Note: Descriptions are shown in the official language in which they were submitted.
1
13-BG-D-23b
Antihert~es Pentaueptide Derivatives Having a
Cycloalkyl Substituted Asyartic Acid Side Chain
Field of the Invention
This invention relates to peptide derivatives having antiviral
properties and to means for using the derivatives to treat viral
infections. More specifically, the invention relates to peptide
derivatives (hereinafter called "peptides") exhibiting activity against
herpes viruses, to pharmaceutical compositions comprising the pep-
tides, and to a method of using the peptides to treat herpes infec-
tions.
Background of the Invention
The family of herpes viruses is responsible for a wide range
of infections that afflict humans and many important domestic ani-
mals. The diseases caused by these viruses range from bothersome
cold sores to highly destructive infections of the central nervous
system (encephalitis). The more common members of this family
include herpes simplex virus (types 1 and 2) responsible for cold
sores and genital lesions; varicella zoster virus which causes chicken
pox and shingles; and Epstein-Barr virus which causes infectious
mononucleosis. Although some significant advances have been made
in the last decade in antiviral therapy, the need for effective, safe
therapeutic agents for treating herpes viral infections continues to
exist. For a recent review of current therapeutic agents in this area,
see M.C. Nahata, "Antiviral Drugs: Pharmacokinetics, Adverse Effects
and Therapeutic Use", J. Pharm. Technol., 3, 100 (1987).
The present application discloses a group of peptide
derivatives having activity against herpes viruses. The relatively
selective action of these peptides against herpes viruses, combined
with a wide margin of safety, renders the peptides as desirable agents
for combating herpes infections.
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2
The association of peptides with anti-herpes activity is
uncommon. Instances of reports of such an association include B.M.
Dutia et al., Nature, 321, 439 (1986), E.A. Cohen et al., Nature, 321,
441 (1986), J.H. Subak-Sharpe et al., UK patent application 2185024,
published July 8, 1987, E.A. Cohen et al., European patent
application 246630, published November 25, 1987, R. Freidinger et
al., European patent application 292255, published November 23,
1988, and R. Freidinger et al., U.S, patent 4,814,432, issued March
21, 1989. The subject peptides of the previous reports can be distin-
guished from the peptides of the present application by characteristic
structural and biological differences.
Summar3~ of the Invention:
The peptides of this invention are represented by formula 1
XNR'-CH(RZ)-C(W')-NH-CR3(R°)-C(Wi)-NRS-CH[CHxC(O)-Y]-C(VV~)-
NH-CR6-[CR'(Ra)-COOH]-C(W4)-NH-CR9(R'°)-Z 1
wherein X is (1-lOC)alkanoyl, (1-lOC)alkoxycarbonyl, benzoyl,
benzoyl monosubstituted or disubstituted with a substituent selected
from halo, hydroxy, lower alkyl, lower alkoxy, phenyl, 2-carboxy-
phenyl or benzyl, 2,2-diphenylacetyl, phenyl(2-lOC)alkanoyl or
phenyl(2-lOC)alkanoyl monosubstituted or disubstituted on the aro-
matic portion thereof with a substituent selected from halo, hydroxy,
lower alkyl, lower alkoxy or phenyl;
R' is hydrogen, lower alkyl or phenyl(lower)alkyl;
RZ is lower alkyl, hydroxy(lower)alkyl or mercapto(lower)alkyl;
R', R5, R6 and R9 each independently is hydrogen or lower alkyl;
R4 is hydrogen, lower alkyl, hydroxy(lower)alkyl,
mercapto(lower)alkyl, methoxy(lower)alkyl, methylthio(lower)alkyl,
lower cycloalkyl or (lower cycloalkyl)methyl;
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3
R' and Ra together with the carbon atom to which they are attached
form a lower cycloalkyl;
R'° is lower alkyl, lower alkenyl or (lower cycloalkyl)-(lower
alkyl);
W', WZ, W3 and W' each independently is oxo or thioxo;
Y is
a. (1-14C)alkoxy, (3-14C)alkenyloxy, CH3(OCHZCH~a O wherein
n is the integer 1, 2 or 3, lower cycloalkyloxy, lower alkoxy
monosubstituted with a lower cycloalkyl, phenoxy, phenoxy
monosubstituted with hydroxy, halo, lower alkyl or lower
alkoxy, phenyl(lower)alkoxy or phenyl(lower)alkoxy in which
the aromatic portion thereof is substituted with hydroxy, halo,
lower alkyl or lower alkoxy, or
b. NR"R'z wherein R" is lower alkyl and R'2 is lower alkoxy,
or
c. NR"R'2 wherein R" is hydrogen or lower alkyl and R'Z is (1-
14C)alkyl, lower cycloalkyl, lower alkyl monosubstituted with
a lower cycloalkyl; phenyl, phenyl monosubstituted with halo,
lower alkyl or lower alkoxy; phenyl(lower)alkyl,
phenyl(lower)alkyl in which the aromatic portion thereof is
substituted with halo, lower alkyl or lower alkoxy; or (Het)-
lower alkyl wherein Het represents a five or six membered
heterocyclic radical containing one or two heteroatoms selected
from nitrogen, oxygen or sulfur, or
d. NR"R'Z wherein R" and R'2 together with the nitrogen to
which they are attached form a pyrrolidino, piperidino,
morpholino, thiomorpholino, piperazino or 4-(lower alkyl)
piperazino; and
Z is hydrogen; COOH; CHZCOOH; CHZOH; 5-1H-tetrazolyl; COOR'3
wherein R" is lower alkyl; CONR'4R's wherein R'4 and R'S each
independently is hydrogen or lower alkyl; or CON(R'6)OH wherein
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4
R'6 is hydrogen or lower alkyl; or a therapeutically acceptable salt
thereof.
A preferred group of the peptides of this invention is
represented by formula 1 wherein X is (1-lOC)alkanoyl; (1-
lOC)alkoxycarbonyl; benzoyl; benzoyl monosubstituted with halo,
hydroxy, lower alkyl, lower alkoxy, phenyl, 2-carboxyphenyl or
benzyl; phenyl(2-lOC)alkanoyl or phenyl(2-lOC)alkanoyl monosub-
stituted or disubstituted on the aromatic portion thereof with a
substituent selected from halo, hydroxy, lower alkyl, lower alkoxy or
phenyl; R' to R'°, inclusive, and W' to W', inclusive, are as defined
hereinabove; Y is (1-14C)alkoxy, (3-14C)alkenyloxy, CH3(OCHZ
CH2)3 O, lower cycloalkyloxy, lower cycloalkylmethoxy, phenyl-
(lower)alkoxy, NRl'R~z wherein R" is lower alkyl and R'Z is lower
alkoxy, or NR11R12 wherein R'1 is hydrogen or lower alkyl and R'Z is
(1-14C)alkyl, lower cycloalkyl, lower cycloalkylmethyl, phenyl,
phenyl monosubstituted with halo, lower alkyl or lower alkoxy,
phenyl(lower)alkyl, phenyl(lower)alkyl monosubstituted with halo,
lower alkyl or lower alkoxy, (Het)-lower alkyl wherein Het is a
heterocyclic radical selected from 2-pyrrolyl, 2-pyridinyl, 4-pyridinyl,
2-furyl, 2-isoxazolyl and 2-thiazolyl, or NR'IR'Z wherein R" and R'2
together with the nitrogen atom to which they are attached form a
pyrrolidino, piperidino or morpholino; and Z is as defined
hereinabove; or a therapeutically acceptable salt thereof.
A more preferred group of the peptides is represented by
formula 1 wherein X, R', R8 and R'° are as defined hereinabove; Rl
is lower alkyl; RZ is lower alkyl or hydroxy(lower)alkyl; R3, R5, R6
and R9 each independently is hydrogen or methyl; R4 is hydrogen,
lower alkyl, hydroxy(lower)-alkyl, methoxy(lower)alkyl, lower
cycloalkyl or (lowercyclo-alkyl)methyl; Wl, WZ and W3 are oxo, W'
is oxo or thioxo, Y is (1-14C)alkoxy, (3-14C)alkenyloxy,
CH3(OCH2CH~3 O, lower cycloalkyloxy, lower cycloalkylmethoxy,
phenyl(lower)alkoxy, N(Me)OMe, NR"R'2 wherein R" is hydrogen or
201000
s
lower alkyl and R'2 is (1-14C)alkyl, lower cycloalkyl, lower
cycloalkylmethyl, phenyl, phenyl(lower)alkyl or pyridinyl(lower alkyl),
or NRllRlz wherein R" and R'Z together with the nitrogen to which
they are attached form a pyrrolidino, piperidino or morpholino; and
s Z is hydrogen, COOH, CHZCOOH, s-1H-tetrazolyl, CHZOH,
CONR'4Rls wherein R'4 and R's each independently is hydrogen or
lower alkyl, or CON(R'6)OH wherein R'6 is hydrogen or lower alkyl;
or a therapeutically acceptable salt thereof.
A most preferred group of the peptides is represented by
formula 1 wherein X is acetyl, 4-methylpentanoyl, octanoyl, Boc,
benzoyl, 2-biphenylylcarbonyl, 2-(2'-carboxy)biphenylylcarbonyl,
phenylacetyl, phenylpropionyl, (4-hydroxyphenyl)propionyl or (3,4-
dihydroxyphenyl)propionyl; R' is methyl; RZ is 1-methylethyl, 1-
methylpropyl, 1,1-dimethylethyl or 1-hydroxyethyl; R3 is hydrogen or
is methyl; R' is hydrogen, lower alkyl, hydroxymethyl, 1-hydroxyethyl,
1- methoxyethyl, cyclopentyl or cyclohexylmethyl; Rs is hydrogen
or methyl; R6 is hydrogen; R' and R8 together with the carbon atom
to which they are attached form a lower cycloalkyl; R9 is hydrogen
or methyl; R'° is 1-methylpropyl, 2-methylpropyl, 3-methylbutyl, 2,2-
dimethylpropyl or 2-cyclohexylethyl; W', VV~ and W3 are oxo; W4
is oxo or thioxo; Y is hexyloxy, 1-methylheptyloxy, decyloxy,
dodecyloxy, trans-3-heptenyloxy, cis-3-octenyloxy, CH3(OCHZCHZ)3
O, cyclopentyloxy, cyclohexyloxy, cyclohexylmethoxy, phenylpropoxy,
N(Me~Me, ethylamino, phenylamino, phenylethylamino, N-methyl-
2s N-phenylethylamino, 2-pyridinylethyl, N,N-dimethylamino, N,N-
diethylamino, N,N-diisopropylamino, N-methyl-N-octylamino, pyrroli-
dino, piperidino or morpholino; and Z is hydrogen, COOH,
CHZCOOH, s-1H-tetrazolyl, CHZOH, CONRI4Rls wherein Rl° and Rls
each independently is hydrogen, methyl, ethyl or propyl, or
CON(R'6)OH wherein Rlb is hydrogen or methyl; or a therapeutically
acceptable salt thereof.
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6
Included within the scope of this invention is a pharma-
ceutical composition comprising an anti-herpes virally effective
amount of a peptide of formula 1, or a therapeutically acceptable salt
thereof, and a pharmaceutically or veterinarily acceptable carrier.
Also included within the scope of this invention is a cosmetic
composition comprising a peptide of formula 1, or a therapeutically
acceptable salt thereof, and a physiologically acceptable carrier
suitable for topical application.
An important aspect of the invention involves a method of
treating a herpes viral infection in a mammal by administering to
the mammal an anti-herpes virally effective amount of the peptide of
formula 1, of a therapeutically acceptable salt thereof.
Another important aspect involves a method of inhibiting the
replication of herpes virus by contacting the virus with a herpes viral
ribonucleotide reductase inhibiting amount of the peptide of formula
1, or a therapeutically acceptable salt thereof.
Processes for preparing the peptides of formula 1 are
described hereinafter.
~01900~
Details of the Invention
GENERAL
Alternatively, formula 1 can be illustrated as:
COY
Rj Wi
qty R 9 R ~o
X ~ ~ :YH
j.~i
Z
R~ ~ R ~ Rs R 7 ~ Rs
i
R
R ~ COOH
The term 'residue' with reference to an amino acid or amino
acid derivative means a radical derived from the corresponding a-
amino acid by eliminating the hydroxyl of the carboxy group and one
hydrogen of the a-amino group.
In general, the abbreviations used herein for designating the
amino acids and the protective groups are based on recommenda-
tions of the IUPAC-IUB Commision of Biochemical Nomenclature,
see European Journal of Biochemistry 138, 9 ( 1984). For instance,
Gly, Val, Thr, Ala, Ile, Asp, Ser and Leu, represent the residues of
glycine, L-valine, L-threonine, L-alanine, L-isoleucine, L-aspartic acid,
L-serine and L-leucine, respectively.
The asymmetric carbon atoms residing in the principal linear
axis (i.e. the backbone) of the peptides of formula 1, exclusive of the
terminal groups, have an S configuration. Asymmetric carbon atoms
residing in the side chain of an amino acid or derived amino acid
residue, including those in terminal groups, may also have the R
configuration. Furthermore, with respect to disubstituted benzoyl and
disubstitued phenyl(1-lOC)allcanoyl as defined for X of peptides of
formula 1, the substituents are selected on the basis that they do not
interfere with each others presence.
The term 'halo' as used herein means a halo radical selected
from bromo, chloro, fluoro or iodo.
20.9005
s
The term "lower alkyl" as used herein, either alone or in
combination with a radical, means straight chain alkyl radicals
containing one to six carbon atoms and branched chain alkyl radicals
containing three to six carbon atoms and includes methyl, ethyl,
propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl
and 1,1-dimethylethyl.
The term "lower alkenyl" as used herein means straight chain
alkenyl radicals containing two to six carbon atoms and branched
chain alkenyl radicals containing three to six carbon atoms and
includes vinyl, 1-propenyl, 1-methylethenyl, 2-methyl-1-propenyl, 2-
methyl-2-propenyl and 2-butenyl.
The term "lower cycloalkyl" as used herein, either alone or in
combination with a radical, means saturated cyclic hydrocarbon
radicals containing from three to six carbon atoms and includes
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term "lower alkoxy" as used herein means straight chain
alkoxy radicals containing one to four carbon atoms and branched
chain alkoxy radicals containing three to four carbon atoms and
includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-
dimethylethoxy. The latter radical is known commonly as tertiary-
butyloxy.
The term "(1-14C)alkyl" as used herein means straight and
branched chain alkyl radicals containing from one to fourteen carbon
atoms, respectively. The terms "(1-lOC)alkoxy" and "(1-14C)alkoxy"
as used herein, either alone or in combination with a radical, mean
straight and branched chain alkoxy radicals containing from one to
ten carbon atoms and one to fourteen carbon atoms, respectively.
The term "(3-14C)alkenyloxy" means straight and branched chain
alkenyloxy radicals containing from three to fourteen carbon atoms
in which the double bond may be cis or trans and is positioned more
~01900~
9
than one carbon atom away from the oxygen atom of the radical; for
example, 3-heptenyloxy and 3-octenyloxy. The term "(1-
lOC)alkanoyl" as used herein means a straight or branched chain 1-
oxoalkyl radical containing from one to ten carbon atoms; for
example, acetyl, 4-methyl-1-oxopentyl (or 4-methylpentanoyl) or 1-
oxooctyl (or octanoyl). The term "phenyl(2-10)alkanoyl as used
herein means phenyl substituted 1-oxoalkyl radicals wherein the 1-
oxoalkyl portion thereof is a straight or branched chain 1-oxoalkyl
containing from two to ten carbon atoms; for example, 1-oxo-3-
phenylpropyl and 1-oxo-S-methyl-6-phenylhexyl.
Additional abbreviations or symbols used hereafter are:
Boc 1,1-dimethylethoxycarbonyl or tertiary
butyloxycarbonyl
DAT desaminotyrosyl or 1-oxo-3-(4-hydroxy-
phenyl)propyl
Ph phenyl
PhCH2CHZC0 1-oxo-3-phenylpropyl
N-Me-Val N-methylvalyl residue
Tbg 2(S)-amino-3,3-dimethylbutanoic acid
residue
Tba 2(S)-amino-4,4-dimethylpentanoic acid
residue
N-Me-Tbg 2(S)-methylamino-3,3-dimethylbutanoic
acid residue
NMe(octyl) N-methyl-N-octylamino
NMe(decyl) N-methyl-N-decylamino
Asp(cyPr) (S)-a-amino-1-carboxycyclopropaneacetic
acid residue
Asp(cyBu) (S)-a-amino-1-carboxycyclobutaneacetic
acid residue
Asp(cyPn) (S)-a-amino- 1-carboxycyclopentaneacetic
acid residue
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to
Asp(cyHx) (S)-a-amino-1-carboxycyclohexaneacetic
acid residue
The symbol "y[CSNH]" used between the three letter
representations of two amino acid residues means that the normal
amide bond between those residues in the peptide, being represented,
has been replaced with a thioamide bond.
The term "pharmaceutically acceptable Garner" or "veterinarily
acceptable carrier" as use herein means a non-toxic, generally inert
vehicle for the active ingredient which does not adversely affect the
ingredient.
The term "physiologically acceptable carrier" as used herein
means an acceptable cosmetic vehicle of one or more non-toxic
excipients which do not react with or reduce the effectiveness of the
active ingredient contained therein.
The term "veterinarily acceptable carrier" as used herein
means a physiologically acceptable vehicle for administering drug
substances to domestic animals comprising one or more non-toxic
pharmaceutically acceptable excipients which do not react with the
drug substance or reduce its effectiveness.
The term "effective amount" means a predetermined antiviral
amount of the antiviral agent, i.e. an amount of the agent sufficient
to be effective against the viral organisms in vivo.
The term "coupling agent" as used herein means an agent
capable of effecting the dehydrative coupling of an amino acid or
peptide free carboxy group with a free amino group of another amino
acid or peptide to form an amide bond between the reactants.
Similarly, such agents can effect the coupling of an acid and an
alcohol to form corresponding esters. The agents promote or
X019005
11
facilitate the dehydrative coupling by activating the carboxy group.
Descriptions of such coupling agents and activated groups are
included in general text books of peptide chemistry; for instance, E.
Schroder and K.L. Lubke, "The Peptides", Vol. 1, Academic Press,
New York, N.Y., 1965, pp 2-128, and K.D. Kopple, "Peptides and
Amino acids", W.A. Benjamin, Inc., New York, N.Y., 1966, pp 33-
51. Examples of coupling agents are thionyl chloride,
diphenylphosphoryl azide, 1,1'-carbonyldiimidazole,
dicyclohexylcarbodiimide, N-hydroxysuccinimide, or 1-hydroxy-
benzotriazole in the presence of dicyclohexylcarbodiimide. A very
practical and useful coupling agent is (benzotriazol-1-yloxy)tris
(dimethylamino)-phosphonium hexafluorophosphate, described by B.
Castro et al., Tetrahedron Letters, 1219 (1975), see also D. Hudson,
J. Org. Chem., 53, 617 ( 1988), either by itself or in the presence of
1-hydroxybenzotriazole.
Process
The peptides of formula 1 can be prepared by processes which
incorporate therein methods commonly used in peptide synthesis such
as classical solution coupling of amino acid residues and/or peptide
fragments, and if desired solid phase techniques. Such methods are
described, for example, by E. Schrbder and K. Liibke, cited above,
in the textbook series, "The Peptides: Analysis, Synthesis, Biology",
E. Gross et al., Eds., Academic Press, New York, N.Y., 1979-1987,
Volumes 1 to 8, and by J.M. Stewart and J.D. Young in "Solid
Phase Peptide Synthesis", 2nd ed., Pierce Chem. Co., Rockford, IL,
USA, 1984.
A common feature of the aforementioned processes for the
peptides is the protection of the reactive side chain groups of the
various amino acid residues or derived amino acid residues with
suitable protective groups which will prevent a chemical reaction
from occurring at that site until the protective group is ultimately
20i900~
12
removed. Usually also common is the protection of an a-amino
group on an amino acid or a fragment while that entity reacts at the
carboxy group, followed by the selective removal of the a-amino
protective group to allow subsequent reaction to take place at that
location. Usually another common feature is the initial protection of
the C-terminal carboxyl of the amino acid residue or peptide
fragment, if present, which is to become the C-terminal function of
the peptide, with a suitable protective group which will prevent a
chemical reaction from occurring at that site until the protective
group is removed after the desired sequence of the peptide has been
assembled.
In general, therefore, a peptide of formula 1 can be prepared
by the stepwise coupling in the order of the sequence of the peptide
of the amino acid or derived amino acid residues, or fragments of the
peptide, which if required are suitably protected, and eliminating all
protecting groups, if present, at the completion of the stepwise
coupling to obtain the peptide of formula 1. More specific processes
are illustrated in the examples hereinafter.
With reference to the preparation of peptides of formula 1 in
which Z is 5-1H-tetrazolyl, the derived amino acid residue containing
the tetrazole can be prepared as follows: Boc-Leu-NHZ, for example,
was converted to its corresponding nitrite derivative by treatment with
p-toluenesulfonyl chloride in methylenedichloride in the presence of
excess pyridine and a catalytic amount of 4-dimethylaminopyridine
(Fieser and Fieser, "Reagents for Organic Synthesis", John Wiley and
Sons, Inc., New York, NY, USA, 1967, vol 1, p 1183). The nitrite
derivative then was mixed with tributyl tin azide, J.G.A. Luijten et
al., Rec. Trav., 81, 202 (1962), giving a tetrazole tin intermediate [cf.
K. Sisido et al., Journal of Organometallic Chemistry, 33, 337 (1971)
and J. Dubois et al., J. Med. Chem., 27, 1230 (1984)]. The latter
was treated with hydrogen chloride in diethyl ether to afford the
desired tetrazole residue as a hydrochloride salt, for example,
zo~~oo~
13
NHZCH[CHZCH(CH3)Z]-5-1H-tetrazole dihydrochloride. The tetrazole
residue, or its hydrochloride salt, was used for coupling with the
appropriate amino acid or protected fragment, leading to the desired
peptide of formula 1.
The peptide of formula 1 of this invention can be obtained in
the form of a therapeutically acceptable salt. In the instance where
a particular peptide has a residue which functions as a base,
examples of such salts are those with organic acids, e.g. acetic, lactic,
succinic, benzoic, salicylic, methanesulfonic or p-toluenesulfonic acid,
as well as polymeric acids such as tannic acid or carboxymethyl
cellulose, and also salts with inorganic acids such as hydrohalic acids,
e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid. If
desired, a particular acid addition salt is converted into another acid
addition salt, such as a non-toxic, pharmaceutically acceptable salt,
by treatment with the appropriate ion exchange resin in the manner
described by R.A. Boissonnas et al., Helv. Chim. Acta, 43, 1849
(1960).
In the instance where a particular peptide has one or more
free carboxy groups, examples of such salts are those with the
sodium, potassium or calcium cations, or with strong organic bases,
for example, triethylamine or N-methylmorpholine.
Antiheroes Activity
The antiviral activity of the peptides of formula 1 can be
demonstrated by biochemical, microbiological and biological
procedures showing the inhibitory effect of the compounds on the
replication of herpes simplex viruses, types 1 and 2 (HSV-1 and
HSV-2), and other herpes viruses, for example, varicella zoster virus
(VZV), Epstein-Barr virus (EBV), equine herpes virus (EHV) and
cytomegalovirus.
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14
Noteworthy is the fact that all of the aforementioned viruses
are dependent on their own ribonucleotide reductase to synthesize
deoxyribonucleotides for their replication. Although this fact may not
be directly linked with the antiviral activity found for the present
peptides, the latter compounds have been shown so far to
have antiviral properties against all viruses dependent on ribonucle-
otide reductase to synthesis DNA for their replication.
In the examples hereinafter, the inhibitory effect on herpes
ribonucleotide reductase is noted for exemplary peptides of formula
1. Noteworthy, in the connection with this specific inhibition of
herpes ribonucleotide reductase, is the relatively minimal effect or
absence of such an effect of the peptides on cellular ribonucleotide
reductase activity required for normal cell replication.
A method for demonstrating the inhibitory effect of the
peptides of formula 1 on viral replication is the cell culture
technique; see, for example, T. Spector et al., 1'roc. Natl. Acad. Sci.
USA, 82, 4254 (1985).
The therapeutic effect of the peptides can be demonstrated in
laboratory animals, for example, by using an assay based on genital
herpes infection in Swiss Webster mice, described by E.R. Kern, et
al., Antiviral Research, 3, 253 (1983).
When a peptide of this invention, or one of its therapeutically
acceptable salts, is employed as an antiviral agent, it is administered
topically or systemically to warm-blooded animals, e.g. humans, pigs
or horses, in a vehicle comprising one or more pharmaceutically
acceptable carriers, the proportion of which is determined by the
solubility and chemical nature of the peptide, chosen route of
administration and standard biological practice. For topical adminis-
tration, the peptide can be formulated in pharmaceutically accepted
vehicles containing 0.1 to 10 percent, preferably 0.5 to 5 percent, of
209005
the active agent. Such formulations can be in the form of a solution,
cream or lotion.
For systemic administration, the peptide of formula 1 is
administered by either intravenous, subcutaneous or intramuscular
5 injection, in compositions with pharmaceutically acceptable vehicles
or carriers. For administration by injection, it is preferred to use the
peptide in solution in a sterile aqueous vehicle which may also
contain other solutes such as buffers or preservatives as well as
sufficient quantities of pharmaceutically acceptable salts or of glucose
10 to make the solution isotonic.
Suitable vehicles or carriers for the above noted formulations
are described in standard pharmaceutical texts, e.g. in "Remington's
Pharmaceutical Sciences", 16th ed, Mack Publishing Company,
Easton, Penn., 1980.
15 The dosage of the peptide will vary with the form of
administration and the particular active agent chosen. Furthermore,
it will vary with the particular host under treatment. Generally, treat-
ment is initiated with small increments until the optimum effect under
the circumstances is reached. In general, the peptide is most desi-
rably administered at a concentration level that will generally afford
antivirally effective results without causing any harmful or deleterious
side effects.
With reference to topical application, the peptide is
administered cutaneously in a suitable topical formulation to the
infected area of the body e.g. the skin or part of the oral or genital
cavity, in an amount sufficient to cover 1 the infected area. The
treatment should be repeated, for example, every four to six hours
until lesions heal.
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16
With reference to systemic administration, the peptide of
formula 1 is administered at a dosage of 10 mcg to 1000 mcg per
kilogram of body weight per day, although the aforementioned varia-
tions will occur. However, a dosage level that is in the range of
from about 50 mcg to 500 mcg per kilogram of body weight per day
is most desirably employed in order to achieve effective results.
Another aspect of this invention comprises a cosmetic
composition comprising a herpes viral prophylactic amount of the
peptide of formula 1, or a therapeutically acceptable salt thereof,
together with a physiologically acceptable cosmetic carrier. Addi-
tional components, for example, skin softeners, may be included in
the formulation. The cosmetic formulation of this invention is used
prophylactically to prevent the outbreak of herpetic lesions of the
skin. The formulation can be applied nightly to susceptible areas of
the skin. Generally, the cosmetic composition contains less of the
peptide than corresponding pharmaceutical compositions for topical
application. A preferred range of the amount of the peptide in the
cosmetic composition is 0.01 to 0.2 percent by weight.
Although the formulation disclosed hereinabove are indicated
to be effective and relatively safe medications for treating herpes
viral infections, the possible concurrent administration of these
formulations with other antiviral medications or agents to obtain
beneficial results is not excluded. Such other antiviral medications
or agents include acyclovir and antiviral surface active agents or
antiviral interferons such as those disclosed by S.S. Asculai and F.
Rapp in U.S. patent 4,507,281, March 26, 1985.
The following examples illustrate further this invention.
Solution percentages or ratios express volume to volume relationship,
unless stated otherwise. Abbreviations used in the examples include
Ac: acetyl; Boc: t-butyloxycarbonyl; BOP: (benzotriazol-1-yloxy)tris-
(dimethylamino)-phosphonium hexafluorophosphate; Bzl: benzyl;
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17
CHZC12: methylenedichloride; DIPEA: diisopropylethylamine; DCC:
N,N-dicyclohexylcarbodiimide; DMF: dimethyl formamide; Et~O:
diethyl ether; EtOH: ethanol; HOBt: 1-hydroxybenzotriazole; HPLC:
high performance liquid chromatography; i-Pr: 1-methylethyl; MeOH:
methanol; NMM: N-methylmorpholine; TFA: trifluoroacetic acid;
THF: tetrahydrofuran. Temperatures are given in degrees centrigrade.
Example 1
Preparation of the Intermediate BOC-Aspf 1(S)-methylheytyloxyl-OH
A solution of Boc-Asp-OBzI (10.2 g, 31.6 mmol) in
acetonitrile was added at 0 ° to a mixture of N,N'-carbonyldiimi-
dazole (5.6 g, 34.7 mmol), DIPEA (8 ml, 46 mmol) and 2(S)-
octanol (6 ml, 37.9 mmol) and 4-dimethylaminopyridine (200 mg).
The mixture was stirred for 3 h and then concentrated to dryness.
The residue was dissolved in EtOAc. The solution was washed with
1N aqueous HC1, 1N aqueous NaHC03, dried (MgSO,) and
concentrated. The resultant oil was purified by chromatography
(Si02, eluent: hexane-EtOAc, 7:3) to give Boc-Asp[1(S)-methyl-
heptyloxy]-OBzI. Hydrogenation of the latter compound in the
presence of 20% Pd(OH)~/C in ethanol solution afforded the title
compound as a solid. NMR(200 MHz, CDC13)S 0.9(m,3H),
1.25(m,lOH), 1.45(s,9H), 2.8(dd,lH), 3.0(dd,lH), 4.6(m,lH),
4.95(m,lH) and 5.55(d,lH).
Analogous esters of Boc-Asp-OH were prepared in the same
manner.
Example 2
Preparation of the Intermediate Boc-Asp(NEt,)-OH
BOP (2.20 g, 5.0 mmol) was added under N2 to a cooled (0 °)
solution of Boc-Asp-OBzI (1.90 g, 4.6 mmol) in CH2C12 (50 ml).
1s 2019005
After 3 min NHEt~.HCI (0.55 g, 5.0 mmol) and DIPEA (2.4 ml,
13.8 mmol) were added. The resultant solution was stirred at 20-
22 ° for 18 h. The solution was washed with 1096 aqueous citric
acid (2 7~, 1096 aqueous NaHCO, (2 ?n and brine (2 7~. The
organic layer was dried (MgSOa and concentrated to give an oil.
After SiO, chromatography of the oil using hexane-EtOAc (7:3) as
the eluent, Boc-Asp(NEt~-OBzI (1.55 g, 8996) was obtained as an oil.
Under a Nl atmosphere, a solution of the latter compound (1.55 g,
4.09 mmol) in MeOH (100 ml) was mixed with 596 PdlC (155 mg).
The mixture was shaken on a Park apparatus under Hs (50 psi) for
90 min. The mixture was filtered through a 45~m membrane and
the filtrate concentrated to give Boc-Asp(NEt~-OH (1.15g, 9896) as
an oil. The structure of the product was confirmed by NMR.
In the same manner, corresponding N-substituted asparagine
analogs were obtained by replacing NHErz.HCl with the appropriate
amine or amine salt (e.g, pynolidine or N,O-dimethylhydroxylamine
hydrochloride).
Example _3
Preparation of (S)-a-Amino-l-carboxycvcloalkvlacetic Acid
Intermediates
These intermediates, which can be used to prepare peptides of
formula 1 in which R' and R' are- joined to form a lower cycloalkyl
can be prepared according to the method of M. Bochenska and J.F.
Biernat, Rocz. Chem., 0 1195 (1976); see Chem. Abstr., 86, 43990r
( 1977).
More specifically exemplified, (t)-Boc-Asp(cyPn)(OBzI)-OH
was prepared as follows: To a solution of 1-bromocyclopentane-
carboxylic acid ethyl ester [17.1 g, 77.3 mmol, described by
D.N. Harpp et al., J. Org. Chem., 46, 3420 (1975)] and freshly
distilled ethyl isocyanoacetate (12.7 g, 122 mmol) in a mixture of
* Trade-mark
A
f. 201900
19
dimethylsulfoxide and EtzO (1:1, 120 ml) was added sodium hydride
(4.5 g, 60% dispersion in mineral oil, 122 mmol) in small portions
over 5 h. The resulting red slurry was stirred at room temperature
for 16 h after which time it was treated with a saturated aqueous
solution of ammonium chloride (5 ml). The mixture was diluted
with water (500 ml). The resulting mixture was extracted (2X) with
ethyl acetate. The ethyl acetate layers were combined and washed
with water (2X) and then with brine. Drying (MgS04), filtering and
concentration of the extract afforded a dark red oil. This material
was subjected to flash chromatography through a 5 x 25 cm column
of silica gel [eluent: EtOAc-hexane (1:10)]. Concentration of the
appropriate fractions provided a-cyano-1-carboxycyclopentaneacetic
acid diethyl ester as a clear colorless viscous liquid (13 g, 66 %).
The latter compound (13 g, 51 mmol) was mixed with 6 N
aqueous HCl (60 ml) at 0 °. After dissolution, the reaction mixture
was heated in a oil bath at 120 ° for 24 h. After this time water
was removed from the mixture using a dry ice rotory evaporator.
The resulting white solid was dried under high vacuum for 18 h.
The dried material was dissolved in a mixture of dioxane (50 ml)
and 3N aqueous NaOH (52 ml). A solution of di(tertiarybutyl)
Bicarbonate ( 14.6 g, 67 mmol) in dioxane (25 ml) was added to the
solution. The mixture was stirred at room temperature for 16 h.
Additional 3N aqueous NaOH was added at intervals insuring a pH
of about 10. The mixture was diluted with water (500 ml) and
extracted (2X) with EtzO (200 ml). The aqueous phase was rendered
acidic (pH = 3) with solid citric acid and extracted (2X) with EtOAc
(300 ml). The combined EtOAc extracts were washed with water
(3x) and brine. Drying, filtering and concentration of the extract
afforded Boc-Asp(cyPn)-OH as a white solid ( 14 g, 96%).
To a solution of the latter compound (7.2 g, 25 mmol) in dry
DMF (50 ml) was added K2CO3 (7.6 g, 55 mmol) and benzyl
bromide (6.6 ml, 55 mmol). The reaction mixture was stirred at
201900
room temperature for about 7 h. Thereafter, the reaction mixture was
poured into a mixture of water (500 ml) and ethyl acetate (350 ml).
The organic phase was washed with water (2X) and brine. Drying,
filtering and concentration of the extract provided a pale yellow
5 viscous liquid. This material was subjected to flash chromatography
through a 5 x 20 cm column of silica gel, eluting with hexane-ethyl
acetate (12:1). Concentration of the appropriate fractions provided
the dibenzyl derivative of Boc-Asp-(cyPn)-OH as a low melting white
solid (11 g, 94%). The dibenzyl product was dissolved in THF (100
10 ml) and an aqueous solution of LiOH (23.5 ml, 1N) was added.
After 4 h, the reaction mixture was poured into water and extracted
(3X) with EtzO. The aqueous phase was rendered acidic with 10%
aqueous citric acid and extracted (2X) with ethyl acetate. The ethyl
acetate layers were combined, dried (MgS04), filtered and
15 concentrated to provide Boc-Asp(cyPn)(OBzI)-OH as a clear color
less gum (7.3 g, 82%).
Example 4
Preparation of the Intermediate Boc-Asp(cyPn)(OBzI)ytfCSNHILeu-
OB zl
20 The title compound is obtained by stirring a mixture of the
protected dipeptide Boc-Asp(cyPn)(OBzI)-Leu-OBzI (5.5 mol) and
Lawesson's reagent (2.7 mmol), see U. Pederson et al., Tetrahedron,
38, 3267 (1982), in toluene at reflux temperature for 2 h; followed
by pouring the cooled reaction mixture onto a column of silica gel
(3.15 x 20 cm) and eluting the column with CH2C12.
Analogous thioamide intermediates are prepared in the same
manner by replacing Boc-Asp(cyPn)(OBzI)-Leu-OBzI with the
appropriate protected dipeptide.
_~ 2019005
21
Example 5
General Procedure for the Solid Phase Preparation of Peptides of
Formula 1
A modified version of the solid phase method of R.B.
Merrifield, J. Am. Chem. Soc., 85, 2149 ( 1963) was used to prepare
the peptides preferably using a BHA-photoresin such as [4-(2-
chloropropionyl)phenoxy]acetamidomethyl-copoly(styrene-1% divinyl-
benzene) resin, see D. Bellof and M. Mutter, Chemia, 39, 317
(1985). Protection of free carboxy groups and hydroxy groups was
provided by the Bzl protective group. Typically, a Boc-amino acid,
representing the C-terminal unit of the desired peptide, e.g. Boc-
Leu-OH, was linked to the above noted BHA-photoresin by the
potassium fluoride method of K. Horiki et al., Chem. Lett., 165
(1978), using 9 molar equivalents of KF and 3.6 molar equivalents
of Boc-Leu-OH, for example in DMF at 70 ° C for 24 hours, to give
[4- { 2-(Boc-leucyl } propionyl } phenoxy]acetamidomethyl-copoly(styrene-
1 % divinylbenzene) resin. The dried amino acid-solid support
typically showed a leucine content of 0.6 to 0.8 mmol/g for the
product, as determined by deprotection of an aliquot, followed by
picric acid titration, B.F. Gisin, Anal. Chim. Acta, 58, 248 (1972).
The latter amino acid-solid support was used to build up the required
sequence of units (i.e. amino acid residues, derived amino acid
residues) of the desired peptide by solid phase methodology. Two
molar equivalents (per mole of the amino-acid solid support) of the
appropriate amino acid residues were coupled serially to the solid
support system using BOP (2 molar equivalents), or BOP (2 molar
equivalents)/HOBt (1 molar equivalent), in the presence of N-methyl-
morpholine (6 molar equivalents) in dry DMF. Completion of
coupling was verified by a negative ninhydrin test, E. Kaiser et al.,
Anal Biochem., 34, 595 (1979). Double coupling was used when
necessary.
2o~oov~
...
22
Cleavage of the protected peptide from the solid support was
accomplished by irradiation at 330 nm in EtOH/DMF (1:4) at 0 °
under an argon atmosphere for 6 to 18 h. Protective groups (Bzl),
if present, were removed from the cleavage product by
hydrogenolysis over 5% or 10% Pd/C or 20°!o Pd(OH)~/C by standard
procedures (c~ example 1). Purification of the final product was
performed by reversed-phase HPLC to better than 95% homogeneity
using 0.06% aqueous TFA/0.06% TFA in acetonitrile gradients.
Example 6
Preparation of PhCH,CH,CO-N-Me-Val-Tb~-Asp(pyrrolidino)-
Asp(cyPn)-Leu-OH
(Example of a solution phase procedure for preparing compounds of
formula 1)
To a solution of Boc-Asp(cyPn)(OBzI)-OH (5.3 g, 14 mmol,
described in example 3) in dry CH2C12 (50 mL), BOP (6.8 g,
16 mmol), NMM (4.6 mL, 42 mmol) and the (4 methyl-
phenyl)sulfonic acid salt of LeuOBzl (6.6 g, 16 mmol) were added
successively. The reaction mixture was stirred at room temperature
for 5 h, after which time it was poured into a two component system
of EtOAc (500 mL) and a saturated aqueous solution of NaHC03
(400 mL). The organic phase was washed with water and brine.
Drying (MgS04), filtering and concentrating of the organic phase
afforded a dark yellow oil. This material was purified by flash
chromatography [Si02, eluent: hexane-EtOAc (6:1)] to provide Boc-
Asp(cyPn)(OBzI)-Leu-OBzI as a clear colorless gum (7 g, 86%,
mixture of diastereoisomers).
The latter compound (7 g, 12 mmol) was mixed with CH2C12
(4 mL). TFA (6 ml) was added to the mixture and the resulting
solution was stirred for 30 min at room temperature. Thereafter, the
majority of the solvent was evaporated and the residue was poured
201 9005
23
into a two component system of EtOAc (200 ml) and a saturated
aqueous solution of NaHCO, (400 mL). Drying (MgSOa, filtering,
and concentrating of the organic phase afforded the amine dipeptide
H-Asp(cyPn)(OBzI)-Leu-OBzI as a clear colorless gum (mixture of
diastercoisomers). The mixture was separated on a Waters LC-500
[2 columns of SiO~, eluent: hexane-EtOAc (1.5:1)]. The first
diastereomer to elute (ca 2g, white solid) proved to provide the more
active end products (peptides of formula 1). 'Ibis observation proved
general for all corresponding cycloalkyl aspartic acid derivatives
made. For convenient storage of material, the pure amine dipeptidc
was treated briefly with 6N HCl/dioxane and concentrated to afford
the hydrochloride salt as a white foam.
The latter hydrochloride salt (400 mg, 0.8 mmol) was coupled
with Boc-Asp(pyrrolidino~OH (250 mg, 0.87 mmol), following the
same general procedure used above for preparing Boc-
Asp(cyPn)(OBzI)-L,eu-0Bzl. The crude product was purified by flash
chromatography [SiO~, eluent: hexane-EtOAc (1:1)] to provide Boc-
Asn(pyrrolidino)-Asp(cyhn)(OBzI)-Leu-0Bzl as a white foam (530
mg, 9196).
The previous material (280 mg, 0.38 mmol) was treated with
6 N HCl/dioxane (4 mL) for 30 min at room temperature. The
solvent was removed and the residue was pumped under high vacuum
for 18 h. The resulting white foam was coupled to Boc-Tbg-0H
(1.1 eq) in essentially the same manner as was done for the previous
coupling. The crude product was purified by flash chromatography
[SiO=, eluent: hexane-EtOAc (1:1)] to provide Boc-Tbg-
Asp(pyrrolidino)-Asp(cyPn)(OBzl~Leu-0Bzl as a white foam
(280 mg, 8596).
The latter compound was coupled to Boc-N-Me-Val-OH in the
same manner as was dons for the previous coupling to provide Boc
* Trade-mark
A
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24
N-Me-Val-Tbg-Asp(pyirolidino)-Asp(cyPn)(OBzI)-Leu-OBzI as a white
foam (86% yield).
The latter compound (88 mg, 0.09 mmol) was treated with
6 N HCl/dioxane ( 1.5 mL) for 20 min at room temperature. The
solvent was removed and the residue was pumped under vacuum for
2 h. This material was dissolved in CH~C12 (0.8 mL) and NMM
(40 L, 0.36 mmol) was added, followed by a premixed solution of
3-phenylpropionic acid (27 mg, 0.18 mmol) and BOP (80 mg,
0.18 mmol) in CHZCIz (0.35 mL). The reaction mixture was stirred
at room temperature for 16 h, after which time it was poured into
EtOAc (30 mL) and a saturated aqueous solution of NaHC03
(20 mL). The organic phase was washed with water and brine, dried
(MgS04), filtered and concentrated to afford a yellow gum. This
material was purified by flash chromatography [Si02, eluent: EtOAc-
hexane (2:1)] to provide PhCHZCH2C0-N-Me-Val-Tbg-
Asp(pyrrolidino)-Asp(cyPn)(OBzI)-Leu-OBzI (80 mg, 90%).
The latter material (80 mg, 0.08 mmol) was dissolved in
MeOH (2 mL). 20% Pd(OH)~/C (50 mg) and ammonium formate
(50 mg) were added to the mixture. The mixture was stirred under
an atmosphere of hydrogen for about 5 h. The reaction mixture was
filtered through diatomaceous earth and the filtrate was concentrated.
The residue was dissolved in water (20 mL), (a few drops of
saturated aqueous solution of NaHC03 were added to insure basicity).
The solution was washed (2X) with Et~O, acidified with solid citric
acid and extracted (2X) with EtOAc. The combined EtOAc extracts
were dried (MgS04), filtered and concentrated to afford the title
compound of this example as a white solid (55 mg, 83%).
The procedures of examples 5 or 6 were used to prepare the
peptides listed in the tables of example 7 with modifications noted
therein in some instances. Commercially available Boc-amino acids
were used. Unnatural amino acids were used in their Boc protected
X01900
form; they were either commercially available, readily prepared from
commercially available corresponding amino acids by reaction with
di-tertiary-butyl carbonate, or prepared by standard methods.
Note that N-alkylated Boc amino acids are commercially
5 available, e.g. Boc-N-methylvaline, or they can be prepared by stan-
dard N-alkylation of corresponding Boc-amino acids. For example,
Boc-N-Me-Asp(NEt~-OH was obtained by reacting Boc-Asp(NEt~)-
OH with 2.5 molar equivalents of methyl iodide and 2.1 molar
equivalents of potassium hydride in THF at 0 ° for 18 h to give a
10 mixture of Boc-N-Me-Asp(NEt~-OH and its corresponding methyl
ester. The mixture was esterified fully (diazomethane) and then
saponified (NaOH/H20/dioxane) to yield the desired compound.
Example 7
Inhibition of Heroes Simplex Virus (HSV, type 1) Ribonucleotide
15 Reductase
a) Preparation of Enzvme
HSV-1 ribonucleotide reductase (partially purified) was
obtained from quiescent BHK-21/C13 cells infected with strain
F HSV-1 virus at 10 plaque forming units/cell as described by
20 E.A. Cohen et al., J. Gen. Virol., 66, 733 (1985).
b) Assay and Results for Exemplified Peptides
By following the procedure described by P. Gaudreau et al.,
J. Biol, Chem., 262, 12413 (1987), the assay results listed in
following tables 1 to 5 were obtained. The assay result for
25 each peptide is expressed as the concentration of the peptide
producing 50% of the maximal inhibition (ICS) of enzyme
activity. The number of units of the enzyme preparation used
in each assay was constant, based on the specific activity of
the enzyme preparation. The results are relative to the
activity obtained in control experiments without peptide and
2019005
26
represent the mean of four assays that varied less than 10°l0
with each other.
TABLE
peptide FAB/MS ICS
M+Na + , .~M
PhCH2CHZC0-N-Me-Val-Tbg- 835 0.06
Asp(pyrrolidino)-Asp(cyBu)-
Leu-OH
PhCHZCHZCO-N-Me-Val-Tbg- 849 0.08
Asp(pyrrolidino)-Asp(cyPn)-
Leu-OH
PhCHZCH2C0-N-Me-Val-Tbg- 908 0.18
Asp[1 (S)-methylheptyloxy]-Asp(cyBu)-
Tba-OH
(4-Hydroxy-Ph)CH2CH2C0-N-Me- 924 0.18
Val-Tbg-Asp[1(S)-methylheptyloxy]-
Asp(cyBu)-Tba-OH
PhCHZCHZCO-N-Me-V al-Ile-Asp(NEtz)-Asp(cyBu)-Leu-OCH3
PhCH2CHZC0-N-Me-Val-Ile-Asp(NEtz)-Asp(cyBu)-Leu-OH
PhCH2CH2C0-N-Me-Val-Ile-Asp(NEt~-Asp(cyPn)-(L-leucinol)
PhCH2CHZC0-N-Me-Val-Ile-Asp(NMe2)-Asp(cyHx)-
NHCH[CHZCH(CH3)~]-5-1H-tetrazole
PhCH2CH2C0-N-Me-Val-Ile-Asp(pyrrolidino)-Asp(cyBu)-
NHCH[CHZCH2CH(CH3)~]COOH 1
PhCH2CHZC0-N-Me-Val-Ile-Asp(NEt,~-Asp(cyPr)-NHCH[CHi
CCH3(=CHZ)]-COOH 2
PhCHZCH2C0-N-Me-Val-Ile-Asp(pyrrolidino)-Asp(cyPn)-
NHCH[CHZC(CH3)3]CHZOH
PhCH2CHZC0-N-Me-Val-Ile-Asp(pyrrolidino)-Asp(cyPn)-
NHCH[CHZCH(CH3)~]CHZCOOH 3
[2-(2~-Carboxy)biphenylyl]carbonyl-N-Me-Val-Ile-Asp(pyrroli-
dino)-Asp(cyHx)-Leu-OH
2~.~9005
27
PhCHZCHzCO-N-Me-V al-NHCH(cyclohexylmethyl)-CO-Asp-
(pyrrolidino)-Asp(cyPn)-Leu-OH
PhCHZCH2C0-N-Me-Val-Ile-Asp(morpholino)-Asp(cyPr)-Leu-
4
2
PhCH2CH2C0-N-Me-Val-Ile-Asp(NHCHZCHZPh)-Asp(cyBu)-
Leu-NEtz s and
PhCH2CHZC0-N-Me-Tbg-Ile-Asp(NMe(decyl)]-Asp(cyPn)-Leu-
OH.
1 The 2(S)-amino-5-methylhexanoic acid methyl ester (Boc-
homoleucine methyl ester) employed for the preparation of this
peptide is obtained by reacting [(CH3)2CHCH~]ZCuLi, prepared by
the method of D. Seebach and H. Neumann, Chem. Ber., 107, 847
( 1974), with the O-tosyl derivative of Boc-Ser-OCH3 according to the
conditions reported by A. Bernardini et al., Tetrahedron Letters, 24,
3717 (1983); followed by Boc deprotection with TFA/CHZCIz of the
resulting Boc-2(S)-amino-5-methylhexanoic acid methyl ester. Subse-
quent coupling of the 2(S)-amino-5-methylhexanoic methyl ester so
obtained with PhCHZCHZCO-N-Me-Val-Ile-Asp(pyrrolidino)Asp(cyBu)-
(OBzI)-OH and deprotection of the coupling product gave the corres-
ponding peptide of formula 1.
2 The 2(S)-amino-4-methyl-5-hexenoic acid methyl ester
employed for the preparation of this peptide is obtained by converting
Boc-Ser-OH to its corresponding 13-lactone by the method of J.C.
Vederas et al., J. Am. Chem. Soc., 107 7105 (1985) and reacting the
B-lactone with the organolithium derivative derived from 2-propylene
bromide according to the method of D. Seebach and H. Neumann,
Chem. Ber., 107, 847 (1974) to give 2(S)-amino-4-methyl-5-hexenoic
acid. Subsequent esterification of the latter compound with
diazomethane gave the desired corresponding methyl ester.
3 The Boc-3(S)-amino-5-methylhexanoic acid employed in the
preparation of this peptide was obtained by an Arndt-Eistert reaction
201900
28
starting from Boc-Leu-OH, W.E. Bachmann and W.S. Struve, Organic
Reactions, 1, 38 (1942).
'' Lucinamide was coupled with the appropriate protected
fragment followed by hydrogenolysis to remove the Bzl protecting
group. More specifically, by coupling PhCH2CH2C0-N-Me-Val-Ile-
Asp(morpholino)-Asp(cyPr)(OBzI)-OH, prepared according to the
procedure of example 6, with leucinamide using BOP in the presence
of DIPEA, followed by hydrogenation to remove the Bzl on the Asp
residue, the desired product is obtained.
5 The corresponding protected C-terminal acid is coupled with
diethylamine hydrochloride using BOP/HOBt in the presence of N-
methylmorpholine, followed by hydrogenation to remove the Bzl on
the Asp residue to give the desired product.
