Note: Descriptions are shown in the official language in which they were submitted.
1
201.9006
13-BG-D-26a
Antiherpes Tetrapeptide Derivatives Having a
Cvcloalkyl Substituted As' artic 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 "peptiides") exhibiting activity against
herpes viruses, to pharmacsutical compositions comprising the
peptides, and to a method of using the peptides to treat herpes
infections.
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 zc~ster virus which causes chicken
pox and shingles; and Epstein-Barn 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).
20.0006
2
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.
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 29'2255, 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
distinguished from the peptides of the present application by
characteristic structural and biologic~~l differences.
Summary of the Invention:
The peptides of this invention are represented by formula 1
X-NH-CHR'-C(W')-NR2-CH[CHZC(O)-Y]-C(V~)-NH-CH[CR3(R4)-
COOH]-C(W3)-NH-CHRs-Z 1
wherein X is (1-lOC)alkanoyl; (1-lOC)alkanoyl monosubstituted with
halo, hydroxy or lower alkoxy; (:l-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;
phenyl(2-lOC)alkanoyl monosubstituted or disubstituted on the
aromatic portion thereof with a substituent selected from halo,
hydroxy, lower alkyl, lower alkoxy or phenyl; phenyl(3-lOC)-
alkenoyl; (lower cycloalkyl)carbom~l; (lower cycloalkyl)carbonyl
201900u
3
substituted with one to four substituents selected from halo or lower
alkyl; cyclohexylcarbonyl substituted at position 2 with lower
alkanoyl, phenyl(lower)alkanoyl or phenyl(lower)alkoxycarbonyl; 3,6-
dimethyl-2-(phenylethoxycarbonyl)cyclohexylcarbonyl; or a straight or
branched chain 1,4-dioxoalkyl containing from five to eleven carbon
atoms;
R' is lower alkyl, hydroxy(lov~rer)alkyl, mercapto(lower)alkyl,
methoxy(lower)alkyl, methylthio(lov~rer)alkyl, benzyloxy(lower)alkyl,
benzylthio(lower)alkyl, carboxy(lowe;r)alkyl, lower cycloalkyl, (lower
cycloalkyl)methyl, phenyl, phenylmethyl, 2-thienyl or 2-thienylmethyl;
RZ is hydrogen, lower alkyl or phenyl(lower)alkyl;
R3 and R4 together with the carbon .atom to which they are attached
form a lower cycloalkyl;
RS is lower alkyl, lower cycloalkyl, or (lower cycloalkyl)methyl;
W', WZ, and W3 each independently is oxo or thioxo;
Y is
a. (1-14C)allcoxy, (3-14)alkenyloxy, CH3(OCH2CH~o O wherein
n is the integer 1, 2 or 3, lower cycloalkyloxy, lower alkoxy
monosubstituted with a lower cycloalkyl, phenoxy, phenoxy
monosubstituted with hydro~:y, 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. NR6R' wherein R6 is lower aalkyl and R' is lower alkoxy, or
c. NR6R' wherein R6 is hydrogen or lower alkyl and R' 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
~~~19006
4
d. NR6R' wherein R6 and R' 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; CH2COOH; CH2CHZCOOH; CHZOH; 5-1 H-
tetrazolyl; COORg wherein Rg is lower alkyl; CONR9R'° wherein R9
and R'° each independently is hydrogen or lower alkyl; or
CON(R")OH wherein R" is hydrogen or lower alkyl; with the
provisos that (1) when X is a (1-10(:)alkanoyl containing one or two
carbon atoms (i.e. formyl or acetyl) then RZ is lower alkyl or phenyl
lower alkyl, and that (2) when Z is hydrogen then R3 is hydrogen or
lower alkyl and R' is lower alkyl or R3 or R4 together with the
carbon atom to which they are attached form a lower cycloalkyl;
or a therapeutically acceptable salt thereof.
A preferred group of the peptides of this invention is
represented by formula 1 whereiin X is (1-lOC)alkanoyl; (1-
lOC)alkanoyl monosubstituted with chloro, fluoro, hydroxy or
methoxy; benzoyl monosubstituted with phenyl, 2-carboxyphenyl or
benzyl; phenyl(2-lOC)alkanoyl; phe:nyl(2-lOC)alkanoyl monosubsti-
tuted on the aromatic portion thereof with a substitutent selected from
halo, hydroxy, lower alkyl, lower alkoxy or phenyl; phenyl(3-
lOC)alkenyl; (lower cycloalkyl)carbonyl; (lower cycloalkyl)carbonyl
monosubstituted, disubstituted, trisubstituted or tetrasubstituted with
methyl; cyclohexylcarbonyl substituted at position 2 with a phenyl-
(lower)alkanoyl; la,2a,313,6~13-3,6-dimethyl-2-(phenyl-
ethoxycarbonyl)cyclohexanecarbonyl or 6-methyl-2-(1-methylethyl)-
1,4-dioxoheptyl; R' is as defined hereinabove; R2 is hydrogen or
lower alkyl; R3 and R4 together with the carbon atom to which they
are joined form a lower cycloalk:yl; Rs is 1-methylethyl, 1,1-
dimethylethyl, 1-methylpropyl, 2-me;thylpropyl, 2,2-dimethylpropyl,
cyclopentyl, cyclopentylmethyl, cycla~hexyl or cyclohexylmethyl; W',
V~ and W3 are as defined hereinabove; Y is (1-14C)alkoxy, lower
cycloalkyloxy, lower cycloalkylmethoxy, phenyl(lower)alkoxy, NR6R'
wherein R6 is lower alkyl and R' is lower alkoxy, or NR6R' wherein
R6 is hydrogen or lower alkyl and R' is (1-14C)alkyl, (3-
20719006
.._
s
14C)alkenyloxy, CH3(OCHZCH~3-O, lower cycloalkyl, lower
cycloalkylmethyl, phenyl, phenyl monosubstituted with halo, lower
alkyl or lower alkoxy, phenyll;lower)alkyl, phenyl(lower)alkyl
monosubstituted with halo, lower alkyl or lower alkoxy, (Het)-lower
s alkyl wherein Het is a heterocyclic radical selected from 2-pyrrolyl,
2-pyridinyl, 4-pyridinyl, 2-furyl, 2-isoxazolyl and 2-thiazolyl, or
NR6R' wherein R6 and R' together with the nitrogen atom to which
they are attached form a pyrrolidino, piperidino or morpholino; and
Z is as defined hereinabove; with the provisos that (1) when X is a
(1-lOC)alkanoyl containing one or two carbon atoms then R2 is
methyl, and that (2) when Z is hydrogen then R3 is hydrogen, methyl
or ethyl and R4 is methyl or ethyl, or R3 and R4 together with the
carbon atom to which they are attached form a lower cycloalkyl; or
a therapeutically acceptable salt thereof.
is A more preferred group of the peptides is represented by
formula 1 wherein X and Rs are as defined in the last instance; R'
is lower alkyl, hydroxy(lowc;r)alkyl, methoxy(lower)alkyl,
benzyloxy(lower)alkyl, lower cycloalkyl, (lower cycloalkyl)methyl,
phenyl, phenylmethyl or 2-thienyl; RZ is hydrogen or methyl; R3 and
R4 together with the carbon atom to which they are attached form a
lower cycloalkyl; W', WZ and W3 acre oxo; Y is (1-14C)alkoxy, (3-
14C)alkenyloxy, CH3(CICHzCH2)3-O, lower cycloalkyloxy, lower
cycloalkylmethoxy, phenyl(lower)alkoxy, NR6R' wherein R6 is lower
alkyl and R' is lower alkoxy, or NR6R' wherein R6 is hydrogen or
2s lower alkyl and R' is (1-14C)alkyl, lower cycloalkyl, lower
cycloalkylmethyl, phenyl, phenyl(lowe;r)alkyl or pyridinyl(lower alkyl),
or NRbR' wherein R6 and R' together with the nitrogen to which they
are attached form a pyrrolidino, pipe;ridino or morpholino; and Z is
hydrogen, CC10H, CH2CC10H, CH2CIH, s-1H-tetrazolyl, CONR9R'°
wherein R9 and R'° each independently is hydrogen or lower alkyl,
or CON(R'1)OH wherein R" is hydrogen or methyl; or a therapeuti-
cally acceptable salt thereof; with the previsos (1) and (2) noted in
the preceding paragraph being applicable.
.~ 209006
6
A most preferred group of the peptides is represented by
formula 1 wherein X is 2-ethyl'.butanoyl, 3-methylbutanoyl, 4-
methylpentanoyl, octanoyl, 2-hydroxy-3-methylbutanoyl, 2-
biphenylylcarbonyl, phenylacetyl, phenylpropionyl, 2-(1-methylethyl)-
6-phenylhexanoyl, 2-(1-methylethyl)-6-phenyl-3-hexenoyl,
cyclopropylcarbonyl, 2,2,3,3-tetramethylcyclopropylcarbonyl, cyclo-
hexylcarbonyl, 2-methylcyclohexylcarbonyl, 2,6-dimethyl-
cyclohexylcarbonyl, 2-(3-phenyl-1-o:KOpropyl)cyclohexanecarbonyl or
1 a,2a,3B,613-3,6-dimethyl-2-(phenyledloxycarbonylkyclohexylcarbonyl;
R' is lower alkyl, hydroxymethyl, 1~-hydroxyethyl, 1-benzyloxyethyl,
cyclopentyl, cyclohexyl, cyclohexylmethyl, phenyl, phenylmethyl or
2-thienyl; R2 is hydrogen or methyl; R3 and R° together with the
carbon atom to which they are attached form a lower cycloalkyl; RS
is 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or
cyclohexylmethyl, Wl, WZ and W3 are oxo, Y is hexyloxy, 1-
methylheptyloxy, octyloxy, decylo:xy, trans-3-heptenyloxy, cis-3-
octenyloxy, CH3(OCHZCHZ)3 O, c:yclopentyloxy, cyclohexyloxy,
cyclohexylmethoxy, phenylpropoxy, :f~(Me)OMe, ethylamino, phenyl-
amino, phenylethylamino, N-methyl-N-phenylethylamino, 2-pyridinyl-
ethyl, N,N-dimethylamino, N,N-dieth~ylamino, N,N-diisopropylamino,
N-methyl-N-octylamino, pyrrolidino, piperidino or morpholino; and Z
is hydrogen, COOH, CHZCOOH, 5-1H-tetrazolyl, CHZOH or
CONR'R'° wherein R9 and R'° each independently is hydrogen,
methyl, ethyl or propyl; with the proviso that when Z is hydrogen
then R3 is hydrogen, methyl or ethyl and R4 is methyl or ethyl, or R3
and R4 together with the carbon atom to which they are attached
form a lower cycloalkyl; or a therapeutically acceptable salt thereof.
2019006
Still another preferred group of the peptides is represented by
formula 1 wherein X is 2-ethylbut<moyl, R' is 1,1-dimethylethyl or
1-methylpropyl, RZ is hydrogen, R3, R', Rs, W', WZ, W3 and Y are as
defined in the last instance, and Z is hydrogen, COOH or CHZOH;
with the proviso that when Z is hydrogen then R3 and R4 each is
methyl or ethyl, or R3 and R' together with the carbon atom to which
they are attached form a lower cycloallcyl; or a therapeutically
acceptable salt thereof.
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 Garner.
Also included within the scoF~e of this invention is a cosmetic
composition comprising a peptide oiP 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.
2011006
g
Details of the Invention
GENERAL
Alternatively, formula 1 c:an be illustrated as:
3
R
NH
x N HN
Z
R R qr
R 4 COOH
R
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 hydroxyll 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-
lions of the IUPAC-IUB Commision of Biochemical Nomenclature,
sew European Journal of Biochemistry 1:38, 9 (1984). For instance,
Gly, Val, Thr, Ala, Ile, Asp, Ser and Leu represent the residues of
glycine, L-valine, L-threonine, L-alanine,1L-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 respexa to disubstituted benzoyl,
disubstitued phenyl(1-lOC)alkanoyl, and disubstituted and
trisubstituted cyclohexane;carbonyl, 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.
209006
9
The symbol "Tbg" represents the amino acid residue of 2(S)-
amino-3,3-dimethylbutanoic acid. ~f'he symbol "Cpg" represents the
amino acid residue of (S)-a-aminocyclopentaneacetic acid. The
symbol "Phg" represents the aumino acid residue of (S)-a-
aminophenylacetic acid. Thr(OBzI) for the residue of 03-benzyl-L-
threonine.
Other symbols used herein acre: Asp(cyPr) for the residue of
(S)-a-amino-1-carboxycyclopropaner~cetic acid; Asp(cyBu) for the
residue of (S)-a-amino-1-carboxycyclobutaneacetic acid; Asp(cyPn)
for the residue of (S)-a-amino-1-carboxycyclopentaneacetic acid; and
Asp(cyHx) for the residue of (S)-a-amino-1-carboxycyclohexaneacetic
acid. Asp(pyrrolidino) is used for the residue of the amide 2(S)-
amino-4-oxo-4-pyrrolidinobutanoic acid; and Asp(morpholino),
Asp(NEtz) and Asp(N-Me-N-octyl) similarly represent the residues of
the corresponding amides wherein the pyrrolidino is replaced with
morpholino, diethylamino and N-methyl-N-octylamino, respectively.
The term 'halo' as used herein means a halo radical selected
from bromo, chloro, fluoro or iodo.
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 atorns 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 s;ix carbon atoms and branched
chain alkenyl radicals containing three to six carbon atoms and
includes vinyl, 1-propenyl, 1-methyle;thenyl, 2-methyl-1-propenyl, 2-
methyl-2-propenyl and 2-butenyl.
-~ X019006
to
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 "lower alkanoyl" as used herein means straight chain
1-oxoalkyl radicals containing from. one to six carbon atoms and
branched chain 1-oxoalkyl radicals containing four to six carbon
atoms and includes acetyl, 1-oxopropyl, 2-methyl-1-oxopropyl, 1
oxohexyl and the like.
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-liDC)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 fourdxn carbon atoms, respectively.
The term "(3-14C)alkenyloxy" mea~~s straight and branched chain
alkenyloxy radicals containing from three to fourteen carbon atoms,
and in which the double bond may be cis or trans and is positioned
more than one carbon atom away from the oxygen atom of the
radical; for example, 2-propenyloxy, :3-heptenyloxy and 3-octenyloxy.
The term "(1-lOC)alkanoyl" as used herein means straight or
branched chain 1-oxoalkyl radicals camtaining from one to ten carbon
atoms; for example, acetyl, 4-methyl-1-oxopentyl or (4-methyl-
pentanoyl) or 1-oxooctyl (or octanoyll). The term "(3-lOC)alkanoyl"
as used herein means straight or braJnched chain 1-oxoalkyl radicals
201900fi
11
containing from three to ten carbon atoms. The term "phenyl(2-
lOC)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 a~ld 1-oxo-5-methyl-6-phenylhexyl.
The term "phenyl(3-lOC)alkenoyl" as used herein means phenyl
substituted 1-oxoalkenyl radicals wherein the 1-oxoalkenyl portion
thereof is a straight or branched chain 1-oxalkenyl containing from
three to ten carbon atoms; for example, 2-methyl-1-oxo-3-phenyl-3-
pentenyl.
The symbol "'~(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 carrier" or "veterinarily
acceptable earner" 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 vehiicle 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 earner" as used herein
means a physiologically acceptable vehicle for administering drug
substances to domestic animals corr~prising one or more non-toxic
pharmaceutically acceptable excipients which do not react with the
drug substance or reduce its effectiveness.
12
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 frEx amino group of another amino
acid or peptide to form an amide bond between the reactants.
Similarly, such agents can effect tihe coupling of an acid and an
alcohol to form corresponding esters. The agents promote or
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, diphenyl-
phosphoryl azide, 1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide,
N-hydroxysuccinimide, or 1-hydroxybenzotriazole in the presence of
dicyclohexylcarbodiimide. A very practical and useful coupling agent
is (benzotriazol-1-yloxy)tris(dimethyhunino)-phosphonium hexafluoro-
phosphate, described by B. Castro ea 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. Schra3er and K. Lubke, cited above,
in the textbook series, "The Peptides: Analysis, Synthesis, Biology",
E. Gross et al., Eds., Academic Press, New York, N.Y., 1979-1987,
20119006
13
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 v~rill prevent a chemical reaction
from occurring at that site until the protective group is ultimately
removed. Usually also common i;s 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 aunino acid residue or peptide
fragment, if present, which is to bea~ome 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
-- ~0~.9006
14
methylenedichloride in the presence of excess pyridine and a catalytic
amount of 4-dimethylaminopyridine (Fieser and Fieser, "Reagents for
Organic Synthesis", John Wiley arid Sons, Inc., New York, NY,
USA, 1967, vol 1, p 1183). The rutrile derivative then was mixed
with tributyl tin azide, J.G.A. Lui,~ten et al., Rec. Trav., 81, 202
(1962), giving a tetrazole tin intermediate [c~ 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, 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 suich 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. hydra~hloric acid, or sulfuric acid, or
phorphoric 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 (19fi0).
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 rations, or with strong organic bases,
for example, triethylamine or N-mett~ylmorpholine.
2014006
a...
is
Antiheroes Activi~
The antiviral activity of the; peptides of formula 1 can be
demonstrated by biochemical, nnicrobiological 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.
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 anti-
viral properties against all viruses dependent on ribonucleotide reduc-
tase to synthesize 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., Proc. Nati. 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
2019006
16
herpes infection in Swiss Webster rnice, described by E.R. Kern, et
al., Antiviral Research, 3, 253 (198:3).
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 t~evtide, 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
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
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
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.
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,
treatment is initiated with small incrE;ments until the optimum effect
under the circumstances is reached. In general, the peptide is most
2010006
17
desirably 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 slan or part of the oral or genital
cavity, in an amount sufficient to cover the infected area. The
treatment should be repeated, for example, every four to six hours
until lesions heal.
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
variations will occur. However, a dlosage level that is in the range
of from about 50 mcg to 500 mcg pier 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 Garner.
Additional 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 pharmacc;utical 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.
20.9006
18
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 ililustrate 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;
CHZC1Z: methylenedichloride; DIPE,A: diisopropylethylamine; DCC:
N,N-dicyclohexylcarbodiimide; DMh: dimethyl formamide; EtzO:
diethyl ether; EtOAc: ethyl acetate; EtOH: ethanol; HOBt: 1
hydroxybenzotriazole; HPLC: high performance liquid chromato
graphy; MeOH: methanol; NMMI: N-methylmorpholine; TFA:
trifluoroacetic acid; THF: tetrahydrofnran. Temperatures are given in
degrees centrigrade.
EXamplf; 1
Preparation of the Intermediate Boc-.Asnfl(S)-methylheptyloxyl-OH
A solution of Boc-Asp-OB~zI (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-dumethylaminopyridine (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 HCI, 1N aqueous NaHC03, dried (MgSO,) and
concentrated. The resultant oil ways purified by chromatography
(SiOZ, eluent: hexane-EtOAc, 7:3) to give Boc-Asp[1(S)-methyl-
2019006
19
heptyloxy]-OBzl (92% yield). Hydr~~genation of the latter compound
in the presence of 20% Pd(OH),/C in ethanol solution afforded a
quantitative yield of the title compound as a solid. NMR(200 MHz,
CDCl3)8 0.9(m,3H), 1.25(m,IOH), 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.
Examvle 2
Preparation of the Intermediate Boc-Asn(NEt,)-OH
BOP (2.20 g, 5.0 mmol) was added under NZ to a cooled (0 °)
solution of Boc-Asp-OBzI ( 1.90 g, 4.6 mmol) in CH2C12 (50 ml).
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 10% aqueous citric
acid (2 X), 10% aqueous NaHC03 (2 X) and brine (2 X). The
organic layer was dried (MgS04) and concentrated to give an oil.
After Si02 chromatography of the oil using hexane-EtOAc (7:3) as
the eluent, Boc-Asp(NEtz)-OBzI (1.55 g, 89%) was obtained as an oil.
Under a NZ atmosphere, a solution of the latter compound ( 1.55 g,
4.09 mmol) in MeOH (100 ml) was mixed with 5% Pd/C (155 mg).
The mixture was shaken on a Parr apparatus under H2 (50 psi) for
90 min. The mixture was filtered tluough a 45 m membrane and
the filtrate concentrated to give Boc-,Asp(NEt,~-OH (1.15 g, 98%) 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 IVI~t~.HC1 with the appropriate
amine or amine salt (e.g. pyrrolidine or N,O-dimethylhydroxylamine
hydrochloride).
2019000
The intermediates of examples 1 and 2 or their analogs can
be incorporated into corresponding peptides of formula 1 according
to the procedures of examples 7 or 8.
Examvle 3
5 Preparation of d,l-cis-6-(3-Phen~rl-1-oxopronvl)-3-cyclohexene-1-
carboxvlic acid
A Grignard reagent was prepared in THF (40 ml) from
phenylethyl bromide (2.75 ml, 20 rnmol) and Mg turnings (0.51 g,
21 mmol) while the mixture was heated at reflux temperature for 1
10 h. A solution of 3a,4,7,7a-tetrahydro-1,3-isobenzofurandione (3.04 g,
20 mmol) in Et~O (100 ml) was cooled to -40°. The solution of the
Grignard reagent was added dropwis~e via a cannula over 15 min to
the stirred cooled solution. The reaction mixture was allowed to
warm to room temperature over 15 min and then stirred at that
15 temperature for 2 h. The reaction mixture was quenched at 0° with
1N aqueous HCl and then extracted with EtOAc. The organic phase
was extracted with 1N aqueous NaOH. The latter aqueous phase was
washed with EtOAc, rendered acidic with 6N aqueous HCl and
extracted with EtOAc. The latter extract was dried (MgSO,) and
20 concentrated to dryness. The crude product was purified by chroma-
tography (Si02; eluent: hexane-EtOAc, 1:1) to give the title com-
pound (2.99 g, 58%). The NMR of the product was in agreement
with the assigned structure.
The title compound was incorporated as a N-terminal residue
into the pentultimate protected interrr~ediate of the desired peptide of
formula 1 according to the procedure of example 7. Subsequent
hydrogenolysis simultaneously removed any benzyl protecting groups
of the pentultimate intermediate and reduced the double bond in the
terminal residue to afford the corresponding peptide of formula 1
wherein X is d,l-cis-2-(3-phenyl-1-oxopropylkyclohexanecarbonyl.
The diastereoisomers can be separated by HPLC.
20.9006
21
Examvle 4
Preparation of la,2a,3B,6B-3.6-Dimethvlcvclohexane-1,2-dicarboxvlic
Acid Monovhenylethvl Ester
A solution of trans,trans-2,4-~hexadiene (1.10 g, 13.4 mmol)
and malefic anhydride ( 1.31 g, 13.4 mmol) in benzene ( 15 ml) was
heated at 85° for 4 h. The mixtm~e was cooled and diluted with
EtOAc. The organic phase was washed with H20, dried (MgSO,)
and concentrated to give 3aa,4B,7B;7aa-tetrahydro-4,7-dimethyl 1,3-
isobenzofurandione (2.00 g, 83%) as a white solid. A solution of the
latter compound (500 mg) in THIF was added to an excess of
magnesium phenylethoxide in THF at -20 °. The stirred reaction
mixture was allowed to come to room temperature and then stirred
for 18 h at that temperature. Isolation of the product in the manner
described in example 4 gave la,2a,3B,6B-3,6-dimethyl-4-cyclohexene-
1,2-dicarboxylic acid monophenylethyl ester (310 mg). Subsequent
hydrogenation [H2, 20% Pd(OH)y~C,EtOH,3h] yielded the title
compound in quantitative yield. The NMR spectrum was in
agreement with the assigned structure for the product.
The title compound was incorporated as a N-terminal reside
according to the procedure of example 7 to yield desired peptides of
formula 1 as a mixture of diastereoi.somers which can be separated
by HPLC.
ExamylE; 5
Preparation of (S)-a-Amino-1-carboxvcycloalkylacetic Acid
Intermediates
These intermediates, which can be used to prepare peptides of
formula 1 in which R' and R4 are joined to form a lower cycloalkyl
can be prepared according to the method of M. Bochenska and J.F.
Biernat, Rocz. Chem., 50, 1195 (197fi); see Chem. Abstr., 86, 43990r
(1977).
20~L9006
22
More specifically exemplifie,i, (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
dimethylsulfoxide and Et~O (1:1, 12CI ml) was added sodium hydride
(4.5 g, 60°k 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 t~~eated 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 (MgSO,), 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: ethyl acetate-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, :il 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)
dicarbonate (14.6 g, 67 mmol) in di~oxane (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 ethyl
acetate (300 ml). The combined ethyl acetate extracts were washed
209006
23
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 KZCO3 (7.6 g, 55 mmol) and benzyl
bromide (6.6 ml, 55 mmol). The reaction mixture was stirred at
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 c;xtract provided a pale yellow
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
ml) and an aqueous solution of Li~OH (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 (2~;) with ethyl acetate. The ethyl
acetate layers were combined, .dried (MgSO,), filtered and
concentrated to provide Boc-Asp(cy:Pn)(OBzI)-OH as a clear color
less gum (7.3 g, 82%).
Example; 6
Preparation of the Intermediate Boc-Asn(cvPn)(OBzI)yr~CSNHILeu-
OBzI
The title compound is obtained by stirring a mixture of the
protected dipeptide Boc-Asp(cyPn)(OBzI)-Leu-OBzI (5.5 mmol) and
Lawesson's reagent (2.7 mmol), see 'U. Pederson et al., Tetrahedron,
38, 3267 (1982), in toluene at reflu~: temperature for 2 h; followed
by pouring the cooled reaction mixture onto a column of silica gel
(3.5 x 30 cm) and eluting the column with CHZC12.
--~ ~~19~~fi
24
Analogs thioamides intermediates are prepared in the same
manner by replacing Boc-Asp(cyPn)(OBzI)-Leu-OBzI with the
appropriate protected dipeptide.
Example 7
General Procedure for the Solid Phase Preyaration of Peytides of
Formula 1
A modified version of the solid phase method of R.B.
Mernfield, J. Am. Chem. Soc., 85, 2.149 (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. Ixtt., 165
(1978), using 9 molar equivalents of KF and 3.6 molar equivalents
of Boc-l:xu-OH, for example in DMF at 70 ° C for 24 hours, to give
{ 4- { 2- { Boc-leucine } 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.,
2019006
Anal Biochem., 34, 595 ( 1979). Double coupling was used when
necessary.
Cleavage of the protected peptide from the solid support was
accomplished by irradiation at 330 nm in EtOH/DMF (1:4) at 0 °
5 under an argon atmosphere for 6 to 18 h. Protective groups (Bzl),
if present, were removed from the cleavage product by
hydrogenolysis over 5°70 or 10%v Pd/C or 20% Pd(OH~/C by standard
procedures (cf. example 1). Purification of the final product was
performed by reversed-phase HPLC to better than 95% homogeneity
10 using 0.06% aqueous TFA/0.6% TFA in acetonitrile gradients.
Example; 8
Preparation of (C,HO,CHO-Tb~-Asp(wrrolidino)-Asp(cyPn)-Leu-OH
(Example of a solution phase proced~.we for preparing compounds of
formula 1 )
15 To a solution of Boc-Asp(cyF'n)(OBzI)-OH (5.3 g, 14 mmol,
described in example 3) in dry CH;zCl2 (50 mL), BOP (6.8 g, 16
mmol), NMM (4.6 mL, 42 mmol) and the (4-methylphenyl)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
20 time it was poured into a two component system of EtOAc (500 mL)
and a saturated aqueous solution of TfaHC03 (400 mL). The organic
phase was washed with water and brine. Drying (MgSO,~, filtering
and concentrating of the organic phase afforded a dark yellow oil.
This material was purified by flash chromatography [SiOz, eluent:
25 hexane-EtOAc (6:1)] to provide Boc-Asp(cyPn)(OBzI)-Leu-OBzI as
a clear colorless gum (7 g, 86%, mi:Kture of diastereoisomers).
The latter compound (7 g, 12 mmol) was mixed with CH2Clz
(4 mL). TFA (6 ml) was added to the mixture and the resulting
solution was stirred for 30 min at room temperature. Thereafter, the
26
20 1 90 06
majority of the solvent was evaF~orated and the residue was poured
into a two component system o:f EtOAc (200 ml) and a saturated
aqueous solution of NaHC03 (400 mL). Drying (MgSO,), filtering,
and concentrating of the organic phase afforded the amine dipeptide
H-Asp(cyPn)(OBzI)-Leu-OBzI as a clear colorless gum (mixture of
diastereoisomers). 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). This observation proved
general for all corresponding c;ycloalkyl aspartic acid derivatives
made. For convenient storage of material, the pure amine dipeptide
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)-Leu-OBzI. The crude product was purified by flash
chromatography [Si02, eluent: hexane-EtOAc (1:1)] to provide Boc
Asn(pyrrolidino)-Asp(cyPn)(OBzI)-Leu-OBzI as a white foam (530
mg, 91%).
The previous material (280 mg, 0.38 mmol) was treated with
6 N HCl/dioxane (4 mL) for 3CI 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-OH
(1.1 eq) in essentially the same manner as was done for the previous
coupling. The crude product was purified by flash chromatography
[Si02, eluent: hexane-EtOAc (1:1)] to provide Boc-Tbg-
Asp(pyrrolidino)-Asp(cyPn)(OBzI)-Leu-OBzI as a white foam
(280 mg, 85%).
The latter material (65 mg, 0.075 mmol) was treated with 6N
HCl/dioxane for 20 min at room temperature. The solvent was
evaporated. The residue was pumped under high vacuum for 2 h
Trade-mark
2019006
27
then dissolved in CH2C12 (1 mL). NMM (30 N.I,, 0.27 mmol) was
added to the solution, followed b~y a premixed solution of 2-
ethylbutanoic acid (12 ~rT., 0.095~ mmol) and BOP (40 mg,
0.09 mmol) in CHZCIz (0.5 mL). The reaction mixture was stirred
at room temperature for 14 h, after which time it was poured into a
two component system of EtOAc (20 mL) and a saturated aqueous
solution of NaHCO, (20 mL). ThE; organic phase was separated,
washed with water and brine, dried (MgSO~, filtered and
concentrated to afford a yellow gum.. This material was purified by
flash chromatography [Si02, eluent: C:H2Cli EtOAc (12:1)] to provide
(CZHs~CHO-Tbg-Asn(pyrrolidino)-Asp(cyPn)(OBzI)-Leu-OBzI as a
solid gum (49 mg, 75%).
The latter material (30 mg, 0.035 mmol) was dissolved in
MeOH (1.5 mL). 20% Pd(OH)~/C (2.0 mg) and ammonium formate
(20 mg) were added to the mixture. The mixture was stirred under
an atmosphere of hydrogen for about 3 h. The reaction mixture was
filtered through diatomaceous earth and the filtrate was concentrated.
The residue was dissolved in water ( 15 mL) containing a few drops
of a saturated aqueous solution of NaHC03. The solution was
washed (2X) with Et~O. The aqueous phase then was 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 (20 mg,
83%), MS: 680(M+1).
The procedure of examples 7 or 8 can be used to prepare the
peptides of formula 1; for example, see the peptides listed in the
table of example 9. Commercially available Boc-amino acids were
used. Unnatural amino acids were used in their Boc protected form;
they were either commercially av;~ilable, readily prepared from
commercially available corresponding; amino acids by reaction with
di-tertiary-butyl carbonate, or prepared by standard methods.
20~~00~
28
With reference to the preparation of peptides of formula 1
wherein RZ is lower alkyl or phenyl(lower)alkyl, the required N-
alkylated Boc amino acids can be prepared by standard N-alkylation
of corresponding Boc-amino acids. For example, Boc-N-Me-Asp-
(NEt~-OH was obtained by reacting Boc-Asp(NEt,~-OH of example
2 with 2.5 molar equivalents of methyl iodide and 2.1 molar
equivalents of potassium hydride in TIC at 0 ° for 18 h to give a
mixture of Boc-N-Me-Asp(NEt~-OH and its corresponding methyl
ester. The mixture was esterified fully (diazomethane) and then
saponified (NaOH/HZO/dioxane) to yield the desired compound.
Example 9
Inhibition of Hert~es Simplex Virus (HSV, type 1) Ribonucleotide
Reductase
a) Preyaration of Enzyme
HSV-1 ribonucleotide reductase (partially purified) was
obtained from quiescent BHK-~21/C 13 cells infected with strain
F HSV-1 virus at 10 plaque forming units/cell as described by
E.A. Cohen et al., J. Gen. Virol., 66, 733 (1985).
b) Assav and Results for Exemylified Peptides
By following the procedure described by P. Gaudreau et al.,
J. Biol, Chem., 262, 12413 (1,987), the assay results listed in
the following table were obtained. The assay result for each
peptide is expressed as the concentration of the peptide
producing 50% of the maxinnal 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. T'he results are relative to the
activity obtained in control experiments without peptide and
represent the mean of four assays that varied less than 10%
with each other.
20~~9~06
29
TABLE
Peptide F~AB/MS ICS
(M+H)+ (p.M)
(C2HS~CHCO-Tbg-Asp(pyrro- 680 0.1
lidino)-Asp(cyPn)-Leu-OH
(see example 8)
(CzHs~CHCO-Tbg-Asp(pyrro- 666 0.08
lidino)-Asp(cyBu)-Leu-OH
(CZHs~CHCO-Tbg-Asp[1(S)- 725 0.54
methylheptyloxy]-Asp(cyBu)-
Leu-OH
(CzHs~CHCO-Tbg-Asp[1(S)- 761 ' 0.37
methylheptyloxy]-Asp(cyPn)-
Leu-OH
(CzHs)zCHO-Tbg-Asp(N-Me-N-738 0.22
octylamino)-Asp(cyBu)-Leu-
OH
(C2H5)zCHO-Tbg-Asp(pyrro- 674 ' 0.5
lidino)-Asp(cyPn)-
(L-leucinol)
(CZHS~CHCO-Tbg-Asp(pyrro- 666 0.27
lidino)-Asp(cyPn)-(L-
leucinol
1. MS: (M+Na)+
Other examples of peptides of formula 1 are:
PhCHZCH2C0-Thr-N-Me-Asp(NEtz)-Asp(cyBu)-Leu-OH
[2-(2'-carboxy)biphenylyl]carbonyl-'Chr-Asp(NMez)-Asp(cyPr)-Leu-
OH
octanoyl-Ile-Asp(octyloxy)-Asp-Leu-IVHz
(CH3)zCHCHzCHzCO-NHCH(cyclohe:Kylmethyl)-CO-Asp(tridecyloxy)-
Asp(cyHx)-Leu-OH
2o~,~oa~
30
[3-methyl-2-(1-methylethyl)-1-oxobutyl]-Ile-Asp(pyrrolidino)-
Asp(cyHx)-Leu-OH
PhCHZCH2C0-Ile-N-Me-Asp(pyrrolidino)-Asp(cyBu)-Leu-OH and
(CH3)ZCHCH2CH2C0-Ile-N-Me-Asp(cxtyloxy)-Asp(cyPn)-Leu-OH.
