Note: Descriptions are shown in the official language in which they were submitted.
z3~7
SARCOSINEl D~HYDROAI.ANINE
ANGIOTENSIN II DER:[VATIVES
The present inven-tion relates to a group oE anyio-
-tensin II deriva-tives of the structure
R Rl
\C/ ',
11
Sar-Arg-Val-Tyr-Ile-His-Pro-NHCCO2H ~I)
whereln R is hydrogen, an alkyl radical contalning 1-8 carbon
atoms or a phenyl radical; Rl is hydrogen or an alkyl radical
containing 1-8 carbon atoms; and the stereochemical configura-
tion of each of tha optically active amino acid residues is
L or DL. ~
The abbreviations connote the amino acids defined ~ ~ ;
in accordance with the nomenclature rules published by the
IUPAC-IUB Commission on Biochemical Nomenclature, Archives
of Biochemistry and Biophysics, 150, 1-8 (1972).
Preferred compounds of the present invention are
those of formula (I) wherein all of the optically active
amino acid resi~ues are o~ the L-stereochemical configuration. ~
The alkyl radicals encompassed by the R and Rl ~; -
substituents are illustrated by methyl, e-thyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, and the branched-
chain isomers thereof.
Also equivalent to the compounds of formula (I)
for the purposes of this invention are the pharmaceutically
acceptable acid addition salts thereof. Such acid addition
salts can be derived from a variety of inorganic and organic
acids such as sulfuric, phosphoric, hydrochloric, hydrobromic,
hydriodic, nitric, sulfamic, citric, lactic, pyruvic, oxalic,
., ,~"
, . ' . .
3:a7
maleic, succinic, tartaric, cinnamic, acetic, trifluoroacetic,
benzoic, salicylic, gluconic, ascorbic and related acids.
The manufacture o-f the instan-t novel compounds is
conveniently`achieved by processes usually adapted to the
syn-thesis of peptides. Thus, -the C-terminal arnino acid,
optionally substituted with protec-ting groups, is coupled
with the appropriate N-protected amino acid to af~ord the
corresponding ~-protec-ted dipeptide. Removal of the N-pro-
tecting group is followed, similarly, by coupling with the
N~protec-ted amino acid required to produce the desired
tripeptide. This sequential procedure is repeated until the
desired octapap-tide derivative is produced. Formation of
the C-terminal ~,~-dehydro amino acid derivative, i.e.
dehydroalanine, dehydrophenylalanine, dehydroalkylalanine
and dehydrodialkylalanine, may be accomplished before coupling,
as exemplified in Example 20, or after coupling, as exemplified
in Example 14.
The aforementioned coupling processes are preferably
carried out in accordance with s-tandard organic chemical
techniques, whereby each intermediate peptide is produced
as described hereinbefore and isolated prior to coupling with
the next appropriate N-protec-ted amino acid active ester.
Alternatively, this sequential coupling process can be con-
ducted by solid phase peptide synthesis, which consists of
first attaching to a polymer support, e.g. chloromethylated
copolys-tyrene-1% divinylbenzene polymer, the optionally N-
protected C-terminal amino acid, followed by removal of the
N-protecting gr~up and coupling, in the presence of a suit-
able reagent, e.g. dicyclohexylcarbodiimide, successively
with each of the appropriate N-protected amino acids.
The compounds of the present invention can also be
~23~
prepared by coupling active esters of -the N protected peptide
blocks of -the appropriate size, which in turn can be obtained
by stepwise synthesis as described in the preceding paragraphs
~ollowed by removal of the protecting groups.
The compounds of the present invention resemble
angiotensin II except for the unnatural amino acids a-t the
N-and C~terminals. These compounds are pharmacological
agents particularly useful as angiotensin inhibitors and are
additionally advan-tageous in view of the fact that they
display antagonist activity without agonist activity. Their
inhibitory proper-ty is demonstrated in the ~ollowing assay
. procedures:
Virgin female Charles River rats weighing 200-250 y.
are injected 24 and 48 hours before use with diethyl
stilbesterol, 1 mg./kg. subcutaneously, dissolved in eorn
oil. The rats are sacrificed by cervical dislocation and
the uterus is removed and a section of the u-terine horns ~`
mounted in a 2 ml. tissue bath containing a modified Tyrode
solution maintained at 30C. and bubbled with 95% oxygen,
5% carbon dioxi~e. A series of control eontractions is
elicited by alternate additions of angiotensin II, anti-
diuretic hormone (~DH) and bradykinin. A solution of the
test compound is then substituted for the plain Tyrode ~-
solution and the treat contractions are obtained after an
equilibration period of 15 or 30 minutes. Regularly timed
contractions are elicited during the equilibration period
in order to maintain the timed sequence of agonis-t additions.
Three control and three treat contractions are averaged to
obtain the mean percent change. The compound is rated
active if it effects a significant decrease in the con-
tractions produced by the action of -the agonist.
~ ~23~
slood pressure is measured in Charles River albino
rats anesthetized with pentobarbital sodium (50 mg./kg.) and
pretreated with phenoxyhenzamine (30 m~./ky ) and propranolol
(15 mg./kg.) while maintaining body -temperature at 32C.
The pressure is recorded from the carotid artery with a P~100
linear core pressure transducer, Physiograph. Both juyular
veins are cannulated, one vein used for infusion of antagonists
and the other for bolus injections of anglotensin II. An
angiotensin II dose response curve is determined before each
tes-t of the antagonists so that each animal serves as its own
con-trol. An additional group of animals is tested to determine
the effects oE a 15-minute placebo infusion of saline on
angiotensin II responses. After de-termination of the angio-
tensin II dose response curve, a placebo or inhibitor infusion
is initiated and maintained for 15 minutes and continued ~
during the second dose response. Immediately after the -
initial 15 minute infusion period the dose response curve
is repeated. After the second dose response curve is obtained,
the infusion of the antagonist is s-topped. Thereafter,
representative doses of angiotensin II which produced
approximately 20-25 mm of mercury response in the control
period are injected at 10-15 minute intervals until the con-
trol response is obtained. The time after infusion for the
same response to appear is considered an indication of the
antagonist's duration of activity. Relative activity is
determined by comparing the ratio of the calculated doses
obtained from the dose response curves of anyiotensin II
necessary to increase blood pressure 25 ~n of mercury before
and after the inhibitor.
The invention will appear more fully from the
examples which follow. These examples are given by way of
illustration only and are no-t to b,e cons-trued as limitiny
the inven-tion ei-ther i'n spiri-t or in scope, as many modi-
fications both in materials and in me-thods will be apparent
to those skilled in the art. In these examples temperatures
are given in dec3rees Celsius, (C.) and quantities of ma-terials
in par-ts by weight unless otherwise noted. The stereo- :
Ghemical confi.guration of each of the opticall,v active amino
acids in the examples is L or DL.
3~7
EXAMPLE 1
__
21.7 g of t-butoxycarbonylvaline was dis~olved in
200 ml of methylene chloride. 22.2ml of N-methylmorpholine
was added to the solution. The solution was cooled to
-70C. 13.1 ml of isobutylchloroformate was then added.
The reaction mixture was warmed up to -15C. and stirred for
5 minutes. The reaotion mixture was again cooled to -70C-
23.2 g of tyrosine methyl ester hydrochloride was added to
- the reaction mixture. The reaction mixture was allowed to
warm ~o room temperature, and then was stirxed for 16 hours.
The reaction mixture was extracted three times with 0.5 M
potassium bisulfate and once with brine. The methylene
~; chloride solution was dried over anhydrous sodium sulfate,
filtered and stripped of solvent The residue was dissolved
in 200 ml of ethyl ether. The ether solution was added to
cold rapidly stirring Skellysolve B. The white precipi
tate was filtered and dried to afford the product t-butoxy-
carbonylvalyltyrosine methyl ester.
EXAMPLE 2
29.6 g of t-butoxycarbonylvalyltyrosine methyl
ester was dissolved in 255 ml. oP acetic acid. To this solu-
tion was added 128 ml of 6N hydrochloric acid in dioxane.
; ~he reaction was allowed to stand for five minutes and the sol-
vPnt was removed under vacuum. The residue was triturated
with ethyl ether. The resultant precipitate was filtered,
wa~hed with ethyl ether and dxied in a vacuum oven at 55C.
to afford valyltyrosine methyl ester hydrochloride.
; EX~UPLE 3
21.5 g of t-butoxycarbonylnitroarginine was dissolved
in 200 ml of methylene chloride~ 15.0 ml of N-methylmorpholine
--6--
",
3~7
was add~d to the solution. 5'he solution was cooled to -70C.
8.8 ml of isobutyl chloroformate was then added. The reac-
tion mixture was warmed up to -15C. and stirred for 5 minutes.
The reaction mi~ture was again cooled to -70~C. 22.3 g of
valyltyrosine methyl ester hydrochloride was added to the
reaction mixture. The reaction mixture was allowed to warm
to room temperature, and then was stirred for 16 hours,
rrhe reaction mixture was extracted three times with 0,5 M
potassium bisulfate and once with brine. The methylene
chloride solutivn was dried over anhydrous sodium sulfate,
filtered and stripped of solvent. The residue was dissolved
in 200 ml of ethyl ethér. The ether solution was added to
cold rapidly stirring Skellysolve B. The white precipitate
was filtered and dried to afford the product t-butoxycarbonyl~
5 nitroarginylvalyltyrosine methyl ester.
EXA~.PLE_4
30~9 g of t-butoxycarbonylnitroarginylvalyl
tyrosine methyl ester was dissolved in 177 ml. of a~atic
acid. ~o this solution was added 88.5 ml of 6N hydrochloriG
acid in dioxane. The reaction was allowed to stand for
five minutes and the solvent was removed under vacuum. The
residue was triturated with ethyl ether. The resultant
precipitate was filtered, washed with ethyl ether and dried
in a vacu~n oven at 55C- to aPfoxd nitroarginylvalyl-
5 tyrosine methyl ester hydrochloride.Ei~PLE 5
8.84 g of t-butoxycarbonylsarcosine was dissolved
in 200 ml of methylen~ chloride. 10.5 ml of N-methyl-
morpholine was added to the solution. rrhe solution was cooled
to ~70 C. 6.1 ml of isobutyl chloroformate was then added.
he reaction mixture was warrned up to -15Co and stirred for
3~7
5 minutes. The reaction mixture was again cooled to -70C~
25.5 9 of nitroarginylvalyltyrosine methyl esthex hydro-
chloride was added to the reaction mixture. The xeaction
mixture was allowed to warm to xoom temperature, and then
was stirred for 16 hours. The reaction mixture was extracted
three times with 0.5 M potassium bisulfats and once with
brine. The methylene chloride solution was dried over anhydrous
sodium sulfate, ~iltered and stripped of solvent. The
residue was dissolved in 200 ml of ethyl ether. The ether
solution was added to cold rapidly stixxing Skellysolve B
The white pxecipitate was filtered and dried to afford the
product t-butoxycarbonylsarcosylnitroarginylvalylt,yrosine
methyl esterD
E~A~PLE 6
10.0 g of t-butoxycarbonylsarcosylnitroarginyl-
valyltyrosine methyl ester was dissol~ed in 30 ml o methanol.
To this solution was added 60 ml of lN lithium hydroxide.
The reaction mixture was ~tirred for three hours, then
neutralized with lN hydrochloric acid to pH 7. The solvent
was removed by vacuum and the residue was dissolved in N,N-
dimethylformamide. The solution was filtered and the solvent
removed by vacuwn. The residue was dissolved in methanol/
2-propanol. The alcohol solution was added to 1 liter of
~old rapidly stirring ethyl ether. The white precipitate
was filtered and dried in vacuo to afford t-butyloxycarbonyl-
sarcosylnitroaryinylvalyltyrosine.
~A~PLE 7
-
21.5 g of t-butoxycarbonylproline was diqsolved in
200 ml of methylene chloride. 22.2 ml of N-methylmorpholine
was added to the solutlon. The solution was cooled to -70C,
13.1 ml of isobutyl chloroformate was then added. The
--8--
, ., ..., .... ~
reaction mixture was warmed up to -15C. and stirred for
5 minu~es. The reaction mixture was again cooled to -70~.
15.6 g of serine methyl ester hydrochloride was added to the
reaction mixture. The reaction mixture was allowed to warm
to room temperature, and then stirred fox 16 hours. The
reaction mixture was extracted three times with 0.5~
pota~sium ~isulfate and once with brine~ The methylene
chloride solution was dried over anhydrous sodium sulfate,
filterPd and stripped of solvent. The residue was dissolved
in 200 ml of ethyl ether. The ether solution was added to
cold rapidly stirring Skellysolve B. The white precipi-
tate was filtexed and dried to afford the product t-butoxy-
carbonylprolylserine methyl ester.
E~A~PLE 8
__
24.4 g of t-butoxycarbonylprolylserine methyl
ester was dissolved in 262 ml o dioxaneO To this solu-
tion was added 141 ml. of 6N hydrochloric acid in dioxane.
The reaction was allowed to stand for ten minutes and the
solvent was removed under vacuum. The residue was triturated
20 with ethyl ethex. The resultant product, prolylserine
methyl ester hydrochloride, was a clear glass.
E~A~PLE 9
23,3 g of t-butoxycarbonylisoleucine was dissolved
in 200 ml of methylene chloride. 33,3 ml of N-methyl-
morpholine was added to the solution. The solution was cooledto -70C. 13.1 ml of isobutylchloroformate was then addedO
The reaction mixture was warmed up to -15C, and stirred for
5 minutes. The reaction mixture wa~ again cooled to -70C-
Z6.0 g o histidine methyl ester dihydrochloride was added
to ~he reaction mixture. The reaction mixture was allowed
to warm to room temperature, and then was stirred or 16
_9_
3~
hours. The r~actlon mixture was extracted three times with
0.5 M potassium bisulfate and once with brin~. The methylene
chloride solution was dried over anhydrous sodium sulfate,
filtered and stripped of solvent. The xesidue was dissolved
in 200 ml. of ethyl ether. The ether solution was added to
cold rapidly stirring Skellysolve B. The white precipi-
tate was filtered and dried to aford the product t-butoxy-
carbonylisoleucylhistidine methyl estèr.
~PLE 10
29.4 g of t-butoxycarbonylisoleucylhistidine
methyl ester was dissolved in 150 ml of methanol. To this
solution was added 308 ml of lN lithium hydroxide. The
reaction was stirred for three hours, then neutralized with
lN hydrochloric acid to pH 3. The solven~ wa~ removed by
vacuum and the residue was dissolved in N,N-dimethylformamide.
The solution was filtered and the solvent removed by vacuum.
The residue was dissolved in methanol/2-propanol. The alcohol
solution was added to 2 liters of cold rapidly stirring,ethyl
ether. The white precipi ate was filtered and dried in vacuo
0 to affoxd t-butoxycarbonylisoleucylhistidine hydrochloride.
E~A~PLE 11
10.1 g of t-butoxycarbonylisoleucylhistidine
hydrochloride and 6.3 g of pxolylserine methyl ester hydxo-
chloride were dissolved in 100 ml of dimethylformamide. The
solution was,cooled to 0C. To the solution was added 3.4 g
of l-hydroxybenzotriazole, 2.8 ml of N-methylmorpholine
and 7.6 g of dicyclohexylcarbodiimide. The reaction
mixture was stirred for two hours at 0C. and then wa~ allowed
to stand at 4C. ~or sixteen hours. Dicyclohexylurea was
filtered from the reaction mixture and was washed with ace-
tone. 300 ml of ethyl acetate was added to the solution.
-10-
,. ~
~7
The solution was extracted three times with 300 ml saturated
potassium bicarbonate and two times with 300 ml of brine.
The product was extracted into 300 ml of 2M potassium bisul-
fate. The acidic solution was neutralized with potassium
caxbonate. The slightly basic aqueous solution was extracted
three times with 300 ml o methylene chlorideO The methylene
chlorlde solu~ion, which contained the product, was dried o~er
sodium sulfat ~ filtered and stripped of solvent under
vacuum. The residue was dissolved in 100 ml of ethyl acetate.
The svlution was added to 1 liter of cold rapidly stirring
Skellysolve B. The white precipitate was filtered and
dried under vacuum at 55C. to afford the product t-but
carbonylisoleucylhistidylprolylserine methyl ester.
EXA~PLE 12
.
8.5 g o t-butoxycarbonylisoleucylhistidylpxolyl-
serine methyl ester was dissolved in 51 ml. o~ dioxane. To
this solution was added 25.5 ml of 6N hydrochloric acid in
dioxane. The reaction was allowed to s,tand for ten minutes
and the solvent was removed under vacuum to afford isoleucyl- -
histidylprolylserine methyl ester hydrochloridea
~ LE 13
9.8 g ~f t-butoxycarbonylsarcosylnitroarginyl-
valyltyrosine and 7.6 g of isoleucylhlstidylprolylserine
methyl ester hydrochloride were dissolved in 100 ml of di-
methylforamide. The solution was cooled to 0C. To thesolution was added 2.03 g o~ l-hydroxybenzotriazole, 1.7 ml
of N-methylmorpholine and 4O64 g of dicyclohexylcarbodiimide.
-~ The reaction mixture was stirred for two hours at 0C. and
then was allowed to stand at 4C. for sixteen hours. Dicyclo-
hexylurea was filtered from the reaction mixture and was
washed with acetone. 300 ml of ethyl acetate was added to
11-
the ~olution. The solution was extracted three times with
300 ml saturated potassium bicarbonate and two times with
300 ml of hrine~ The product was extracted into 300 ml o~
2~ potassium bisulfate. The acidic solution was neutralized
with potassium carbonate. The slightly basic aqueous solu~
tion was extracted three times with 300 ml of methylene
chloride. The methylene chloride solution, which contained
the product, was dried over sodium sulate, filtered and
stripped of solvent under vacuum. The residue was dissolved
in 100 ml of ethyl acetate. The solution was added to 1
liter of cold rapidly stirring Skellysolve B. The white
precipitate was filtered and dried under vacuum at 55C.
to afoxd the product t-butoxycarbonyl~arcosylnitroarginyl-
valyltyrosylisoleucylhistidylprolylserine methyl ester.
lS PLE 14
3.0 g of t-butoxycarbonylsarcosylnitroarginyl- -
valyltyrosylisoleucylhistidylprolylserine methyl ester was
dissolved in 20 ml of pyridine and 10 ml of triethylamine.-
The solution was cooled in an ice bath. 0.51 g of tosyl
chloride was added to the solution. At ten minute inter-
vals, three additional 0.51 g batches of tosyl chloride were
added. After standing ~or two hours, the reaction mixture
was filtered. 200 ml of methylene chloride was added to the
reaction. The solution was extracted three times with 5%
ammonium hydroxide in water. ~he solution was then dried
over sodium sul~ate and stripped under vacuum~ The residue
was taken up in 2% methanol/methylene chloride and passed
through a silica gel column to remove the colored impurities
and a~ford the pure product t-butoxycarbonylsarcosylnitro-
arginylvalyltyrosylisoleucylhistidylprolyldehydroalaninemethyl ester.
-12-
~1~23~
~ ~ PLE 15
2.09 g of t-butoxycaxbonylsarcosylnitroaryinyl-
valyltyrosylisoleucylhistidylprolyldehydroalanine methyl
~ster was dissolved in 30 ml of dioxane. To this solution
was added 60 ml. of lN lithium hydroxide. The reaction
mixture was stirred for three hours, then neutralized with
1~ hydrochloric acid to p~ 7. The solvent was removed
by vacuum and the residue was dissolved in N,N-dimethyl
ormamide. The solut,ion was filtered and the solvent removed
by vacuum. The residue was dissolved in dioxane and added
to 300 ml of cold rapidly s~irring ethyl ether. The preci-
; ` pitate was filtered and dried in vacuo to afford t-butoxy-
carbonylsarcosylnitroarginylvalyltyrosylisoleucylhistidyl-
prolyldehydroalanine.
E~AMPLE 16
1.07 g of t-butoxycarbonylsarcosylnitroarginyl- ,~
valyltyrosylisoleucylhistidylprolyldehydroalanine was stirred
for thixty minutes at C. in 10 ml of hydrogen fluoride
and 1 ml of anisole. The crude product was run in a counter-
current distribution system of n-butanol:acetic acid:water
(4 :1 :5) for 200 transfers. The solvent was removed under
vacuum. The residue'was dissolved in water and lyophilized
to af~ord the product sarcosylarginylvalyltyrosylisol~ucyl-
histidylprolyldehydroalanine diacetic acid salt.
' A~PLE 17
52.8 g o ~-phenylserine was dried under vacuum at
C for four hours. Thè anhydrous amino acid was s,uspended
-13-
... . , . , .. ~
~VZ~7
in 250 ml of methanol and the mixture was cooled to 10C.
96.4 ml of ~hionyl chloride was added gradually, keeping
the temperature below 10C. The mixture was stirred for
24 hour~ a~ room temperature. The clear solution was stripped
to dryness and the residue shaken with ether to af~ord
~-phenylserine methyl ester hydrochloride~
~MPLE 18
61.0 g of ~-phenylserine methyl ester hydrochloride
was finely powdered and suspended in 500 ml of methylene
chloride. 62.3 g of t-butoxycarbonylproline was then added,
followed by 30~2 ml of N-methylmorpholine. A clear solu-
tion was obtained after stirring for one-half hour under
reflux. The solution was cooled to oC. and 59.6 g of
dicyclohexylcarbodiimide in lO0 ml of methylene chloride
was added~ with stirring~ After stand:ing for 16 hours at
room temperature, the dicyclohexylurea was removed by filtra-
tion. The iltrate was washed twice with 0.5 M potassium
bisul~ate. The solution was then dried over sodium sulfatè
and the solvent removed in vacuo. The product was shaken
with Skellysolve B, and the product was filtered off and
dried to afford t-butoxycarbonylprolyl-~-phenylserine
methyl ester.
EXAMPLE l9
3.92 g o~ t-butoxycarbonylprolyl-~-phenylserine
methyl ester was dissolved in 40 ml o~ pyridine and the
solution was cooled to 0C. 2.30 g of p-toluenesulfonyl
chloridé was added and the mixture was stirred at 0C. until
a clear solution resulted. The solution was allowed to stand
for 16 hours at room temperature. The pyridine was stripped
off under vacuum at room temperature. The residue was
dissolved in chloroform, and the chloroform solution was
washed three times with 0.5 M potassium bisulfate. The
-14-
, . .... . _
solution was dried over sodium sulfate and the solven-t was
stripped off to afford th~ product, a brittle foam, t-butoxy-
carbonylprolyl-o-tosyl~ phenyl) serine methyl ester.
~XA~PLE 20
5.00 g of t-butoxycarbonylprolyl-o-tosyl-(~-
ph~nyl) sexine methyl ester was dissolved in 50 ml o ethyl
acetate and 1.81 g of dicyclohexylamine was added. A~ter
standing for 16 hours, the dicyclohexylammonium tosylate
was removed by filtration and washed with ethyl acetate
The combined filtrates were washed twice with 0.5 M potassium
bisulfate, dried over sodium sulfate, and the solvent was
stripped off. The residual oil was chromatographed on
neutral sili¢a using benzene as the eluting solvent to
afford the purified solid product t-butoxycarbonylprolyl-
dehydrophenylalanine methyl est~r.
~PLE 21
3.74 g of t-butoxycarbonylprolyldehydrophenyl-
alanine methyl ester was di solved in 75 ml of ether. The
solution was cooled to 0C. and saturated with hydrogen chlo~
ride gas. After a few minute~, the produck, prolyldehydro-
phenylalanine methyl ester hydrochloride, began to
crystallize. The mixture was allowed to stand for one
houx at room temperature, and the product was filtered
off and washed with ethyl ether.
A~PLE 22
6.20 g of proly~dehydrophenylalanine methyl
ester hydrochloride was di~solved in 100 ml of dimethyl-
formamide and 7.36 g o~ t-butoxycarbonylisoleucylhistidine
was added, followed by 2.70 g of l-hydroxybenzotriazole,
The solution was cooled to 0C and 4.16 g o dicyclohexyl-
carbodiimide was added in one portion. Th~ mixture was
~V~L7
stirred ~or one houx at room temperature, and then allow~d
to stand for 16 hours. The dicyclohexylurea which crystal-
lized was removed by filtration and the solvent was removed
under high vacuum at 40C. ~he viscous liquid which
S remained was shaken with a large volume of water. The water
was then decanted and the gummy product was rubbed with
ether. The resulting product was purified by countercurrent
distribution, 400 transfers, in a system o~ methanol:water:
chloroform:carbon tetrachloride (37:10:26:27) to afford
t-butoxycarbonylisoleucylhistidylprolyldehydrophenylalanine
methyl ester.
A~PLE 23
6.24 g of t-butoxycarbonylisoleucylhistidylprolyl-
dehydrophenylalanine methyl ester was dissolved in 125 ml
of ethyl acetate. The solution was cooled in an ice bath ,
and dry hydrogen chloride gas was bubbled in for one hour.
The product began to separate after a few minutes. At
the conclusion of the reaction, the salt was filtered off
and washed with ethyl,acetate to yield pure isoleucyl-
histidylprolyldehydrophenylalanine methyl ester dihydro-
- chloride.
E~A~PLE 24
6.52 y of ~-butoxycarbonylsarcosylnitroarginyl-
valyltyrosine (Example 6) was dissolved in 130 ml of methanol:
acetic acid:water (8 :1:1). O.i g of palladium black was
added and the mixture was hydrogenated at 60 psi and room
temperature for 24 hours. The solvents were stripped off,
the residue was dissolved in 50 ml of water, and the solu~
tion was lyophilixed. The residual powder was dissolved in
50 ml of water. 10 ml o lN hydrochloric acid was added
and the solution lyophilized to give t-butoxycarbonyl-
-16-
sarcosylarginylvalyltyrosine hydrochloride
EXA~PL~ 25
.
6.44 9 of -t-butoxycarbonylsarcosylarginylvalyl-
tyrosine hydrochloride and 5.97 g sf isoleucylhistidyl-
prolydehydxophenylalanine methyl ester dihydrochloride were
dissolved in 100 ml of dimethylformamide. 1.01 m~l of N-
methylmorpholine was added, follvwed by 2.70 g of l-hydroxy-
benzotriazole. The solution was cooled in an ice bath and
2.29 g of dicyclohexylcarbodiimide was added. The solution
was stirred for 16 hours at room temperature. The dicyclo-
hexylurea was filtered o and the filtrate was stripped to
dryness under high vacuum. The residue was shaken with a
large volume of water~ The product was then filtered and
washed thoroughly with water to afford the pure product
t-butoxycarbonylsarcosylarginylvalyltyrosylisoleucylhisti-
dylprolyldehydrophenylalanine methyl ester dihydrochloride.
EX~PLE_26
11.86 g of t-bu~oxycarbonylsarcosylarginylvalyl-
tyrosylisoleucylhistidylprolyldehydrophenylalanine methyl
ester dihydrochloride was dissolved in 330 ml of methanol
and the solution was cooled in an ice bath. 110 ml of lN
sodium hydroxide was added dropwise with rapid stirring over
one-half hour. The solution was removed from the ice bath
and the stirring was continued at room temperature for one
additional hour. 6.0 y o acetic acid was added and the
solution was stripped to dry~ess. The residue was shaken
with water and the crude product was filtered and washed with
water to afford t-butoxycarbonylsa'rcosylarginylvalyltyro-
sylisoleucylhistidylprolyldehydrophenylalanine hydrochloride.
The product was purified by partition chromato-
graphy on LH-20 as follows: 200 g of dry gel was swollen
-17-
,, ~
3~'7
by s-~irring the gel for 16 hours in two liters of the lower
phase from ~utanol:acetic acid:w~ter ~4:1:5~. The swollen
gel was packed into a column and washed with 2 liters of the
upper pha~e. The product was dissolved in a minimum of upper
phase and pipet-ted onto the col~nn in a thin band, which
was allowed to soak in. The column was eluted with upper
phase at a flow rate of 60 ml per hour~ and 25 ml fractions
were collected. The fractions containing the pure product
were pooled and stripped to dryness.
E~A~PLE 27
5.68 g of t-butoxycarbonylsarcosylarginyl-
valyltyrosylisoleucylhistidylprolydehydrophenylàlanine
hydrochloride was dissolved in 25 ml of acetic acid, and
50 ml of 6N hydrogen chloride in dioxane was added. The sol-
ution was allowed to stand for one hour at room tempera-
ture and the solven was removed under vacuum. The residue
was stirred with ether and the product was filtered and
washed with ether. The final product, sarcosylarginyl-
trihydrochlo~ide, was homogeneous on thin layer chromato-
graphy in n-butanol:acetic acid:water (7:1:2).
EX~PLE 28
2-Phenyl-4-isopropylidene-5-oxazolinone and other
2-phenyl-4-alkylidene-5-oxazolinones were prepared
accordiny to the method described in E. Baltazzi, et.
al., Chemistry and Industry, February 13, 1954, p. 191
4.6g of sodium metal were added to 1200 ml f
methanol. The sodium was allowed to react with the
methanol, then 149g of benzyl mercaptan were added
followed by 201g of 2 phenyl-4-isopropylidene-5-oxazolinone.
The reaction mixture was stirred for six hours then
neutralized to pH5 with glacial acetic acid. The
methanol was removed under vacuum. The residue was
-18-
~C12~7
subjected to colun~ chromatography on silica gel in 50%
hexane-50~ chloroform to remove the excess benzyl mercaptan.
This aEforded pure procluct, methyl N-benzoyl-S-benzyl
pen:icillaminate.
~X~PLE 29
1000 ml of concentxated hydrochloric acid, 1000 ml of
90% formic acid and 1000 ml of distilled water were added
to 286 g methyl N-benzoyl~S-benzylpenicillaminate. The
reaction mixture was refluxed for forty-eight hours. The
solvents were removed under vacuum, and residue was washed
copiously with hot diethyl ether. The resultant precipitate
was pure product, S-benzylpenicillamine hydrochloride.
EXAMPLE 30
1000 ml of methanol were cooled to -20C. To this was
added dropwise 171 g of thionyl chloride. After the thionyl
chloride had been added, 199 g of S-benzylpenicillamine
hydrochloride were added. The reac-tion mixture was re- -
fluxed for sixteen hours. The methanol was removed under
vacuum. Fresh methanol was added to the reaction mixture
then removed under vacuum. llhe residue-was triturated
wlth die-thyl ether. The precipitate was filtered and washed
with diethyl ether. This resulted in pure product, methyl
S-benzylpenicillaminate hydrochloride.
EXAMPLE 31
:
146 g of t-butoxycarbonyl proline were dissolved in
1500 ml of methylene chloride. 150 ml of N-methylmorpholine
were added to the solution. The solution was cooled to
-70C. 89 ml of isobutylchloroformate were added. The
renction mix-ture was warmed to ~15C. and then cooled to
~70C. 197 g of methyl S-benzylpenicillarninate hydrochloride
--19--
~z~
were added to the reaction mixture. The reaction mixture
was stirred for sixteen hours at room temperature. The
reaction mixture was extracted three times with l~OM
potassum bisulfate and once wit~l brine. The methylene
chloride solution was dried ovex anhydrous sodiurn sulfate~
~iltered and stripped of solvent. The residue was dissolved
in a minimum of diethyl ether. The ether solution was added
to cold rapidly stirring hexane. The white precipitate
was filtered and dried to afford the product, methyl
t-butoxycarbonylprolylpenicillaminate.
EXAMPLE 3
230 g of methyl t-butoxycarbonylprolylpenicillaminate
were dissolved in 1000 ml methylene chloride. 167 g of
methyl trifluoromethylsulfonate were added to the solution.
The reac-tion mixture was stirred for six hours. 500 ml of
eth~nol were added to the reaction mixture. The solvents
were removed from the reaction under vacuum.
The residue was dissolved in 1000 ml of methylene
chloride. 1~2 ml of triethylamine were added to the
solution. The reaction mixture was allowed to stand for
two hours. The solvent was removed under vacuum. The
residue was subjected to column chromatograplly on silica
gel in 20% hexane-80-% chloroform. This afforded pure
product, methyl t-butoxycarbonylprolyl-2,3-dehydrovalinate.
E~MPL~ 33
.. .. _ .
16.3 g o~ methyl t-butoxycarbonylprolyl-2,3-dehydro-
valinate were dissolved in 100 rnl of methanol. 150 ml of
lM sodium hydroY.ide were added to the solution. The
solution was stirred for six hours. The solution was
neutralized to p~l3 with lM potassium blsulfate. The solvent
-20-
~C12~
was removed under vacuum. The residue was dissolved in
ethyl acetate~ 'Lhe inorganic salts were filtered from
the ethyl acetate solu-tlon. The ethyl acetate was
removed under vacuum to yield the product, t-butoxycar~onyl-
prolyl-2,3-dehydrovaline.
_Y~MPLE 34
- 1.2 g of t-butoxycarbonylprolyl-2,3-dehydrovalille
were dissolvecl in 70 ml of dimethylformamide. 0.62 g of
cesium carbonate and 10 g of chloromethylated polystyrene
2% divinylbenzene resin (0.35 milliequivalents/gram) were
added to the solution. The reaction mixture was stirre~
for thirty-six hours at 60C~ The resin was fil-tered and
washed with dimethylformamide, water, 2-propanol, and
metllylene chloride. The resin was dried at 70C. under
vacu~n to yield the diepeptide t-butoxycarbonylprolyl-2,3-
dehydrovaline which was attached to the resin by a benzyl
ester linkage.
EX~MPLE 35
To attach t-butoxycarbonyl-im-tosylhistidine to the
butoxycarbonylprolyl-2,3-dehydrovaline resin an~ add
the subsequent amino acids to obtain the desired peptide the
following sequence was repeated for each amino acid on an
automated peptide synthesizer.
The t-butoxycarbonyl protecting group of lOg oE
t~butoxyprolyl-2,3-dehydrovalyl resin was removed with 40
trifluoroacetic acid-60~ methylene chloride. This resin
was treated twice with the acid solution to ensure the removal
of the t-butoxycarbonyl group. The excess acid was rinsed
off with methylene chloride followed by 2-propanol and the
sequence of rinsing with methylene chloride followed by
2-propanol was repeated sevcral times.
-21-
Tile trifluoracetate salt of the peptide resin was
neutralized Wi-til N,N-diisopropylethyl amine. The excess
amine was rinsed off with methylene chloride followed by
2-propanol as above.
The deblocked pep-tide resin had attached to i-t
t-butoxycarbonyl-im-tosylhidtidine via dicyclohexylcar-
bodiimide. t-Butoxycarbonyl-im-tosylhistidine and dicyclo-
hexylcarbodiimide were added in two-fold excess. 2.36 g of
t-butoxycarbonyl-im-tosylhistidine dissolved in 2S ml of
methylene chloride and 1.46 y of dicyclohexylcarbodiimide
dissolved in 25 ml methylene chloride were added to the
resin. The reaction was agitated for two hours. -The coupling
was repeated. The resin was rinsed as before with methylene
chloride followed by 2-propanol. The resin was acetylated
two times with 0.4M acetic anhydride in methylene chloride.
The resin was rinsed as before with methylene chloride
followed by 2-propanol.
The above sequence was r~peated for the addition of
each amino acid until the desired pept:ide, t-butoxycarbonyl-
sarcosyl-N -nitroarginylvalyl-0-2-bromobenzyloxylcarbonyl-
tyrosylisoleucyl-im-tosylhistidylprolyl-2,3-dehydrovalyl
resin was obtained.
EXAMPLE 36
100 ml of liquid hydrogen fluoride was added to 10 g
of the protected peptide resin from the previous example
in 10 ml of anisole. The reaction was stirred for twenty
minutes at 4C. The hydrogen fluoride was removed under
water aspirator vacuum with a stream of nitrogen gas. The
resin was washed with eidthyl ether to remove the anisole.
The resin was then washed three times with 25 ml of distilled
water. The water was removed from the product under vacuum.
-22-
3~
~he residue was subjected to column chromatography on siLica
gel which had been modified with octadecyltrichlorosilane.
This afforded pure product, sarcosylargirlylvalyltyrosyl-
isoleucylhistidylprolyl-2,3-dehydrovaline.
EXAMPLE 37
. Substitution of 2-phenyl-4-methylene-5-oxazolinone for
the 2-phenyl-4-isopropylidine-5-oxazolinone in Example 28
and substantial repetitlon of the procedures described in
Examples 28 through 36 gave sarcosylaryinylvalyltyrosyl~
isoleucylhistidylprolyldehydroalanine.
~ bstitution of 2-phenyl-4-benzylidine-5-oxazol.inone
for the 2-phenyl-4-isopropylidine-5-oxazolinone in ~xample
28 and subs:tantial repetition of the procedures described
in Examples 2~ through 36 gave sarcosylarginylvalyltyrosyl-
isoleucylhistidylprolyldehydrophenylalanine.
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Z3~7
EXAMPL~ 38
Described below are typical pharmaceutical composi-
tions con-taining the compounds of this invention.
SUPPOSITORY
,
Inyredient Amount (mg.)/Suppository
, ~ ,
A compound of the instant invention
(e.g., sarcosyl-L-arginyl-~valyl-
L-tyrosyl-L-isoleucyl-L-histidyl-L-
prolyl-dehydroalanine 10.0 mg.
10Theobroma Oil (Cocoa Butter) 990.0 mg.
The cocoa butter was melted, preferably on a
water or steam bath to avoid overheating, then the active
ingredient was suspended in the melt. Finally, the mass
was poured into cooled metal molds, whi.ch were chrome
plated and the melt was readily solidified. -
Other acceptable pharmaceutical carriers for a
suppository product are exemplified by trigylcerides of
oleic, palmitic and stearic acids (cocoa butter),
partially hydrogenated cottonseed oil, branched saturated
fatty alcohols such as Suppository base G, hydroyenated
coconut oil triglycerides of C12-C18 fatty acids, water
dispersible vehicles such as the polyethylene glycols
glycerin, gelatin, polyoxyl 40 stearates, polyethylene-4-
sorbitan monostearates, and materials which can raise the
melting poi.nt of the suppository base, such as beeswax,
spermaceti, eta.
.
-24-
L'AI~LN'LI,L L
Ingredlent ~mount (my.) /5 cc.
A comi~ound of the instant invention
(e.g., sarcosyl-L-arginyl~L-valyl-
L-tyrosyl-L-iso]eucyl-L-histidyl-L-
prolyl-dehydroalanine 1.0 my.
Phosphate buffer 1.0 ml.
Water for Injection, U.S.P. q.s. 5.0 ml.
The active ingredient was dissolved in the phosphate
buffer and water for lnjection, the solution was filtered
and filled into ampuls and ampuls were sealed. Sterili-
zation of the ampuls was achieved by an appropriate
sterilization procedure~
Other acceptable pharmaceutical carriers for a
parenteral product are exemplified by vegetable oils such
as peanut, corn, cottonseed, sesame oil, benzyl alcohol,
saline, phosphate buffer, ethylene gl~col polymers,
urea, dimethylacetamide, triton, dioxolanes, ethyl car-
bonate, ethyl lactate, glycerol formal, isopropyl myristate,
surfactants (nonionic, cationic, anioIIic), polyalcohols,
ethanol.
In the compositions of the type described above,
the novel compounds of this invention are present in an
amount envisioned to produce the desired effect. ~lthough 1.0 or
10.0 mg.per unit dose is often convenient, considerably
more or less active inyredient can be incorporated into
each dosage unit if so desired. The daily dosage of these
compounds is dependen-t upon various factors such as the
particular compowld ernployed, the condi-tion ~or which the
compound is administered and the patient's individual
response.
The matter contained in each of the eollowing
claims is to be read as part of the general description
of the present invention.
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