WO1999020290A1

WO1999020290A1 - Use of a buxus sempervirens alkaloid extract for making a medicine with an anti-il 2 activity

Info

Publication number: WO1999020290A1
Application number: PCT/FR1998/002204
Authority: WO
Inventors: Pierre Dellamonica; Jacques Durand; Alain Rebouillat; Max Rombi
Original assignee: Universite De Nice-Sophia-Antipolis; Laboratoires Arkopharma
Priority date: 1997-10-17
Filing date: 1998-10-14
Publication date: 1999-04-29
Also published as: FR2769840A1; AU9545698A

Abstract

The invention concerns the use of a Buxus sempervirens alkaloid extract for making a medicine with an anti-IL 2 activity.

Description

USE OF AN ALKALOID EXTRACT OF BUXUS SEMPERVIRENS FOR THE MANUFACTURE OF A DRUG WITH ANTI-IL 2 ACTIVITY

The present invention relates to the use of an alkaloid extract of Buxus sempervirens for the manufacture of a medicament with anti-IL 2 activity.

In general, the present invention relates to the use of Buxus sempervirens for the manufacture of medicament with anti-EL 2 activity. It may especially be the powder of the plant which has revealed such activity.

Preferably, the invention relates to an extract rich in alkaloids obtained under the conditions described by VOTICKY Z. et al.- Alkaloids from Buxus sempervirens. Coll. Czech. Comm. 1975, 40, 3055-3060, with a gradient of acid solutions.

On such an alkaloid extract, different separations were carried out on chromatographic columns so as to purify and / or separate different alkaloids whose anti-IL 2 activity was determined in comparison with that of cyclosporin Λ according to the operating protocol described by RANDAK C. et al. - Cyclosporin A suppresses the expression of the interleukin-2 gene by inhibiting binding of lympohcyte specifies factors to the 11 2 enhanecr. EMBO J. 1990, 9, 2529-2536.

The results obtained within the framework of this experiment have been recorded in the table below:

(1) IC 50 = inhibitory concentration 50% of the production of Interleukin 2.

(2) HENDERSON D.J. et al. -Comparison of the cfiect of F 506, cyclosporin Λ and rapamycin on IL 2 production. Immunology 1991, 73, 316-321.

In order to describe more fully the object of the present invention, reference is made below to the nonlimiting examples of isolation and identification of some main alkaloids of common boxwood or Buxus sempervirens.

The examples reported below consist first of all in the extraction of total alkaloids according to the method described by VOTICKY (acidic hydroalcoholic solutions), see reference above.

The total alkaloids were then fractionated using a solution of decreasing pH.

After separation of the alkaloids by a succession of chromatographs on an open column, the structure of the various isolated compounds was determined by different spectral methods, nuclear magnetic resonance and mass spectrometry. EXAMPLES:

Extraction of total alkaloids from Buxus scmncrvircn.s

800 g of green powder of small granulomctric Buxus sempervirens are extracted with 4 liters of a hydromethanolic solution (50:50 v / v) containing 5% (v / v) of acetic acid.

The solution is stirred for 2 hours at room temperature, the maceration continuing to stand overnight.

After centrifugation and filtration, the residue is again extracted with 4 liters of a hydromethanolic solution (50:50 v / v) containing 5% (v / v) of acetic acid (the operation is repeated up to the amount of 4 successive extractions).

A total of 4 kg of Buxus sempervirens powder is treated with this method, which makes it possible to recover 80 liters of acidic hydroalcoholic extract solution. After evaporation of the methanol under reduced pressure, about 25 liters of acidic aqueous solution remain.

After transfer to a separating funnel, this solution is made alkaline with 4 liters of ammonia and exhausted with 4 x 8 liters of dichloromethane. We thus reach an SI solution.

Fractionation of total alkaloids as a function c i pfi (

To avoid handling of excessive solvents, the chloromethylenic solution S I is concentrated by evaporation under reduced pressure until approximately 10 liters of solution are obtained.

This solution is transferred to a separatory funnel, extracted with 4 x 3 liters of pH 7 buffer solution.

After ammonia alkalization, the aqueous phases are exhausted with 3 3 liters of dichloromethane. The dichloromethane is evaporated to dryness under reduced pressure - Fraction A (16.2 g).

The solution S 1, partially exhausted, is subjected to a new extraction with x 3 liters of pH 5.5 buffer solution.

After alkalization with ammonia, the aqueous phases are exhausted with 3 x 3 liters of dichloromethane. The dichloromethane is evaporated to dryness under reduced pressure - »Fraction B (17.4 g).

The rest of the SI solution is evaporated to dryness under reduced pressure - Fraction C (154.9 g) -

The two buffer solutions used (pH 7 and pH 5.5) are prepared according to a protocol described in the French Pharmacopoeia * "'° edition.

Phosphate buffer solution pH 7 (0.067M):

Solution I: dissolve 0.908 g of monopotassium phosphate R in water and make up to 100.0 ml with the same solvent.

Solution II: dissolve 2.38 g of disodium phosphate R in water and make up to 100.0 ml with the same solvent

Mix 38.9 ml of solution I with 61.1 ml of solution II and adjust the pH if necessary.

Phosphate buffer solution pH 5.5:

Solution I: dissolve 13.61 g of monopotassium phosphate R in water and complete with

1000 ml with the same solvent.

Solution II: dissolve 35.81 g of disodium phosphate R in water and make up to 1000 ml with the same solvent. To 96.4 ml of solution I add 3.6 rai of IL solution

The three fractions A, B and C are then chromatographed on an open column, various supports being used (silica, alumina).

The fractionation is followed by chromatography on a thin layer: 20 × 20 cm aluminum plates coated with silica gel 60 F ₂₅₄ , thickness of the layer 0.2 mm (Merck).

Migration solvent: Toluene-Acetone-Ethanol-Ammonia (40/40/6/2). Revelation with the Dragendorff reagent prepared according to the protocol described in the French Pharmacopoeia X * edition: Solution A: Bismuth sub-nitrate 0.850 g

Distilled water 40 ml

Glacial acetic acid 10 ml

Solution B: potassium lodurc 8 g

Distilled water 20 ml

Stock solution: Mix solution A and solution B

Diluted solution for use: Stock solution 20 ml

Acetic acid 20 ml

Distilled water 60 ml Fractionation of A

15 g of fraction A are chromatographed on an open column of alumina gel 90 (degree of activity III-HI, 70-230 racsh ASTM. Merck). The column consists of 450 g of alumina suspended in dichloromethane.

Since fraction A is not completely soluble in dichloromethane, it is necessary to carry out a "dry mounting" beforehand by adsorption of A onto 45 g of alumina.

The fractions are eluted with dichloromethane with a methanol gradient (0 to 100%).

36 fractions, with a volume of 400 ml, are eluted. In fraction A7 (3.54 g), eluted with a dichloromeihane-methanol mixture (99/1), two major alkaloids are highlighted.

In fractions A8 to A10 (2.72 g), eluted with the same solvent as A7, two major alkaloids are highlighted, the first of them already identified in A7.

The fractions A6 to A16 ^" in which these three alkaloids are highlighted represent approximately 2/3 of the fraction A,

Splitting Λ7

The fraction A7 is chromatographed on an open column of silica gel 60H (Merck).

The column consists of 100 g of silica in suspension in an ethyl acetate-

Methanol-Diethylamine (90/10 1.5). The column is fitted with a sintered glass filter (3).

A "dry assembly" is carried out with 2.5 g of fraction and 5 g of 60H silica.

140 fractions of 7 ml, eluted with the same solvent, are collected.

One of the major alkaloids is identified in fractions A7.95 to A7.106 (350 mg), but it is from fractions A7.98 to A7.100 that about 80 mg of this monobasic alkaloid already described in Buxus are isolated. sempervirens: cycloprotobuxinc D

In fractions A7.81 to A7.96 (640 mg) buxaminc G is identified and in fractions

A7.68 to A7.76 (230 mg) is identified buxamine E. Splitting from A 8 to A 1 0

Fractions A8 to A10 (2.7 g) are chromatographed on an open column of 60H silica gel and fitted with a sintered glass filter (3).

The column consists of 80 g of silica suspended in a mixture of ethyl acetate.

Methanol-Diethylarainc (90/10 2).

A "dry mounting" using 8 g of silica is necessary.

111 fractions, with a volume of 7 ml. are elected.

Fractions A8.53 to A8.5S (225 mg). by precipitation in acetone are isolated about 80 mg of an alkaloid in the form of an amorphous white powder: buxaminc G (Ref. 21. 50, 51).

This alkaloid is present in fractions A8.43 to Λ8.61 (900 mg).

Fractions A8.76 to A8.85 (380 mg) is isolated in the same way as above _, approximately 200 mg of an amorphous white powder which appears to be a mixture of two alkaloids described in Buxus sempervirens: cyciobuxinc D and In cyclovirobtixinc D

These two alkaloids, in the thin layer chromatography system used, have the same frontal ratio and it has not yet been possible to separate them. The mixture of these two alkaloids is present in Λ8.68 fractions A8.100 _(1, 0 g).

Ff splitting

15 g of the fraction B _t its gel chromatography ouvcπc alumina ^alumina column

90 (degree of activity H-iπ. 70-230 mesh ASTM. Merck).

_T are the column consisting of 450 g of alumina suspended in dichloromethane. A

"dry mounting" is carried out with 45 g of alumina.

The fractions, with a volume of 400 ml. are eluted with dichloromethane and gradients CALEA _t e acetate (0-100%) and methanol (0 to 100%).

59 fractions are collected.

Both alkaloids majori _t fraction B areas are identified in the fractions B42 to B56

(7.5 g), eluted with ethyl acetate-methanol mixtures (90/10. 80/20. 50/50) then pure methanol. B56 split

Fraction B56, eluted with pure methanol. is chromatographed on an open column of 60H silica gel. The column consists of 25 g of silica in suspension in a mixture of ethyl acetate-Methylianol-Diethylamine (90/10/2). It is fitted with a friπé glass filter (3). A "dry assembly" is prepared with 450 mg of B56 and 1.4 g of 60H silica. 82 fractions with a volume of 7 ml are reconstructed.

The fraction B56.9 (50 mg) consists for the most part of an alkaloid previously described in Buxus sempervirens: buxaminc E This alkaloid is also identified in the fractions BS6.7 to B56.10 (I40mg).

Fractionation from B42 to B46

Fractions B42 to B46 (2 g), eluted with ethyl acetate-methanol mixtures (90/10 and 80/20), are chromatographed on a 60 H silica gel column. The column, fitted with a sintered glass (3) consists of 75 g of silica suspended in an ethyl acetate-methanol-diethylamine mixture (90/10 / 1.5). A "dry assembly" is prepared with 5 g of 60H silica. 209 fractions with a volume of 6 ml are collected.

The major alkaloid of these fractions is identified in fractions B42.86 to B42.125 (650 mg).

Purification of these fractions is undertaken by chromatography on a 60H silica gel column (15 g of silica in suspension in an ethyl acetate-methanol-diethylaminc mixture (90/10/1).

A "mounting to sεc" is prepared with 1 g of silica. 107 fractions of 2 ml are collected. The fractions B42.86.64 to B42.86.72 (70 mg) consist for the most part of an alkaloid of structure for the moment undetermined. Its molar mass is 580 which is not the case with any alkaloid described in Buxus sempervirens (generally lower molar mass).

The spectral studies carried out to date (mass, 1 H NMR and RN ¹³ C) do not allow for the moment to give the structure of this alkaloid. Splitting from B47 to B55

Fractions B47 B55 (4 g), eluted with ethyl acetate-methanol mixtures (80/20 and 50/50), are chromatographed on a 60 H silica gel column. The column, fitted with a glass filter fritt (3) consists of 120 g of silica in suspension in an ethyl acetate-methanol-diethyl arainc mixture (90/10 / 1.5). A "dry assembly" is prepared with 10 g of 6UII silica. 150 fractions with a volume of 6 ml are collected.

The main alkaloid in this fraction is mainly eluted in fractions B47.97 to B47.113 (520 mg). It appears that this alkaloid is buxamine E mixed with buxaminc G.

Another alkaloid, of undetermined structure, is identified in fractions B 7.81 to B47.85 (60 mg).

Fra ctction of C

15 g of fraction A are chromatographed on an open column of alumina gel 90 (degree of activity III-ILI, 70-230 mesh ASTM. Merck).

The column consists of 450 g of alumina in suspension in dichloroméihanc.

Fraction C is chromatographed on a column of alumina gel 90 (degree of activity D.-III, 70-

230 mesh ASTM, Merck).

The column consists of 4 kg of alumina suspended in the cyclohcxanc. A "dry assembly" is prepared with 150 g of C and 400 g of alumina.

The fractions, with a volume of 400 ml, are eluted with cyclohexanc ci gradients of dichloromethane (0 to 100%) then of methanol (0 to 100%).

64 soni fractions eluted (28.2 g), fractions C30 to C36 eluted with a dichloromethane-methanol mixture (95/5) alone represent 20.2 g. The alumina is then recovered and exhausted with 4 x 3 liters of methanol at 2% (v / v) of hydrochloric acid.

After concentration and alkalization with sodium hydroxide, the solution is flooded with 3 liters of water and exhausted with 3 x 1 liter of dichloromethane.

The chloromethylenic solution is evaporated to dryness under reduced pressure, which gives the fraction C65 (3.33 g) which does not contain alkaloids. Splitting from C32

The fraction C32 (7.3 g) is the largest of the fractions obtained from C. It is chromatographed on a column of silica gel 60 (70-230 esh ASTM. Merck). The column consists of 200 g of silica in suspension in dichloromethane and the fracuons are eluted with dichloromedianceand raétiianol gradients (0 to 15%).

A "mounting to s c" is prepared with 20 μg of silica.

76 fractions, with a volume of 200 ml, are eluted. Up to fraction 44 there are no or very few alkaloids.

From fraction C32.18 (170 mg), eluted with a dichloromethane-methanol mixture

(99.5 / 0.5), is isolated and identified a triierpenic acid previously described in Buxus sempervirens: betulin.

From fraction C32.55 (178 mg), eluted with a dichloromethane-methanol mixture (90/10). is isolated by successive crystallization from acetone 20 mg of an alkaloid also described: cyclobuxophylline M.

C31 split

The C31 fraction (5.8 g) is eluted under the same conditions as the C32 fraction. It is chromatographed on a column of silica gel 60. The column consists of 180 g of silica suspended in dichloromethane and the fractions are eluted with dichloromethane and methanol gradients (0 to 157 °).

A "dry assembly" is prepared with 18 g of silica. 61 fractions, with a volume of 200 ml, are collected. Fractions C31.53 to C31.57 (800 mg), eluted by dichloromethane / methanol mixtures (90/10 and 85/15), is isolated by successive crystallizations from acetone 1 10 mg of the same alkaloid as that present in C32.55: cyclobuxophylline M. This alkaloid exists in two forms, the majority rrααj derivative and its cis isomer.

Fractions C31.42 to C31.44 (400 mg), eluted with a dichlororaethane-methanol mixture

(95/5) contain a majority alkaloid which is purified by chromatography on a 60H silica gel column.

The column consists of 12 g of 60H silica in suspension in an ethyl acetate-methanol-diethylamine mixture (90/10/2). It is fitted with a fried glass filter (3). A "dry assembly" is prepared with 1 g of 60H silica.

22 fractions with a volume of 2 ml are collected.

C31 fractions. 2.10 and C31. 2.11 is isolated 130 mg of an alkaloid already described in

Buxux sempervirens: N-Benzoyl-O-Acetylbuxadicnin E. Identification of compounds i.spLcs_d.c ... UJLXUX ι.c // ιμcrγjr ns

£ _Ω πxαtûliu XÛLCJÛ

j »î-i

Mass

The molecular ion appears at m / z 386 (6) which indicates the presence of an even number of nitrogen atoms (dibasic alkaloids).

Us ions M-15 (m / z 37 1; 10), M-30 (m / z 356; 18). M-56 (m / z 330; 6) and to m z 70 (25). m z 53 (100), m / z 57 (17) and m / z 44 (9) are characteristic of the methylamino groups substituting carbons 3 and 20 /

R H

It appears, on the ¹ H NMR spectrum, 2 singles at 2.38 and 2.4-4 ppm each integrating for 3 protons and corresponding to the two N-CHj groups carried by the molecule. At 0.31 and 0.52 ppm, 2 doublets are visible (J = 4Hz). integrating each for 1 proton and characteristics of the cyclopropyl group (protons carried by carbon 19). The three protons of the C21 méthyliquc group appear as ^a doublet 1, 04 ppm (J = 6Hz) then- than other méthyliqucs protons (18. 30 carbons, 3 1 and 32) appear as singulcts ( no vicinal protons) at 0.75, 0.90, 0.94 and 0.98 ppm.

ea

enough

The molecular ion appears at ra z 370 (100), this compound is a dibasic alkaloid,

The ion at m z 327 (52) is important and is due to the loss of the ethylamino radical carried by carbon 17. Carbon 20 carries a primary arαine.

The ions M- 15 (m / z 355; 12) and to m z 70 (46). m / z 58 (22). m / z 57 (16) and m / z 44 (78) are characteristic of the raethylarαino group substituting carbon 3.

NMR

On the ¹ H NMR spectrum, a singulct appears at 2.43 ppm integrating for three protons and corresponding to the N-CHj group carried by carbon 3.

Two signals are of great importance: the singulct at 5.94 ppm, integrating for a proton, ci the doublet at 5.51 ppm (J = 2.5 Hz), also integrating for a proton. These correspond respectively to the protons H 18 and Hl 1. These chemical shifts are characteristic of ethylenic protons and, for the alkaloids of Buxus sempervirens, of nor- \ -

/ ιomσ-B-5-α-prcgnanεs.

As for cycloprotobuxinc D. the methyl protons are easily recognized: doublet at 1.07 ppm (J = 6 Hz) for the methyl protons at C21. singulct at 0.68, 0.69, 0.73 and 1.00 ppm for methyl protons at C30, C32. Cl 8 and C31.

Another signal is easily detected on the spectrum; it is a ^"split quadruplci, integrating to a proton, and which corresponds to the proton H20. This couples with the methyl protons at C21 (J = 6Hz) and the proton borne by the carbon 17 (J = 9.5 L ).

On the ¹³ C NMR spectrum, the most characteristic signals appear at 128.7. 128.8, 136.0 cl 138.3 ppm ci correspond to ethylenic carbons, respectively C18. C it.

C l0 ct C9.

Study of the rest of the ¹ H NMR and ⁿ C NMR spectra as well as correlation spectra

(Cozy, XH and HMBC) confirms the structure of 1a buxaminc G. Cvdnhuxiπc D and CYclovirobuxinc ^' P.

These two compounds being in mixture, it appears on the specires of mass and nuclear magnetic resonance, signals characteristic of each to cook eαx.

Cyclobuxinc D

Jo h

Cyclovirobuxinc D

^j iiS5 £

The molecular ions appearing m / z 402 ci ra z 336, these two compounds are dibasic alkaloids.

As for your cycloprotobuxinc D, heap ions M- 1 5 (m z 3S7; 36 and m / z 37 1; 19). M-30 (m / z

372; 36 ci m / z 356; 18), M-56 (m z 346; 23 and m / z 330; 5) and a m / z 70 (19), m z 5S (100). m / z

57 (15) and ra / z 44 (5) are characteristic of the methytamlno- groups substituting carbons 3 and 20. E ri

The protons H19 of the cyclopropyl group appear in the form of doublets (J = 4 Hz) at 0.05 and 0.27 ppm for cyclobuxinc D and 030 and 0.53 ppm for cyclovirobuxinc D. The chemical shifts of carbon 19 being respectively at 28.0 and 30.4 ppm.

The protons of the methyl group carried by the amine at C20 appear as a singulct at 2.44 ppm for the two compounds, while those of the methyl group carried by the amine at C3 appear as a singulct at 22.44 ppm for cyclovirobuxinc D and

2.49 ppm for cyclobuxinc D.

The methyl carbon carried by the amine in C20 appears 34.0 ppm, that carried by the amin in

C3, at 34.7 ppm for cyclobuxinc cl 35.8 ppm for cyclovirobuxinc D.

The presence of a hydroxyl function on carbon 16 is confirmed by the chemical displacement of the proton H16 which appears in the form of a multiplet at 4.10 ppm and that of carbon

16 to 78.8 ppm.

Among the other signals common to the two molecules, it is easy to spot those of the proton

H19 (multiplet at 2.49 ppm), methyl protons at C21 (doublet at 1.09 ppm. J = 6.5

Hz), in C18 and C32 (singulct 0.96 and 1.1 1 ppm).

What clearly marks the difference between the two molecules is the chemical displacement of ethylenic protons for cyclobuxine D (wide singulec at 4.80 and 4.57 ppm) and methylenic protons for cyclovirobuxinc D (singlet at 0.74 and 0, 94 ppm).

The chemical shifts of carbons 3 and 5 were also modified (63.9 and 44.9 ppm for cyclobuxine D against 68.8 and 48.8 ppm for cyclovirobuxinc D) as well as that of the proton H3 (doublet doubled 2.88 ppm , Jl = 10 Hz and J2 = 3 Hz, for cyclobuxinc D against 1.95 ppm for cyclovirobuxinc D).

Correlation spectra (Cozy, XH. HMBC) confirm these structures.

I> Vitamin E

(H ₃

Jύ 31

The spectra have a very great similarity with those of your bυxaminc G since there is only one methyl group of difference.

On the mass spectrum, the molecular ion appears at m / z 384 ci the ions have a mass difference of 14 with those observed for buxaminc G.

CvclohuxnphvUlnc M

30 hrs

My S £

The molecular ion appearing m / z 369, fdvèlc that this compound is a monobasic alkaloid (odd number of nitrogen atoms).

On the mass spectrum, we find the characteristic ions, methylamino-substituted carbon 3 groups; the ions M-15 (m / z 354; 1 1) and a m z 70 (48), m z 58 (9). m / z 57

(100) and m / z 44 (10).

Also characteristic of this compound, the ion M-43 (m / z 326, 6), NMR

The displacement of the H19 protons which appear as a 0.3S doublet and 0.63 ppm (J

= 4Hz). reveals the presence of a cyclopropyl group and makes it possible to attach this compound to the group of 9 [U9-cyclo-5-α-prcgnancs.

The H20 proton appears in the form of a quadrupla (J ≈ 7.5 Hz), by coupling with the median protons at C21. 6.55 ppm (trans derivative) or 5.7 1 ppm (cis derivative). The C21 methyl protons appearing as a doublet (J = 7.5 Hz) 1.32 ppm (trans derivative) or 20.9 ppm (cis derivative).

The other methyl protons. in C I S, C30. C31 and C32. have chemical shifts from

0.76. 0.94. 0.95 and 1.32 ppm except those of the methyl group poπé by nitrogen in

C3 and which appear at 2.44 ppm in the form of a singulct.

On the NMR spectrum C. carbon 16. carrying the keto function. appears at 207 ppm.

N-PcnzovI- O-Acctyl bnxflf i pjn F

30 hrs

^The molecular ion was at m / z 546 (8). this compound is therefore a dibasic alkaloid. The ⁱ on at m / z 503 (1 1) results from the loss of a dimethylamino group on carbon 3 or carbon 20.

^The ion at m / z 105 (50) is characteristic of a bcnzoyle group (C ₆ H ₅ CO ^+). This allows this bcnzoyl group to be placed (nitrogen at C3 or C20). it is the ion at m / z 148 which corresponds to the ion C <jH ₅ CONΗCHCH ₃ ⁺ . This ion appears only when the _t groupemen bcnzoyle is attached to ^nitrogen poπé by carbon 20. NMR

The two protons Hl l and H19 appear respectively as a singulct enlarged to

5.52 ppm and a singlet at 5.91 ppm and indicate that this compound has a structure of πσr-19-

/ ισm £ -B-5-α-prcgnane. The aromatic proions are easily identifiable: doublet at 7.72 ppm (J = 7 Hz) for protons in ort o, triplet at 7.40 ppm (J = 7 Hz) for protons in meta and doublet at 7.46 ppm

(J = 7 Hz) for the proton in para.

The nitrogen-borne proton appears as a doublet at 6.37 ppm (J = 8 Hz) and the proton

H2O as a mutiplet. At 4.37 ppm. The twin proton with the acetyl function has a chemical shift of 5.09 ppm (triplet. J = 8

Hz).

The signals and chemical shifts of methyl proions are also very characteristic: those of the acetyl function appear in the form of a singulct at 1.78 ppm, those on carbon 21 in the form of a doublet at 1.28 ppm (J = 6.5 Hz), those of the dimethylamino group in the form of a singlet at 2.28 ppm and the others in the form of singlets at

0.71 and 1.00 ppm (methyl 30 and 31). 0.86 and 0.87 ppm (methyl 18 and 32).

In the ¹³ C NMR spectrum, several carbons are important: Its aromatic carbons (134.7 ppm for Cl ^' . 126.5 ppm for C2' and C6 '. 128.3 ppm for C3' _and C5 'and _t 131.1 ppm for C4 ^' ). ethylene carbons (138.2 ppm for C10. 128.2 ppm for C19. 136.6 ppm for C9 and 127.6 ppm for Cl 1) and carbons carrying a carbonyl function (170.3 ppm for acetyl ci 165.9 ppm for bcnzoyle).

Also characteristic of the chemical shifts of carbons 3 (71.7 ppm) _, 16 (79.3 ppm) and 17 (56.7 ppm).

The structure of this compound was confirmed by the study of the rest of the 1 H NMR and ¹³ C NMR spectra as well as correlation spectra (Cozy. XH and HMBC).

Seven majority alkaloids have been isolated, pure or mixed, from Buxus sempervirens.

They are four alkaloids of type 9β, 19-cyclo-5-α-pregnane, cycloprotobuxine D, cyclobuxine D, cyclovirobuxine D and cyclobuxophylline M, and three alkaloids of type nor-19-homo-B -5-α-pregnane, buxamine G, buxamine E and N-benzoyl-O- acetylbuxadienine E.

Among these compounds, only cyclobuxophylline M is a monobasic alkaloid, the others being dibasic alkaloids.

While some of these alkaloids, such as buxamine E, cyclobuxine D or cyclovirobuxine D, are according to the bibliographic data held to be the majority, others, such as cyclobuxophylline M and N-benzoyl-O-acetylbuxadienin E do not did not appear to be important compounds.

Total alkaloids (A, B and alkaloid fraction of C) represent about 1% of the drug treated, but none of the compounds identified seem to exceed 2% of the total alkaloids.

However, buxamine G and cyclobuxophylline M seem to be a little more important than the others. These are also the alkaloids which presented the least difficulties for their isolation.

In view of the thin layer chromatography plates, it appears that there are a very large number of alkaloids in Buxus sempervirens. However, it would be possible to isolate 1 or 2 new important alkaloids.

The drugs prepared by using enriched extracts of Buxus sempervirens according to the invention can be used for the prevention of graft rejection in organ and tissue transplants, as well as for the treatment of rejection after bone marrow transplant.

These drugs can also be used to treat nephrotic syndromes, psoriasis, rheumatoid arthritis, uveitis, and bone marrow aplasia.

More generally, such drugs demonstrate anti-IL 2 activity and can therefore promote numerous immune mechanisms.

Claims

1. Use of Buxus sempervirens for the manufacture of a medicament with anti-IL 2 activity.

2. Use of an extract of Buxus sempervirens enriched in alkaloids for the manufacture of a medicament endowed with an anti-IL 2 activity.

3. Use of Buxus sempervirens according to claim l, characterized in that the extract of Buxus sempervirens has been enriched in alkaloids by extraction using a gradient of acidic hydroalcoholic solutions.

4. Use of Buxus sempervirens according to claim 3, characterized in that the extract enriched in alkaloids contains one or more alkaloid (s) obtained (s) by separation methods on a chromatographic column.

5. Use of Buxus sempervirens according to claim 4, characterized in that the said alkaloid (s) are chosen from the various alkaloids: Cycloprotobuxine D, Buxamine G, Cycloprotobuxine A, Buxamine E, N-benzoyl-O-acetyl buxadienin E, Cyclomicrophylline B , Cyclobuxophylline M, Cyclovirobuxinc D, and their derivatives and mixtures.