained was filtered and washed with several times with cold water to remove the traces of cyanide. The solid was subsequently dried and recrystallised from aqueous alcoholic solution. The yield of the product n the aforesaid reaction was found to be 86%. The 5,5'-spirocyclopentane hydantoin thus obtained was characterized by I.R spectroscopy (stretching bands characteristic of the carbonyl group were observed at 1710-1740 cm1 and 1760-1780 cm1 respectively)
The 5,5'-spirocyclopentane hydantoin (O.05mol) was dissolved in 45ml of 60% H2SO4 and refluxed at 150-160 C for about 28hrs. The reaction mixture was cooled to room temperature and diluted with water (150ml). The diluted solution was filtered to remove the charred particles. The clear solution was chilled in ice cold water and neutralised with ammonia solution. The solution was further concentrated and cooled. Shining white precipitate was obtained The precipitate thus obtained was filtered and dried. The amino acid i.e.l-aminocyclopentane carboxylic acid (Ac5c) was confirmed by I.R spectroscopy (1610-1640 cm1 for COO" group and 3060-3090 cm1 for -NH3 group)
Preparation of Fmoc-Asn(trt)-Resin
A typical preparation of the Fmoc-Asn(trt)-Resin was carried out using 0.5g of 4-(2',4'-Dimethoxyphenyl- Fmoc- aminomethyl) phenoxymethyl- derivatized polystyrene 1% divinylbenzene (Rink Amide ) resin ( 0.47 mM / g ) (100-200 mesh), procured from Calbiochem-Novabiochem Corp., La Jolla, U.S.A. Swelling of the resin was typically carried out in dichloromethane measuring to volumes 10-40ml /g resin. The resin was allowed to swell in methylene chloride (2 X 25 ml, for 10 min.). It was washed once in dimethylformamide (DMF) for 1 min All solvents in the protocol were added in 20 ml portions per cycle. The Fmoc-protecting group on the resin was removed by following steps 3-7 in the protocol. The deprotection of the Fmoc group was checked by the presence of blue beads in Kaiser test. For loading of the first amino acid on the free amino (NH2) group of the resin, the first amino acid, Fmoc-Asn(trt)-OH, was weighed in four fold excess, along with a similar fold excess of HOBt, in the amino acid vessel of the peptide synthesizer. These were dissolved in dimethylformamide (A.C.S. grade) (J.T.Baker, Phillipsburg, New Jersey, U.S. A.) and activated with DIG, just prior to the addition to the resin in the reaction vessel of the peptide synthesizer. HOBt was added in all coupling reactions, especially in the case of Arg, Asn, Gin and His. The coupling reaction was carried out for a period ranging from 1-3 hours. The loading of the amino acid on the resin was confirmed by the presence of colorless beads in the Kaiser Test. The loading efficiency was ascertained by the increase of weight of the resin after the addition of the amino acid.
Synthesis of SEP. ID. NO: 1: (Aib4.4-Cl-D-Phe6,Leu17, )-VIP
The synthesis of (Aib4,4-Cl-D-Phe6,Leu17, )-VTP, amidated at the carboxy- terminus, was initiated by using all of the resin loaded with Fmoc-Asn(trt)-OH as prepared in example (a) above. This was subjected to stepwise deprotection and coupling steps as in steps 1-10 of the synthesis cycle. In each coupling reaction, a four- fold excess of amino- acid, DIC and HOBt were used.
Upon completion of synthesis and removal of the N-terminal Fmoc protecting group (steps 1-6 of the synthesis cycle), the peptide- resin was washed twice with methanol, dried and weighed to obtain 0.649g. This was subjected to cleavage in a cleavage mixture consisting of trifluoroacetic acid and scavengers, crystalline phenol, ethanedithol, thioanisole and water for a period of 3-5 hours at room temperature with continuous stirring. The peptide was precipitated using cold dry ether to obtain ~ 330 mg of crude peptide. The crude peptide was purified on a C18 preperative reverse phase HPLC column (250X10) on a gradient system comprising of acetonitrile and water in 0.1% TFA as described previously, in the art. The prominent peaks were collected and lyophilized, reanalysed on analytical HPLC and subjected to mass spectrometry. There was a good agreement between the observed molecular weight and calculated molecular weight. The pure peptide was then used for bioassays.
Synthesis of SEP. I.D. NO: 4 : [Aib4]-VIP:
A 0.255g portion of Fmoc-Asn (trt)-Rink Amide resin from Example 2 was subjected to solid phase synthesis using the protocol stated in "Detailed Description of the Invention". All couplings were performed using the appropriate molar excess of the required Fmoc-amino acids. Coupling reagents and additives were used as well known to those skilled in the art. After the assembly of the peptide was complete the Fmoc group was removed from the resin, as mentioned earlier. The peptide was cleaved, lyophilized, purified and characterized according to the protocols described in the previous section.
Synthesis of Analog SEP. I.D. NO: 8: [Aib4. Dpgl7]-VIP:
A 0.255g portion of Frnoc-Asn (trt)-Rink Amide resin from Example 2 was subjected to solid phase synthesis using the protocol stated in "Detailed Description of the Invention". All couplings were performed using the appropriate molar excess of the required Fmoc-amino acids. Coupling reagents and additives were used as well known to those skilled in the art. In a preferred embodiment of the invention, twenty seven coupling cycles were performed using appropiately protected amino acids as according to the sequence mentioned above. After the assembly of the peptide was complete the Fmoc group was removed from the resin, as mentioned earlier. The peptide was cleaved, lyophilized, purified and characterized according to the protocols described in the previous section.
Biological activity of peptide
The biological activity of synthesized peptide SEQ ID: 1 was tested on different human cancer cell lines such as HT-29 (colon), A549 ( non small cell lung), KB (oral), HuTu80 (duodenum) and MIAPaCa 2 (Pancreas) at various molar concentrations.
The percent cytotoxicity induced by different concentrations of the peptide SEQ ID: 1 is summarized in the following table.
The cytotoxicity of the peptide analog listed in the table was carried out by two day MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. MTT assay is based on the the principle of uptake of MTT,a tetrazolium salt.by metabolically active cells where it is metabolized by active mitochondria into a blue colored formazon product, which can be read spectrometrically (Ref: J.of Immunological Methods 65: 55-63, 1983). To prepare the MTT stock
1. A vasoactive intestinal peptide analog of the formula (I)
Rl is Aib, Deg or Ac5c,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg
wherein Aib is α-amino-isobutyric acid, Deg is diethylglycine, Ac5c is 1-amnocyclopentane carboxylic acid and Dpg is di-n-propylglycine.
2. The peptide as claimed in claim 1 wherein Rl=Aib, R2=4-D-Cl-Phe, and R3=Leu
and the said compound is:
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lyr-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:1)
3. The peptide as claimed in claim 1 wherein Rl=Deg, R2=4-D-Cl-Phe, and R3=Leu
and the said compound is:
His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lyr-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:2)
4. The peptide as claimed in claim 1 wherein Rl=AcSc, R2=4-D-Cl-Phe, and R3=Leu
and the said compound is:
His-Ser-Asp-Ac5c-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lyr-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:3)
5. The peptide as claimed in claim 1 wherein Rl=Aib, R2=Phe, and R3=Met and the
said compound is:
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:4)
6. The peptide as claimed in claim 1 wherein Rl=Aib, R2=Phe, and R3=Leu and the
said compound is:
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:5)
7. The peptide as claimed in claim 1 wherein Rl=Ac5c, R2=Phe, and R3=Leu and the said
His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:6)
8. The peptide as claimed in claim 1 wherein Rl=Deg, R2=4-D-Cl-Phe, and R3=Leu and
the said compound is:
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:7)
9. The peptide as claimed in claim 1 wherein Rl=Aib, R2=Phe, and R3=Dpg and the said
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:8)
10. The peptide as claimed in claim 1 wherein Rl=Aib, R2=4-D-Cl-Phe, and R3=Dpg and
the said compound is:
His-Ser-Asp-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:9)
11. The peptide as claimed in claim 1 wherein Rl=Deg, R2=Phe, and R3=Dpg and the said
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 10)
12. The peptide as claimed in claim 1 wherein Rl=Ac5c, R2=Phe, and R3=Dpg and the said
His-Ser-Asp-Ac5c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 11)
13. A solid phase synthesis process for the preparation of a peptide analog as claimed in
any preceding claim which comprises sequentially loading the corresponding protected
α-α-dialkylated amino acids in sequential cycles to the amino terminus of a solid phase
resin, coupling the amino acids in the presence of conventional solvents such as herein
described and reagents in a manner herein described to assemble a peptide-resin
assembly, removing in a manner herein described the protecting groups and cleaving the
peptide from the resin to obtain a crude peptide analog.
14. A process as claimed in claim 13 wherein said α- α-dialkylated amino acids are
protected at their a- amino groups by a 9-fluorenyl methoxy carbonyl (Fmoc) group.
15. A process as claimed in claim 13 or 14 wherein the coupling is carried out in the
presence of activated agents selected from the group consisting of BOP, PyBOP, HBTU,
TBTU, TNTU, TSTU, PyBROP, HOBt.
16. A process as claimed in claim 15 wherein the coupling was carried out in the presence
of a solvent selected from the group consisting of DMF, DCM, NMP or any mixtures
17. A process as claimed in anyone of claims 13 to 16 wherein said crude peptide is
cleaved from said peptide-resin assembly by treatment with trifluoracetic acid,
crystalline phenol, ethanedithiol, thioanisole and deionised water for 1.5 to 5 hours at
18. A vasoactive intestinal peptide analog substantially as herein described with reference
to the foregoing examples.
19. A solid phase synthesis process for the preparation of a peptide analog as claimed in
claim 1 and substantially as herein described with reference to the foregoing examples.
|Indian Patent Application Number||136/DEL/2000|
|PG Journal Number||48/2007|
|Date of Filing||18-Feb-2000|
|Name of Patentee||DABUR RESEARCH FOUNDATION|
|Applicant Address||22, SITE IV, SAHIBABAD, GHAZIABAD 201010, UTTAR PRADESH, INDIA.|
|PCT International Classification Number||C07K 14/575|
|PCT International Application Number||N/A|
|PCT International Filing date|