CA2162703A1 - Novel lupane derivatives, their preparation and pharmaceutical compositions containing same - Google Patents

Abstract

Lupane derivatives of general formula (I) in which R is: (II), wherein X is a bond or an optionally substituted carbamoyl or aminocarbonyl, Y is a bond or phenylene, R DEG and R DEG DEG , which are the same or different, are H or methyl and n is 6 to 12, m is 0 to 3 and p is 0 to 2, with 6 </= m+n+p </= 14, and R1 is methyl, hydroxymethyl, -CH2OR' or -CH2SR' (R' is alkyl or hydroxyalkyl), the alkyl radicals (1 to 4C) are straight or branched. The invention also concerns the salts of said derivatives and their preparation. The products according to the invention are especially useful in the treatment of AIDS and in infections induced by the herpes family of viruses.

Description

2 ~ 2 ~ 3 WO 94/26725 PCT / I; R94 / 00533 NOUV ~ UX D ~ RIV ~ DU T ~ upAN ~ T ~ T`uR PRT'PARATION F ~ 'T TT` ~ COMPOSITIONS
PHARMA ~ T ~ `UTIOUT '~ YES TT ~' ~ CONTIT ~` NNT'NT

The present invention relates to lupane derivatives, of formula general:

CH3 ~ CONH-R
(I) . Hl, H CH3 HO

their salts, their preparation and the pharmaceutical compositions which contain them.

In the ~ Pm ~ n ~ Japanese patent J 01 143 832 have been described betulin derivatives of general formula:

H 2 C = l "

`H2 - R2 in which R1 and R2 are hydroxy or acyloxy and R3 is in particular methyl. These derivatives are useful in the ~ o ~ -ne anticancer.

It has now been found that the lupane derivatives of the general formula rale ~ I) in which:

R represents a radical of general formula:

WO 94/26725 PCT ~ R94 / 00533 2 ~ 627 ~ 3 2 NOT
(CH2) n - X - (CH2) m - Y (C) p ~ / ll (II) RRN

X is a bond or represents a carbamoyl radical, N-methyl carbamoyl, aminocarbonyl or N-methylaminocarbonyl, Y is a bond or represents a meta or para phenylene radical, R and R identical or different are hydrogen atoms or methyl radicals (it being understood that all the -CRR units are not not necessarily identical) and, n is an integer from 6 to 12, m is an integer from 0 to 3 and p is an integer from 0 to 2 with the understanding that m + n + p is com-taken between 6 and 14, and R1 is a methyl, hydroxymethyl radical or a -CH2OR 'radical or -CH2SR 'for which R' is alkyl or hydroxyalkyl, as well as their pharmaceutically acceptable salts, exhibit a cytoprotective effect of cells infected with an HIV virus (Human Immunodeficiency Virus) and inhibitory activity production of reverse transcriptase of an HIV virus.

In the general formula above, it is understood that the radicals alkyl are straight or branched and contain 1 to 4 carbon atoms bone.

In cases where R and R are different, the products of formula general (I) have stereoisomeric forms, it is also understood that the stereoisomeric forms as well as their mixtures Also within the scope of the present invention.

According to the invention, the lupane derivatives of general formula (I) can can also be prepared by the action of an azide on a nitrile of general formula:

j ~) 94/26725 ~ 16 2 ~ ~ 3 PCT / FR94 / 00533 ~ ~ ~ CoNH- (cH2 \ nX- (CH2) mY- (C) p-CN

¦ H ~, H CH3 RR

HO ~
H3C CH3 (III) where R1 R, R, X, Y, n, m, and p are defined as previous ~ ^ m ~ ^ nt.

The azide is advantageously chosen from the sorting azide n.butyltin, trimethylsilyl azide or an alkali azide.
When using tributyltin azide the reaction is carried out in an organic solvent such as 1,2-dimethoxyethane, 2-ethoxyethanol or a mixture of such solvents, at the temperature reflux of the reaction mixture. When the azide of trimethylsilyl, the operation is carried out in the presence of aluminum trichloride, in a chlorinated solvent such as, for example, dichloromethane, at a temperature between 10 and 40C.

According to the invention the lupane derivative of general formula (I) for which X (in the radical R) represents a carbamoyl radical or optionally N-methylated aminocarbonyl and the other radicals are defined as above ~ ^ mm ~ nt, can also be obtained from a lupane derivative of general formula:

CH3 ~ CONH-R (IV) ¦ Hl, H CH3 HO

WO 94/26725 PCT ~ R94 / 00533 ~ 1627 ~ 3 in which R1 is defined as pr ~ ce ~ -rt and R "represents a radical of general formula:
(CH2) n - COOH (IVa) or (CH2) n-NH-R "'(IVb) in which n is defined as above ~ f ~ rt and R "'is an atom of hydroqene or a methyl radical, by the action respectively of a amine or acid of general formulas:
N ~ N
R "'- HN - (CH2) m - Y / C) P ~ N ~ N (V a) RRH
or N - N
HO-CO - (CH2) m - Y (C) p ~ ~ N (V b) RRH

in which R, R, m and p are defined as above ~ Pmm ~ nt and R "'is defined as above.

It is understood that the hydroxy radicals of the lupane derivative of general mule (IV) are preferably protected by radicals compatible which can be set up and fli mi n ~ S without touching the rest of the molecule. For example, protective groups can be chosen from the radicals described by TW GREENE, Protective Groups in Organic Synthesis, J. WILEY-Interscience Publication (1991) or by Mc OMIE, Protective Groups in Organic Chemistry, Plenum Press (1973). We will choose radicals that can be ~ limin ~ 5 in neutral, basic or acidic medium.

In particular, hydroxy radicals can be protected ~ the state of ter (formyloxy, acetoxy, i.butoxy, trichloracetyloxy, phenoxyacetyl-oxy, benzyloxy) i in this case, ~ liminAtion is carried out by hydrolysis in basic medium, in particular in the presence of soda at a temperature between 10 and 50C. Protection can also be performed killed ~ ketone state, in the form of carbonate by a radical wo 94.26725 2 I 6 ~ 7 0 ~ PCT ~ R94 / 00533 -COORa in which Ra is an optional alkyl or benzyl radical-ment substituted or also by a trialcoylsilyl radical.

The reaction is carried out according to the usual condensation methods of an amine on an acid. In particular, we operate in the presence of an accept-acid such as an organic nitrogen base (trialcoylamine, pyri-dine, N-methyl-morpholine, diaza-1,8 bicyclo [5.4.0] 11n ~ supper-7, diaza-1.5 bicyclo [4.3.0] nonene-5 for example) in an organic solvent as a chlorinated solvent (dichloromethane, dichlorethane, chloroform for example) or an amide (dimethylformamide for example). It is also It is also possible to operate in the presence of a condensing agent such as a carbodiimide (dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for example) and optionally-ment in the presence of a catalyst such as N-hydroxybenzotriazole or N-hydroxysuccinimide, at a temperature between -20 and 40C.

The amine of the general formula (Va) is condensed by preferably from the amine hydrochloride.

The nitrile of general formula (III) can be prepared by dehydration of the corresponding amide, by any known method which does not alter the rest of the molecule.

One operates in particular by means of trifluoroacetic anhydride in a ether (tetrahydrofuran for example), at a temperature between 10 and 66C

The amide corresponding to the nitrile of general formula (III) can be itself obtained from the acid according to known methods and / or cited below in the examples.

The corresponding acid can be prepared by the action of a ~ tno ~ cide of general formula:
H2N - (cH2) nx- (cH2) my- ~ c ~ p-cooH (VI) - RR

WO 94/26725 PCT ~ R94 / 00533 ~ lS2703 6 where R, R, X, Y, n, m, and p are defined as above ment and whose acid function is pr ~ AlAhl ~ -nt protected, on the acid chloride of general formula:

CH3 ~ COOH (VII) ~ Hl, H CH3 HO

in which Rl is defined as above ~ t and whose radicals hydroxy are protected beforehand, followed by the elimination of the protective radicals.

The reaction of the amine on the chloride of lupen-20 (29) oic acid-28 of general formula (VII) is carried out according to the usual methods which do not affect the rest of the molecule. We operate in particular-ment in the presence of an organic nitrogenous base such as a trialkyla-mine (triethylamine for example) in an organic solvent such as chlorinated solvent (chloroform, 1,2-dichloroethane, dichloromethane) or in tetrahydrofuran or in a mixture of these solvents, at a temperature between 15 and 30C.

It is understood that the hydroxy radical (s) are preferably previously protected. Protection and liberation of radicals protectors are made as described above ~ rt.

For example, the acid protecting radicals can be chosen from alkyl radicals (methyl, ethyl, t.butyl), al-substituted coyl (trichlorethyl, haloethyl, p.toluenesulfonyl-ethyl, benzyl, benzyl substituted by a nitro radical, benzhydryl, triphenylmethyl, benzyloxymethyl ...), alkyloxyalkyl (methoxymethyl-thyle), tetrahydropyranyl or trimethylsilyl. The establishment and the ~ minAtion of these radicals is carried out according to the methods usual cited above.

~ o 94,26725 ~ ~ 6 ~ PCT ~ R94 / 00533 When the radical X (in R) is a carbamoyl radical or inoc ~ rbonyl optionally N-methylated, the nitrile of the general formula rale (III) can also be prepared by condensation of an amine or an acid of general formula:
R "'- NH - (CH2) m ~ Y- (C ~ p - CN tVIIa) RR
or HO-CO - (CH2) mY- (C) p - CN (VIIb) RR

where R "', R, R, Y, m, and p are defined as above ment, on a lupane derivative of general formula (IVa) or (IVb).

The reaction is carried out under conditions identical to those of preparation of a lupane derivative of general formula (I) ~ from an amine or an acid of general formula (Va) or (Vb) and a lupane derivative of general formula (IV).

Acid derived from lupane, of general formula (IVa) can be prepared by analogy with the reaction of Amino ~ cide of general formula (VI) on the acid chloride derived from lupane of general formula (VII), by the action of an amino acid of general form:

H2N ~ (CH2) n ~ CH (VIII) in which n is defined as above ~ ~ nt and whose function acid is previously protected.

The amine derived from lupane, of general formula (IVb), for which R '''is a hydrogen atom, can be prepared from a lupane derivative of general formula (VII), by the action of a ~ i ~ ine of general formula:

H2N- (cH2) n-NH2 (IX) in which n is defined as prec ~ e cnt ~

The derivatives (IVb) for which R '''is a methyl radical may be obtained by N-methylation according to the method described by WO 94/26725 21 ~ 2 7 ~ 3 PCT ~ Rg4 / 00533 A. KATRITSKY, J. Chem. Soc. Perkin Trans. I, 2539 (1987), from product of general formula (IVb) for which R '''is an atom hydrogen.

The products of general formula (Va) or (Vb) can be prepared according to or by analogy with the methods cited or described below in the examples.

The lupane derivatives of general formula (IV) or (VII) for which Rl is a hydroxymethyl radical can be prepared by the action of silver acetate on the corresponding brominated derivative for which R1 is bromomethyl and for which the hydroxy radical is previously protected. The reaction is advantageously carried out in an organic solvent.
for example toluene, at a temperature between 20C and the reflux temperature of the reaction mixture. The derivative starting bromine can be obtained by action of a bromination agent such as tetrabutyl tribromide ~ mm ~ r - um or N-bromosuccinimide on the corresponding derivative of lup-20 acid (29) -en-28-oic. The reaction is carried out in a chlorinated solvent such as methylene chloride, chloroform, or carbon tetrachloride at a temperature close to 25C.

The lupane derivatives of general formula (VII) for which R1 is a radical -CH2OR 'or -CH2SR' can be prepared from the de-riveted lupane for which R1 is bromomethyl, by action of the corresponding alcoholate or thiolate.

The acid chloride of the lupane derivative of general formula (VII) can be prepared according to known methods ~ s For example, operates by the action of oxalyl chloride or thionyl chloride, in a chlorinated solvent such as chloroform, dichloroethane or dichloromethane The new lupane derivatives of general formula (I) can be purified if necessary by physical methods such as crystallization or chromatography.

When the lupane derivative of general formula (I) exhibits stereoisomeric forms, the preparation is carried out by means of rivets O 94/26725 2 1 ~ 2 ~ ~ ~ PCT ~ R94 / 00533 chirals of general formula (Va), (Vb), (VI), (VIIa) or (VIIb). he it is understood that when one wants to obtain a chiral intermediary, the separation is carried out according to the usual methods which do not affect not the rest of the molecule, such as for example the chromatographic phie on chiral phase, or by conversion of l ~] ~ racemic endiastereoisomers and separation by crystallization or chromatography phie.

The products according to the invention can be transformed into metallic salts.
metallic with strong bases according to methods known per se.
These salts can be obtained by the action of a metal base in a suitable solvent. The salt formed precipitates after concentration possible solution; it is separated by filtration.

As examples of pharmaceutically acceptable salts can be cited the salts with alkali metals (sodium, potassium, lithium).

The new lupane derivatives according to the present invention are particularly useful for prophylaxis and treatment of AIDS
(acquired immunodeficiency syndrome) and associated syndromes [ARC
(AIDS related complex)]. By prophylaxis we mean the treatment of subjects who have been exposed to HIVs, in particular link asymptomatic HIV-positive people who are at risk of developing the disease in the coming months or years after primary infection.

The products according to the invention, which are inhibitors of the effect HIV cytopathogen and inhibitors of transcriptase production reverse in cell culture at ineffective concentrations cytotoxic or cytostatic, are particularly interesting.

It has also been demonstrated that the products according to the invention are active for the treatment of conditions in which viruses of the herpes family are involved.

The herpes family is indeed the source of many conditions some of which can be very serious. She understands Herpes simplex, varicella-zoster, cytomegalovirus and virus of Epstein-Barr. Herpes simplex can vary from mild forms like WO 94/26725 PCT ~ R94 / 00533 ~
2 ~ 9 ~ 7 ~ 3 ~ o cold sores with more serious forms such as genital herpes and may even be responsible for life-threatening encephalitis in danger. Varicella zoster is the virus responsible for chickenpox and shingles, it can also cause illnesses more serious including encephalitis. Infections cytomegaloviruses are generally asymptomatic in subjects healthy, but can cause serious eye infections (retinitis) which can lead to blindness, morbidity and mortality in immunocompromised subjects (people with AIDS or any other immunodeficiency, for example after transplantation organs or after cancer chemotherapy). Cytomegalovirus is also responsible for severe clinical manifestations for the fetus or newborn in the case of a primary infection during pregnancy or when transferring seropositive blood to a new-born HIV negative.

The activities were highlighted in the following tests:

Activity vis-à-vis the cytopathic effect ~ wine ene ~ HIV

The powdered products were dissolved in a proportion of 2 mg of produced by 2 ml (approximately 4 x 10 M) in dimethylformamide (DMF) to obtain a 100% DMF product stock solution. The test is carried out on the lymphoh1 ~ tode CEM 4 line. In a microplate of 96 wells 25 ~ l / well of a solution of product to be tested are deposited in isotonic phosphate buffer (TPI) or TPI alone in the case controls. The products are studied at 8 concentrations, of 6 wells per concentration. 125 ~ l of a suspension are then added C ~ M cells (8 x 10 cells per ml) in RPMI medium containing 10% fetal calf serum, 100 IU / ml penicillin, 100 ~ g / ml streptomycin and 2 ~ moles / ml glutamine and microplates are incubated for one hour at 37C, under an atmosphere containing 5% carbon dioxide. For each concentration, the test is divided into two parts: one part (3 wells) on cells infected, for determining antiviral activity and the other part (3 wells) on uninfected cells, to determine the product cytotoxicity. We then infect the first series with HIV-1 (100 ~ l per well of a suspension of LAV-1-BRU virus containing ~ 0 94/2672 ~ 21 6 ~ ~ ~ 3 PCT ~ R94 / 00533 200-300 TCID50) while the other series receives 100 ~ l of RPMI medium as defined above ~ m ~ nt. After 5 days of incubation, 100 ~ l cells are taken to measure cell viability [determined according to a modification of the technique described by R.
Pauwels et al., J. Virol. Meth., ~ Q, 309-321 (1988)]. We add to this sample 10 ~ l of a solution containing 7 mg of M ~ T [bromide

3- (4,5-dimethyl 2-thiazolyl) -2,5-diphenyltetrazolium] per ml of isotonic phosphate buffer. After 3 hours of incubation at 37C the supernatant is removed. MTT is converted to a formazan salt (blue) only inside living cells. We add then 100 ~ l of isopropanol (containing 0.04 mole / l of acid hydrochloric) and the microplates are agitated until the solubilization of formazan blue. Absorbance at 540 nm is read with an automatic microplate ELISA reaction reader. This absorbance is proportional to the amount of living cells.

The protection rate (in ~) of a given product is determined by-drawing of the optical densities (OD) by the formula:

DO ~ cell. treated and inf.) - DO (cell not treated and inf.) DO (cell treated and not inf.) - DO (cell not treated and inf.) If necessary, the inhibitory concentration 50% is determined.

The results demonstrate that for concentrations of product tested between 0.01 and 10 ~ g / ml, we obtain a ~ nution signifi-cative of the cytopathogenic effect.

The protection rate provided by the products according to the invention is 25 between 20 and 100%

The inhibitory concentration 50 ~ of the products according to the invention is between 0.05 and 50 ~ g / ml when it can be determined.

D ~ ter ~ in ~ tion ~ viral multiplication by ~ bone ~ e ~ e the tran ~ criD-HIV virs reverse tase The reverse transcriptase activity is measured in 10 ~ l of culture supernatant Using the poly r (A) reverse assay kit transcriptase [3H] -SPA enzyme assay (RPQN 0100, Amersham, GB) according to WO 94/26725 2 ~ 6 ~ q 0 3 12 PCT ~ R94 / 00533 the procedure recommended by the manufacturer. The reaction time is 6 hours and radioactivity counted using a microbeta counter tTOPCOUNT ~ PACKARD].

Inhibitions of reverse transcriptase production are observed associated with the virus, between 20 and 100% at concentrations tested (0.01 to 10 ~ g / ml).

Action on herbal family viruses:

The action of derivatives of general formula (I) on the viruses of family of herpes was highlighted in the described technique by NEYTS et al., Virology, 179, 41-50 (1990).

The technique used consists of measuring the effect cytopathogen of the virus and its protection by the use of products of general formula (I). Antiviral activity is appreciated by measuring the EC50 (concentration necessary to inhibit 50 virus-induced cytopathic effect).

The results obtained in this technique are given below:

Activity on cytomegalovirus was studied on Davis and DER 648 (strain resistant to Ganciclovir):

, Strain Davis DER 648 Example n EC50 (~ g / ml) EC50 (~ g / ml) 1 0.86 0.57 0.8 0.76 13 sup. 2 extra 0.5 14 sup. 2 2 Table I

The activity on varicella zoster was studied on the strain resistant thymidine kinase negative (TK-) YS / R:

O 94/26725 ~ 1 6 2 7 0 ~ PCT ~ R94 / 00533 N of the example YS / R strain EC50 (~ g ~ ml) 1 sup. 2 13 sup. 2 Table II

The following examples, given without limitation, illustrate the pre-invention.

~ mD on 1 To a solution of 0.5 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- acid aminooctanoic in 90 cm of dichloromethane, we have ~ 125 mg of 5- ~ min ~ hydrochloride thyl-tetrazole, 141 mg hydrate 1-hydroxybenzotriazole, 380 mq of 1- (3-dimethylamino-) hydrochloride propyl) -3-ethylcarbodiimide and 0.48 cm of triethy1 ~ in ~. The solution is stirred for 24 hours at 20C then the reaction mixture is washed 5 times with 100 cm of distilled water. The organic phase is then dried over magnesium sulfate and evaporated to dryness under pressure reduced sion (2.7 kPa) at a temperature close to 40C. The residue obtained (600 mg) is chromatographed on a silica column (0.02-15 0.045 mm) eluted first with 2 liters of a ~ nge of cycloh ~ Y ~ ne and ethyl acetate (30-70 by volume), then with 2 liters of one mixture of methanol and ethyl acetate (30-70 by volume); the product-containing fractions are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue is taken up with 10 cm of ethyl ether, filtered, washed twice with 5 cm of ethyl ether and dried under reduced pressure (13.5 Pa) at 20C. 290 mg of 5- {N '- [N- (3 ~ -acetoxylup-20 (29) - ~ n- are thus obtained.

WO 94/26725 ~ 7 ~ 3 PCT ~ Rg4 / 00533 28-oyl) -8-aminooctanoyl] -Am; n ~ m ~ thyl} -tetrazole in the form of a hlAnch ~ powder (Rf = 0.26; thin layer chromatography of silica; eluent: chloroform-methanol-A ~ m ~ n-Aque at 20% (12-3-0.5 in volumes)).

A solution of 1.07 g of 5- {N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] - ~ minomethyl} -tetrazole obtained as described below-above, 5.7 cm of 5N sodium hydroxide, 18.4 cm of tetrahydrofuran and 36.9 cm3 of methanol is stirred for 20 hours at a temperature in the region of 20C. The mixture is cooled to 5C, then acidified with chlorhy-drique 5N, and diluted with 100 cm of distilled water. After 1 hour under stirring at 5C, the solid obtained is separated by filtration, washed 5 times with 25 cm of distilled water and dried under reduced pressure (15.5 Pa) at a temperature in the region of 20C. 880 mg is thus obtained of 5- {N '- [N- (3 ~ -hydroxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino-methyl} -tetrazole, in the form of a white solid melting towards N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoic acid can be prepared as follows:

After heating at reflux for 3 hours, a mixture of 330 mg of 8-aminooctanoic acid and 410 mg of chlorotrimethylsilane in 50 cm of dichloromethane, the solution is cooled to around 20C and add 1 g of 3 ~ -acetoxylup-20 (29) -en-28-oyl chloride (prepared at from 3 ~ -acetoxylup-20 (29) -en-28-oic acid) then 0.81 cm3 of triethylamine. Stirring is continued for 15 hours at a time temperature close to 20C and the solvent is evaporated to dryness under pressure reduced sion (2.7 kPa) at a temperature close to 35C. The residue is chromatographed on an eluted silica column (0.02-0.045 mm) with a mixture of cyclohexane and ethyl acetate (60-40 in vo-lumes). The fractions containing the product are collected ~ hl ~ and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 35C. 1.1 g of N- (3 ~ -acetoxylup-) acid are thus obtained.
20 (29) -en-28-oyl) -8-aminooctanoic in the form of a meringue hlAnche (Rf - 0.4; thin layer silica chromatography;
eluent: cyclohexane, ethyl acetate (60-40 by volume)).

O 94/26725 ~ 1 S 2 ~ ~ 3 PCT ~ R94 / 00 ~ 33 5- ~ min ~ m ~ thyl-tetrazole hydrochloride can be prepared according to the method described in the ~ m ~ n ~ of patent DE 3,626,130.
F ~ mnle 2 To a solution of 640 mg of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) acid -8-aminooctanoic in 50 cm of chloroform, 134 mg of 5-amino-1H-tetrazole, 168 mg of 1-hydroxybenzotriazole hydrate, 50 cm3 of dimethylformamide, 692 mg of 1- (3-dimethyl-) hydrochloride laminopropyl) -3-ethylcarbodiimide, and 0.70 cm of triethylamine as solution in 5 cm of chloroform. The solution is stirred for 2 10 days at 20C then the reaction mixture is washed 3 times with 50 cm3 1N hydrochloric acid, then 5 times with 100 cm of distilled water.
The organic phase is dried over magnesium sulfate and evaporated dry under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (730 mg) is chromatographed on a column 15 of silica (0.02-0.045 mm) eluted with 800 cm of ethyl acetate then with 800 cm of a mixture of dichloromethane and methanol (95-5 in volumes). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue is taken up in 15 cm of isopropyl ether, filtered, 20 dried under reduced pressure (13.5 Pa) at 20C. 265 mg are thus obtained of 5- {N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino} -tetrazole in the form of a beige powder (Rf 0.40; chromatography phie on a thin layer of silica; eluent: chloroform-methanol-ammo-niac at 20% (12-3-0.5 by volume)).

25 260 mg solution of 5- {N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino} -tetrazole, 0.92 cm3 of hydroxide 1N thium, 2.5 cm3 of tetrahydrofuran and 5 cm of methanol is stirred 20 hours at a temperature close to 20C. The mixture is acidified with lN hydrochloric acid, and diluted with 80 cm of distilled water lée. The mixture is then extracted with 100 cm of ethyl acetate then 2 times with 25 cm of ethyl acetate. The organic phases are combined, washed 4 times with 40 cm of distilled water, dried on magnesium sulfate and evaporated ~ dry under reduced pressure (2.7 kPa) at a temperature close to 45C. The residue obtained 35 (170 mg) is chromatographed on a silica column (0.02-WO 94/26725 PCT ~ R94 / 00533 ~ 27 ~ 3 16 0.045 mm) eluted with a mixture of chloroform, methanol and A -ni ~ only at 20% t12-3-0.5 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) to a temperature close to 40C. The residue is taken up by 5 cm isopropyl ether, filter, wash 2 times with 1 cm of oxide of isopropyl and dries under reduced pressure (15.5 Pa) at 20C. We thus obtains 80 mg of 5- {N '- [N- (3 ~ -hydroxylup-20 (29) -en-28-oyl) -8-aminooctanoyl] -amino} -tetrazole, as a white solid melting around 165-170C.

~ mnle 3 Has a solutlon of 100 mg of N- (3 ~, 30-diacetoxylup-20 (29) -en-28- acid) oyl) -8-aminooctanoic in 50 cm of dichloromethane, we add 22 mg of hydrochloride of 5- ~ min ~ thyl-tetrazole, 25 mg of hydrate of 1-hydroxybenzotriazole, 70 mg of 1- (3-dimethylamino-) hydrochloride propyl) -3-ethylcarbodiimide and 0.09 cm of triethylamine. The solution is stirred for 24 hours at 20C then the reaction mixture is washed successively 3 times with 50 cm of distilled water, 2 times with 25 cm of 1N hydrochloric acid, then 3 times with 50 cm of water distilled. The organic phase is then dried over sulphate of magnesium and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (110 mg) is chromato-written on a silica column (0.02-0.045 mm) eluted with a metal lange of chloroform, methanol and Ammnni ~ that at 20% (12-3-0.5 in vo-lumes). The fractions containing the product are combined and concentrated trees under reduced pressure (2.7 kPa) at a temperature close to 40C. This gives 65 mg of 5- {N '- [N- (3 ~, 30-diacetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl] -Amin ~ thyl} -tetrazole in the form a white meringue (Rf = 0.40; thin layer chromatography silica; eluent: chloroform-methanol- ~; ~ only 20% (12-3-0.5 in volumes)).

A 160 mg solution of 5- {N '- [N- (3 ~, 30-diacetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl ~ - ~ min ~ m ~ thyl} -tetrazole, 1.3 cm 5N sodium hydroxide, 6 cm of tetrahydrofuran and 3 cm of methanol is stirred for 20 hours at a temperature close to 20C. The mixture is cooled to 5C, then acidifies with 5N hydrochloric acid, and diluted with 30 cm3 of water ~ 0 94/2672 ~ 21 6 2 7 0 3 PCT ~ R94 / 00533 distilled After 1 hour with stirring at 5C, 150 cm are added distilled water and the mixture is extracted 5 times with 50 cm of a mixture mixture of dichloromethane and methanol (9-1 by volume ~. The phases organics are combined, dried over maqnesium sulfate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C The residue obtained (140 mg) is chromatographed on a silica column (0.02-0.045mm) eluted with a ~ el ~ nqe of chloro-form, methanol and ~ m ~ Q ~ i ~ only 20% (12-3-0.5 by volume). The frac-tions containing the product are combined and concentrated under pressure reduced (2.7 kPa) at a temperature close to 40C. The residue is taken up in 2 cm of acetonitrile, filtered and dried under pressure pick (15.5 Pa) at 20C. 48 mg of 5- {N '- [N- (3 ~, 30-di-hydroxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -aminomethyl} -tetrazole, in the form of a white solid melting around 145-150C

15 The N- (3 ~, 30-diacetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoic acid is synthesized by analogy with N- (3 ~ -acetoxylup-20 (29) -èn-28- acid oyl) -8-aminooctanoic from 3 ~, 30-diacetoxylup- chloride 20 (29) -en-28-oyl and methyl 8-aminooctanoate (R = 95%; Rf =
0.34; thin layer chromatography on silica; eluent: cyclo-hexane-ethyl acetate (50-50 by volume)).

The chloride of 3 ~, 30-diacetoxylup-20 (29) -èn-28-oyle is prepared from as follows:

To a solution of 560 mg of acid 3 ~, 30-diacetoxylup-20 (29) -èn-28-oic in 25 cm of dichloromethane, 0.22 cm of chloride is added of oxalyl. After 15 hours of stirring at a temperature close to 20C, the solvent is evaporated under reduced pressure (22 kPa) and at a temperature close to 40C We obtain 600 mg of a meringue hlAn ~ he which is used without further purification.

Acid 3 ~, 30-diacetoxylup-20 (29) -èn-28-oïque is obtained from ma-Next 30:

A suspension of 1.8 g of 3 ~ -acetoxy-30-bromolup-20 (29) -èn-28- acid oic and 800 mg of silver acetate in 50 cm of toluene is agitated 48 hours at a temperature close to 20C then filtered. The solution obtained is then concentrated to dryness under reduced pressure (2 kPa).
--WO 94l26725 PCT ~ R94 / 00 ~ 33 ~
~ 2 ~ ~ 3 to 18 The residue is dissolved in 50 cm3 of ethyl acetate. The organic phase is decanted and washed with a total of 60 cm of water distilled, dried over anhydrous magnesium sulfate, then concentrated reduced pressure (2.7 kPa) at an Vosine temperature of 40C. The white solid obtained (1.7 g) is chromatographed on a co-lonne 1.7 cm in diameter and containing 35 g of silica (0.02-0.045 mm) eluted with a mixture of cycloh ~ n ~ and ethyl acetate 80-20 ten volumes), with fractions of 15 cm. The 4 first fractions obtained are ~ l imin ~, the next 10 are concentrated to dryness under reduced pressure (13.5 Pa) at a temperature close to 40C. 1.1 g of 3 ~, 30-diacetoxylup- acid are thus obtained.
20 (29) -èn-28-oique in the form of a white meringue sufficient ~ Amment - pure for subsequent transformations.

3 ~ -acetoxy-30-bromolup-20 (29) -en-28-oic acid is obtained from as follows:

A solution of 2.5 g of 3 ~ -acetoxy-lup-20 (29) -en-28-oic acid and 2.41 g of tetrabutyl tribromide ~ mm ~ nium in 50 cm3 of chloroform is stirred for 5 days at a temperature in the region of 25C. The mixture re-active is washed successively twice with 25 cm of distilled water, 2 times with 25 cm of a 0.1N sodium thiosulfate solution and with 2 times 25 cm of distilled water then dried over magnesium sulfate anhydrous. The organic phase is concentrated to dryness under pressure reduced (2.7 kPa) and at a temperature close to 40C. We get a yellow pasty solid (4 g) which is chromatographed on a column of 3.2 cm in diameter containing 100 g of silica (0.02-0.045 mm) and eluted with a mixture of cyclol- ~ x ~ n ~ and ethyl acetate 85-15 (in volumes) by collecting 20 cm fractions. The first 5 fractions obtained are ~ l; mi n ~, the next 10 are evaporated under reduced pressure (13.5 Pa) at a temperature close to 40C. We thus obtains 2.7 g of 3 ~ -acetoxy-30-bromolup-20 (29) - ~ n-28-oic acid in the form of a white solid melting at a temperature close to l 90C

3 ~ -acetoxylup-20 (29) -en-28-oic acid can be prepared according to BRUCXNER, KOVACS and KOCZKA, J. Chem. Soc., 948 (1948).

_ ~ YO 94/26725 PCT ~ R94 / 00533 `~ 2 ~ 6 ~ 0 ~
, 9 ~ m ~ the 4 To a solution of 400 mg of N- [3 ~ -acetoxylup-30- (2'-aCetoxy-thylthio) -20 (29) -èn-28-oyl] -8-aminooctanoique, 90 mg hydrochloride of 5-aminomethyltetrazole, 100 mg of 1-hydroxybenzotria- hydrate zole, 250 mg of 1- (3-dimethylaminopropyl) -3-ethyl- hydrochloride carbodiimide in 100 cm of dichloromethane, is added in approximately 10 minutes, 0.32 cm of triethylamine. The solution is stirred for 15 hours at a temperature close to 20C, plus 50 cm distilled water then acidified to a pH close to 1 per 15 cm of a so-aqueous hydrochloric acid N. The organic phase is decanted tée, the aqueous phase is extracted with 3 times 50 cm of dichlorom ~ -thane. The combined organic phases are then dried on sul-magnesium fate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (400 mg) is 15 chromatographed on a silica column (0.02-0.045 mm), eluted with a mixture of dichloromethane and methanol (90-10 by volume). The fractions containing the product are combined and concentrated under pressure reduced sion (2.7 kPa) at a temperature close to 40C. We obtain thus 300 mg of 5- {N '- [N- (3 ~ -acetoxylup-30- (2'-acetoxyethylthio) -20 20 (29) -en-28-oyl) -8-aminooctanoyl] aminomethyl} -tetrazole under the form of a white meringue (Rf = 0.40; layer chromatography thin silica; eluent: chloroform-methanol- ~ mmoniAque at 20%
(12-3-0.5 by volume)).

A 300 mg solution of 5- {N '- [N- (3 ~ -acetoxylup-30- (2'-acetoxy-25 thylthio) -20 (29) -en-28-oyl) -8-aminooctanoyl] aminomethyl} -tetrazole, 2.2 cm of sodium hydroxide, 10 cm of t ~ trahydrofuran and 20 cm of methanol is stirred for 48 hours at a temperature in the region of 20C. The 1 year is acidified to pH 1 by addition of 4 cm of an aqueous solution of 4N hydrochloric acid then diluted with 70 cm of distilled water.
After 2 hours with stirring at a temperature close to 20C, the solid is filtered and washed 5 times with 10 cm of distilled water. The O-the solution is dissolved in 20 cm of ethanol. The solution is filtered and the - filtrate is evaporated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (300 mg) is chromatographed on 35 silica column (0.02-0.045 mm), eluted with a mixture of chloro roform, methanol and ~ mmQni ~ only 20% (24-6-1 by volume). The frac-WO 94/26725 PCT ~ R94 / 00533 ~
~ 6 ~ 7 ~ 20 the product containing product are combined and concentrated under pressure reduced (2.7 kPa) at a temperature close to 40C. The solid obtained (150 mg) is treated with 5 cm of isopropyl ether, the solid is filtered, washed twice with 1 cm of isopropyl ether and then dried over potassium hydroxide. 100 mg of 5- {N '- [N- (3 ~ -hydroxylup-30- (2'-hydroxyethylthio) -20 (29) -èn-28-oyl) -8-aminoocta-noyl] -A ~ tnom ~ thyl} -tetrazole in the form of a melting white solid around 140C.

N- [3 ~ -acetoxylup-30- (2'-acetoxyethylthio) -20 (29) -èn-28-oyl] acid -8-aminooctanoic can be obtained in the following way:

To a solution of 500 mg of N- [3 ~ -acetoxylup-30- (2'-hydroxy-thylthio) -20 (29) -èn-28-oyl] -8-aminooctanoic acid and 600 mg of acetate sodium in 35 cm of acetic anhydride is brought to a temperature close to reflux for about 3 hours. The reaction mixture is cooled to 20C then concentrated under reduced pressure (2.7 kPa) to a temperature close to 40C. The residue is treated with a mixture of 15 cm of pyridine and 15 cm of water and stirred for 12 hours at a temperature close to 20C. The reaction mixture is concentrated to dry under reduced pressure (2.7 kPa) at a temperature close to 50C. The residue is treated with 50 cm of distilled water and 10 cm of a aqueous solution of hydrochloric acid, e N. The organic phase is decanted, the aqueous phase is extracted with 3 times 40 cm3 of dichloromethane. The combined organic phases are washed 3 times with 20 cm of distilled water, dried over magnesium sulfate and then evaporated to dryness under reduced pressure (2.7 kPa) at a temperature sine of 40C. The residue obtained (500 mg) is recrystallized from 5 cm3 an ethanol-water mixture (75-25 by volume). The solid obtained is filtered, then washed twice with 1 cm of an ethanol-water mixture (75-25 volumes) and dried under reduced pressure (13.5 Pa) on pentoxide phosphorus 400 mg of N- [3 ~ -acetoxylup-30- (2'-acetoxyethylthio) -20 (29) -en-28-oyl] -8-aminooctanoic in the form a white meringue (Rf = 0.25; chromato ~ raffia on a thin layer silica; eluent: chloroform-methanol-Amm ~ n; ~ only 20% (24-6-1 in volumes)).

~ O 94/26725 ~ ~ ~ 2 7 ~ 3 PCT ~ Rg4 / 00533 N- [3 ~ -acetoxylup-30- (2'-hydroxyethylthio) -20 ~ 29) -èn-28-oyl acid - -8-aminooctanoic can be obtained in the following way:

To a solution of 1.3 q of N- [3 ~ -acetoxylup-30-bromo-20 (29) -en- acid 28-oyl ~ -8-aminooctanoic in 10 cm of ethanol, a solution is added.
tion of 0.60 q of sodium 2-hydroxyethanethiolate in 35 cm3 ethanol. The reaction mixture is allowed to stir for 48 hours at a temperature close to 20C. We add drop by drop in 10 minutes approximately, 5 cm of an aqueous solution of hydrochloric acid N then diluted with 350 cm of water. The reaction mixture is extracted 3 times with 50 cm of dichloromethane. The combined organic phases are washed 3 times with 30 cm of distilled water and then dried over sulfate of maqnesium and evaporated to dryness under reduced pressure (2.7 kPa) at a vol temperature 40C The oil obtained (1.1 q) is chromatoqra-phied on a silica column (0.02-0.045 mm), eluted with a mixture of dichloromethane and methanol (98-2 by volume). Fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. This gives 500 mg N- [3 ~ -acetoxylup-30- (2'-hydroxyethylthio) -20 (29) -èn-28-oyl] acid -8-aminooctanoic in the form of a white meringue (Rf 0.40;
thin layer chromatography on silica; eluent: chloroform-m ~ thanol-ammonia 20% (12-3-0.5 by volume) ~.

N- [3 ~ -acetoxylup-30-bromo-20 (29) -èn-28-oyl] -8-aminooctanoic acid can be obtained as follows:

To a mixture of 640 mg of N- [3 ~ -acetoxylup-20 (29) -èn-28-oyl] -8- acid aminooctanoic and 30 cm of carbon tetrachloride, add in 350 mg N-bromosuccinimide only once. The suspension is agitated 12 hours at a temperature close to 20C. The solid is filtered, washed with 3 x 10 cm carbon tetrachloride. The filtrate is evaporated under reduced pressure (2.7 kPa) at a temperature close to 30 40C. 800 mg of N- [3 ~ -acetoxylup-30-bromo-20 (29) -en- acid are obtained.
28-oyl] -8-aminooctanoic in the form of a meringue hl ~ n ~ he (Rf -0.25; thin layer chromatography on silica; eluent: chlo-roform-methanol-ammonia at 20 ~ (24-6-1 by volume)).

N- [3 ~ -acetoxylup-30-bromo-20 (29) -èn-28-oyl] -8-aminooctanoic acid is prepared as described in Example 1.

WO 94/26725 PCT ~ R94 / 00533 Sodium hydroxyethanethiolate is prepared by the action of ethyl-sodium late on 2-hydroxyethanthiol in solution in ethanol.

3 ~ -acetoxylup-20 (29) -en-28-oyl chloride is prepared according to J.
PROVITA and A. VYSTRCIL, Collect. Czech. Chem. Common. 41, 1200 (1976).

~ mn on 5 To a solution of 1.9 g of N - [[3 ~ -acetoxylup-20 (29) -an-28-oyl] acid -8-aminooctanoic in 55 cm of dichloromethane, 2.2 cm3 are added thionyl chloride. The solution is stirred for 24 hours a 20C then the reaction mixture is concentrated to dryness under pressure reduced (2.7 kPa) at a temperature close to 35C. The residue obtained is dissolved in 25 cm of dichloromethane and the solution is again evaporated to dryness under reduced pressure (2.7 kPa) at a temperature erasure close to 35C. The solid obtained (1.97 g) and 485 mg of 5- (4-aminophenyltetrazole) are dissolved in 60 cm of dichlorome-thane and 1.41 cm of triethylamine are added dropwise. The solution obtained is stirred for 24 hours at a temperature close to 20C. The solution obtained is concentrated under reduced pressure (2.7 kPa) at a temperature close to 35C. To the solid obtained is added 50 cm of dichloromethane and 25 cm of an acid solution hydrochloric N The organic phase is decanted, washed twice with 10 cm of a hydrochloric acid solution N then 2 times with 10 cm distilled water The organic phase is dried over sodium sulfate then concentrated to dryness under reduced pressure (2.7 kPa) to a temperature close to 35C. 2.0 g of a solid are thus obtained, which is chromatographed on a silica column (0.02-0.045 mm) eluted, with a mixture of methanol and ethyl acetate (10-90 by volume), the product-containing fractions are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The 30 residue (280 mg) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloroform, methanol and Ammon; Aque (24-6-1 by volume), the fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 35C. 310 mg of 5- ~ 4- {N- [N'-35 (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino} -phenyl] -té-~ 0 94/26725 21 ~ 2 7 ~ 3 PCT ~ R94 / 00533 trazole in the form of a white powder (Rf 0.32; chromatography phie on a thin layer of silica; eluent: chloroform-methanol-am-moniac ~ 20 ~ (24-6-1 by volume)).

A solution of 0.31 q of 5- [4- {N- [N '(3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminoctyll-amino} -phenyl] -tetrazole, 1 cm 4N sodium hydroxide, 3 cm3 of tetrahydrofuran and 1.5 cm of methanol is stirred for 26 hours at temperature close to 20C. The mixture is evaporated under pressure reduced (2.7 kPa) to a temperature close to 35C, then diluted with cm of distilled and acidified water with 1.2 cm of a solution 4N hydrochloric acid. After 1 hour of stirring, at a temperature ture close to 20C, the solid is separated by filtration, washed 6 times with 5 cm of distilled water and dried under reduced pressure (15.5 Pa) at a temperature close to 30C. 250 mg are thus obtained of 5- [4- {N- [N '- (3 ~ -hydroxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino} -phenyl] -tetrazole, as a white, melting solid around 200C.

5- (4-aminophenyl) tetrazole can be prepared according to JM McMANUS
and R. HERBST, J Org. Clem, 24, 1044 (1959).

Ex ~ mole 6 To a solution of 1.9 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- acid aminooctanoic in 55 cm of dichloromethane, 2.2 cm 3 of thionyl chloride. The solution is stirred for 24 hours at 20C
then the reaction mixture is concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 35C. The residue obtained is put dissolved in 25 cm of dichloromethane and the solution is new dry concentrate under reduced pressure (2.7 kPa) at a temperature erasure close to 35C. The solid obtained (1.97 g) and 485 mg of 5- (3-aminophenyltetrazole) are dissolved in 60 cm of dichlorome-thane and 1.41 cm of triethylamine are added dropwise. The solution obtained is stirred for 24 hours at a temperature close to 20C. The solution obtained is concentrated under reduced pressure (2.7 kPa) at a temperature close to 35C. To the solid obtained is added 50 cm of dichloromethane and 25 cm of an acid solution hydrochloric N. The organic phase is decanted, washed twice with 10 cm of a hydrochloric acid solution N then 2 times with 10 cm WO 94/26725 ~ PCT ~ R94 / 00533 ~
7 ~ ~ 24 distilled water. The organic phase is dried over sodium sulfate then concentrated to dryness under reduced pressure (2.7 kPa) ~ one temperature close to 35C. 2.2 g of a solid are thus obtained, which is chromatographed on a silica column (0.02-0.045 mm) eluted, with a mixture of chloroform, methanol and ammonia (24-6-1 in volumes), the fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a similar temperature 35C. 840 mg of a solid are thus obtained, which is again chromatographed on an eluted silica column (0.02-0.045 mm), first with ethyl acetate then with a mixture of chloroform, methanol and Ammoni ~ that (24-6-1 by volume), the product-containing fractions are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 35C. We thus obtains 390 mg of 5- [3- {N- [N '- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino} -phenyl] -tetrazole in the form of a powder white (Rf = 0.36; thin layer chromatography on silica;
eluent: chloroform-methanol-A ~; aque to 20 ~ (24-6-1 by volume)).

A solution of 380 mg of 5- [3- {N- [N '- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctalloyll-amino} -phenyl] -tetrazole, 1.2 cm 4N sodium hydroxide,

4 cm of tetrahydrofuran and 2 cm of methanol is stirred for 22 hours at a temperature close to 20C. The mixture is evaporated under pressure reduced (2.7 kPa) to a temperature close to 35C, then diluted with 25 cm of distilled and acidified water with 1.4 cm of a solution 4N hydrochloric acid. After 1 hour of stirring, at a temperature ture close to 20C, the solid is separated by filtration, washed 6 times with 5 cm of distilled water and dried under reduced pressure (15.5 Pa) at a temperature close to 30C. 270 mg are thus obtained of 5- [3- {N- [N '- (3 ~ -hydroxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -amino} -phenyl] -tetrazole, as a white, melting solid around 170C.

5- (3-aminophenyl) tetrazole can be prepared according to JM McMANUS
and R. HERBST, J. Org. Chem., 24, 1044 (1959).

~ n on 7 670 mg N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -11-aminonn ~ c ~ n ~ nitrile and 1 g of tributyltin oture are dissolved in 5 cm3 of ~ 0 94/2672 ~ 2 1 ~ 2 ~ o ~ PCT ~ R94 / 00533 1 ~ 2-dimethoxyethane ~ The mixture is heated at reflux for 48 hours. 0.5 g of tributyltin azide is added and the mixture is again heated at reflux for 48 hours. The reaction melanqe is diluted with 50 cm of distilled water and then extracted with 100 cm of ethyl acetate. The organic phase is then washed with 75 cm of distilled water, dried over magnesium sulfate and evaporated dry under reduced pressure ~ 2.7 kPa) at a similar temperature 40C. The residue obtained ~ 2.2 g ~ is chromatographed on a column silica ~ 0.02-0.045 mm) eluted with a mixture of chloroform, thanol and ammonia at 20% ~ 12-3-0.5 by volume). Fractions containing the product are combined and concentrated under pressure pick (2.7 kPa) at a temperature close to 40C. We thus obtain 700 mg of 5- ~ N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -10-aminodecyl] -té-trazole in the form of a white meringue (Rf - 0.32; chromato-written on a thin silica layer; eluent: chloroform-methanol-Amm ~ niAque at 20% (12-3-0.5 by volume)).

A 290 mg solution of 5- [N- (3 ~ -acetoxylup-20 ~ 29) -èn-28-oyl) -10-aminodecyl] -tetrazole, 0.82 cm 5N sodium hydroxide, 3 cm tetrahydrofu-rane and 6 cm 3 of methanol is stirred for 20 hours at a temperature sine of 20C. The mixture is acidified with 5N hydrochloric acid, and diluted with 40 cm of distilled water. The 51 Year ~ e is extracted by 50 cm, then twice 25 cm, of ethyl acetate. Organic phases are collected, washed with 100 cm of distilled water, dried on magnesium sulfate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (280 mg) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloroform, methanol and A --lAqUe at 20% (12-3-0.5 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue is taken up by 30 cm3 of isopropyl ether, filtered, washed with 10 cm of isopropyl ether and dried under reduced pressure (15.5 Pa) at 20C. We thus obtain 185 mg of 5- [N- (3 ~ -hydroxylup-20 (29) -èn-28-oyl) -10-aminodécyl] -tetrazole, as a white solid melting around 200C.

Tributyltin azide can be prepared according to HR KRICHELDORF
and E. LEPPERT, Synthesis, 329 (1976).

WO 94/2672

5 ~ PCT / FR94 / 00533 ~
7 ~ 3 26 N- (313 - acetoxylup-20 (29) -èn-28-oyl) -11-amino ~ ln ~ l ~ CcAnF ~ nitrile can be obtained as follows:
To a solution of 2.5 g of N- (3¦3-acetoxylup-20 (29) -èn-28-oyl) -11-ami-noundecanamide in 300 cm of dry tetrahydrofuran, drop added 5 to drop 1.92 cm of trifluoroacetic anhydride. The solution is agi-ted 15 hours at room temperature then an additional 1.9 cm of trifluoroacetic anhydride are added and the solution is stirred 48 hours at room temperature. The solution is neutralized by addition of saturated sodium bicarbonate solution then 10 concentrated under reduced pressure (2.7 kPa). The residue is taken up by 100 cm of distilled water and the mixture is extracted twice with 150 cm ethyl acetate. The organic phases are combined, washed 3 times with 100 cm of distilled water, dried over magnesium sulfate sium and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature 15 rature close to 40C. The residue obtained (2.5 g) is chromatographed on a silica column (0.02-0.045 mm) eluted with dichlorome-thane. The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. We 1.6 g of N- (313-acetoxylup-20 (29) -en-28-oyl) -11- are thus obtained 20 amino - n ~ lécAn ~ nitrile in the form of a blAn meringue ~ he ~ Rf - 0.6;
thin layer chromatography on silica; eluent: cycl ~ h ~ cAne ethyl acetate (60-40 by volume)).

N- (3 ~ 3 - acetoxylup-20 (29) -èn-28-oyl) -11-aminonn ~ l ~ c ~ n ~ Tnide can be obtained as follows:

25 To a solution of 2.6 g of N- [313-acetoxylup-20 (29) -èn-28-oyl] acid -11-aminonndécAnoïque in 240 cm of dichloromethane, we add 3.5 cm of Amm ~ njAque at 20%, 580 mg of hydrate of 1 -hy-droxybenzotriazole, and 1.45 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. The solution is stirred for 30 days 2 days at 20C then the organic phase is decanted, washed successively afterwards with 50 cm of distilled water, 3 times 25 cm of chlorine acid lN water, twice 40 cm of distilled water, then dried over sul-magnesium fate, filtered and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. 2.7 g are thus obtained.
35 of N- (3 ~ 3-acetoxylup-20 (29) -èn-28-oyl) -11-aminol ~ nll ~ cAnA ~; from under ~ o 94/26725 ~ 3 pct ~ R94 / 00533 form of a cream-colored meringue (Rf! 0.15; chromatography on thin layer of silica; eluent: cyclohPxAne-ethyl acetate (30-70 in volumes)).

The N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -11-amino acid ~ ecAnoic acid is synthesized by analogy with N- (3 ~ -acetoxylup-20 (29) -èn-28- acid oyl) -8-aminooctanoic from 3 ~ -acetoxylup-20 chloride (29) -en-28-oyl and methyl 11-aminoundecanoate (R = 38%; Rf 0.57; thin layer chromatography on silica; eluting dichloromethane-methanol (90-10 by volume)).

~ m ~ the 8 1 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanenitrile and 1.6 g of tributyltin azide are dissolved in 25 cm of 1,2-dimethoxyethane. The mixture is heated at reflux for 24 hours. 35 cm of 2-ethoxyethanol are added, the 1,2-dimethyl is distilled thoxyethane and the mixture is again heated to reflux for 48 hours. The reaction mixture is cooled to temperature room, poured into a 100 cm solution of hydrochloric acid lN. After 15 minutes of stirring, the mixture is extracted with 3 times 50 cm of ethyl acetate. The organic phases are gathered, washed with 7 times 30 cm of distilled water, dried over sulphate magnesium and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (2.7 g) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloroform, methanol and A ~ oniAque at 20% (12-3-0.5 in volumes). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature vo; ci n ~
40C. The residue obtained is washed with 5 times 75 cm of pentane, filtered, dried under reduced pressure (2.7 kPa) at 40C. We obtain thus 920 mg of 5- [N- (3 ~ -acetoxylup-20 ~ 29) -èn-28-oyl) -7- ~ inoheptyl]
r 30 tetrazole in the form of a creamy white powder (Rf = 0.28 thin layer chromatography on silica; eluent: chloroform-methanol-ammonia at 20 ~ (12-3-0.5 by volume)).

A 900 mg solution of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -7-Aminoheptyl] -tetrazole, 2.7 cm 5N sodium hydroxide, 10 cm tetrahydrofu-35 rane and 20 cm of methanol is stirred for 15 hours at a temperature WO 94/26725 PCT ~ R94 / 00533 2 ~ 27 ~ 3 close to 20C. The mixture is acidified with chlorhy-drique 1N, and diluted with 175 cm of distilled water. The mixture is ex-treated with 3 times 100 cm of ethyl acetate. Organic phases are rAcse ~ hlées, washed with 250 cm of distilled water, dried on magnesium sulfate and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained ~ 865 mg) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloroform, methanol and ~ only 20% (12-3-0.5 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue is taken up by 10 cm3 of isopropyl ether, filtered, washed with 6 cm of isopropyl ether and - dried under reduced pressure (15.5 Pa) at 20C. The powder obtained (510 mg) is again chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of dichloromethane and methanol (95-5 by volume) The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a similar temperature 40C. The residue is taken up in 5 cm of isopropyl ether, filtered, washed with 2 cm3 of isopropyl ether and dried under reduced pressure 20 (15.5 Pa) at 20C. This gives 224 mg of 5- [N- (3 ~ -hydroxylup-20 (29) -èn-28-oyl) -7- ~ minoheptyl] -tetrazole, in the form of a solid white melting around 150-155C.

The N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanenitrile can be obtained in the following way:

25 To a solution of 2.4 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanamide in 200 cm of dry tetrahydrofuran, we add drop by drop 1.97 cm of trifluoroacetic anhydride. The solution is stirred for 15 hours at room temperature then 5.0 cm additional trifluoroacetic anhydride are added and the solution is stirred for 48 hours at room temperature. The solution is neutralized by adding a saturated solution of bicarbonate sodium, then diluted with 100 cm of distilled water. Tetrahydrofuran is evaporated under reduced pressure (2.7 kPa) at 40C. The mixture is then taken up in 200 cm3 of ethyl acetate. The aqueous phase is extracted with 50 cm of ethyl acetate. The organic phases are combined, washed twice with 100 cm of distilled water, dried over ~ O 94/2672 ~ 216 2 7 0 ~ PCT ~ R94 / 00533 magnesium sulfate, filtered and evaporated to dryness under pressure reduced ~ 2.7 kPa) at a temperature close to 40C. We thus obtain 2.2 g of N- (3 ~ -acetoxylup-20t29) -èn-28-oyl) -8-aminooctanenitrile under the shape of a creamy white meringue (Rf - 0.23; chromatography on thin layer of silica; eluent: cycloh ~ An ~ -ethyl acetate (80-20 in volumes)).

The N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanamide can be obtained as follows:

To a solution of 2.4 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- acid 10 aminooctanoic acid in 240 cm of dichloromethane, 3.8 cm3 are added of Armo ~ iAque at 20%, 580 mg of 1-hydroxybenzotriazole hydrate, and 1.45 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodii- hydrochloride mide. The solution is stirred for 24 hours at temperature A ~ hi ~ nte then the organic phase is decanted, washed with 5 ~ 250 cm distilled water drained, dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. We 2.4 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooc- are thus obtained tanamide in the form of a hlAnche meringue (Rf 0.1; chromato-written on a thin layer of silica; eluent: cycloh ~ An ~ -acetate of ~ thyle (50-50 by volume)).

1.3 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -12-aminodo ~ cAn ~ nitrile and 1.27 g of tributyltin azide are dissolved in 50 cm of 2-ethoxyethanol. The mixture is heated at reflux for 25 48 hours. The reaction mixture is poured into 300 cm3 of distilled water.
frozen tillée. After 5 minutes of stirring, the r ~ lAnqe is acidified with a 2N hydrochloric acid solution then extracted with 3 times 80 cm of dichloromethane. The organic phases are combined, washed until neutral with distilled water, dried over sulphate magnesium and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (1.4 q) is chromato-graphiated on a silica column (0.02-0.045 mm) eluted with a metal lange of chloroform, methanol and A ~ Aque at 20% (12-3-0.5 in vo-lumes). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to WO 94/26725 PCT ~ R94 / 00533 _ 2 ~ 3 40C. 1.2 g of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -11-aminoundecyl] -tetrazole in the form of a white meringue (Rf - 0.45; thin layer chromatography on silica; eluent:
chloroform-methanol- ~ ~ ni ~ only 20% (12-3-0.5 by volume)).

5 A solution of 1.2 q of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -11-aminoundecyl ~ -tetrazole, 3.3 cm 5N sodium hydroxide, 12 cm tetrahydrofu-rane and 24 cm of methanol is stirred for 20 hours at a temperature close to 20C. The mixture is acidified with chlorhy-drique 5N, and diluted with 150 cm of distilled water. The mixture is ex-milked with 3 times 60 cm of dichloromethane. Organic phases are collected, washed until neutral with distilled water, dried over magnesium sulfate and evaporated to dryness under pressure reduced (2.7 kPa) at a temperature close to 40C. The residue obtained (1.1 g) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of dichloromethane, methanol and ~ rmoniAque a 20% (12-3-0.5 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature ture close to 40C. The residue is taken up in 15 cm of ethyl ether.
lique, filtered, washed with 12 cm of ethyl ether and dried under pressure 20 reduced ion (15.5 Pa) to 20C. 340 mg of 5- [N- (3 ~ -hydroxylup-20 (29) -en-28-oyl) -11-aminoundecyl] -tetrazole, under the form of a white solid melting around 200-205C.

N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -12-aminodo ~ P ~ AnPnitrile can be obtained as follows:

25 To a solution of 1.5 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -12-ami-nodod ~ cAn ~ mlde in 150 cm of dry tetrahydrofuran, we have ~ drop all drop 1.9 cm of trifluoroacetic anhydride. The solution is agi-t ~ e 60 hours at room temperature, then diluted with 30 cm3 of water distilled. The tetrahydrofuran is evaporated ~ dry under pressure re 30 pick (2.7 kPa) at 40C. The mixture is taken up in 100 cm3 of water tilled, neutralized by adding a saturated bicarbonate solution sodium then extracted with 3 times 50 cm3 of dichloromethane. The organic phases are combined, washed with 3 times 40 cm3 of water distilled, dried over magnesium sulfate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. We O 94/26725 ~ ~ ~ 2 7 ~ 3 PCT ~ R94 / 00533 thus obtains 1.36 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -12-ami-nodo ~ 5c ~ ne ~ itrile in the form of a meringue hl ~ nche (Rf ~ 0.35;
thin layer chromatography on silica; eluent: cycloh ~ YAne-ethyl acetate (80-20 by volume)).

The N- (3 ~ -acetoxylup-2o (29) -èn-28-oyl ~ -12-aminodo ~ c ~ n ~ mlde can be obtained as follows:

To a solution of 1.5 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) acid -12-aminododecanoic acid in 250 cm of dichloromethane, we add 2.15 cm3 of ammonia ~ 20%, 330 mg of 1-hydroxybenzo hydrate triazole, and 826 mg of 1- (3-dimethylaminopropyl) -3- hydrochloride ethylcarbodiimide. The solution is stirred for 15 hours at temperature ambient then the organic phase is decanted, washed successively with 5 times 80 cm of distilled water, 3 times 80 cm of chlorhy-drique lN, 5 times 80 cm of distilled water, then dried over sulfate 15 mg of magnesium, filtered and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. 1.5 g are thus obtained.
of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -12-aminododecanamide under the form of a cream-colored meringue (Rf = 0.47; chromatography on thin layer of silica; eluent: dichloromethane-methanol (90-10 in 20 volumes)) ~ N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -12-aminodod ~ ecanoic acid is synthesized by analogy with N- (3 ~ -acetoxylup-20 (29) -èn-28- acid oyl) -8-aminooctanoic from 3 ~ -acetoxylup-20 chloride (29) -en-28-oyl and methyl 12-aminododecanoate (R = 75%; Rf =
0.45; thin layer chromatography on silica; eluting dichloromethane-methanol (90-10 by volume)).

~ X ~ mDle 1 0 490 mg N'- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -3-r aminopropanenitrile and 700 mg of tributyltin azide are used 30 solution in 10 cm of 1,2-dimethoxyethane. The mixture is heated - at reflux for 6 days. The reaction mixture is cooled to tem-ambient temperature, diluted with 100 cm of dichloromethane and acidified with 25 cm of 2N hydrochloric acid. After 5 minutes of agitation, the organic phase is decanted, washed with 5 times 40 cm3 of water WO 94/26725 PCT ~ R94 / 00533 ~
2 ~ ~ 7 ~ 3 32 distilled, dried over magnesium sulfate, filtered and concentrated dry under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained is washed with 5 times 10 cm of pentane, chroma-tographed on a silica column ~ 0.02-0.045 mm) eluted with a mixture of ethyl acetate and methanol (80-20 by volume). The fractions containing the product are combined and concentrated under reduced sion (2.7 kPa) at a temperature close to 40C. We obtain thus 435 mg of 5- {N '- [N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooc-tanoyl] aminoethyl} -tetrazole in the form of a meringue hlAnrh ~, (Rf 0.39; chromatography on a thin layer of silica; eluent:
chloroform-methanol-Ammo ~ i ~ only 20% (12-3-0.5 by volume)).

A 600 mg solution of 5- {N'- ~ N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl] aminoethyl} -tetrazole, 1.6 cm 5N sodium hydroxide, 10 cm tetrahydrofuran and 20 cm of methanol is stirred for 15 hours at a temperature close to 20C. The mixture is acidified with acid hydrochloric lN, and diluted with 120 cm of distilled water. The mixture is stirred for 1 hour at 20C, filtered, washed until neutral with distilled water, then with 5 times 5 cm of pentane, and dried under pressure reduced sion (15.5 Pa) at 20C The powder obtained (553 mg) is chroma-tographed on a silica column (0.02-0.045mm) eluted with a mixture of chloro ~ elm, methanol and ammonia at 20 ~ (12-3-0.5 in volumes) The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue is taken up in 15 cm of isopropyl ether, filtered, and dried under reduced pressure (15.5 Pa) at 20C. We thus obtain 430 mg of 5- {N '- [N- (3 ~ -hydroxylup-20 (29) -en-28-oyl) -8-aminoocta-noyl] aminoethyl} -tetrazole, in the form of a melting white solid around 155-160C.

N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -3-ami-nopropanenitrile can be obtained as follows:

To a solution of 960 mg of N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -3-aminopropanamide in 70 cm of tetrahydrofuran dry, 0.94 cm of trifluoroacid anhydride is added dropwise tick. The solution is stirred for 15 hours at room temperature, then the tetrahydrofuran is evaporated under reduced pressure (2.7 kPa) at ~ 0 94/2672 ~ 216 2 7 0 3 PCT ~ R94 / 00 ~ 33 40C. The mixture is then taken up in 70 cm of dichloromethane. The organic phase is decanted, washed with 3 times 50 cm of distilled water lated, dried over magnesium sulphate and evaporated to dryness under pressure reduced sion (2.7 kPa) at a temperature close to 40C. The residue obtained (887 mg) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of ethyl acetate and cyclohexane (70-30 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature sine of 40C. This gives 692 mg of N '- [N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl] -3-amino propanenitrile in the form a white meringue (Rf = 0.27; thin layer chromatography silica; eluent: cycloh ~ x ~ ne-ethyl acetate (30-70 in ~ volumes)).

N '- [N- (3 ~ -acétoxylup-20 ~ 29) -èn-28-oyl) -8-aminooctanoyl] -3-ami-nopropanamide can be obtained as follows:

To a solution of 967 mg of acid N '- [N- (3 ~ -acétoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyll-3-aminopropanoic acid in 150 cm of dichloro-methane, 1 cm3 of ~ m ~ oni ~ is added to 20 ~, 208 mg of hydrate 1-hydroxybenzotriazole, and 521 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. The solution is agitated 50 hours at room temperature then the organic phase is decanted, washed with 60 cm of 1N hydrochloric acid, 5 times 50 cm3 distilled water, dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature 25 close to 40C. This gives 960 mg of N '- [N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl] -3-aminopropanamide in the form a creamy white meringue (Rf = 0.68; layer chromatography thin silica; eluent: chloroform-methanol-A i ~ only at 20%
(12-3-0.5 by volume)).

N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -3- acid aminopropanoic can be obtained in the following way:

To an anhydrous solution of 2.3 g of lithium iodide and 100 cm3 of collidine, 1.3 g of N - [(3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- are added methyl aminooctanoyl] -3-aminopropionate. The solution is heated at reflux for 3 hours, then concentrated under reduced pressure WO 94/26725 PCTn ~ R94 / 00533 ~

(2.7 kPa) at a temperature close to 40C. The residue is taken up by 150 cm of distilled water and 150 cm of ethyl acetate, then cooled at 0C. The organic phase is extracted, washed with 3 times 50 cm hydrochloric acid 1N, then with distilled water to neutral lity, dried over magnesium sulfate and evaporated to dryness under pressure reduced pressure ~ 2.7 kPa) at a temperature close to 40C. The residue is taken up in 20 cm of ethyl acetate and the mixture is concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. We 1.07 g of N '- [N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) - acid is thus obtained 8-aminooctanoyl] -3-aminopropanoic in the form of a meringue beige (Rf = 0.22; thin layer chromatography on silica eluent: chloroform-methanol-ammonia at 20 ~ (12-3-0.5)).

N '- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl] -3-amino methyl propionate can be prepared as follows:

To a solution of 2 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- acid aminooctanoic acid in 260 cm of dichloromethane, 524 mg of 3-amino methyl propionate hydrochloride, 480 mg hydrate 1-hydroxybenzotriazole, 1.2 g of 1- (3-dimethylamino-) hydrochloride propyl) -3-ethylcarbodiimide, and 1.1 g of triethylamine in solution in 15 cm of dichloromethane. The solution is stirred for 50 hours a room temperature then the organic phase is decanted, washed with

6 times 150 cm of distilled water, dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure (2.7 kPa) at a tem-temperature close to 40C. The residue obtained (2.4 g) is chromatographed phied on a silica column (0.02-0.045 mm) eluted with a mixture ethyl acetate and cyclohexane (50-50 by volume). The frac-tions containing the product are combined and concentrated under pressure reduced (2.7 kPa) at a temperature close to 40C. We thus obtain 2 g of N '- [N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl] -3-methyl amino propionate in the form of a hlAnche meringue (Rf = 0.62; thin layer chromatography on silica; eluent: aces-ethyl tate) ~ emn the 11 720 mg of [N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl-methyl-amino] -acetonitrile and 1.1 g of tributyltin azide are 0 94/26725 ~ 1 ~ 2 7 Q 3 PCT ~ R94 / 00533 ,,.

solution in 15 cm of 1,2-dimethoxy ~ thane. The mixture is heated at reflux for 3 days. The reaction mixture is diluted with 40 cm of distilled water and 15 cm of 2N hydrochloric acid, then extracted with 3 times 80 cm of dichloromethane. The organic phase is dried over magnesium sulfate, filtered and evaporated to dryness under pressure reduced (2.7 kPa) at a temperature close to 40C. The residue obtained (1.8 g) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloroform, methanol and ~ oni ~ only at 20%
(12-3-0.5 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at one temperature close to 40C. The residue is washed 5 times with 20 cm of pentane, then dried under reduced pressure (15.5 Pa) at 20C. We thus obtains 820 mg of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl- ~ etllyl-alnillomethyl] -tetrazole in the form of a white meringue (Rf = 0.33; thin layer chromatography of silica; eluent: chloroform-methanol- ~ m ~ oni ~ only at 20% (12-3-0.5 in volumes)).

To an 800 mg solution of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-A ~ inom ~ thyl] -tetrazole, 2.7 cm 5N sodium hydroxide, 10 cm3 of tetrahydrofuran and 20 cm of methanol is stirred for 48 hours at a temperature close to 20c. The mixture is acidified with lN hydrochloric acid, and diluted with ~ 100 cm of distilled water. The mixture is stirred for 1 hour at 20C, filtered, washed until neutral with distilled water, then with 5 times 5 cm of pentane, and dried under reduced pressure (15.5 Pa) at 20C. The product obtained (740 mg) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloroform, methanol and 20% ammonia (12-3-0.5 in volumes). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a similar temperature 40C. The residue is taken up in 15 cm of isopropyl ether, filtered, and dried under reduced pressure (15.5 Pa) at 20C. We obtain thus 360 mg of 5- [N- (3 ~ -hydroxylup-Z0 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-aminomethyl] -tetrazole, in the form of a white solid melting around 151C.

[N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-amino] -acetonitrile can be obtained as follows:

WO 94/26725 PCT ~ R94 / 00533 ~ 36 To a solution of 1.2 g of [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-amino ~ -acetamide in 70 cm of tetrahydrofuran dry, 1 cm of trifluoroacetic anhydride is added dropwise.
The solution is stirred for 15 hours at room temperature, then neutralized with a saturated sodium bicarbonate solution. The tetrahydrofuran is evaporated under reduced pressure (2.7 kPa) at 40C.
The mixture is then taken up in 200 cm of distilled water and extracted with 3 times 80 cm of dichloromethane. The organic phases are re-united, washed with 3 times 50 cm of distilled water, dried on sul-magnesium fate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 40C. The residue obtained (1.0 g) is chroma-tographed on a silica column (0.02-0.045 mm) eluted with a mixture of ethyl acetate and cycloh ~ x ~ ne (30-70 by volume). The fractions containing the product are combined and concentrated under reduced sion (2.7 kPa) at a temperature close to 40C. 720 mg of [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl- are thus obtained.
methyl-amino] -acetonitrile in the form of a meringue blAn ~ h ~ (Rf -0.32; thin layer chromatography on silica; eluting cyclohexane-ethyl acetate (50-50 by volume)).

[N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-amino] -acetamide can be obtained as follows:

To a solution of .00 mg of acid [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-metllyl-amino] -acetic in 100 cm di-chloromethane, 0.72 cm of 20% A ~ oniAque, 150 mg of hy-1-hydroxybenzotriazole drate, and 380 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. The solution is agitated 15 hours at room temperature then the organic phase is decanted, washed with 30 cm of 1N hydrochloric acid and then with 5 times 40 cm of distilled water, dried over magnesium sulfate, filtered and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (630 mg) is chromatographed on a silica column (0.02-0.045 mm) eluted with a mixture of chloromethane and methanol (97-3 by volume). The fractions the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. This gives 500 mg of [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-amino] -O 94/26725 ~ ~ ~ 2 7 ~ 3 PCT ~ R94 / 00533 acetamide in the form of a creamy white meringue (Rf - 0.32 thin layer chromatography of silica i eluent: dichlorome-thane-methanol (95-5 by volume)).

[N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl-methyl- acid - 5 amino] -acetic can be obtained as follows:

A solution of 167 mg of sarcosine, 406 mg of chlorotrimethylsilane and 75 cm of dichloromethane is heated at reflux for 15 hours. This solution is then added to a solution containing 1 g of N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminoocta- acid 10 noic, 239 mg of 1-hydroxybenzotriazole hydrate, 600 mg of chlor-1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrate, and 75 cm3 dichloromethane. 1.5 cm of triethylamine are then added in solution in 10 cm of dichloromethane. The solution is agitated 50 hours at room temperature then the reaction mixture is washed 15 per 70 cm of 2N hydrochloric acid, then 3 times 80 cm of water distilled, dried over magnesium sulfate, filtered and concentrated to dry under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (2.4 q) is chromatographed on a column of silica (0.02-0.045 mm) eluted with a mixture of dichloromethane, 20 methanol and ammonia at 20 ~ (12-3-0.5 by volume) The fractions containing the product are combined and concentrated under pressure pick (2.7 kPa) at a temperature close to 40C. We thus obtain 712 mg of acid [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctanoyl-methyl-amino] -acetic in the form of a meringue hlAn ~ he (Rf 0.32; thin layer chromatography on silica; eluting chloroform-methanol-Ammonia at 20 ~ (12-3-0.5 by volume)).

E8 ~ mnl e 12 To a solution of 1.4 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctylcarbamoylacetonitrile in 20 cm of dichloromethane, we 30 adds 0.8 cm of trimethylsilyl azide and 270 mg of trichloride aluminum. The reaction mixture is stirred for 72 hours at 20C
then poured dropwise over 30 minutes onto 50 cm of a solution aqueous at 10% sodium hydrogencarbonate. The organic phase is decanted, the aqueous phase is extracted with 2 times 50 cm3 of dichloromethane. The combined organic phases are washed 3 times -WO 94l26725 PCT ~ R94 / 00533 ~
~ 2 ~ ~ 3 38 with 50 cm of distilled water, dried over magnesium sulfate and then evaporated to dryness under reduced pressure (2.7 kPa) at a temperature sine of 30C. The residue obtained (1.9 g) is chromatographed on co-ton of silica (0.02-0.045 mm), eluted with a mixture of chloro-form, methanol and 20% ammonia (24-6-1 by volume). The frac-tions containing the product are combined and concentrated under pressure reduced (2.7 kPa) at a temperature close to 40C. We thus obtain 1 g of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-aminooctylcarba-moylméthyl] -tétrazole in the form of a meringue hl ~ nrh ~ (Rf =
0.25; thin layer chromatography on silica; eluent: chloro-form-methanol-ammonia at 20% (24-6-1 by volume)).

A 1 g solutlon of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8-ami-nooctylcarbamoylmethyl ~ -tetrazole, 5.4 cm of sodium hydroxide N, 20 cm of tetrahydrofuran and 40 cm of methanol is stirred for 72 hours at a temperature close to 20C. The mixture is poured into 10 cm of a hydrochloric acid solution N then diluted with 250 cm of water tilled. After 1 hour with stirring at a temperature close to 20C, the solid is filtered and washed 4 times with 10 cm of distilled water then dissolved in 30 cm of ethanol. The solution is filtered, the wire-trat is evaporated under reduced pressure (2.7 kPa) at a temperature close to 50C The residue obtained (140 mg) is taken up by 15 cm isopropyl oxide The solid obtained is lumpy, washed twice 5 cm of isopropyl ether then dried under vacuum (13.5 Pa) at a tem-temperature close to 40C. 700 mg of 5- [N- (3 ~ -hy-25 droxylup-20 (29) -èn-28-oyl) -8-aminooctylcarbamoylmethyl] -tetrazole as a white solid melting around 175C

N- (3 ~ -acétoxylup-20 (29) -èn-28-oyl) -8-aminooctylcarbamoylacétoni-trile can be obtained as follows:

To a solution of 3.6 g of N- [3 ~ -acetoxylup-20 (29) -èn-28-oyl] -1,8-30 ~ i ~ minooctane ~ 500 mg of cyanoacetic acid, 1.06 g of hydrate 1-hydroxybenzotriazole, 2.76 g of 1- (3-dimethylamino-) hydrochloride propyl) -3-ethylcarbodiimide in 75 cm of dichloromethane, is added in about 10 minutes, 3.6 cm of triethylamine. The solution is stirred for 15 hours at a temperature close to 20C then the reaction mixture is washed 3 times with 40 cm of distilled water.

~ O 94/26725 21 ~ 62 ~ ~ ~ PCT ~ R94 / 00533 The organic phase is then dried over magnesium sulfate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature sine of 40C. The residue obtained (4.8 g) is chromatographed on co-ton of silica (0.02-0.045 mm), eluted with a mixture of dichlorome-thane and metharlol (99-1 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. This gives 3.7 g of N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctylcarbamoylacetonitrile under the shape of a yellow meringue (Rf = 0.40; layer chromatography thin silica; eluent: chloroform-methanol- ~ mm ~ niac at 20%
(12-3-0.5 by volume)) N- (3 ~ -aceto: ~ ylup-20 (29) -erl-28-oyl) -1,8-diaminooctane can be obtained from the following ma ~ re:

50 cm of a solution in dichloromethane chloride 3 ~ -acaceous-15 toxylup-20 (29) -en-28-oyl (prepared from 5 g of 3 ~ -acid- acid toxylup-20 (29) -en-28-oic) are added dropwise in approximately 3 hours to a solution of 8.6 g of 1.8 ~; n ~ octane in 20 cm of dichloromethane. The reaction mixture is stirred for 2 hours at a temperature close to 20C then concentrated to dryness under pressure reduced (2.7 kPa) at a temperature close to 40C. The residue obtained is chromatographed on a silica column (0.02-0.045 mm), eluted with a mixture of chloroform, methanol and A -ni ~ only 20% (24-6-1 in volumes). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 25 40C. 5.9 g of N- [3 ~ -acetoxylup-20 (29) -èn-28-oyl] are thus obtained -1,8-diaminooctane in the form of a yellow meringue (Rf G 0 ~ 25;
thin layer chromatography on silica; eluent: chloroform-methanol-ammonia at 20 ~ (24-6-1 by volume)).
~ n the 13 30 To a solution of 2.1 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -7-ami-noheptycarbamoylacetonitrile in 25 cm of dichloromethane, we add 1.07 cm of trimethylsilyl azide and 410 mg of trichloride aluminum. The reaction mixture is stirred for 72 hours at 20C
then poured dropwise over 30 minutes onto 50 cm of a solution aqueous at 10% sodium hydrogencarbonate. The organic phase is WO 94/26725 I PCT ~ R94 / 00533 ~
211 ~ 2703 40 decanted, the aqueous phase is extracted with 2 times 60 cm of dichloromethane. The combined organic phases are washed 3 times with 50 cm of distilled water, dried over magnesium sulphate pUi5 concentrated to dryness under reduced pressure (2.7 kPa) ~ one temperature close to 40C. The residue obtained (2.5 g) is chromatographed on co-lonne of silica ~ 0.02-0.045 mm), eluted with a mixture of chloro-form, methanol and ~ nmoni ~ only 20% (24-6-1 by volume). The frac-tions containing the product are combined and concentrated under pressure reduced (2.7 kPa) at a temperature close to 40C. We thus obtain 1.4 g of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -7-Am1nohPptylcarba-moylméthyl] -tétrazole in the form of a meringue hl ~ n ~ h ~ (Rf 0.25; thin layer chromatography on silica; eluent: chloro-form-methanol-ammonia at 20% (24-6-1 by volume)).

A solution of lg of 5-rN- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -7-aminoheptylcarbamoyllnétllyl] -tetrazole, 8 cm of sodium hydroxide, 25 cm of tetrahydrofuran and 50 cm of methanol is stirred for 48 hours at a temperature close to 20C. The reaction mixture is evaporated by-partially under reduced pressure (2.7 kPa) at a temperature close to from 40C until the appearance of a disorder. The mixture is then aci-dified has a p ~ between 1 and 2 by addition of an aqueous solution of hydrochloric acid N. After 2 hours with stirring at temperature close to 20C, the solid is filtered and washed 5 times with 5 cm of distilled water and then dissolved in 50 cm3 of ethanol. The solution is filtered, the filtrate is evaporated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained is taken up by 20 cm of isopropyl ether. The solid obtained is lumpy, washed in pairs times 5 cm of isopropyl ether and then dried under reduced pressure (13.5 Pa) has a temperature close to 40C. The residue obtained is chromatographed on a silica column (0.02-0.045 mm), eluted with a mixture of chloroform, metllanol and ~ ni ~ only 20% (24-6-1 in volumes) The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a similar temperature 40C. The solid obtained is dissolved in 20 cm3 of chloroform, the solution is filtered, the filtrate is evaporated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained is taken up in 20 cm of isopropyl ether. The solid obtained is matured, washed with 2 times 5 cm of isopropyl ether then O 94/26725 ~ 16 2 ~ ~ 3 PCT ~ R94 / 00533 dried under vacuum (13.5 Pa) at a temperature close to 40C. We thus obtains 900 mg of 5- [N- (3 ~ 3-hydroxylup-20 (29) - ~ n-28-oyl) -7-Aminoheptylcarbamoylmethyl] -tetrazole as a white solid melting around 160C.

5 N- (3 ~ 3-acetoxylup-20 (29) -èn-28-oyl) -7- ~ minohPptylcarbamoyl-acet nitrile can be obtained as follows:

To a solution of 2.45 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1,7-~ i ~ minoh ~ ptane ~ 340 mg of cyanoacetic acid, 730 g of hydrate 1-hydroxybenzotriazole, 1.9 g of 1- (3-dimethylamino-) hydrochloride propyl) -3-ethylcarbodiimide in 120 cm of dichloromethane, is added in about 10 minutes, 2.5 cm of triethylamine. The solution is stirred for 15 hours at a temperature close to 20C then the reaction mixture is washed 3 times with 50 cm of distilled water.
The organic phase is then dried over magnesium sulfate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature sine of 40C. The residue obtained (2.7 g) is chromatographed on co-ton of silica (0.02-0.045 mm), eluted with a dichlorome mPlAnge thane and methanol (99-1 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at 20 a temperature close to 40C. This gives 3.7 g of N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -7- ~ mi n ~ hpptylcarbamoylacetonitrile under the shape of a white meringue (Rf r 0.40; chromatography on thin layer of silica; eluent: chloroform-methanol- ~ m ~ on1 ~ than at 20 ~ (12-3-0.5 by volume) ~.

25 The N- [3 ~ 3-acetoxylup-2o (29) -èn-28-oyl] -l ~ 7- ~ iAminoheptane can be obtained as follows:

1000 cm of a solution in dichloromethane chloride 3 ~ 3-acetoxylup-20 (29) -èn-28-oyle ~ prepared from 30 g of acid 3 ~ -acetoxylup-20 (29) -en-28-oic) are added dropwise, approximately 30 ron 10 hours, to a solution of 46.8 g of 1.7- ~ i ~ minohPptane in 120 cm of dichloromethane The reaction mixture is stirred for 15 hours at a temperature close to 20C then concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The re-sidu is taken up in 2000 cm of distilled water. The solid is filtered, washed with 3 times 200 cm of distilled water, then dissolved in WO 94/26725 ~ 16% 7 0 3 42 PCT ~ R94 / 00533 ~

500 cm of ethanol. The solution is filtered, the solid is washed 2 times with 50 cm of ethanol and the filtrates are evaporated to dryness under pressure.
reduced sion (2.7 kPa) at a temperature close to 50C. The residue obtained is chromatographed on a silica column (0.02-0.045 mm), eluted with a mixture of chloroform, methanol and ~ m ~ on; ~ only 20%
(24-6-1 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature sine of 40C. This gives 28 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1.7- ~ in ~ heptane in the form of a yellow meringue (Rf =
0.25; thin layer chromatography on silica; eluent: chloro-form-methanol-ammonia at 20 ~ (24-6-1 by volume)).

~ em ~ the 14 To a solutlon of 2.3 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -7-ami-noheptylcarbamoylpropionitrile in 25 cm3 of dichloromethane, we add 1.30 cm of trimethylsilyl azide and 450 mg of trichloride aluminum. The reaction mixture is stirred for 48 hours at 20C then 32 hours at a temperature close to reflux. We add to new 1.30 cm of trimethylsilyl azide and 450 mg of trichlo-aluminum coating and heating for 24 hours to a temperature close to reflux. The reaction mixture is cooled to 20C and then poured dropwise dropwise in 30 mlnutes over 40 cm of a 10% aqueous solution of hy-sodium drocarbonate. The organic phase is decanted, the phase aqueous solution is extracted with 3 times 50 cm of dichloromethane. The phases combined organics are washed 3 times with 50 cm of distilled water, dried over magnesium sulfate and then evaporated to dryness under pressure reduced ~ 2.7 kPa) at a temperature close to 40C. The residue obtained (2.5 g) is treated with 25 cm of isopropyl ether, then washed 3 times with 5 cm of isopropyl ether. The solid obtained (1.8 g) is put in solution in 25 cm of dichloromethane. The solution is filtered on celite. The filtrate is concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The solid is processed by 20 cm of isopropyl ether then dried under reduced pressure (2.7 kPa) at a temperature close to 40C. 1.5 g are thus obtained.
of 5- [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -7-A ~ i noheptylcarbamoyla-thyl] -tétraæole in the form of a meringue hl ~ n ~ he (Rf 0.25;

WO 94/26725 ~ 1 ~ 2 7 0 3 PCT / FR94 / 00533 . . .

thin layer chromatography on silica; eluent: chloroform-methanol- ~ ni ~ only 20% ~ 24-6-1 by volume)).

A solution of 1 g of 5- [N- (313-acetoxylup-20 (29) -èn-28-oyl) -7-aminoheptylcarbamoylethyl] -tetrazole, 20 cm of sodium hydroxide, 25 cm of tetrahydrofuran and 50 cm of methanol is stirred for 72 hours at a temperature close to 20C. The mixture diluted with 250 cm of water tilled and acidified to pl ~ l by addition of an aqueous acid solution hydrochloric N After 1 hour with stirring at a room temperature sine of 20C, the solid is filtered and washed 5 times with 10 cm of water distilled and then treated with 5 times 40 cm of ethyl acetate. The combined organic phases are evaporated under reduced pressure (2.7 kPa) at a temperature close to 40C. The residue obtained (700 mg) is chromatographed on a silica column (0.02-0.045 mm), eluted with a mixture of chloroform, methanol and ~ mmor ~ i aque at 20%
(24-6-l by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. The solid obtained (600 mg) is treated with 15 cm isopropyl ether, the solid is filtered, washed twice with 5 cm isopropyl ether then dried over potassium hydroxide. We thus obtains 140 mg of 5-tN- (313-hydroxylup-20 (29) -èn-28-oyl) -7-aminoheptylcarbamoylethyl] -tetrazole as a white solid melting around 110C.

N- (3f ~ -acetoxylup-20 (29) -èn-28-oyl) -7-aminoheptylcarbamoylpropio-nitrile can be obtained as follows:

To a solution of 2.45 g of N- (3 ~ 3-acetoxylup-20 (29) -èn-28-oyl) -1,7-diaminoheptane, 400 mg of 2-cyanopropionic acid, 730 g of hydrate 1-hydroxybenzotriazole, 1.9 g of 1- (3-dimethylamino-) hydrochloride propyl) -3-ethylcarbodiimide in 120 cm of dichloromethane, is added in about 10 minutes, 2.5 cm of triethylamine. The solution is stirred for 15 hours at a temperature in the region of 20C. The reaction mixture is washed 3 times with 50 cm of distilled water. The the organic phase is then dried over magnesium sulfate and evaporated to dryness under reduced pressure (2.7 kPa) at a temperature sine of 40C. The residue obtained (3.5 g) is chromatographed on co-ton of silica (0.02-0.045 mm), eluted with a mixture of dichlorome-WO 94/26725, PCT ~ R94 / 00533 ~ 1 & ~ 7 ~ 3 44 thane and methanol (99-1 by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature close to 40C. 2.3 g of N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -7-Aminoheptylcarbamoylpropionitrile under the shape of a white meringue (Rf = 0.40; chromatography on thin layer of silica; eluent: chloroform-methanol-A ~ niAque to 20 ~ (12-3-0.5 by volume)).

N- (3 ~ -acetoxylup-2o (29) -èn-28-oyl) -1 ~ 7- ~ iAmin ~ h ~ ptane can be obtained as follows:

1000 cm3 of a solution in dichloromethane chloride 3 ~ -acetoxylup-20 (29) -èn-28-oyl (prepared from 30 g of acid 3 ~ -acetoxylup-20 (29) -en-28-oic) are added dropwise approximately ron 10 hours in a solution of 46.8 g of 1.7- ~; ~ minoh ~ ptane in 120 cm of dichloromethane. The reaction mixture is stirred for 15 hours at a temperature close to 20C then concentrated to dryness under reduced pressure (2.7 kPa) at a temperature close to 40C. The re-sidu is taken up in 2000 cm3 of distilled water. The solid is filtered, washed with 3 times 200 cm of distilled water, then dissolved in 500 cm of ethanol. The solution is filtered, the solid is washed 2 times with 50 cm3 of ethanol and the filtrates are evaporated to dryness under pressure.
reduced sion (2.7 kPa) at a temperature close to 50C. The residue obtained is chromatographed on a silica column (0.02-0.045 mm), eluted with a mixture of chloroform, methanol and ammonia at 20 ~
(24-6-1 by volume) The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) at a temperature sine of 40C. This gives 28 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1.7- ~ iArinoheptane in the form of a yellow meringue (Rf 2 0.25; thin layer chromatography on silica; eluent: chloro-form-methanol- ~ -niAque at 20 ~ (24-6-1 by volume)).

Example 15 By operating by analogy with Example 10, 5- {N - {[3 ~ -hydroxylup-20 (29) -èn-28-oyl] -8-aminooctanoyl} -2-aminopropyl (R, S)} tetrazole a was prepared from N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- acid aminooctanoyl} -2-aminobutanoic (R, S)}. The product comes in the shape of a white powder; pF = 130C.

WO 94/26725 2 ~ 6 2 7 o 3 E ~ r ~ Rg4 / 00533 N- (3 ~ -acetoxylup-20 (29) -en-28-oyl) -8-aminooctanoyl} -3- acid aminobutanoic (R, S)} can be prepared by analogy with [N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -8- ~ inooctanoyl} -methyl- acid amino ~ acetic (example 11). N- (3 ~ -acetoxylup-20 (29) -en-28- acid oyl) -8-aminooctanoyl} -3-aminobutanoic (R, S)} occurs under the form of a white meringue (Rf = 0.44; layer chromatography thin silica; eluent: dichloromethane, ethyl acetate and methanol 40-40-20 (by volume)).
~ pmnle 16 By operating by analogy with Example 13, 5- {N- [3 ~ -hydroxylup-20 (29) -en-28-oyl] -7-aminoheptyl- (N-methylcarbamoyl)} methyltetrazole a was prepared from N-methyl N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1,7- ~ inoheptane, pFs183C

N-methyl-N '- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1,7-di ~ minoheptane can be obtained as follows:

To a solution of 15.2 g of N-methyl 2-methylthiobenzothia iodide-zolium in 300 cm3 of dichloromethane, a solution of 28.7 g of N- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1.7- ~; ~ m; noheptane in 50 cm3 of dichloromethane then in approximately 15 minutes, 6.6 cm3 of triethylamine. The solution is stirred at a temperature close to 20C for 2 hours then the reaction mixture is concentrated under atmospheric pressure at a temperature close to 60C. To the oil obtained (35.6 g), 150 cm 3 of iodide of methyl and the reaction mixture is brought for 30 hours to a temperature close to reflux. The reaction mixture is concentrated under reduced pressure (2.7 kPa) at a temperature close to 50C. The oily residue is taken up in 300 cm3 of diethyl ether and tritur ~.
39.8 g of an orange solid are thus obtained which is dissolved in 100 cm3 of dichloromethane. To the solution obtained, 4.3 is added.
cm3 of n-butylamine. The reaction mixture is stirred for 4 hours at a temperature close to 25C, then diluted with 300 cm3 of dichloromethane and washed with 450 cm3 of distilled water. The sentence organic is then dried over magnesium sulfate and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature WO 94/26725 PCT ~ R94 / 00533 ~ 627 ~ 3 46 close to 40C. The residue obtained is chromatographed on a column silica (0.02 - 0.045 mm) eluted with a mixture of dichloromethane and methanol 19-1 (by volume). The fractions containing the product are combined and concentrated under reduced pressure (2.7 kPa) ~ one temperature close to 40C. 7.1 g (26%) of N- are thus obtained.
methyl-N '- (3 ~ -acetoxylup-20 (29) -èn-28-oyl) -1,7- ~ i ~ minoheptane under the shape of a white meringue ~, pF ~ 158C.

~ m ~ on the 17th By operating by analogy with Example 14, the 5- {3- [N- (3 ~ -hydroxylup-20 (29) -èn-28-oyl) -7-AminohPptylcarbamoyl] -2,2-dimethylpro-pyl} tetrazole as a white solid; pF = 155C.

The present invention also relates to pharmaceutical compositions.
ticks containing at least one product of general formula (I) even in the form of salt, ~ pure or in the form of an asso-ciation with one or more compatible diluents or adjuvants and pharmaceutically acceptable, or with another agent intended for treatment of AIDS, an antiviral, immunosuppressive or anti-Crobian.

The composition according to the invention is capable of keeping alive the cells infected with an HIV virus and therefore reduce progression towards AIDS or to decrease its severity in subjects already infected reducing the mortality of infected cells. The essays can be used orally, parenterally or rectally.

The compositions can be used for curative purposes or as preventive in subjects with immunodeficiency and / or infected with an HIV virus. Of course, the constitution of these components will be adapted to the specific case of the digestive tract of the immu-depressed.

As solid compositions for oral administration may be used tablets, pills, capsules, powders or granul ~ s. In these compositions, the active product according to the invention is mixed with one or more inert diluents or adjuvants, such as s ~ h ~ Arose, lactose or starch.

WO 94/26725 2 ~ ~ 2 7 ~ 3 PCT ~ R94 / 00s33 These compositions can include substances other than thinners, for example a lubricant such as magnesium stearate or a coating for controlled release.

As liquid composites for oral administration, use pharmaceutically acceptable solutions, suspensions sions, emulsions, syrups and elixirs containing inert diluents such as water or paraffin oil. These compositions may also include substances other than thinners, for example wetting, sweetening or flavorings.

The compositions for parenteral administration can be sterile solutions or emulsions. As solvent or vehicle, can use propylene glycol, polyethylene glycol, oils vegetable, in particular olive oil, organic esters injectables, for example ethyl oleate.

These compositions can also contain adjuvants, in particular particularly wetting, isotonizing, emulsifying agents, dispersants and stabilizers.

Sterilization can be done in several ways, for example using a bacteriological filter, by irradiation or by heating.
They can also be prepared in the form of compositions sterile solids which can be dissolved at the time of use in sterile water or any other sterile injectable medium.

The compositions for rectal administration are suppositories or rectal capsules, which contain, in addition to the active ingredient, excipients such as cocoa butter, semi-glycerides synthetic or polyethylene glycols.

In human therapy, the doctor will determine the dosage he considers the most appropriate based on preventive treatment or curative, depending on age, weight, degree of infection and other factors specific to the subject to be addressed. Generally, doses are between 10 and 100 mg / kg orally for a adult.

WO 94/26725 PCT ~ R94 / 00533 The present invention also relates to associations formed one or more lupane derivatives of general formula (I), and / or the where appropriate their salts, and of another active principle known for its anti-retrovirus activity, possibly in the presence of excipients pharmaceutically acceptable.

The anti-retrovirus agents which can be combined are chosen from compatible and inert agents vis-à-vis the lupane derivative of general formula (I). Without limitation, these agents are chosen among reverse transcriptase inhibitors [zidovudine (AZT), didanosine (DDI), dideoxycytidine (DDC), TIBO, nevirapine, PMEA, D4T, pyridones, ~ -APA, HEPT ...], among protease inhibitors [such as RO 31-8959 or A 77003], or among inhibitors tat proteners as for example RO 24-7429].

The following example illustrates a composition according to the invention.

Example ~

Product tablets are prepared according to the usual technique.
active ingredient with the following composition:

- 5- [N '- [N- [3 ~ -hydroxylup-20 (29) -en-28-oyl] -8-aminooctanoyl] -Aminom ~ thyl] -tetrazole, 100 mg - starch 332 mg - silica 120 mg - magnesium stearate 12 mg

Claims

1 - A lupane derivative of general formula:

in which :

R represents a radical of general formula:

X is a bond or represents a carbamoyl radical, N-methyl carbamoyl, aminocarbonyl or N-methylaminocarbonyl, Y is a bond or represents a meta or para phenylene radical, R° and R°°, identical or different, are hydrogen atoms or methyl radicals (it being understood that not all -CR°R°° units are not necessarily identical to each other) and, n is an integer from 6 to 12, m is an integer from 0 to 3 and p is an integer from 0 to 2, it being understood that m + n + p is composed of taken between 6 and 14, and R1 is a methyl or hydroxymethyl radical or a -CH2OR' radical or -CH2SR' for which R' is alkyl or hydroxyalkyl, it being understood that the alkyl radicals are straight or branched and contain 1 to 4 carbon atoms, as well as their salts and the case optionally their stereoisomeric forms and their mixtures.

2 - A derivative of lupane according to claim 1, characterized in that that it is 5-{N'-[N-(3.beta.-hydroxylup-20(29)-en-28-oyl)-8-amino-octanoyl]-aminomethyl}-tetrazole.

3 - A derivative of lupane according to claim 1, characterized in that that it is 5-[4-{N-[N'-(3.beta.-hydroxylup-20(29)-en-28-oyl)-8-amino-octanoyl]-amino}-phenyl ]-tetrazole.

4 - A derivative of lupane according to claim 1, characterized in that that it is 5-[N-(3.beta.-hydroxylup-20(29)-en-28-oyl)-7-aminoheptyl-carbamoylmethyl]-tetrazole.

- A lupane derivative according to claim 1, characterized in that that it is 5-[N-(3.beta.-hydroxylup-20(29)-en-28-oyl)-7-aminoheptyl-carbamoylmethyl]-tetrazole 6 - Process for preparing a lupane derivative according to claim tion 1, characterized in that an azide is made to act on a nitrile with general formula:

where R1 R°, R°°, X, Y, n, m, and p are defined as in of claim 1, then removes, where appropriate, radicals protectors and eventually transforms the product obtained into a salt.

7 - Process for preparing a lupane derivative according to claim tion 1, for which X (in the radical R) represents a radical car-optionally N-methylated bamoyl or aminocarbonyl and the others radicals are defined as in claim 1, characterized in what an amine or an acid of the general formulas is made to act:

Where wherein R°, R°°, m and p are defined as in claim cation 1 and R"' is a hydrogen atom or a methyl radical, on a lupane derivative of general formula:

wherein R1 is defined as in claim 1 and R"
represents a radical of general formula:
(CH2)n-COOH
Where (CH2)n-NH-R"' wherein n is defined as in claim 1 and R"' is defined as above, then removes any radicals protectors and eventually transforms the product obtained into a salt.

8 - Pharmaceutical composition characterized in that it contains at least one product according to claim 1, in the pure state or in as-association with any compatible diluent or adjuvant and pharmaceutical only acceptable and/or in combination with another active ingredient antiviral, immunomodulator or antimicrobial.