WO1994021648A1

WO1994021648A1 - Novel carpaine monoamides, their preparation and their use as drugs

Info

Publication number: WO1994021648A1
Application number: PCT/FR1994/000268
Authority: WO
Inventors: Jacques Fahy; Stylianos Mamatas; Dennis Bigg; Anna Kruczynski; Robert Kiss
Original assignee: Pierre Fabre Medicament
Priority date: 1993-03-15
Filing date: 1994-03-14
Publication date: 1994-09-29
Also published as: FR2702768B1; AU6211594A; ZA941763B; FR2702768A1

Abstract

Novel carpaine derivatives of general formula (2), wherein A is a direct bond, a -(CH2)n- chain wherein n is 1, 2 or 3, or a -CH2-X- chain wherein X is an oxygen or sulphur atom; and Aryl represents a phenyl, naphthyl, furyl, thienyl, benzofuryl or benzothienyl ring optionally substituted by one or more substituents selected from a halogen atom, a C1-14 alkyl group, a C1-4 alkoxy group, a nitro group, a cyano group, a trifluoromethyl group or a trifluoromethoxy group; and therapeutically acceptable salts of such molecules. The therapeutical uses of such compounds and a method for preparing same are also disclosed.

Description

"New mono-amides of Carpaine, their preparation and their use as medicaments"

Carpaine (J, main alkaloid of Carica papaya (family Caricaceae), is known for its biological effects. For example, an antiparasitic activity has been described by JD Phillipson and MJ O 'Neill [Acτa Pharm. Nord., 131 (1989) ], cardiovascular activity by CA Hornick, LL Sanders and YC Lin. fRes. Commun. Chem. Pathol. Pharmacol .. 22. 277 (1978)], antimicrobial activity by FM Hashem, MY Haggag and AMS Gabal fEgypt. T. Pharm. Sci .. 2,199 (1982), 22, 23 (1983)], and cytotoxic activity has been demonstrated in vitro by L Oliveros-Belardo et al. FAsian 1. Pharm .. 2. 26 (1972 )].

Methods of extracting Carpaine from the leaves of Carica papaya and other plants have also been studied [US Patents 3,248, 300 (1964), US 3,274,072 (1964)].

Carpaine J

We have confirmed the cytotoxic activity of Carpain on a range of cancer cell lines and we have unexpectedly found that certain derivatives of this molecule have much more potent cytotoxic activity than that of Carpain. Also, the present invention relates to new chemical compounds derived from Carpain, their preparation and their therapeutic application. The compounds of the invention correspond to general formula 2:

in which

A represents

* either a direct link:

* or a string - (CH ₂ ) _n -, where n can take the values of 1, 2 or 3:

* s o i t u n e c h a în e - C H? -X-, where X represents an oxygen atom or a sulfur atom.

Aryl represents a phenyl, naphthyl, furyl, thienyl, benzofuryl, benzothienyl ring; these rings can be substituted or not by one or more substituents chosen from a halogen atom, a C1-C4 alkyl group, a C ₁ -C ₄ alkoxy group, a nitro group, a cyano group, a trifluoromethyl group, or a trifluoromethoxy group.

The invention also relates to the salts of the compounds of general formula 2 with pharmaceutically acceptable mineral or organic acids. The acid used can be, by way of nonlimiting example, hydrochloric acid or 2, -toluenesulfonic acid.

The present invention relates more particularly to the compounds of general formula 2 chosen from the following compounds: N-benzoylcarpain

N- (2-methyl benzoyl) carpain

N- (4-methyl benzoyl) carpain

N- (4-methoxy benzoyl) carpain N- (3,4-dimethoxy benzoyl) carpain

N- (3,4,5-trimethoxy benzoyl) carpain

N- (4-chloro benzoyl) carpain

N- (4-fluoro benzoyl) carpain

N- (4-nitro benzoyl) carpain N- (3-trifluoromethyl benzoyl) carpain

N- (4-cyano benzoyl) carpain

N- (trifluoromethoxy-2-benzoyl) carpain

N- (naphthoyl-2) carpain

N- (furoyl-2) carpain N- (4-butyl benzoyl) carpain

N- (7-methoxy benzo [b] furoyl-2) carpain

N- (5-chloro-4-methoxy-2-thénoyl) carpain

N- (3-chloro benzo [b] thenoyl-2) carpain

N- (phenylacetyl) carpain N- (2-thienyl acetyl) carpain

N- (3-phenyl propionyl) carpain

N - [(4-methoxyphenyl) -4 butyryl] carpain

N- (phenoxyacetyl) carpain

N- (thiophenoxy acetyl) carpain

as well as their salts with therapeutically acceptable inorganic or organic acids, in particular the hydrochloride and the paratoluenesulfonate.

The compounds of general formula 2 can be prepared by the action of an acid chloride of general formula 3, or of an acid anhydride of general formula 4, or also by reaction with a carboxylic acid of general formula 5, in the presence of a dehydrating agent, on the Carpaine according to the reaction scheme: Carpain 1_

1 3

, O 4

H 5

where A and Aryle are defined as above.

The reaction between Carpaine (J and an acid chloride of formula 3 is carried out in an inert solvent such as dichloromethane, ether or tetrahydrofuran in the presence of a base such as a tertiary amine. reaction is carried out at a temperature between 0 ° C and the reflux of the solvent.

The reaction between Carpaine (J and an acid anhydride of formula 4 is carried out in an inert solvent such as dichloromethane, ether, or tetrahydrofuran at a temperature between 20 ° C and the reflux of the solvent The reaction can be accelerated by the addition of a catalyst such as dimethylaminopyridine (DMAP). The reaction between Carpaine (JJ and a carboxylic acid of general formula 5. is carried out in an inert solvent such as dichloromethane or tetrahydrofuran, at a temperature between 0 ° C and room temperature. This reaction is carried out in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC).

The following examples illustrate the invention without, however, limiting its scope.

Elementary analyzes and IR and NMR spectra confirm the structure of the compounds obtained according to the invention.

Example 1: N - benzoylcarpain (2a)

In a 25 ml flask surmounted by a condenser, 1 g (2 mmol) of Carpaine (JJ and 0.47 g (2 mmol) of benzoic anhydride are dissolved in 5 ml of dichloromethane. 0.025 g (0, 2 mmol) of dimethylaminopyridine, and the mixture is heated to reflux under a nitrogen atmosphere, after approximately 12 hours, the reaction crude is poured into 50 ml of a 10% sodium carbonate solution and then extracted with 25 ml of dichloromethane The organic phase is separated, which is dried over MgSO ₄ .

After filtration and evaporation, the product thus obtained is purified by chromatography on a column of silica eluted with an Et ₂ mélange mixture: AcOEt:

MeOH (4: 2: 1).

The N-benzoylcarpain (2a) (0.22 g; 37%) thus recovered is then dissolved in 5 ml of ethyl ether, and salified by bubbling gaseous hydrochloric acid. The desired product, obtained in the form of hydrochloride monohydrate, is then filtered and dried under vacuum.

C ₃₅ H ₅₄ N ₂ 0 ₅ . HC1, H ₂ 0: 637.29

Melting point: 208-212 ° C

IR (KBr): 1738 cm-i (C = O lactone); 1634 cm-i (C = O amide) 1 H NMR (CDC1 ₃ ): 1.1-1.8 (m, 36H); 2.3 (m, 4H); 2.6 (m, 1H); 3.07 (m, 1H);

3.33 (m, 1H); 3.6-4.2 (m, 1H); 4.85-5.06 (m, 5H); 7.36 - 7.40 (m, 5H). Elementary analysis:

96 Wedge. C 65.96 H 9.01 N 4.39% Tr. C 66.00 H 8.88 N 4.40

Example 2:

N - (trimethoxy - 3,4,5 benzoyl) carpain (2f)

5 g (10.4 mmol) of Carpaine are introduced into a 100 ml flask

(DD dissolved in 50 ml of dichloromethane. 1.45 ml (10.4 mmol) of triethylamine are added thereto, then under an inert atmosphere (nitrogen or argon), a solution of 2.40 g is poured dropwise ( 10.4 mmol) of trimethoxy-3,4,5 benzoic acid chloride in 10 ml of dichloromethane.

After 24 hours of stirring at room temperature, the reaction medium is washed with 2 times 25 ml of 3% tartaric acid in water. The organic phase is separated, dried over MgSθ ₄ , filtered and evaporated. The solid obtained is purified by chromatography on a silica column eluted with a CHCl _{3 3} : EtOH mixture (95: 5).

1.80 g (26%) of N - (trimethoxy - 3,4,5 benzoyl) carpain (2f) are recovered and salified by bubbling gaseous hydrochloric acid in isopropyl ether.

The white precipitate is filtered and dried under vacuum.

C ₃₈ Heo N ₂ Og. HC1: 709.36 melting point: 224-227 ° C IR (KBr): 1720 cm-i (C = 0 ester), 1621 cm-i (C = 0 amide).

1 H NMR (CDCI ₃ ): 1.0 - 2.0 (solid, 39 H); 2.25 - 2.55 (solid, 5 H); 2.65 (m,

1H); 3.07 (m, 1H); 3.64 (s, 6H); 3.65 (s, 3H); 4.77 (m, 2H); 4.91 (s, 1 H, echJ; 6.53 (s, 2 H) Elementary analysis:% Cale. C 64.34 H 8.66 N 3.95

% Tr. C 64.10 H 8.63 N 3.92

Example 3:

N - (4-butyl benzoyl) carpain (2o) In a 50 ml flask placed in an ice bath, 0.74 g (417 mmol) of 4-butyl benzoic acid and 0.36 ml (4.17 mmol) of oxalyl chloride are mixed in 5 ml of dichloromethane. 1.7 ml (12.51 mmol) of triethylamine diluted in 5 ml of the same solvent are then poured in therewith, with effective stirring. After 30 min, a solution of 2 g (4J7 mmol) of Carpaine (1) dissolved in 15 ml of dichloromethane is added, and the mixture is left to stir at ambient temperature for 24 hours.

The reaction medium is then washed with 2 times 25 ml of 3% tartaric acid in water. The organic phase is separated, dried over MgS0 ₄ , filtered and evaporated. The solid obtained is purified by chromatography on a column of silica eluted with a CHCl ₃ : EtOH mixture (95: 5).

0.80 g (30%) of N - (4-butyl benzoyl) carpain (2o) is thus obtained. which is salified by the addition, in 5 ml of ethyl acetate, of 0.24 g of para-toluenesulfonic acid monohydrate. The white precipitate, obtained after slight concentration of the solvent and standing overnight at + 4 ° C, is filtered and dried under vacuum.

C ₃₉ H _{62 2} Os. ^ Hs Qs S: 811.12 Melting point: 223-224 ° C

IR (KBr): 1738 cm-i (C≈O lactone), 1645 cm-i (C = 0 amide). ¹ H NMR (CDCI ₃ ): 0.92 (t, j = 7.1 Hz, 3 H); 1.05 - 2.03 (massive, 42 H); 2.31 -2.66 (solid, 8 H); 2.88 (m, 1H); 3.90 (solid, 1 H); 4.81 (massive, 3 H); 7.22 (m, 4H). Elementary analysis:

96 Wedge. C 68J1 H 8.70 N 3.45 96 Tr. C 67.89 H 8.73 N 3.45

Example 4: N - (3-chloro benzo [b] thénoyl- 2) carpain (2r)

2 g (4J7 mmol) of Carpaine (j dissolved in 15 ml of dichloromethane) are introduced into a 50 ml flask. 0.6 ml (4.2 mmol) of triethylamine is added thereto, then under an inert atmosphere (nitrogen or argon) ), a solution of 0.97 g (4J7 mmol) of acid chloride is poured in dropwise 3-chloro benzo [b] 2-thiophene carboxylic acid in 5 ml of dichloromethane.

After 24 hours of stirring at room temperature, the reaction medium is washed with 2 times 25 ml of 3% tartaric acid in water. The organic phase is separated, dried over MgSO ₄ , filtered and evaporated. The solid obtained is purified by chromatography on a column of silica eluted with a CHCl ₃ : EtOH mixture (97: 3), then on a column of basic alumina eluted with

And ₂ 0: AcOEt: MeOH (4: 2: 1).

0.60 g (21%) of N - (3-chloro benzo [b] thénoyl-2) carpain (2r) is recovered and salified by addition, dissolved in 5 ml of ethyl acetate, of 0J5 g (0.75 mmol) of para-toluenesulfonic acid monohydrate. The white precipitate, obtained after slight concentration of the solvent and standing overnight at + 4 ° C, is filtered and dried under vacuum.

C ₃₇ H ₅₃ Cl N ₂ O ₅ S. C ₇ Hs O5 S: 845.54

Melting point: 260 - 263 ° C

IR (KBr): 1732 cm-i (C = 0 lactone), 1635 cm-i (C = 0 amide).

1 H NMR (CDCI ₃ ): 1.05 - 1.30 (solid, 25 H); 1.52 - 2.02 (massive, 14 H); 2.25

-2.55 (solid, 5 H); 2.90 (m, 1H); 3.66 - 4.16 (m, 1H); 4.79 - 4.96 (solid, 3 H); 7.45 (m, 2H); 7.81 (m, 2H). Elementary analysis:

% Calc. C 62.50 H 7.27 N 3.31

96 Tr. C 62.83 H 7.27 N 3.33

Example 5:

N - (2-thienyl acetyl) carpain (2t)

1.9 g (3.9 mmol) of Carpain are introduced into a 50 ml flask

(1) dissolved in 20 ml of dichloromethane. 0.56 ml (3.9 mmol) of triethylamine is added thereto, then under an inert atmosphere (nitrogen or argon), a solution of 0.49 ml (10.4 mmol) of chloride is poured dropwise thiophene-2 acetic acid in 5 ml of dichloromethane.

After 72 hours of stirring at room temperature, the reaction medium is washed with 2 times 25 ml of 3% tartaric acid in water. The organic phase is decanted, dried over MgSU ₄ , filtered and evaporated. The solid obtained is purified by chromatography on a column of silica eluted with a mixture CHC1 ₃ : EtOH (95: 5). 0.45 g (19.96) of N - (2-thienyl acetyl) earpain (2t) is recovered, which is salified by adding, in solution in 5 ml of ethyl acetate, 0J4 g (0.75 mmol) of para-toluenesulfonic acid monohydrate. The white precipitate, obtained after slight concentration of the solvent and standing overnight at + 4 ° C, is filtered and dried under vacuum.

C ₃₄ H ₅₄ N ₂ 0 ₅ S. C ₇ Hβ O ₃ S: 775.07 Melting point: 169-173 ° C

IR (KBr): 1736 cm-i (C = 0 lactone), 1643 cm-i (C = 0 amide). 1 H NMR (CDCI3): 1.03 - 1.66 (solid, 38 H); 2.01 (m, 1H); 2.22 -2.57 (massive,

6H); 2.88 (m, 1H); 3.92 (clump, 2H); 4.36 - 5.05 (solid, 3 H); 6.92 (m, 2H); 7.20 (m, 1H). Elementary analysis:

% Calc. C 63.53 H 8.06 N 3.61% Tr. C 63.45 H 7.99 N 3.61

Example 6:

N - (3-phenyl propionyl) carρaine (2u)

3 g (6.27 mmol) of Carpaine (1.) dissolved in 15 ml of dichloromethane are introduced into a 50 ml flask. 0.9 ml (6.27 mmol) of triethylamine is added thereto, then under an inert atmosphere (nitrogen or argon), a solution of 0.93 ml (6.27 mmol) of chloride is poured dropwise 3-phenyl propionic acid in 5 ml of dichloromethane. After 24 hours of stirring at room temperature, the reaction medium is washed with 2 times 30 ml of tartaric acid at 396 in water. The organic phase is separated, dried over MgSθ ₄ , filtered and evaporated. The solid obtained is purified by chromatography on a column of silica eluted with an Et ₂ 0: AcOEt: MeOH (4: 2: 1) mixture. 1.1 g (28%) of N - (ρhenyl-3 propionyl) carpain (2u) are recovered and taken up in ethyl ether to salify it by bubbling gaseous hydrochloric acid. The desired product, obtained in the form of a partially hydrated hydrochloride, is then filtered and dried under vacuum.

C ₃₇ H ₅₈ N ₂ 0 ₅ . HO, _ H ₂ 0: 656.33 Melting point: 194 - 196 ° C IR (KBr): 1734 cm-i (C = 0 lactone), 1629 cm- ⁱ (C≈O amide).

1 H NMR (CDC1 ₃ ): 1J6 - 1.43 (solid, 25 H); 2.01 (m, 12 H); 2.00 -2.80 (solid,

8H); 2.97 (m, 3H); 3.20 - 3.66 (m, 2H); 4.17 - 4.77 (m, 2H); 5.02 (m, 1H); 7.22 (m, 5H).

Elementary analysis:

96 Wedge. C 67.71 H 9.21 N 4.26 Cl 5.48

96 Tr. C 67.75 H 9.29 N4.31 Cl 5.53

Example 7:

N - [(4-methoxyphenyl) -4 butyryl] carpain (2v)

1.61 g are mixed in a 50 ml flask placed in an ice bath

(8.34 mmol) (4-methoxyphenyl) -4 butyric acid, 1.72 g (8.34 mmol) of N, N'-dicyclohexylcarbodiimide (DCC) then 2g (4J7 mmol) of

Carpaine (1) in 20 ml of dichloromethane. The mixture is left to stir at ambient temperature for 10 days.

The reaction medium is then washed with 2 times 25 ml of tartaric acid at 396 in water. The organic phase is separated, dried over MgSθ ₄ , filtered and evaporated. The residue obtained is purified by chromatography on a column of silica eluted with a CHCl3: EtOH mixture (98: 2) then (95: 5). 0.55 g (20%) of N - [(4-methoxyphenyl) -4 butyryl] carpain (2v) is thus recovered in hygroscopic form, in the form of a partially hydrated oil.

C ₃₉ Hβ ₂ N ₂ Oe. 1.5 H ₂ 0: 681.95

Melting point: (oil)

IR (KBr): 1732 cm-i (C = 0 lactone), 1614 cm-i (C = 0 amide). 1 H NMR (CDCI ₃ ): 1.03 - 2.00 (solid, 43 H); 2.20 -2.65 (solid, 6 H); 2.88 (m, 1H); 3.66 (s, H ₂ O, 3H); 3.70 (solid, 2 H); 3.78 (s, 3H); 4.50 - 4.94 (solid, 3 H);

6.82 (d, j = 8.0 Hz, 2H); 7.10 (d, j == 8.0 Hz, 2 H). Elementary analysis:

96 Wedge. C 68.69 H 9.60 N 4.10

% Tr. C 68.67 H 9.42 N 3.92 Table 1 below summarizes the main synthesized products which illustrate the invention without, however, limiting its scope.

TABLE 1

m rπ -o

É.

C5

hydrate hemi-hydrate

The compounds of the invention have been subjected to pharmacological tests which have demonstrated their advantage as substances with therapeutic activity. Also, the cytotoxic activity of the products was evaluated using the MTT test [T. Mosman, T. Immunol. Method. £ 5_, 55 (1983)] on different tumor cell lines. The MTT test is based on the ability of living cells to reduce, by the action of their mitochondrial enzymes, a yellow tetrazolium salt to a blue violet compound, formazan, measurable by spectrophotometry after dissolution in dimethylsulfoxide. The amount of formazan formed (and therefore the intensity of the coloration obtained) is directly proportional to the number of living cells present in the culture medium at the time of the test. The lines used, of human origin, are marketed by the American Type Cell Collection (ATCC), a benchmark organization for the supply of standardized strains.

The experimental protocol used is essentially that described by C. Carmichael, W.G. De Graff, A.F. Gazdor, D. Minna, and B. Mitchell [Cancer Res., 47_, 936 (1987)]. The results are expressed as a percentage of growth inhibition relative to the controls.

Table 2 gives, by way of example, the results obtained for certain derivatives of the invention. Table 2

In view of their pharmacological properties, the compounds of the present invention can be used in human therapy in the treatment of cancer pathology.

Pharmaceutical preparations containing these active ingredients can be shaped for administration by the oral, intravenous or subcutaneous route.

Claims

1) New Carpaine derivatives corresponding to the general formula 2

in which :

A represents

* either a direct link: * either a chain - (CH2) _n -_ where n can take the values of 1, 2 or 3

* or a chain -CH ₂ -X-, where X represents an oxygen atom or a sulfur atom.

Aryl represents a phenyl, naphthyl, furyl, thienyl, benzofuryl, benzothienyl ring; these rings may be unsubstituted or substituted by one or more substituents selected from a halogen atom, an alkyl group in C ₁ -C ₄ alkoxy group, C ₁ -C ₄ alkyl, nitro, cyano, a group trifluoromethyl, or a trifluoromethoxy group.

as well as their salts with therapeutically acceptable mineral or organic acids.

2) Compounds of general formula 2 according to claim 1, characterized in that they are chosen from: N-benzoylcarpain

N- (2-methyl benzoyl) carpain

N- (4-methyl benzoyl) carpain

N- (4-methoxy benzoyl) carpain N- (3,4-dimethoxy benzoyl) carpain

N- (3,4,5-trimethoxy benzoyl) carpain

N- (4-chloro benzoyl) carpain

N- (4-fluoro benzoyl) carpain

N- (4-nitro benzoyl) carpain N- (3-trifluoromethyl benzoyl) carpain

N- (4-cyano benzoyl) carpain

N- (trifluoromethoxy-2-benzoyl) arpain

N- (naphthoyl-2) carpain

N- (f uroy 1-2) carpain N- (4-butyl benzoyl) carpain

N- (7-methoxy benzo [b] furoyl-2) carpain

N- (5-chloro-4-methoxy-2-thénoyl) carpain

N- (3-chloro benzo [b] thenoyl-2) carpain

N- (phenylacetyl) carpain N- (2-thienyl acetyl) carpain

N- (3-phenyl propionyl) carpain

N - [(4-methoxyphenyl) -4 butyryl] carpain

N- (phenoxyacetyl) carpain

N- (thiophenoxyacetyl) carpain

3) Process for the preparation of compounds according to claims 1 and 2, characterized in that the carpaine is reacted with an acid chloride of general formula 3, according to the scheme:

Λryle - A - COCl 3

It's soul i

where A and Aryle are defined as above. 48

16

4) Process for the preparation of compounds according to claim 3, characterized in that the reaction of Carpaine with a compound of general formula 3 is carried out in an inert solvent such as dichloromethane, ether, or tetrahydrofuran at a temperature between 0 ° C and the reflux of the solvent and in the presence of a base such as a tertiary amine.

5) Process for the preparation of compounds according to claims 1 and 2, characterized in that the carpaine 1_ is reacted with an acid anhydride of general formula 4 according to the scheme:

where A and Aryle are defined as above.

6) Process for preparing the compounds according to claim 5, characterized in that the reaction of Carpaine with a compound of general formula 4 is carried out in an inert solvent such as dichloromethane, ether, or tetrahydrofuran at a temperature between 20 ° C and the reflux of the solvent, and optionally in the presence of a catalyst such as dimethylaminopyridine (DMAP).

7) Process for the preparation of compounds according to claims 1 and 2, characterized in that the carpaine is reacted with a carboxylic acid of general formula according to the scheme:

where A and Aryle are defined as above. 8) A preparation method according to claim 7, characterized in that the reaction of Carpaine with a compound of general formula 5, is carried out in an inert solvent such as dichloromethane or tetrahydrofuran at room temperature, in presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC).

9) As new drugs useful, for example, in human therapy in the treatment of cancer pathology, the compounds defined according to one of claims 1 and 2.

10) Pharmaceutical composition characterized in that it contains, as active principle, at least one compound according to one of claims 1 and 2, associated with an acceptable pharmaceutical vehicle.