FR2673626A1 - Process for synthesis of cucurbitine (cucurbitin) using 1-benzyl-3-pyrrolidone as starting material - Google Patents

Abstract

The invention relates to a process for the synthesis of cucurbitine. This synthetic process is characterised in that 1-benzyl-3-pyrrolidone is used as starting material. Cucurbitine can then be used to prepare a cosmetic or pharmaceutical, especially dermatological, composition having an anti-allergic activity or in the preparation of a cosmetic or pharmaceutical composition with a reduced allergising risk.

Description

Process for the synthesis of cucurbitine using as starting material 1-benzyl-3-pyrrolidinone.

 The present invention essentially relates to a process for the synthesis of cucurbitine from 1-benzyl-3-pyrrolidinone.

 The invention also relates to the use of cucurbitine for the preparation of cosmetic or pharmaceutical composition, especially dermatological, anti-allergic, and a process involving application.

est un acide aminé naturel soluble dans L'eau que l'on trouve dans les cucurbitacées (voir V.H. Mihranian et al., LLOYDIA (1968), 31 (1) 23-29). Cucurbitine, or 3-amino-3-alkyltrinylcarboxyllic acid of formula I, which follows

is a water-soluble natural amino acid found in cucurbits (see VH Mihranian et al., LLOYDIA (1968), 31 (1) 23-29).

 Cucurbitine is known as an anti-parasite, particularly as an anthelmintic against Schistosoma japonicum.

(Morimoto Y. et al., Chem Pharm Bull (1987) 35 (9) 3845-3849).

 Cucurbitine can be obtained by extraction in laevorotatory form or by synthesis in racemic form.

Among the various methods of cucurbitine synthesis, mention may be made particularly of the method of synthesis of H.J. Monteiro, J.

Chem. Soc., Chem. Common. (1973) 2. This method only leads to relatively low yields of racemic cucurbine. Another method of synthesis makes it possible to separately obtain one and
The other optical isomer of cucurbitine. This is the method of Morimoto et al., Chem. Pharm. Bull. (1987) 35 (9) 3845-3849, which is a stereospecific enzymatic synthesis method by the use of pig liver esterase. This method is however complicated, and requires a relatively large number of steps.

 It has now been unexpectedly discovered that cucurbitine inhibited the formation of histamine, a well-known mediator of allergies, and thus exhibited valuable hypohistaminemic activity. This hypohistaminemic activity results from the inhibitory action of cucurbitine on the histidine decarboxylase which is the enzyme responsible for the transformation of histidine into histamine. As a result, the administration of cucurbitine helps to lower the concentration of histamine in blood serum and tissues.

 This is an important technical advance because allergic manifestations, especially lung and skin allergies, pose many problems for therapists with a limited number of active substances, and some of these substances may have side effects. . Thus, the development of a new preventive and curative composition of allergies is therefore an important need.

 Thus, the main object of the present invention is to solve the technical problem of providing a solution which makes it possible to inhibit the formation of histamine, a mediator in the context of allergic manifestations, in order to allow the prevention and treatment of go anywhere.

 According to another aspect, the main object of the present invention is to solve the technical problem of providing a solution for preventing and treating allergic manifestations.

 The present invention also aims to solve the new technical problem consisting in providing a solution for reducing the allergenic potential of cosmetic or pharmaceutical compositions, especially dermatological.

 The present invention also aims to solve the new technical problem consisting in providing a solution for carrying out the synthesis of cucurbitine by a simple synthesis process, requiring a minimum of steps, in good yields.

 The present invention makes it possible to solve all these technical problems in a simple, reliable and reproducible manner that can be used on an industrial scale.

 Thus, according to a first aspect, the present invention relates to the use of cucurbitine or one of its salts or esters, or a plant extract containing it, for the preparation of a cosmetic or pharmaceutical composition, in particular dermatological, antiallergic activity.

According to a particular variant embodiment, it concerns the preparation of a cosmetic or pharmaceutical composition, especially a dermatological composition, intended for the prevention or the symptomatic treatment of allergic manifestations, whatever may be the case.
The origin and the point of application, especially the bronchi, the skin and the eye. Thus, said composition is intended in particular for the prevention or the symptomatic treatment of allergic bronchial asthma or stress, hay fever, spasmodic rhinitis and tracheitis, urticaria, other allergic rashes, eczema, redness or allergic skin irritations, pruritus, angioedema, allergic conjunctivitis, and allergic reactions of medicinal origin.

 In the more particular field of cosmetology, said composition is advantageously intended for lines of hypoallergenic products or for sensitive or irritable skin.

 Cucurbitine can be used either in free form, or in the form of one of its salts or its cosmetically or pharmaceutically, including dermatologically, acceptable esters.

The aforementioned salts and esters may be prepared by conventional methods well known to those skilled in the art. Among the salts are mono- and dibromohydrate and mono- and dihydrochloride. Esters include methyl ester and ethyl ester.

 According to an advantageous variant, the above-mentioned plant extract containing cucurbitine is an extract of cucurbitaceae, in particular Cucurbita maxima Duch., Cucurbita pepo L. or Cucurbita moschata Duch; preferably, it is a seed extract or fruit pulp.

 According to a second aspect, the present invention also relates to a cosmetic composition, characterized in that it comprises, as active ingredient, cucurbitine or a cosmetically acceptable salt or ester thereof or a plant extract containing it.

 According to an advantageous variant, the aforesaid plant extract is a cucurbitaceae extract as defined above.

 According to one preferred embodiment, the cucurbitine or one of its salts or esters is present in an amount effective to exhibit an antiallergic activity, in particular at a concentration of from 0.001% to 10%, preferably from 0.01% to 5%. % by weight of the total composition.

 According to a third aspect, the present invention relates to a pharmaceutical composition, especially dermatological, characterized in that it contains cucurbitine or a pharmaceutically acceptable salt or ester thereof in an amount effective to exhibit an antiallergic activity, particularly for prevent or treat allergic manifestations.

 According to one variant, the above-mentioned cosmetic and pharmaceutical compositions contain an extract of plant origin as defined above.

 According to a fourth aspect, the present invention also relates to a process for reducing the allergenic potential of a cosmetic or pharmaceutical composition, especially a dermatological composition, characterized in that an effective amount of cucurbitine is incorporated in the said composition in free form or in the form of one of its cosmetically or pharmaceutically acceptable salts or esters, or a plant extract containing it as previously defined, so that the final composition presents a reduced allergenic risk.

 In the context of any one of the preceding aspects, the preferred concentration by weight of cucurbitine, or its salts or esters for compositions for topical use, is between 0.001% and 10%, more preferably between 0.01% and 5%.

 For the compositions intended for administration by the general route (such as orally, parenterally, rectally, inhalations, ...) the concentration of cucurbitine is not critical and can reach for example 60% of the composition. The dosage in humans will generally be between 0.1 mg / kg / day and 20 mg / kg / day and preferably between 1 mg / kg / day and 15 mg / kg / day.

In addition, it is generally used natural cucurbitine, levorotatory, or its salts or esters. Particularly interesting sources of levorotatory cucurbitine are the pulp and seeds of cucurbitaceae, particularly species
Cucurbita pepo L., Cucurbita maxima Duch., Cucurbita moschata Duch.

However, it is also possible to use the racemic form of cucurbitine or its salts or esters.

In addition, cucurbitine can be used in pure form, or in the form of extracts, according to any of the extraction procedures known to those skilled in the art. Extremely interesting extraction procedures are described in the publication by Valentine H. MIHRANIAN et al. in Lloydminster 1968, 31, (1) 23-29 which is incorporated herein by reference, particularly page 24. A preferred source of cucurbitine is the pulp and especially the seeds of the species.
Cucurbita, such as Cucurbita pepo L., Cucurbita maxima Duch. and
Cucurbita moschata Duch.

 According to a fifth aspect, the present invention also covers a method of treating humans or an animal, characterized in that said human being or said animal is administered an effective amount of cucurbitine or one of its pharmaceutically acceptable salts or esters, or a plant extract containing it as previously defined, for preventing or treating allergic manifestations.

 In particular, the above-mentioned treatment is applicable to the prevention or the symptomatic treatment of allergic bronchial asthma or stress, hay fever, spasmodic rhinitis and tracheitis, urticaria and other allergic rashes. eczema, redness or allergic skin irritations, pruritus, angioedema, allergic conjunctivitis, as well as allergic reactions of medicinal origin.

 According to an alternative embodiment, cucurbitine or one of its pharmaceutically acceptable salts or esters is administered topically to a concentration of preferably between 0.01% and 10% by weight.

 According to another embodiment, cucurbitine or one of its pharmaceutically acceptable salts or esters is administered generally at a dosage in humans of between 0.1 mg / kg / day and 20 mg / kg / day and preferably between 1 mg / kg / day and 15 mg / kg / day.

 The invention also relates to a process for the preparation of a cosmetic or pharmaceutical composition, especially a dermatological composition, characterized in that cucurbitine or one of its cosmetically or pharmaceutically acceptable salts or esters or a plant extract thereof is incorporated. containing as defined above, in a carrier, vehicle or excipient cosmetically or pharmaceutically acceptable.

 According to one embodiment variant, cucurbitine or one of its cosmetically or pharmaceutically acceptable salts or esters is incorporated into a complete cosmetic or pharmaceutical formulation to reduce the allergenic risk thereof.

 According to another embodiment, a composition with antiallergic activity is prepared. Preparation variants also result from the foregoing description.

According to a particular embodiment of the invention in the context of one of the aspects described above, the above-mentioned composition containing cucurbitine or one of its salts or esters or
The above-mentioned plant extract also contains liposome-type vesicles. According to a particular variant, the cucurbitine, its salt or ester is encapsulated, at least in part, in the liposome-type vesicles. The expression "liposome-type vesicle" is intended to mean both hydrated lipidic lamellar phases and lipid vesicles formed from ionic or nonionic amphiphilic lipids. Also, by the expression "incorporate at least partially into vesicles of liposome type "is understood in the present description and claims that cucurbitine, its salt or ester is combined with liposome-type vesicles irrespective of the form of this combination. However, a preferred combination resides in the encapsulation of cucurbitine, its salt or ester in liposome-type vesicles. But it is not necessary for the whole to be incorporated or encapsulated to obtain the desired antiallergic effect according to the invention.

 It is known that liposome-type vesicles are prepared from lipid substances. The term "lipidic" covers all substances comprising a so-called fatty carbon chain, generally comprising more than 5 carbon atoms, this substance usually being called "lipid".

 According to the invention, it is used as lipid, to form either the lipidic lamellar phases or the liposome-type vesicles, amphiphilic lipids, that is to say constituted by molecules having a hydrophilic group indifferently ionic or nonionic and a lipophilic group, these amphiphilic lipids being capable of forming lipidic lamellar phases or liposome-type vesicles in the presence of an aqueous phase.

 In particular, among these lipids, mention may be made of: phospholipids, phosphoaminolipids, glycolipids, polyoxyethylene fatty alcohols, optionally polyoxyethylenated polyol esters. Such substances are, for example, constituted by an optionally hydrogenated egg or soy lecitin, a phosphatidylcholine, a phosphatidylserine, a sphyngomyelin, a cerebroside or an oxyethylenated polyglycerol stearate.

The incorporation into hydrated lipidic lamellar phases or into liposomes of the compounds used according to the present invention can be carried out according to known preparation techniques, described for example in the document
US-A-4,508,703 and possibly in combination with the document
US-A-4,621,023.

 According to a seventh aspect, the present invention also covers a process for the synthesis of cucurbitine, characterized in that 1-benzyl-3-pyrrolidinone is used as starting material.

 According to a particular variant of this synthetic process, 1-benzyl-3-pyrrolidinone is treated with an ammoniacal solution of ammonium chloride and potassium cyanide to obtain (+) 3-amino-1-benzyl -3-cyano-pyrrolidine. This compound is then converted by acid or basic hydrolysis into (+) 3-amino-1-benzyl-3-pyrrolidinecarboxylic acid, and finally catalytic hydrogenolysis is carried out to obtain (+) 3-amino-3-pyrrolidinecarboxylic acid. or (+) cucurbitine.

 According to a preferred variant, the above-mentioned ammoniacal solution is a hydroalcoholic solution, the alcohol being advantageously isopropanol or methanol.

 According to another preferred variant, the aforesaid hydrolysis is carried out using a 6 N aqueous solution of hydrobromic acid.

 According to yet another variant, the catalytic hydrogenolysis is carried out in water under hydrogen at atmospheric pressure in the presence of a catalyst such as palladium on carbon dispersed in the aqueous reaction medium.

 According to a preferred embodiment of the synthesis method, 1-benzyl-3-pyrrolidinone is allowed to react with ammonium chloride and potassium cyanide in a 1/4/4 molar ratio at room temperature for at least 48 hours. h.

 According to another particular embodiment, the optical isomers of the racemic mixture are separated, according to any separation technique known to those skilled in the art, and in particular via the preparation of diastereoisomers.

 The process for synthesizing cucurbitine according to the invention leads to particularly high yields of racemic cucurbitine, on average 2 to 3 times higher than those of the known processes.

 Other objects, features and advantages of the present invention will become apparent from the following explanatory description with reference to various examples of cucurbitine preparation, as well as various examples relating to the results of pharmacological tests, as well as as various examples of cosmetic or pharmaceutical formulation. In the examples, percentages are by weight unless otherwise indicated.

Example 1
Synthesis of cucurbitine as a racemic mixture
The procedure is as follows: a) Synthesis of (+) 3-amino-1-benzyl-3-cyano-pyrrolidine
0.5 g of 1-benzyl-3-pyrrolidinone (2.85 mmol) dissolved in 3 ml of 2-propanol are added to a solution of 0.741 g (11.4 mmol) of potassium cyanide and 0.615 g (11 g). 4 mmol) of ammonium chloride in 7 ml of 28% ammonia. The mixture remains at room temperature with stirring for 3 days. The solution is washed with 15 ml of a 10% potassium carbonate solution and extracted with dichloromethane (3 x 15 ml).

After drying over magnesium sulphate and evaporation of the solvents, an oil (0.475 g) is obtained.

 It is purified on a silica column with solid deposition. Eluted with a 4: 2 ether-petroleum ether mixture.

 0.402 g of a beige solid (yield: 70%) consisting of 3-amino-1-benzyl-3-cyano-pyrrolidine having the following NMR spectrum are obtained: 1 H NMR, 300 MHz, CDCl 3 1.8 (bs) , 2H, NH 2); 1.97 (ddd, 1H, J4 4, = 13.4, J4 = 8, J4.5, = 5.4); 2.5 (ddd, 1H, J4.4, = 8, J4.5, 84, 5 '= 5.4); 2.64 t 1H, J2.2, = 9w4); 3.04 (d, 1H, J2 '2 = 9.4); 67 (s, 2H, CH2-C6H5); 7.33 (m, 5H, aromatic protons).

b) Synthesis of (+) 3-amino-1-benzyl-3-pyrrolidinecarboxyli acid
that or (+) 1-benzyl-cucurbitine
The hydrolysis of the previously obtained compound can be carried out either in an acid medium or in a basic medium.

Hydrolysis in acidic medium
0.3 g (1.49 mmol) of 3-amino-1-benzyl-3-cyano-pyrrolidine, obtained in step a) dissolved in 5 ml of 48% hydrobromic acid, are brought to 40-50. C for 4 h. After evaporation of the acid, it is purified on a silica column. The impurities are removed by 10% CH 2 Cl 2 / MeOH and the amino acid is removed by
15% MeOH / H2O.

 After removing the methanol, the compound is decolorized to the animal black in a minimum of hot water and then lyophilized.

A dark yellow solid (yield: 80%) is obtained consisting of (+) 3-amino-1-benzyl-3-pyrrolidinecarboxylic acid in mono- or dihydrobromide form having the NMR spectrum according to H NMR, 300 MHz, D 2 O 2 47-2.69 (m, 1H, H4); 2.75-2.91 (m, 1H, Ho '); 3.69-3.94 (m, 3H, H5, H5, H2); 4.12 (d, J2.2, = 14.2, 1H, H2,); 4.58 (2d,
J = 13.8, 2H, CH2-C6H5>; 7.62 (s, 5H, protonsaromatic).

Hydrolysis in a basic medium
280 mg (1.39 mmol) of 3-amino-1-benzyl-3cyano-pyrrolidine of step a) are dissolved in 2 ml of ethanol. 5 ml of a 10% sodium hydroxide solution are added and the mixture is refluxed for 5 hours. After cooling, the mixture is acidified with 48% hydrobromic acid.

 Purified on a silica column: 10% MeOH / H 2 O. It is discolored with animal black then freeze-dried. The presence of sodium bromide salts results in a yield greater than 100%.

 To eliminate the salts present, 200 mg of pyrrolidine are taken and brought to pH = 8 with a 10% sodium hydroxide solution. The Duolite Cl is charged with a 2N hydrobromic acid solution. The salts are removed with distilled water. To remove the pyrrolidine, a solution of 0.1 N hydrobromic acid is used.

 After lyophilization, 120 mg of (+) 3 amino-1-benzyl-3-pyrrolidinecarboxylic acid (yield: 40%) are obtained.

c) Synthesis of '+) 3-amino-3-pyrrolidinecarboxylic acid or
(+) cucurbitine
300 mg of (+) 3-amino-1-benzyl-3-pyrrolidinecarboxylic acid hydrobromide obtained in step b) are dissolved in 10 ml of water. 0.5 mg of 10% charcoal of palladium is dispersed and this suspension is then placed in a hydrogen atmosphere at atmospheric pressure. Stirring is maintained for 18 h. After filtration on filter paper and lyophilization, a yellow solid (98%) consisting of (+) 3-amino-3-pyrrolidinecarboxylic acid or (+) cucurbitine having the following NMR spectrum is obtained
1 H NMR, 300 MHz, D 2 O 2.45 (m, 1H, H 4); 2.69 (m, 1H, H4,); 3.61 (d, 1H, J2.2 '= 13.4); 3.67-3.78 (m, 2H, H5, H5,); 4 (1H, J21.2 = 13.4).

Example 2
Optimization of the yield of the synthesis of (+) cucurbitine
The operation is carried out as indicated in Example 1, choosing in step b) the hydrolysis with 6 N hydrobromic acid, however varying the proportion of reactants of step a).

 The yields obtained are shown in Table I below.

TABLE I

<tb><SEP> Number <SEP> equivalents
<tb><SEP> moles <SEP> Conditions <SEP> Yield
<tb> NBP <SEP> NH4Cl <SEP> KCN <SEP> Experimental <SEP> CN <SEP> COOH
<tb><SEP> of <SEP> step <SEP> a)
<tb><SEP> 1 <SEP> 1-1 <SEP> 1 <SEP> 6 <SEP> h <SEP> temperature <SEP> ambient <SEP> * <SEP><<SEP> 10 <SEP>%
<tb><SEP> 1 <SEP> 1-1 <SEP> 1 <SEP> 4 <SEP> h <SEP> 40 -50 <SEP> * <SEP> 4 <SEP> 10 <SEP>%
<tb><SEP> 1 <SEP> 4 <SEP> 4 <SEP> 4 <SEP> h <SEP> 40 -50 <SEP> * <SEP> 20 <SEP><SEP>%<SEP>
<tb><SEP> 1 <SEP> 8 <SEP> 8 <SEP> 4 <SEP> h <SEP> 40 -50 <SEP> * <SEP> 20 <SEP>%
<tb><SEP> 1 <SEP> 4 <SEP> 4 <SEP> 48 <SEP> h <SEP> temperature <SEP> ambient <SEP> 66 <SEP>% <SEP> 44 <SEP>%
<tb><SEP> 1 <SEP> 4 <SEP> 4 <SEP> 72 <SEP> h <SEP> temperature <SEP> ambient <SEP> 70 <SEP>% <SEP> 49 <SEP>%
<tb><SEP> 1 <SEP> 4 <SEP> 4 <SEP> 4 <SEP> 84 <SEP> h <SEP> temperature <SEP> ambient <SEP> 70 <SEP>% <SEP> 56 <SEP> %
<Tb>
NBP: 1-benzyl-3-pyrrolidinone *: non-isolated amino nitrile
CN: yield of 3-amino-1-benzyl-3-cyano-pyrrolidine
COOH: overall yield of 3-amino-3-pyrrolidinecarboxy acid
lic
The last column of Table I contains the yields of (+) cucurbitin relative to the starting reagents.

 It is observed that the yields are maximum if the reaction is conducted at room temperature for a period greater than 48 hours using 1/4/4 proportions between the reagents: 1-benzyl-3-pyrrolidone, ammonium chloride and potassium cyanide.

 Hydrolysis with hydrobromic acid has also been found to lead to better yields than when hydrochloric acid is used at the same concentration as the hydrolysis agent.

Example 3
Separation of the optical isomers of cucurbitine
The separation method, known in itself, is based on the preparation of diastereoisomeric derivatives by coupling of (+) cucurbitine with certain optically active reagents, after protection of the acid function by esterification and the cyclic amine function. Each form of these stereoisomers, corresponding to one or the other isomer of cucurbitine, will be isolated by chromatography, in particular by high performance liquid chromatography or on a silica column. To then regenerate the two enantiomers of cucurbitine, it is sufficient to saponify the protective ester and then hydrolyze to release the acid function, and release the amine function, for example by hydrogenolysis as described in Example 1 in the case where the amine function was protected by substitution with a benzoyl radical .

As reagents for the present method, optically active levogenic S compounds can be used, such as
- camphanyl-1S chloride, - (S) - (-) - -methoxy-trifluoromethyl-phenyl-acetic acid,
N- (tert-butoxycarbonyl) -L-phenylalanine.

 This example describes the resolution of the racemic cucurbitine by coupling with camphanyl chloride.

a) Protection of the acid function of La (t) 1-benzyl-cucurbitine:
synthesis of its methyl ester
0.1 g of (+) 1-benzyl-cucurbitine (0.20 mmol), obtained in step b of Example 1, is added slowly to an ice-cold solution of thionyl chloride (0.28 mmol) and of methanol (2 ml).

The temperature of the reaction should not rise above -50C. The mixture is stirred at OOC for 2 h and then cooled to room temperature for two days.

 After evaporation, it is purified on a silica column with solid deposition. (Eluent CH 2 Cl 2 / MeOH from 10 to 50%).

 A yellow product is obtained with a yield of 70%, consisting of the methyl ester of (+) 1-benzyl-cucurbitine.

b) Coupling with Camphanyl Chloride (C)
1 mol of (+) 1-benzylcucurbitine methyl ester in solution in 1.5 ml of methylene chloride is neutralized with 1 mole of triethylamine. 1.1 equivalents of 1S (-) camphanyl chloride are added. The mixture is stirred at room temperature for 16 hours.

 It is purified on a silica column with solid deposition (eluent: CH2Cl2 / MeOH 10%).

The separation of the diastereoisomers was carried out by
HPLC on various columns including grafted ZorbaR3NH2.

Example 4
Preparation of a pulp extract of Cucurbita pepo
Fresh fruits of Cucurbita pepo are cut in half, the seeds that can be used to make seed extracts are removed. The resulting pulp is ground and lyophilized. The powder is recovered which is degreased with petroleum ether at a rate of 1 I per 100 g of powder. The insoluble material is recovered by filtration which constitutes the desired Curcurbita pepo pulp extract. The proportion of cucurbitine in this extract is determined by HPLC and a concentration of 0.03% by weight of cucurbitine is obtained in the defatted dry extract.

Example 5
Preparation of a seed extract of Cucurbita pepo
1.5 kg of seeds, previously peeled, are ground
Cucurbita pepo. The powder obtained is extracted three times with hexane (3 liters, 2 liters and 1.8 liters) to remove the fat. The dried cake obtained is extracted with an aqueous solution of hydrochloric acid maintained at about pH 4. This extraction is made in three times: two at room temperature - about 220C - and the third at 700C. Each time, keeping the cake-solution in contact with the hydrochloric acid solution (2 liters the first time and 2.5 liters the next two times) is 24 hours.

 After wringing, the solid residue is removed and the aqueous phase is collected, which is partially evaporated and centrifuged. The centrifugation pellets are washed with distilled water and then removed. The washing waters are combined with centrifugation concentrates.

 The aqueous fraction is reconcentrated and then treated weight for weight with ethanol. A white precipitate is formed which is removed by centrifugation. The hydro-alcoholic supernatant, which is neutralized for example with sodium hydroxide, constitutes an extract containing (-) - cucurbitine, which can be used as it is.

 It is also possible to evaporate the alcohol and then atomize the aqueous solution obtained, so as to obtain a powder titrating 3 to 5% (-) - cucurbitine, according to the seed lots used.

Example 6
Demonstration of the inhibitory activity of histamine formation by cucurbitine
1 - A by enzymatic test
This test is based on the inhibitory action of cucurbitine on the enzyme histidine decarboxylase (HDC) which transforms
Histidine in histamine, compared to that of tritoqualine, which is a known inhibitor of HDC (see Carpi C., Maggi GC

Bull. Soc Ital. Sper. 1968, 44 (6,543-4), which is used therapeutically as a hypohistaminemic agent under the name Hypostamine.
Inhibitory activity on HDC can be easily performed by colorimetric assay on the basis of the following chemical reaction
Colorimetric assay

<tb><SEP> I <SEP> 1 <SEP> CH, -CH-COOH
<tb> HN <SEP> CHz-CH-COOH <SEP> N <SEP> D <SEP> C <SEP> NM2 <SEP> Enzyme <SEP> 2
<tb><SEP> HNN <SEP> 2 <SEP> Enzyme <SEP> HN <SEP> N <SEP> + <SEP> CO
<tb><SEP> L-Histidine <SEP> Assayed <SEP> 2
<tb><SEP> Histamine
<tb><SEP> - <SEP> 1- <SEP> CH2CH2NHz <SEP> Diamine <SEP> Oxidase <SEP> 77CH2-CHO
<tb> HN <SEP> N <SEP> enzyme <SEP>'HN<SEP> iHZ02 <SEP> + <SEP> NH3
<tb><SEP> Histamine <SEP> Auxiliary
<tb> H2O2 + MBTH Peroxidase Blue Color
DMAB indicator enzyme
# = 595 nm.

MBTH = 3-methyl-2-benzothiazolinone hydrazone
DMAB = 3- (dimethylamino) benzoic acid
In practice, it is observed that the formation of the blue color is proportional to the concentration of histidine consumed. It has thus been possible to define initial reaction rates and establish so-called Mickaelis curves as described in Fundamentals of Enzymology, 2nd Ed. Oxford Univ. Press, 1989.

 The initial velocity is expressed in absorbance variation per minute.

 The results obtained with various initial concentrations of histidine, namely at 132 nmol, 265 nmol, and 530 nmol, respectively without effector, with tritoqualine as a known HDC inhibitor, and with cucurbitine in racemic form as an inhibitor according to the invention are listed in Table II below and are the subject of the Mickaelis curve, the object of Figure 1.

Table II

<tb><SEP> Histidine <SEP> 132 <SEP> nmol <SEP> 265 <SEP> nmol <SEP><SEP> 530 <SEP> nmol
<tb> Without <SEP> effector <SEP> 0.062 <SEP> 0.089 <SEP> 0.1132
<tb> Tritoqualine <SEP> 0.021 <SEP> 0.0374 <SEP> 0.0604 <SEP>
<tb> (previous <SEP> art)
<tb> Cucurbitine <SEP> 0.013 <SEP> 0.0242 <SEP> 0.042 <SEP>
<tb><SEP> (invention)
<Tb>
It is clear from Table II and from your
Mickaelis object of Figure 1, cucurbitine is a much stronger HDC inhibitor than tritoqualine, which is a result quite surprising for a man of the art.

1 - B by RIA dosage
The antihistaminic activity of cucurbitine can also be demonstrated by radioimmunoassay (Radio Immuno Assay or "RIA") as follows.

 This assay is carried out by assaying the histamine directly produced by the action of the enzyme HDC, by the RIA method, well known to those skilled in the art, and in particular described in the instructions for use of a kit. dosage form called HISTAMINE Radioimmunoassay kit (Cat * 1302) marketed by IMMUNOTECH (Marseille-France).

 The quantity of histamine released (in nanomoles) for a concentration of 16 .mu.-3 molar of histidine in phosphate buffer at pH 6.3, without effector, is then measured over time with tritoqualine as a comparison effector and with racemic cucurbitin synthesis as antihistamine agent according to the invention. Tritoqualine and cucurbine were used at a concentration of 2.10 3 molar.

 The results obtained expressed in nanomoles of histidine released are listed in Table III below and are the subject of the curve of FIG. 2, where lton has indicated on the ordinate the number of nanomoles of histamine released and on the abscissa the time expressed in minutes. . The effector-free curve is drawn in continuous line, the curve obtained with tritoqualine is drawn in dotted line and the curve obtained with cucurbitine is drawn in dotted line.

TABLE III

<tb><SEP> Time <SEP> 5 <SEP> min ** <SEP> 10 <SEP> min ** <SEP>% <SEP> I * (at <SEP> 5 <SEP> min) <SEP> % <SEP> I * <SEP> (at <SEP> 10 <SEP> min)
<tb> Without <SEP> effector <SEP> 32.66 <SEP> 140.79 <SEP> 0 <SEP> 0
<tb> Tritoqualine <SEP> 16.48 <SEP> 49.27 <SEP> 49 <SEP> 65
<tb> Cucurbitine <SEP> 14.85 <SEP> 1 <SEP> 38.29 <SEP> 55 <SEP> 73
<Tb>
* I = Inhibition
** = nanomoles of histamine released
It is clear from Table III that the amount of histamine released is clearly the lowest in the presence of cucurbitine.

 The results obtained by the RIA method thus confirm that cucurbitine has a much more potent histamine-forming inhibitory activity than tritoqualine after a few minutes.

Various examples of formulation of cosmetic or pharmaceutical compositions, especially dermatological compositions according to the invention are the following:
Example 7
tablets
Per tablet for oral administration:
- (+) cucurbitine 100 mg
- starch 38 mg
- lactose 75 mg
- talcum 10 mg
- other excipients for tablets
(including magnesium stearate) qs 250 mg
Indications: preventive and curative treatment of allergic manifestations, especially cutaneous and respiratory.

 Dosage: 1 to 10 tablets per day in adults.

Decrease doses by 2 in children up to 15 years.

Example 8
Aerosol powder
For 100 g in a pressurized bottle
- (+) cucurbitine 3g
- mannitol 1g
- 96 g pulsating gases
Indications: preventive and curative treatment of all respiratory allergic manifestations such as bronchial asthma and asthmatic bronchitis.

 Dosage: intrabronchial administration, at a rate of 4 to 6 aspirations per day on average.

Example 9
Softening emulsion for sensitive skin extracted from seeds according to Example 5
titrating 0.5 X in (-) cucurbitine 10 g - cosmetic emulsion
classic for sensitive skin
(fatty alcohols, polyoxy fatty alcohols
ethylenated mineral oil, palmitate
isopropyl, glycerin, gelling agent,
preservatives, perfumes, water) qs 100 g
Example 10
Hypoallergenic foundation extract of cucurbita pulp
pepo of example 4 10 g - conventional composition for a background
complexion (fatty esters, squalane,
soy lecithin, volatile silicone,
propylene glycol, xanthan gum,
solar filter, pigments, conserva
water, perfume, water) qs 100 g
Example
Anti-irritant mascara - (+) - cucurbitine 1.0 g - blue overseas 9.0 9 - hexadecyl alcohol 7.4 g propylene glycol 9.0 g - stearic acid 11.3 g - glycerol monostearate 4.4 g - triethylamine 3.6 g - preservative 0.3 g - water qs 100 g

Claims (4)

 Process for the synthesis of cucurbitine, characterized in that 1-benzyl-3-pyrrolidinone is used as starting material.  2. Process according to claim 1, characterized in that the 1-benzyl-3-pyrrolidinone is treated with an ammoniacal solution of ammonium chloride and potassium cyanide, obtaining (+) 1-benzyl-3- amino, 3-cyano-pyrrolidine which is then converted to (+) 3-amino-3-pyrrolidinecarboxylic acid by acid or basic hydrolysis followed by catalytic hydrogenolysis.  3. Process according to claim 1 or 2, characterized in that the reaction of 1-benzyl-3-pyrrolidinone with ammonium chloride and potassium cyanide is carried out in a molar ratio 1/4/4 to room temperature for at least 48 hours.  4. Method according to one of claims 1 to 3, characterized in that isolates the levorotatory isomer of 3-amino-3-pyrrolidinecarboxylic acid from the racemic mixture, in particular via the preparation Somerous asteroids.