FR2662359A1 - Novel diuretic and natriuretic peptides - Google Patents

Abstract

Diuretic and natriuretic composition containing as active substance a dipeptide of structure H-Asp-Glu-OH, in which Asp represents L-aspartic acid and Glu represents L-glutamic acid, or one of its salts.

Description

 The present invention relates to a new hexapeptide, the production of the synthesis of this new hexapeptide, - the demonstration of the diuretic and natriuretic properties of this new hexapeptide, as well as the diuretic and natriodiuretic pharmaceutical compositions containing the new hexapeptide or some of its fragments and derivatives.

 Certain experiments carried out on natural extracts of the dog lymph have made it possible to demonstrate that these extracts exhibited a diuretic activity.

 According to the present invention, a hexapeptide was extracted from the dog lymph, H-Ala-Ser Asp -Gl u-Thr-Gly-OH, whose sequence H-Ala-Ser-Asp -Glu-Thr-Gly corresponds to the terminal C sequence of ceruloplasmin. According to the present invention, the synthesis of this new hexapeptide has been carried out, and its diuretic and natric properties have been demonstrated.

The new hexapeptide according to the invention has the following structure: H-Ala-Ser-Asp-Glu-Thr-Gly-OH in which Ala represents L-Alanine, Ser represents L-Serine, Asp represents acid
L-Aspartic, Glu represents L-Glutamic acid, Thr represents L-Threonine, Gly represents Glycine, and in which the amino acid chain has an amino terminus at its left end and a carboxylic terminus at its right end.

The developed formula is as follows

The molecular weight of the hexapeptide according to the invention
is 578.5.

The proton nuclear magnetic resonance spectrum
at 300 MHz of the hexapeptide of the invention in solution in Dimé
deuterated thylsulfoxide (DMSO) highlights according to the table
given below the presence of all the functions of the hexapeptide.

Proton NMR spectrum at 300 MHz of the hexapeptide in solution in deuterated DMSO
Chemical shifts found 6 (in ppm relative to TMS), line allocation and comparison with references given by: "K. WUTRRICH NMR in Biological Research Peptides and Proteins
North Holland Publishing Co. (Amsterdam) ".

number allocation reference #
8.60 1
8.35 1 7.95 2 5 NH 7.83 to 8.21
7.80 1
4.55 1
4.401
4.30 1 5 α CH 4.34 to 4.63
4.15 1
4.00 1
3.90 1 ss CH Thr 3.93
3.70 2 α CH2 Gly 3.76
3.60 2 ss CH2 Ser 3.60
2.55 2.44
2.65 2 ss CH2 Asp 2.70
2.25 2 γ; CH2 Glu 2.26
1.95 2 13 CH2 Glu 1.87
1.30 3 F CH3 Ala 1.22
1.05 3 T CH3 Thr 1.07
Furthermore, the aminogram of the hexapeptide of the invention after hydrochloric hydrolysis as shown in the figure.
1, with ordinate optical density under a monochroma light
wavelength tick at 440 nm, and on the abscissa the time of
retention, clearly shows the presence of 6 amino acids as well
than their equimolecular proportions. References 1 to 6 correspond
tooth respectively to Asp, Thr, Ser, Glu, Gly, Ala acids.

According to the present invention, it has also been demonstrated
diuretic or natriodiuretic activity of dipeptide fragments
of the new hexapeptide, namely, the dipeptides H-Thr-Gly-OH, -H-Asp-Glu-OH, H-Ala-Ser-OH.

We also studied the diuretic or natriodiuretic activity
prodrug derived salts of 1 hexapeptide and its segments - or residues, isomers of position Ot and P on aspartic acid
that in which the terminations have been replaced by an acid
amino, peptide, ester, acyl, amide, or any other
protecting group. Similarly, the activity of copper salts of
compounds above.

The experiments also focused on a derivative gone
dipeptide H-Asp-Glu-OH, namely, N acetyl-L aspartyl-L-glutamic acid, in its form or, and in its form, and
in the form of its salts. The copper salts of the oC form have
have been particularly studied.

We have also studied the activity of certain
o dipeptide (H-Asp-Glu-OH, like the derivative (N-acetyl-Asp-Asp
obtained by the replacement in o & N acetyl-Asp-Glu of the acid
glutamic by aspartic acid, and o (N acetyl
Asp-Arg obtained by replacing glutamic acid
by arginine, as well as their positional isomers.

) de Merrifield
Le dérivé

= 9-fluorenylmethoxycarbonyl) contenant 0,6 millimole de glycine par gramme fut le point de départ pour la réalisation de cinq cycles de couplage analogue au premier qui nous est décrit complètement ci-après. Il comprend les étapes sui vantes - déprotection du FMOC par une solution de pipéridine a 50% dans
le chlorure de méthylene.The synthesis of the hexapeptide could be carried out from
derivatives of suitably protected amino acids. Reactions
chosen couplings have been made so that there is no
possibility of alteration of the asymmetry centers.
thodes can be used and three of them are described
below in examples of preparations of the hexapeptide, and
of its dipeptide segments, H-Thr-Gly-OH, H-Asp-Glu-OH, and
their salts and derivatives, such as N-acetyl -L-aspartyl-L-glutami-
that in its forms o (and P.,
Example No. 1: Preparation of the hexapeptide L-Alanyl-L-Seryl
L-aspartyl-L-glutamyl-L-Threonyl-Glycine (H-Ala
Ser-Asp-Glu = Thr-Gly-OH) by the solid support method (noted

) from Merrifield
The derivative

= 9-fluorenylmethoxycarbonyl) containing 0.6 millimole of glycine per gram was the starting point for carrying out five coupling cycles similar to the first which is described to us completely below. It includes the following steps - deprotection of the FMOC with a 50% piperidine solution in
methylene chloride.

- washes with methylene chloride, dimethylformamide, water / dioxane
1/1, dimethylformamide and methylene chloride - coupling with FMOC-Thr- (tBu) -OH, 2.5 times in excess for 2
hours in the presence of dicyclohexylcarbodiimide (DCCI 2.5 equi
valents) and dthydroxybenzotriazole (HOBT 3.5 equivalents) in
methylene chloride with a small amount of dimethyl
formamide to dissolve HOBT - washes with methylene chloride, dimethylformamide, chloride
of methylene, ethanol and new methylene chloride - recoupling under the same conditions as coupling with aupara
before possible deprotonation of the free amine functions by the
diisopropylethylamine in solution at 0.75 96 in chloride of
methylene, then washes with chloroform and methylene chloride - acetylation with acetic anhydride in the presence of DIPAE for
20 minutes in the DMF / CH2CL2 mixture - washes with DMF, ethanol, methylene chloride.

The yield of each coupling was estimated at 99.5% after the Kaiser colorimetric test.

In the fifth cycle we use the BOC-Ala-OH derivative instead of the derivative
FMOC and we finally obtain the following resin peptide derivative
BOC-Ala-Ser (tBu) -Asp (OtBu) -Glu (OtBu) Thr (tBu) -Gly-

The cut-off of the resin peptide and the final deprotection were carried out in a single one hour operation in 80% trifluoroacetic acid (TFA) in methylene chloride.

 The solution in: the TFA was concentrated under good vacuum, the residue dissolved in acetic acid and precipitated in a 1/1 ethyl ether / petroleum ether mixture.

 The proton nuclear magnetic resonance spectrum at 300 MHz in the deuterated DMSO of the product obtained corresponds well to that of the hexapeptide. The same is true for the aminogram.

Example No. 2: Preparation of the dipeptide-L Threonyl-Glycine
(H-Thr-Gly-OH)
The coupling was carried out in methylene chloride between the benzyloxycarbonyl L Threonine (Z Thr OH) and the benzyl glycinate tosylate (TosOH, H-Gly-OBZl) by addition of an equivalent of dicyclohexylcarbodiimide (DCCI) and an equivalent triethylamine (TEA). Deprotection was carried out with hydrogen in the presence of 10% palladium on carbon in a methanol-acetic acid mixture.

The free peptide was crystallized from anhydrous acetone.

Example No. 3: Preparation of the L-Aspartyl-L-Gluta Acid Peptide
mique (H-Asp-Glu-OH)
This dipeptide was obtained from N formyl-L aspartic anhydride by coupling with glutamic acid L in solution in water at 0 ° C., the pH being maintained at 8.5 by automatic addition of 10 N sodium hydroxide.

 The i N formyl peptide obtained crystallizes with separation of the tC isomer after demineralization of the solution.

The final deformylation was carried out at 65 ° C. in the presence of N / 5 hydrochloric acid.

The o (dipeptide was crystallized as a sodium salt.

Example No. 4: Preparation of the N-acetyl-L-Aspartyl-L dipeptide
Glutamic (N-Ac-Asp-Glu)
This derivative was prepared in the same way as the previous peptide described in Example No. 3, starting from N anhydride acetyl aspartic.

 The mixture of isomKeres omits and (OL 70%, t 30%) was separated to isolate the pure ss isomer.

 The diuretic and natriodiuretic activity of the compounds of the invention was determined by measuring the group diuresis of two awake Wistar male rats (200 g), fasted for 18 hours but hydrated.

 These rats were previously chosen. For this eight days before the test, they underwent a diuresis test. Animals that deviated from the standard were not used for activity measurements. On the day of the test, each rat was gavaged by an esophageal probe with a solution of CINa at 9 / oo at the rate of 4% of the body weight (V / weight).

Immediately afterwards, these animals received intraperitoneally - the control batch 2 ml of NaCl at 9 "/ 00 - the test batches 2 ml of a solution of NaCl 90 / oo containing
the peptide to be tested.

 The dosage is reported in mg / kg; the effect on diuresis is expressed as a percentage relative to the diuresis of the control batch measured for 4 hours after intraperitoneal administration. The sodium excretion is expressed in mEq / h / kg. The sodium measurement was carried out with a flame photometer after diluting the urine to 1/100.

 The natriuretic activity is calculated as a percentage relative to the sodium excretion of the control rats.

 The results obtained are presented in the following table.

RESULTS OBTAINED
Dosage - Dose 0.1 mg / Kg / day
Products experienced activity activity
natriuretic diuretic
H-Ala-Ser-Asp-Glu-Thr-Gly-OH + 50 to 60% + 33%
(TFA)
H-Asp-Glu-OH, 2 Na + + 50% + 30%
HBr, H-Thr-Gly-OH + 30% 0
Mixture of the ot and p isomers of
-N-Ac-Asp-Glu + 60% + 50% (α 70 8, ss 30%)
Mixed Copper Salts
N-Ac-Asp-Glu + 60% + 60%
(α70%,> 30%) Isomer pure
N-Ac-L-Asp-L-Gl u
N-Ac-Asp-Asp-OH 0 0
N-Ac-Asp-Arg-OH - 8% 0
It appears from the table of the results of the experiments that the hexapeptide according to the invention has very good diuretic activity (+ 50 to 60%) and good natriuretic activity (+ 33%), that the dipeptide derivative H-Asp-Glu- OH has the same activity as the hexapeptide and therefore corresponds well to the active sequence of the hexapeptide.

 The other dipeptide fragments of the hexapeptide, such as the dipeptide H-Thr-Gly-OH, exhibit less diuretic activity (+ 30%) and do not exhibit natriuretic activity.

Furthermore, the results of the experiments show
although the derivatives of the active dipeptide H-Asp-Glu-OH fragment retain the same remarkable diuretic and natriuretic activity as the dipeptide H-Asp-Glu-OH fragment itself. In particular, the acyl derivative in its mixture of o (and F isomers shows excellent diuretic activity and excellent natriuretic activity.

The copper salts of the latter compound exhibit an even better diuretic and natriuretic activity.

By contrast, when the structure of the H-Asp-Glu-OH dipeptide is modified by replacing glutamic acid with another amino acid such as aspartic acid or arginine, the compounds obtained no longer exhibit any interesting activity. , as shown by the results obtained for the Ac derivatives
Asp-Asp-OH and Ac-Asp-Arg-OH.

 Likewise, it clearly appears that only the Go isomers of the peptides of the present invention can exhibit interesting diuretic and natriuretic activity, while the isomers show no activity.

 Finally if we compare the diuretic and natriuretic activity of the products of the present invention with that of a known diuretic agent such as furosemide, it is found that in order to obtain an activity comparable to that of the products of the present invention administered at a daily dose of 0.1 mg / kg, it is necessary to administer 10 to 15 mg / kg of furosemide. This means that the peptides according to the present invention are 100 to 150 times more active than furosemide, which is quite remarkable.

 The peptides according to the present invention can be used as active compounds in pharmaceutical preparations having diuretic or natriodiuretic properties.

 Since these peptides are natural products made up of L-amino acids and are gradually hydrolyzed in the body to produce L-amino acids or other peptides, they have very low toxicity. They do not stimulate the production of antibodies and do not cause anaphylactic shock.

 Their remarkable diuretic and natriodiuretic properties and this absence of toxicity, allow them to be the active ingredients of pharmaceutical compositions.

 The daily dose of active compounds which can be administered is between 0.1 mg / kg and 0.6 mg / kg and is preferably 0.3 mg / kg.

 The diuretic and natriodiuretic pharmaceutical compositions containing the peptides of the invention can be administered as are the other pharmaceutical compositions containing peptides, in particular parenterally, for example intraperitoneally, intravenously or intramuscularly. The therapeutic unit for intraperitoneal administration contains approximately 5 mg of active substance.

 The pharmaceutical compositions can also be administered orally.

Claims (6)

1. Diuretic and naturrietic composition containing as active substance a dipeptide of structure H-Asp-Glu-OH in which Asp represents 11 acid L-Aspartic, Glu represents L-Glutamic acid, or a salt thereof. 2. diuretic or natriodiuretic compositions containing as active substance the prodrug derivatives of the dipeptide according to claim 1, in which the terminations have been replaced by an amino acid, a peptide, an ester, an acyl, an amide, or any other protective group. 3. Diuretic and natriuretic compositions containing as active substance 1 0 -N-acetyl-L-aspartyl-L-glutamic acid, or its salts. 4. Diuretic or natriodiuretic compositions containing as active substances the copper salts or complexes of the active compounds present in the compositions according to claims 1 and 2. 5. Diuretic and natriuretic compositions containing as active substances the copper salts or complexes of o-N-acetyl-L-aspartyl-L-glutamic acid. 6. Diuretic or natriodiuretic compositions according to any one of claims 1 to 5, characterized in that the administrable therapeutic unit contains approximately 5 mg of active substance.