Note: Descriptions are shown in the official language in which they were submitted.
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
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VASOACTIVE INTESTINAL PEPTIDE ANALOGS
FIELD OF THE INVENTION
The present invention encompasses novel analogs of vasoactive
intestinal peptide (VIP), containing substitutions at appropriately selected
amino
S acids. The invention particularly relates to the design and synthesis of
novel
biologically active VIP analogs containing a, a-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 peptides, compositions
containing
the peptides and the pharmacological applications of these peptides especially
in the
treatment and prevention of cancer.
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
nonstandard
amino acids like D-amino acids, N-alkyl and N-hydroxy-amino acids, a-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.
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
Hi s-S er-Asp-Ala-V al-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-
Gln-Met-Ala-V al-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NHz
(SEQ ID NO: 1).
VIP is known to exhibit a wide variety of biological activities in the
WO 01/60862 CA 02405689 2002-10-08 PCT/LTS00/20871
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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 et 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-differentiated
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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 VIP
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.
The design of conformationally constrained bioactive peptide
derivatives has been one of the widely used approaches for the development of
peptide-based therapeutic agents. Non-standard amino acids with strong
1 S 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 a, a-dialkylated amino
acids
have been well studied. The incorporation of these amino acids restricts the
rotation
of ~, 'Y, angles, within the molecule, thereby stabilizing a desired peptide
conformation. The prototypic member of a, a-dialkylated aminoacids, a-amino-
isobutyric acid (Aib) or a, a-dimethylglycine has been shown to induce ((3-
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 a, a-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 (AcSc), 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane
carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (AcBc) have also
been shown to induce folded conformation (Prasad et al., 1995; Karle et al.,
1995).
a, a-dialkylated amino acids have been used in the design of highly potent
chemo-
tactic peptide analogs (Prasad et al., 1996). The applicants are not aware of
any
prior art for the synthesis of novel peptide analogs, encompassed in the
present
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
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invention. The present invention exploits the conformational properties of a,
a-
dialkylated amino acids for the design of biologically active peptide
derivatives,
taking VIP as the model system under consideration.
REFERENCES
Domschke, S. et al. (1977) Gastroenterology, 73, 478-480.
Dutta, A.S. (1993) Small Peptides: Chemistry, Biology and Clinical
Studies, Elsevier,
Pharmacochemistry Library, 19, pp 293-350.
Karle, LL. et al. (1995) J. Amer. Chem. Soc. 117, 9632-9637.
Karle, LL. 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. 153, 1787-1796.
Pasternak, A. et al. (1999) Biorg. Med. Chem. 9, 491-496.
Prasad, BVV and Balaram, P. (1984) CRC Crit. Rev. Biochem. 16,
307-347.
Prasad, S et al. (1995) Biopolymers 35, I 1-20
Prasad, S et al. ( 1996) Int. J. Peptide Protein Res. 48, 312-318.
Reubi, J.C. et al. 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.
Scholar E.M. Cancer 67(6): 1561-1569, 1991.
Scoff, C. et al. (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 & Co.
Tang, C. et al., (1997a) Gut, 40, 267-271.
Tang, C. et al., (1997b) Br. J. Cancer, 75, 1467-1473.
Xia, M. et al., J. Clin. Immunol., 16 (1), 21-30, 1996
Throughout the specification and claims the following abbreviations
are used with the following meanings:
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BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexfluorophosphate
PyBOP: Bena:otriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexofluorophospate
HBTU: O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-
hexofluoro-phosphate
TBTU: 2-(1H-Benzotriazole-1-yl)-1, 1, 3, 3-tetra-
methyluronium tetrafluoroborate
HOBt: 1-Hydroxy Benzotriazole
DCC: Dicyclohexyl carbodiimide
DIPCDI: Diisopropyl carbodiimide
DIEA: Diisopropyl ethylamine
DMF: Dimethyl formamide
DCM: Dichloromethane
NMP: N-Methyl-2-pyrrolidinone
TFA: trifluoroacetic acid
Throughout the specification
and claims the amino
acid residues are
designated by their
standard abbreviations.
Amino acids denote
L-configuration
unless otherwise indicated
by D or DL appearing
before the symbol and
separated
from it by a hyphen.
SUMMARY OF THE INVENTION
The present invention comprises VIP antagonists of the following
general formula, wherein appropriate amino acids in VIP have been replaced by
a, a-dialkylated amino acids in a specific manner. The invention also
comprises
the pharmaceutically acceptable salts of the antagonists of the following
general
formula:
His-S er-Asp-Rl-V al-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys
Gln-R3-Ala-V al-Lys-Lys-Tyr-Leu-Asn-S er-I le-Leu-Asn-NHz
wherein
Rl is Aib, Deg or AcSc,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg or a hydrolyzable carboxy protecting group;
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or pharmaceutically acceptable salt of the antagonists.
DETAILED DESCRIPTION OF THE INVENTION
The present invention comprises VIP antagonists of the following
general formula, wherein appropriate amino acids in VIP have been replaced by
a, a-dialkylated amino acids in a specific manner. The invention also
comprises
the pharmaceutically acceptable salts of the antagonists of the following
general
formula:
Hi s-S er-Asp-Rl-V al-R2-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-
Gln-R3-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NHZ
wherein
R1 is Aib, Deg or AcSc,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, Leu or Dpg or a hydrolyzable carboxy protecting group;
or pharmaceutically acceptable salt of the antagonists.
A hydrolyzable carboxy protecting group are those groups which on
hydrolysis converts to carboxy group such as -CONH2, -COOMe, etc.
Salts encompassed within the term "pharmaceutically acceptable salts"
refer to non-toxic salts of the compounds of this invention. Representative
salts and
esters include the following:
acetate, ascorbate, benzenesulfonate, benzoate, bicarbonate, bisulfate,
bitartrate, borate, camsylate, carbonate, citrate, dihydrochloride,
methanesulfonate,
ethanesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinate, edetate,
edisylate,
estolate, esylate, fumarate, gluconate, glutamate, glycerophophates,
hydrobromide,
hydrochloride, hydroxynaphthoate, lactate, lactobionate, laurate, malate,
maleate,
mandelate, mesylate, mucate, napsylate, nitrate, n-methylglucamine, oleate,
oxalate,
palmoates, pamoate (embonate), palmitate, pantothenate, perchlorates,
phosphate/
diphosphate, polygalacturonate, salicylates, stearate, succinates, sulfate,
sulfamate,
subacetate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and
valerate.
Other salts include Ca, Li, Mg, Na, and K salts; salts of amino acids
such as lysine or arginine; guanidine, diethanolamine or choline; ammonium,
substituted ammonium salts or aluminum salts.
The salts are prepared by conventional methods.
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
The preferred VIP antagonists of the present invention are as follows:
(R1 = Aib, R2 = 4-D-CI-Phe, and R3 = Leu):
His-Ser-Asp-Aib-Val-4-CI-D-Phe-Thr-Asp-Asn-Tyr-
Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-V al-Lys-Lys-Tyr-
S Leu-Asn-Ser-Ile-Leu -Asn-NHZ (SEQ ID NO: 2);
(R1 = Deg, R2 = 4-CI-D-Phe, and R3 = Leu):
His-Ser-Asp-Deg-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-
Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-V al-Lys-Lys-Tyr-
Leu-Asn-Ser-Ile-Leu-Asn-NHZ (SEQ ID NO: 3);
(R1 = AcSc, R2 = 4-CI-D-Phe, and R3 = Leu):
Hi s-S er-Asp-Ac5 c-V al-4-CI-D-Phe-Thr-Asp-Asn-Tyr-
Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-
Leu-Asn-Ser-Ile-Leu-Asn-NHz (SEQ ID NO: 4);
(R1 = Aib, R2 = Phe, and R3 = Met):
Hi s-S er-Asp-Aib-V al-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-Met-Ala-V al-Lys-Lys-Tyr-Leu-Asn-
Ser-Ile-Leu-Asn-NHZ (SEQ ID NO: 5);
(R1 = Aib, R2 = Phe, and R3 = Leu):
Hi s-S er-Asp-Aib-V al-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
Ser-Ile-Leu-Asn-NHz, (SEQ ID NO: 6);
(R1 = AcSc, R2 = Phe, and R3 = Leu):
His-Ser-Asp-AcSc-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
Ser-Ile-Leu-Asn-NHZ (SEQ ID NO: 7);
(R1 = Deg, R2 = Phe, and R3 = Leu):
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
-g_
His-Ser-Asp-Deg-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-Leu-Ala-V al-Lys-Lys-Tyr-Leu-Asn-
Ser-Ile-Leu-Asn-NHz (SEQ ID NO: 8);
$ (R1 = Aib, R2 = Phe, and R3 = Dpg):
His-S er-Asp-Aib-V al-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-Dpg-Ala-V al-Lys-Lys-Tyr-Leu-Asn-
Ser-Ile-Leu-Asn-NHz (SEQ ID NO: 9);
(R1 = Aib, R2 = 4-Cl-D-Phe, and R3 = Dpg):
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-
Thr-Arg-Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-
Leu-Asn-Ser-Ile-Leu-Asn-NHZ (SEQ ID NO: 10);
1$ (R1 = 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-NHZ (SEQ ID NO: 11);
(R1 = Ac$c, R2 = Phe, and R3 = Dpg):
His-Ser-Asp-Ac$c-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
Ser-Ile-Leu-Asn-NHZ (SEQ ID NO: 12).
2$ 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.
Suitable routes of administration are those known in the art and
include oral, rectal, transdermal, vaginal, transmucosal, or intestinal
administration;
parenteral delivery, including intramuscular, subcutaneous, intramedullary
injections,
as well as intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal,
or intraocular injections.
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Pharmaceutical ~;ompositions suitable for use in present invention
include compositions wherein ~ he active ingredients are contained in an
effective
amount to achieve its intended pizipose. The term "an effective amount" means
that
amount of a drug or pharmaceutical agent that will elicit the biological or
medical
S response of a tissue, system, animal or human that is being sought.
In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable carriers
excipients,
diluents, solvents, flavorings, colorants etc. The preparations may be
formulated in
any form including but not limited to tablets, dragees, capsules, powders,
syrups,
suspensions, slurnes, time released formulations, sustained release
formulations,
pills, granules, emulsions, patches, injections, solutions, liposomes and
nanoparticles.
The exact formulation, route of administration and dosage can be
chosen by the individual physician in view of the patient's condition.
Toxicity and therapeutic efficacy of the peptides of this invention can
be determined by standard pharmaceutical procedures in cell cultures or
experimental animals.
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 a, a-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 using a strong acid or base, preferably HzS04, HCI, NaOH, or
Na2C03 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 (Stewart and Young, 1969).
In a preferred embodiment of the present invention the peptides were
synthesized using the Fmoc strategy, on a semi automatic peptide synthesizer
(CS
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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:
TABLE I
STEP REAGENT MIX TIME (MIN) NO.
OF
CYCLES
1. Methylene chloride 1 2
2. Dimethyl formamide 1 1
3. 20 % Piperidine in 1 1
Dimethyl formamide
4. 20 % Piperidine in 29 1
Dimethyl formamide
5. Dimethyl formamide 1 3
6. Isopropanol 1 2
7. Methylene chloride 1 2
8. Amino Acid Variable 1
9. Dimethyl formamide 1 2
10. Stop or Return for next
cycle
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 NHZ/g resin).
The present invention also provides a solid phase synthesis process
for the preparation of a peptide analog of formula (I):
His-Ser-Asp-Rl-Val-R2-Thr-Asp-Asn-Tyr-Thr-Arg-
Leu-Arg-Lys-Gln-R3-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
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Ser-Ile-Leu-Asn-NHz
wherein
R1 is Aib, Deg or AcSc,
R2 is Phe or 4-Cl-D-Phe,
R3 is Met, 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-fluorenylmethoxy-
carbonyl 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-sulfonyl (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 DCC, DIPCDI, DIEA, BOP, PyBOP,
HBTU, TBTU, and 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
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Atherton, E. et al., 1988, J. Chem.Soc., Perkin Trans. I, 2887; Bodansky, M in
"The
Peptides, Analysis, Synthesis and Biology (E. Gross, J, Meienhofer,eds) Vol.
1,
Academic Press, New York, 1979, 106.
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 a cleavage
mixture
of 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~ C,8 (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 C,8 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/or
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
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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
S which on hydrolysis with strong acid or alkali such as HZS04, HCI, NaOH or
NazC03 gave the respective amino acids.
EXAMPLE 1
Cyclopentanone (0.1 mol), KCN (0.3mo1) and (NH4)zC03 were
dissolved in 300m1 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 HCI.
The
precipitate thus obtained was filtered and washed several times with cold
water to
remove the traces of cyanide. The solid was subsequently dried and
recrystallized
from aqueous alcoholic solution. The yield of the product in the aforesaid
reaction
was found to be 86%. The 5,5'-spirocyclopentane hydantoin thus obtained was
characterized by LR. spectroscopy (stretching bands characteristic of the
carbonyl
group were observed at 1710-1740 cm-' and 1760-1780 cm' respectively).
The 5,5'-spirocyclopentane hydantoin (O.OSmol) was dissolved in
45m1 of 60% HzS04 and refluxed at 150-160°C for about 28 hrs. The
reaction
mixture was cooled to room temperature and diluted with water (150 ml). The
diluted solution was filtered to remove the charred particles. The clear
solution was
chilled in ice cold water and neutralized 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. 1-
aminocyclopentane carboxylic acid (AcSc) was confirmed by LR spectroscopy
(1610-1640 cm-' for COO- group and 3060-3090 cm' for NH3+ group).
EXAMPLE 2
Preparation of Fmoc-Asn(trt)-Resin
A typical preparation of the Fmoc-Asn(trt)-Resin was carned out
using O.Sg 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.
WO 01/60862 CA 02405689 2002-10-08 PCT/LTS00/20871
- 14-
Swelling of the resin was typically carried out in dichloromethane measuring
to
volumes 10-40 mL/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 the Kaiser test. For loading of the first amino acid on the free
amino
(NHZ) 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
DIPCDI,
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, Gln 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.
EXAMPLE 3
Synthesis of SEO ID NO: 2
(Aib4, 4-Cl-D-Pheb, Leu",~-VIP
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Art-Lys
Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NHZ
The synthesis of (Aib4, 4-Cl-D-Phe6,Leu",)-VIP, amidated at the
carboxy terminus, was initiated by using all of the resin loaded with Fmoc-
Asn(trt)-
OH as prepared in Example 2 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, DIPCDI 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,
WO 01/60862 CA 02405689 2002-10-08 PCT/L1S00/20871
-15-
crystalline phenol, ethanedithol, thioanisole and water for a period of 3-S
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
C~g preperative reverse phase HPLC column (250 X 10) on a gradient system
comprising acetonitrile and water in 0.1 % TFA as described previously, in the
art.
The prominent peaks were collected and lyophilized, reanalyzed 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.
EXAMPLE 4
Synthesis of SEO ID NO: 5 [Aib4]'-VIP
His-Ser-AsL-Aib-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Art-Lys-
Gln-Met-Al a-V al-Lys-Lys-Tyr-Leu-Asn-S er-Ile-Leu-Asn-NHZ
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.
EXAMPLE S
Synthesis of Analo~y SEO ID NO: 9 IAib4, Dpi"1-VIP
Hi s-S er-Ash Aib-V al-Phe-Thr-Ash Asn-Tyr-Thr-Art-Leu-Art-Lys-
2 S Gln-D~ ~-Al a-V al-Lys-Lys-Tyr-L eu-Asn-S er-Ile-Leu-Asn-NHZ
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. In a preferred embodiment
of
the invention, twenty seven coupling cycles were performed using appropriately
protected amino acids as according to the sequence mentioned above. After the
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
-16-
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.
EXAMPLE 6
The cytotoxic activity of synthesized peptides was tested on six human tumor
cell lines namely PA-1 (ovary), SW620 (colon) HuTu80 (duodenum), L132 (lung),
U87MG (glioblastoma), KB (oral), MIAPaCa2(pancreas), A549(non small cell lung)
and HT-29(colon). The tumor cells were collected at exponential growth phase
and
resuspended in medium (1.5 x 106 cells/ml in RPMI 1640 containing 10% FBS).
1501 of medium was added to the wells of a 96-well tissue culture plate (Nunc,
Derunark) followed by 30p,1 of cell suspension. The plate was left in
incubator
(37°C, 5% COZ) overnight. 20p,1 of the peptide (100 pM to luM
concentration) was
added to marked wells of the 96-well plate. Each concentration was plated in
triplicates. 201 of medium alone was added to control wells while wells
without
cells served as blanks. A total volume of 2001 was ensured in each well and
plate
was left in incubator (37°C, 5% COZ). After 72 hours of incubation an
MTT assay
was performed and percentage cytotoxicity was calculated with respect to
control
cells. Following Tables show the maximum cytotoxicity achieved on various cell
lines.
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
-17-
His-Ser-Asp-Aib -Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-
Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-
NHZ (SEQ ID NO: 2)
Cell Lme Percentage
cytotoxicity
at different
concentrations
1pM 100nM IOnM 1nM 100pM
PA1 16.53.4 18.94.2 28.95.5 30.06.7 163.3
SW620 18.55.1 233.8 304.5 286.6 16.94.5
HuTu80 394.5 245.6 184.5 205.5 103.5
L132 15.97.5 18.95.0 30.97.0 284.5 18 2.3
U87MG 144.5 19.07.0 28.95.6 127.6 10.54.5
KB 430.5 375.0 346.0 428.0 478.5
MIAPaCa2 36 0.5 32 4.5 35 3.5 31 5.0 20 6.5
A549 45 5.5 416.0 215.5 19 4.5 165.5
HT29 385.5 305.8 254.5 254.5 265.5
His-Ser-Asp-Deg-Val-4-C1-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-
Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-
NHZ (SEQ ID N0:3)
Cell Line Percentage
cytotoxicity
at different
concentrations
1 pM 1 OOnM 1 OnM 1 nm 1 OOpM
PAl 22.14.5 23.55.2 22 4.5 295.8 154.5
SW620 163.4 227.3 275.5 295.6 15.96.6
HuTu80 14.57.1 247.8 284.7 296.2 147.8
L132 146.5 266.5 296.7 233.5 145.6
U87MG 254.6 266.7 287.5 166.6 117.8
KB 143.4 188.5 228.2 24.5.9.5 138.5
WO Ol/f)0862 CA 02405689 2002-10-08 PCT/US00/20871
-18-
His-Ser-Asp-AcSc-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-
Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-
NHZ (SEQ ID N0:4);
Cell Line Percentage
cytotoxicity
at different
concentrations
1~M lOOnM IOnM 1nm IOOpM
PA1 21S.5 22:34.5 233.5 306.0 16.10.0
SW620 154.5 236.7 28.54.5 316.5 16.95.5
HuTu80 15.56.5 277.9 275.0 307.0 118.0
L132 134.5 285.5 305.5 274.5 118.0
U87MG 275.5 287.7 296.7 157.8 104.6
KB 274.3 265.6 277.8 307.8 138.0
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-NHz (SEQ
ID N0:9);
Cell Lme Percentage
cytotoxicity
at different
concentrations
1 pM 1 OOnM 1 OnM 1 nM 1 OOpM
PA1 15.12.3 17.43.2 25.6 4.5 27.84.3 15.53.3
SW620 16.74.1 213.6 273.4 285.0 15.94.5
HuTu80 14.65.1 236.0 265.5 28.53.2 14.62.7
L132 13.65.6 17.55.5 18.96.0 200.0 172.3
U87MG 12.56.5 18.96.5 26.73.5 13.73.6 11.55.0
KB 10.56.5 16.75.1 16.73.2 21.54.5 135.0
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
-19-
His-Ser-Asp-Aib-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-
Arg-Lys-Gln-Dpg-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-
NHZ (SEQ ID NO:10)
Cell Lme Percentage
cytotoxicity
at different
concentrations
1~M 100nM lOnM lnm 100pM
PA1 236.5 255.0 263.0 316.0 280.0
SW620 165.5 24 7.3 295.0 306.5 174.0
HuTu80 164.5 288.0 285.0 317.0 157.8
L132 175.0 295.5 316.0 286.0 157.0
U87MG 294.0 297.0 30.14.0 167.8 125.0
KB 305.0 276.0 267.8 32.7.8 12 8.0
All publications referenced are incorporated by reference herein,
including the nucleic acid sequences and amino acid sequences listed in each
publication. All the compounds and methods disclosed and referred to in the
publications mentioned above are incorporated by reference herein, including
those
compounds disclosed and referred to in articles cited by the publications
mentioned
above.
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
SEQUENCE LISTING
<110> BURMAN C, ANAND
PRASAD, SUDHANAND
MUKHERJEE, RAMA
SINGH T, ANU
MATHUR, ARCHNA
GUPTA, NEENA
<120> VASOACTIVE INTESTINAL PEPTIDES ANALOGS
<130> 128002
<140>
<141>
<150> 136/DEL/2000
<151> 2000-02-18
<160> 12
<170> PatentIn Ver. 2.0
<210> 1
<211> 28
<212> PRT
<213> Sus barbatus
<400> 1
His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 2
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
<222> (4)
1
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
<223> /product = alpha-aminoisobutyric acid/label = Aib
<220>
<221> MOD_RES
<222> (6)
<223> /product =9-chloro-D-phenylalanine/label =
4-C1-D-Phe
<400> 2
His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Leu Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 3
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
<222> (4)
<223> /product = di-ethyl glycine/label = Deg
<220>
<221> MOD_RES
<222> (6)
<223> /product = 4-chloro-D-phenylalanine/label =
9-C1-D-Phe
<400> 3
His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Leu Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 4
<211> 28
<212> PRT
<213> Artificial Sequence
2
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
<222> (4)
<223> /product = 1-Aminocyclopentane carboxylic
acid/label = AcSc
<220>
<221> MOD_RES
<222> (6)
<223> /product = 4-chloro-D-phenylalanine/label =
4-C1-D-Phe
<400> 4
His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Leu Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 5
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD RES
<222> (4)
<223> /product = alpha-aminoisobutyric acid/label =Aib
<400> 5
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 6
3
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
<222> (4)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 6
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Leu Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 7
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD RES
<222> (4)
<223> /product = 1-Aminocyclopentane carboxylic
acid/label = AcSc
<400> 7
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Leu Ala Val Lys Lys Tyr Leu Arg Ser Ile Leu Asn
20 25
<210> 8
<211> 28
<212> PRT
<213> Artificial Sequence
4
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
<220>
<223> Description of Artific_al Sequence: This peptide
was synthetically generated.
<220>
<221> MOD RES
<222> (4)
<223> /product = di-ethyl glycine/label = Deg
<400> 8
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Leu Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 9
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
<222> (4)
<223> /product = alpha-aminoisobutyric acid/label =Aib
<220>
<221> MOD RES
<222> (17)
<223> /product = di-n-propylglycine/label = Dpg
<400> 9
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Xaa Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 10
<211> 28
<212> PRT
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD RES
<222> (4)
<223> /product = alpha-aminoisobutyric acid/label =Aib
<220>
<221> MOD_RES
<222> (6)
<223> /product = 4-chloro-D-phenylalanine/label =
9-C1-D-Phe
<220>
<221> MOD RES
<222> (17)
<223> /product = di-n-propyglycine/label = Dpg
<400> 10
His Ser Asp Xaa Val Xaa Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Xaa Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 11
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD RES
<222> (4)
<223> /product = di-ethylglycine/label = Deg
<220>
<221> MOD_RES
<222> (17)
<223> /product = di-n-propylglycine/label = Dpg
6
WO 01/60862 CA 02405689 2002-10-08 PCT/US00/20871
<400> 11
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Xaa Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
<210> 12
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated
<220>
<221> MOD RES
<222> (4)
<223> /product = 1-Aminocyclopentane carboxylic
acid/label = AcSc
<220>
<221> MOD_RES
<222> (17)
<223> /product = di-n-propylglycine/label = Dpg
<400> 12
His Ser Asp Xaa Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln
1 5 10 15
Xaa Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn
20 25
7
