Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
KX37
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This invention relates to novel esters and amides
of the nonapeptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro
which have been found to inhibit the hypertensive effect
of angiotensin I.
The action of the enzyme renin on renin substrate,
a pseudoglobulin in blood plasma, produces a polypeptide
angiotensin I, also known as hypertensin I. The latter is
converted by an enzyme to angiotensin II, also known as
hypertensin II or angiotonin. Angiotensin II is an active
pressor substance which is present in the plasma of indivi~
duals with hypertension in quantities sufficient to maintain
elevated blood pressure. Inhibition of the enzyme responsible
for the conversion of angiotensin I to angiotensin II serves
to remove a cause of essential hypertension.
U. S. Patent No. 3,832,337 to Ondetti et al. dis-
closes various peptides and acylated peptides which inhibit
enzymatic conversion of angiotensin I into angiotensin II,
among such peptides being
Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro.
Studies on structure-activity relationships concerning pep-
tides as disclosed in U. ~. Patent. No. 3,~32,337 indicate
that a ~ree terminal carboxyl group is needed to obtain
potent inhibitors ln vitro and ln vivo. For example, see
the paper by Cushman et al., "Inhibition of Angiotensin-
Converting Enzyme by Analogs o~ Peptides from Bothrops
jararaca Venom", Experienta 29, 1032 (1973), Birkhauser
Verlag, Basel, Switzerland. However, it has now been ~ound
that not only is the above nonapeptide an inhibitor of angio-
tensin I induced hypertension, but esters and amides of such
nonapeptide are useful for such purpose as well. This is
KX37
~6~3S~3
surprising inasmuch as such esters and amides show ver~ low
or no detectable enzyme inhibition activity in vitro.
Generally, ln vitro activity of peptides in this area usu-
ally corresponds to in vivo activity thereof. Accordingly,
.
it is indeed unexpected that such esters and amides are
potent inhibitors of angiotensin I induced hypertension.
The present invention provides a peptide having the
formula
Pyr-Trp-Pxo-Arg-Pro-Gln-Ile-Pro-Pro-
~
wherein Y is -NH2 or -OR where R is an alkyl group contain-
ing 1 to 10 carbon atoms.
qlhe present invention also provides a process for
preparing a peptide having the formula
Pyr-l'rp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-Y
wherein Y is -NH2 or -OR where R is an alkyl group contain-
ing 1 to 10 carbon atoms which comprises coupling or reacting
suitable amino acids and peptides or their reactive deri~a-
~ives in a per se conventional procedure adopted for peptide
2.0 s~nthesis to form the peptide
Pyr-'rrp-Pro-Arg-Pro-(31n-~le-Pro-Pro
and before, during or after the formation of this peptide,
adding an alkyl ester group or an amide group to the terminal
Pro by conventional procedures.
Unless otherwise indicated in the following speci~i-
cation and claims, all amino acids are of the L-configuration.
In describing the peptides of the present invention,
the following abbreviations will be used throughout the
specification and claims:
KX37
~L~6~ 8
Arg - Arginine
Gln - Glutamine
Ile - Isoleucine
Pro - Proline
Pyr - Pyroglutamic acid
Trp - Tryptophane
The following peptides of the present invention have
been found to be effective in inhibiting angiotensin I in~
duced hypertension:
Ia - The esters having the formula
Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OR, ~
wherein R is alkyl :
II - The amide having the formula
Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-NEI2
The alkyl group o the above esters may comprise a
straight or branched chain hydrocarbon fragment containing
1 to 10 carbon atoms, and preferably 1 to 6 carbon atoms,
such as methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, -
t-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl, as
well as the various isomers of the latter six groups. The
methyl and t-butyl esters are particularly preferred.
The esters of the lnvention are pre~erably prepared
by reaction o~ a di-, tri-, tetra-, penta-, hexa-, hepta-
or octa-peptide including a Pro- terminal group protected
by an alkyl ester with one or more peptides, as is necessary, ~
employing conventional peptid0 preparatory techniques, to :
form the nonapeptide alkyl ester (Ia) of the invention. In
this manner, the ester of the invention may be formed directly
without having to first form the parent peptide ~:
(III) Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro
--3--
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~0~58S8 KX37
The amide of the invention is preferably prepared
by reaction of a di-, tri-, tetra-, penta-, hexa-, hepta-
or octa-peptide including a Pro-terminal group protected
by an amide group with one or more peptides, as is necessary,
employing conventional peptide preparatory techniques, to
form the nonapeptide amide (II) of the invention. In this
manner, the amide of the invention may be formed directly
- without having to first form the parent peptide (III).
However, it wlll be appreciated that the esters as
well as the amide of the invention may be formed by first
forming the parent peptide III in accordance with the pro-
cedures as outlined in U. S. Patent No. 3,832,337 and there-
after adding the alkyl ester or amide group to the terminal
Pro employing conventional procedures.
The compounds of the present invention inhibit the
conversion of angiotensin I into angiOtensin II in vivo and
thereby antagonize the hypertensive effect of angiotensin I.
The present compounds are capablé of inhibiting the hyper-
tensive effect of angiotensin I when administered to mammals
such as rats, mice or dogs, in a dosa~e level of from about
0.5 to about 10 mg/ky. For the latter purpose they may be
administered parenterally by incorporating the appropriate
dosage with a physiologically acceptable carrier.
~ KX37
~5~
he following examples illustrate the present
invention without, however, limiting the same thereto.
All temperatures are expressed in degrees Centigrade.
Example 1
Benzyloxycarbonyl-isoleucyl-proline-proline
Prolyl-proline hydrobromide (4.24 g) is dissolved
in a mixture of dimethylformamide (40 ml) and triethylamine
(2O8 ml). Benzyloxycarbonyl-isoleucine p-nitrophenyl ester
(9.27 g) and l-hydroxybenzotriazole (3 g) are added and the
mixture is stored at room temperature for 16 hours. Dimethyl-
aminopropylamine (1 ml) is added and after 2 hours the solvent
is removed ln vacuo. The residue is dissolved in ethyl acetate
(200 ml) and washed se~uentially with 0.1 N hydrochloric '
acid, and water. The organic phase is dried over magnesium
sulfate and concentrated to dryness ln vacuo to yield 13.8 g
solid. This material is applied to a silica gel column ~-~
(300 g) in ethyl acetate and eluted with the same solvent.
The fractions containing the desired above-titled tripeptide
are pooled and concentrated to dryness.
Example 2
Methyl benzyloxYcarbonyl-isoleucyl-proly-l-prolinate
The tripeptide acid oE Example 1 (~.6 g) is dissolved
in methanol and treated with an ethereal solution of diazo-
methane until a persistent yellow color is obtained. After
0,5 hour a few drops of acetic acid are added to discharge
. ,, , , ~
KX37
6~
the yellow color; the solvent is removed in vacuo and replaced
with ethyl acetate. This solution is washed sequen-tially with
0.1 N ilCl, water, sa-turated sodium bicarbonate and water.
The organic phase is dried over magnesium sulfate and
concentrated to dryness to yield the title compound.
xample 3
~thyl benzyloxycarbonyl-c3lutaminyl-isoleucyl-prolyl-prolinate
The tripeptide of Example 2 (1.4 g) is dissolved in 95
ethanol (30 ml) and N hydrochloric acid (3 ml.) Palladium on
charcoal (0.3 g) is added and the suspension is stirred
under a positive hydrogen pressure until no more carbon
dioxide is detected in the outcoming gases. The catalyst
is removed by filtration and the filtrate is concentrated to
dryness in vacuo. The residue is dissolved in a mixture of
.. ...
dimethylformamide (6 ml) and triethylamine (0.5 ml). Benzyloxy-
carbonyl-glutamine p-nitrophenyl ester (1.6 g) and l-hydroxy-
benzotriazole (0.5 g) are added and the reaction is allowed to
proceed at room temperature until the ninhydrin test is
negative. Dimethylaminopropylamine (0.5 ml) is added and the
reaction is allowed to proceed for another hour. Ethyl
acetate (200 ml) is added and -the enSuinCJ solu-tion is washed
neutral. The organic phase is dried over magnesium sulfate
and the solvent :is removed in vacuo to yield thc title co~pound.
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31L(~6~
Example 4
Methyl benzyloxycarbonyl-~-nitroarqinyl-prolyl-qlutaminyl-
isoleucyl-prolyl-prolinate
The tetrapeptide of Example 3 (14 y) is dissolved in
absolute ethanol (325 ml) and N hydrochloric acid (23 ml).
Palladium on charcoal (3.0 g) is added and the mixture~is
stirred under positive hydrogen pressure until no more carbon
dioxide is liberated. The catalyst is filtered off and the
filtrate is concentrated to dryness. The residue is dissolved
in dimethylformamide (43 ml) and triethylamine (3.2 ml).
senzyloxycarbonyl nitroarginyl-proline 2,4,5-trichlorophenyl
ester (15.7 g) and l-hydroxybenzotriazole (3.1 g) are added
immediately and the reaction is allowed to proceed until the
ninhydrin test is negative. Dimethylamino propylamine (5.4 ml)
is added and after two hours the solvent is removed ln vacuo.
The residue is dissolved in ethyl acetate and the solution is
washed neutra].. The e-thyl acetate is concentrated ln vacuo
to ca 70 ml and the solution poured into 1 liter of vigorously
stirred ether. The precipitate is filtered and dried to give
the title compound.
Example 5
~ethyl benzyloxycarbonyl-tryptophyl-proLyl-arqinyl-prolyl-
ql~
The hexapeptide of Example 4 (1.8 g) is dissolved in
absolute ethanol (27 ml) and N hydrochloric acid (4 ml).
Palladium on charcoal (0.38 y) is added and the mixture is
stirred under a positive hydrogen pressure until the ultraviolet
absorption of the nitroguanidine chromophore can no longer
~65~5~ KX37
be detected. The catalyst is filtered off and the filtrate
is concen-trated to dryness ln vacuo. The residue is dissolved
in dimethylformamide (10 ml) and triethylamine (0.28 ml).
Benzyloxycarbonyl-tryptophyl-proline 2,4,5-trichlorophenyl
ester (1.5 g) and l-hydroxybenzotriazole (0.3 g) are added
immediately and the reaction is allowed to proceed until the
ninhydrin test is negative. The triethylamine hydrochloride
precipitate is filtered off and the precipitate is poured
into 250 ml of vigorously stirred ethyl acetate. The
precipitate is filtered and dried to give the title compound.
Example 6
Pyroqlutamyl-tryptophyl-prolyl-arqinyl-prolyl-qluta-
minyl-isoleucyl~prolyl-proline, methyl ester
The octapeptide of Example 5 (10 g) is dissolved in
absolute ethanol (150 ml) and N hydrochloric acid (9 ml).
Palladium on charcoal (2 g) is added and the suspension is
stirred under positive hydrogen pressure until no more carbon
dioxide is evolved. The catalyst is removed by filtration and
the filtrate is concentrated to dryness ln vacuo. The residue is
dissolved in dimethylformamide (~0 ml) and l-hydroxybenzotriazole
(1.2 y), triethylamine (1.2 ml) and pyro~lutamic acid 2,~,5-tri-
chlorophenyl ester are added in rapid succession. The reaction
is allowed to proceed until the ninhydrin test is negative. The
triethylamine hydrochloride precipitate i~s removed by filtration
and the filtrate is poured into 1.1 liters of vigorously stirred
ethyl acetate. The precipitate is filtered and dried to give
the title compound. This material is purified by
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:. :
~;
. .
1~65858 KX37
chromatography on Sephadex G-25 in 0.01 M ammonium bicar-
bonate.
Amino Acid Analysis - Molar Ratios:
Found (Calculated): NH3 0.92 (1.0); Arg 0.86 (1.0);
Pro 4.0 (4.0); Trp 0.78 (1.0); ; ~ -
Gln 2.12 (2.0); Ile 0.96 (1.0)
Thin layer chromatography - Silica gel (Butyl alcohol 30,
Pyridine 20, Acetic Acid 6, Water 24) Rf = 0.53.
~ xample 7
n-Butyl benzyloxycarbonyl-isoleucyl-proline-prolinate
A solution of benzyloxycarbonyl-isoleucyl-prolyl- ;
proline (4.5 g) in chloroform (30 ml) is added slowly to a
solution of l-n-butyl-3-p-tolyltriazene (1.9 g) in chloro-
form ~15 ml). When the reaction is complete, the chloroorm
solution is washed neutral and the organic phasa is dried
over magnesium sulfate and concentrated to dryness in vacuo
to give the title compound.
Example 8
n-Butyl-benzyloxycarbonyl-glutaminyl-isoleucyl-prolyl-
prolinate
This compound is prepared by the procedure o~ Exam-
ple 3, starting with the tripeptide in Example 7.
,
Example 9
n-Butyl benzyloxycarbonyl-~-nitroarginyl-prolyl-glutaminyl-
isoleucyl-prolyl-prolinate
This compound is prepared by the procedure of Exam
,
ple 4, starting with the tetrapeptide obtained in Bxample 8.
~ ., .
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~0658S8 KX37
~xample 10
n-Butyl benzyloxycarbonyl-tryptophyl-prolyl-arginy;l-prol~l-
.
glutaminyl-isoleucyl-prolyl-prolinate
This compound is prepared by the procedure o Exam- '
ple 5, starting with the hexapeptide of Example 9.
Example ll
Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl
isoleucyl-prolyl-proline, n-butyl ester
This compound is prepared by the procedure of Exam-
ple 6, starting with the octapeptide of Example lO.
'
Example 12
Benzyloxycarbonyl-isoleucyl-prolyl-proline amide
Benzyloxycarbonyl-isoleucyl-prolyl-proline (4.5 g)
is dissolved in a mixture o tekrahydrofuran (30 ml) and
triethylamine (l.4 ml). The so1ution is chilled in a -5
cooling bath and isobutyl chloroformate (1.6 ml) is added.
The solution is allowed to warm to room temperature (10
minutes) and concentrated aqueous ammonia (lO ml) is added.
After Eour hours stirring at room temporature, the reaction
is concentrated ln vacuo, diluted with ethyl acetate and
wa~hed 9equentially with ~aturated sodium bicarbonate, water,
O.l N hydrochloric acid and water. The organic phase is
dried over magnesium sulate and concentrated to dryness
in vacuo to give the title compound.
Example 13
Benzyloxycarbonyl-glutaminyl-isoleucyl-prolyl-proline amide ''
This compound is prepared by the procedure of Exam~
30 ple 3, starting with the tripeptide of Example 12. ,
~ 10--
1~X37
5~
Example 14
Benzyloxycarbonyl-~nitroarqinyl-prolyl-qlutaminyl-isoleucyl-
prol~l-proline amide
This compound is prepared by the procedure of Example 4,
startiny with the tetrapeptide of Example 13.
Example 15
BenzYloxYcarbonyl-tryptoPhyl-Prolyl-arqinyl-prolyl-qlutamin
'~
isoleucyl-pro~y~-proline amide
This compound is prepared by the procedure of Example 5,
starting with the hexapeptide of Example 14.
Example 16
Pyroqlutamyl-tryptophYl-prolYl-arqinYl-prolYl-qlutaminyl-
isoleucyl-prolyl-prolinamide
This compound is prepared by the procedure of Example 6,
starting with the octapeptide o:E Example lS.
Example 17 .
BenzyloxycarbonYl-tryptophyl-prolyl--arqinyl-prolyl-
qlutaminyl-isoleucyl-prolyl~proline, ter-t-butyl es-ter
t-Butyl benzylo~ycarbonyl nitroarginyl-proline-
glutaminyl-isoleucyl-proline-prolinate (9.42 g,~ 10 mmoles)
is dissolved in 135 ml absolute ethanol and 20 ml N HCl and
1.9 g l~/o palladium on carbon with stirring under positive
hydrogen pressure for 22 hours. It is filtered through hyflo
and taken to dryness ln vacuo. The crude residue is dissolved
in 50 ml of dimethylformamide and 1.4 ml triethylamine followed : :
--11--
: . . . : :
. .
- .... .
~ KX37
~651~5~
immediately by (12 mmoles) 7.4 g of benzyloxycarbonyl-tryp-
tophyl-prolyl-2,4,5-trichlorophenyl ester. Over a 30 hour
period an additional 1.5 mmoles of benzyloxycarbonyl-
tryptophyl-prolyl-2,4,5-trichlorophenyl ester and 1.5 mmoles
of triethylamine are added. After 50 hours the triethyl-
amine-HCl is filtered and the ~iltrate added to 1.2 1
of vigorously swirling ethyl acetate. The precipitate is
filtered and washed with ethyl acetate to give the title
compound. Yield ~ 10.64 g.
Example 18
Pyroqlutamyl=trypto~hy---prolyl-arqinyl-prolyl-qlutamin
isoleucyl-prolyl-proline, t-butyl ester
10 g (~ 8.5 mrnoles) of the peptide of Example 17 is
dissolved in 150 ml of absolute ethanol and 8.5 ml N HCl
and 2 g of 10~/~ palladium on carbon and s-tirred under positive
hydrogen pressure for eight hours. It is filtered through
hyflo and the filtrate concentrated to dryness ln vacuo to :
yield 9.2 g of crude residue.
The crude residue is dissolved in 40 ml of dimethyl-
formamide and 1.14 g (8.5 mmoles) of l-hydroxy benzotriazole .
and (8.5 mmoles) 1.2 ml of triethylamine followed immediately
by 3.14 g pyroglutamic acid 2,4,5-trichlorophenyl ester
(2~/q excess~. ~n additional 0.6 ml triethylamine is added~
The reaction is allowed to run overnight. The triethylamine HCl .
.is filtered and the filtrate poured into 1.1 1 of vigorously
swirling ethyl acetate. The recovered precipitate is filtered
throuyh paper~ 42 and washed with ethyl acetate to yield
9.0 g of the title compound.
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KX37
1~65858
,,
Amino Acid Analysis - ~olar Ratios;
Found (Calculated: NH3 0.85 (1.0); Trp 0.66 (1.0);
Arg 1.0 (1.0); Glu 2.03 (2.0);
Pro 4.0 (4.0); Ile 0.9 (1.0)
Thin layer chromatography - Silica gel (Butyl alcohol 30,
pyridine 20, acetic acid 6, water 24) Rf = 0.58.
Purification is achieved on a DEAE Sephadex A-25
column by elution with 0.005 M NH4HCO3.
Example 19
Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl-
isoleucyl-prolyl-proline, methvl ester
Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-gluta-
minyl-isoleucyl-prolyl-proline (1.6 g) is dissolved in 80 ml
of methanol. To this, excess ethereal diazomethane is added
and the solution is kept for seven hours at room temperature.
Acetic acid is added to destroy the excess diazomethane and
the resulting mixture is concentrated to dryness in vacuo.
Purification is effected on a ~ephadex G-25 column
eluting with 0.01 M (NH4)HCO3, followed by a DEAE*Sephadex
A-25 column eluting with 0.005 M NH~HCO3 to yield 1.1 g of
the title compound.
Example 20
Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-glutaminyl-
isoleucyl-prolyl-prolinamide
Pyroglutamyl-tryptophyl-prolyl-arginyl-prolyl-
glutaminyl-isoleucyl-prolyl-proline (1 g) is dissolved in
6 ml of dimethyl sulfoxide with gentle warming. The solu-
tion is cooled to room temperature and 0~14 ml of triethyl-
amine and 0.16 ml of isobutyl chloroformate are added. The
-13-
*Trade Mark
`- xx37
5851
reaction mixtuxe is stirred for 15 minutes. One ml of
concentrated ammonium hydroxide is added and the reaction
proceeds for four hours prior to slow addition to 200 ml
of vigorously swirling ethyl acetate. A precipitate is
formed wh.ch is ~iltered and washed with ethyl acetate to
yield 1.375 g of solids.
This material is purified on a 225 ml coluDu~ of
DEAE Sephadex A-25 by elution with 0.005 M NH4HCO3 to yield
924 mg of the title compound~
Amino Acid Analysis - Molar Ratios:
Found (Calculated): NH3 1.71 (2.0); Arg 0.88 (1.0);
Pro 4.13 (4.0); Trp 0.80 (1.0);
Gln 2.12 (2.0); Ile 0.93 (1.0)
Thin layer chromatography - Silica gel (Butyl alcohol 30,
pyridine 20, acetic acid 6, water 20) Rf = 0.53.
.'.""' ' .
Example 21
For determination of I50 values (concentration of
peptide expressed in micrograms/ml producing a 50% inhibition
of angiotensin-converting enzyme), varying concentrations of
2~ the peptide of Example 19 are added to 13 x 100 mm assay tubes
containing a final volume of 0.25 ml containing 100 mM potas- ~ ;
sium phosphate buffer, pH 7.5, 30 mM NaCl, and 0.3 mM angio- ;~
tensin I. En~ymatic reactions are init~ated by addition of
enzyme and incubation is carried out at 37. The concentra-
tion of the peptide of Example 19 of the present invention
which inhibits conversion of 50% of angiotensin I into
angiotensin II is found to be 7 ~g/ml as opposed to an I50
(~g/ml) of 0.9 for the parent peptide Pyr-Trp-Pro-Arg-Pro-
Gln-Ile-Pro-Pro. This ln vitro test clearly demonstrates
that the Example 19 peptide of the invention is substantially
B~
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KX37
~L~6585~?,
less potent than the parent control peptide in vitro in
inhibiting conversion of angiotensin I into angiotensin II.
Example 22
The peptide of Example 19 and the parent control
peptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro are administered
intravenously in two atropinized anesthetized male rats
infused with pentolinium followed by an injection of 0.10
~g/kg of angiotensin~I. On the basis of the degree of
inhibition of angiotensin I induced pressor response, it
is found that the in vivo activity of the Example lg pep-
tide of the invention is roughly equivalent to that of the
parent control peptide.
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-15-
