FR2721826A1 - Use of 2,6-di-t-butyl:phenyl:carboxylic acid derivs. - Google Patents

Abstract

Use of 2,6-di-t-butylphenylcarboxylic acids of formula (I) or their salts or esters to form a medicament for combatting HIV (esp. HIV-1 or HIV-2) infections is claimed. In (I), R is COOH or A-COOH; and A is aliphatic hydrocarbon gp.. Also claimed is pharmaceutical compsn. comprising (I). Compsn. contains 3,5-di-t-butyl-4-hydroxybenzoic acid (Ia).

Description

USE OF 2,6-DI-T-COMPOUNDS
BUTYLPHENOLS SUBS'I'l'ULLES IN POSITION 4 IN 'l'hAI'l'EMEN'l'
AND AIDS PREVENTION.

 The present invention relates to 2,6-di-t-butylphenol compounds substituted in the 4-position by a COOM group as well as their salts and esters. The invention relates to the use of these compounds for obtaining drugs for the treatment and prevention of AIDS, as well as the pharmaceutical compositions containing them.

4-Substituted 2,6-di-t-butylphenols, also named according to another nomenclature 3,5-di-tert-butyl-4-hydroxybenzenes substituted in the 1-position, have been known for a long time. The preparation of some of them by oxidation of 2,6-di-t-butyl-4-methylphenol has in particular been described by GR Yohe et al., In J. Org. Path. (1956), 21, 1289-1292. Because of their antioxidant property, compounds of this family were initially used in petroleum products and then as a food additive due to an activity on animal fats (JC Dacre, Biochem J., 1961, vol 78, n04 , pp. 758-766). It was then shown that 2,6-di-t-butylphenol compounds substituted in the 4-position by an aliphatic hydrocarbon residue, designated BHT for the English name "butylated hydroxytoluene", had antiviral properties against capsid viruses. Lipids (W. Snipes et al., Science, 1975, Vol.188, No. 4183, pp. 64-65), therefore it has been proposed, for example, in the patent application
French published under number 2 507 891 or in the in the International Patent Application DCT / FR91 / 00882, to use these compounds for obtaining medicaments for the treatment of diseases related to an infection of an individual with viruses , especially herpes viruses.

 The toxicity of BHT has been discussed, in particular by J. G. Llaurodo (West J. Med., 1983, vol.

139, No. 2, pp. 229-230), DM Shlian (N. Engl J. Pied., 1986, vol 314, No. 10, pp. 648-649) and MW Grogan
(West J. Med., 1986, Vol 145, No. 2, pp. 245-246), and novel 2,6-di-t-butylphenol derivatives have been proposed especially as antiallergic agents in the Patent Application. European Publication No. 0 212 848 and US Patent No. 4,968,710.

3,5-di-tert-butyl-4-hydroxybenzoic acid, as well as its halides, in particular bromide and chloride, have also been described in the European patent application published under the number 0 269 981 and the patent. United States of America 4,708,966 as a synthetic intermediate. It has been proposed in
International Patent Application PCT / FR91 / 00882 to use this acid, among other 4-substituted 2,6-di-tbutylphenols compounds, to prepare antiviral drugs for the treatment of diseases related to an infection of an individual lipid-capsid type viruses and more particularly herpes viruses, or by papillomaviruses.

dans laquelle R est un groupe alkyle en Cl-Cl2 un groupe alcényle en C2-C12, un groupe alcynyle en C2-C12, un groupe alcoxy en C1-C12, un groupe formyle, un groupe alkanoyle en C2-C12, un groupe hydroxyalkyle en Cl-Cl2 un groupe amine primaire, secondaire ou tertiaire, un groupe OCH3, CH2OH, CElO ou un groupe COOH ou un groupe A - COOH dans lequel A est un reste hydrocarboné aliphatique en Cl-Cll. Cette
Demande de Brevet envisage aussi les sels et esters des composés précédents dans lesquels R est COOH ou A-COOH.The International Patent Application
PCT / FR91 / 00882 supra discloses the 4-substituted 2,6-di-tert-butylphenol compounds of the following formula (I)

in which R is a C1-C12 alkyl group, a C2-C12 alkenyl group, a C2-C12 alkynyl group, a C1-C12 alkoxy group, a formyl group, a C2-C12 alkanoyl group, a hydroxyalkyl group; C1-C12 a primary, secondary or tertiary amine group, OCH3, CH2OH, CE10 or a COOH group or A-COOH group wherein A is a C1-C11 aliphatic hydrocarbon residue. This
Patent Application also contemplates the salts and esters of the foregoing compounds wherein R is COOH or A-COOH.

 The Applicant has now demonstrated that the 2,6-di-t-butylphenol compounds substituted in the 4-position by a COOH group or an A-COOH group, where A is an aliphatic hydrocarbon residue, and their salts and esters, are active against HIV-like viruses that cause AIDS.

 The isolation and characterization of a first retrovirus called LAV, which had been assigned responsibility for the development of AIDS, was described for the first time in an article by F.

Sinus Bar in 1983 (Science, Vol 220, No. 45-99, 20, pp. 868-871). Since then, other similar strains and variants of LAV have been isolated. In application of the rules of nomenclature adopted by the scientific community, retroviruses likely to induce in humans lymphadenopathies and AIDS are designated globally by "HIV", abbreviation of the English expression "Human Immunodeficiency Virus". All viruses belonging to the category of LAV have been designated HIV, the two major classes are HIV-1 and HIV-2 viruses.

 The acquired immunodeficiency syndrome, referred to as AIDS, is characterized by repeated opportunistic infections, which develop in patients due to a collapse of their immune system, which is due to proliferation in the body. lymphocytes and macrophages of an HIV-type virus. Thus, the immune capacity of the subject infected by the HIV virus against pathogenic microorganisms decreases gradually, until becoming unable to respond to traditionally common infections.

 Today, scientists are beginning to have more specific ideas about the mechanisms involved in HIV infection and many teams are working on vaccine development. At the same time, on the therapeutic side, antivirals have already been offered to patients and clinical trials of new molecules are increasing. But the discovery and development of these new molecules to fight against HIV infection are long and costly and often uncertain; also, a complementary strategy consists in testing known molecules, on experimental models of HIV virus target cells, in order to highlight a potential activity of these molecules in the treatment of AIDS.

Thus, the activity of BHT against lipid-enveloped viruses has led several teams to test this product on the HIV-1 virus. But the results of these tests concluded that there was no activity of BHT on this virus (H. Claeys, M. Mercken, C. Vermylen,
Medical Assumptions 1988, 27, 145-146; E. Reimund,
Medical Hypotheses 1987, 23, 39-42).

 However, the Applicant has just demonstrated quite surprisingly a virucidal effect of 3,5-di-t-butyl-4-hydroxybenzoic acid on the infectivity of the HIV-1 virus.

dans laquelle R représente un groupe COOH ou un groupe
A-COOH, où A est un reste hydrocarboné aliphatique, ainsi que leurs sels et esters, pour l'obtention d'un médicament destiné au traitement et à la prévention chez l'homme d'une infection par les virus du type HIV, et plus particulièrement du type HIV-1 et HIV-2.The subject of the present invention is therefore the use of compounds of formula

wherein R represents a COOH group or a group
A-COOH, where A is an aliphatic hydrocarbon residue, and their salts and esters, for obtaining a medicament for the treatment and prevention in humans of infection with HIV-type viruses, and more particularly of the type HIV-1 and HIV-2.

 Advantageously, in formula (I), A is a C 1 -C 11 aliphatic hydrocarbon radical.

 In the context of the invention, mention should be made more particularly of the compounds of formula I in which R represents a COOH group, and also their salts and esters.

 Among these compounds, the invention specifically envisages the use of 3,5-di-t-butyl-4-hydroxybenzoic acid and its salts and esters, for obtaining a medicinal product intended for the treatment and the prevention in humans of an infection with HIV-type viruses, and more particularly of the HIV1 and HIV-2 type.

Among the salts of the compounds used according to the invention, mention may in particular be made of the inorganic salts obtained by reaction of the acid of formula (I) with a mineral base. These inorganic salts are compounds of formula (I) in which R = COOX or A-COOX, X representing NH4 + or a cation 1 / mMm +, M being a metal of groups Ia, Ib, IIa and IIb of the periodic table and m valence, such as Na +, K +, 1 / 2Ca2 +, 1 / 2zon2 +, 1 / 2Mg2 +,
Cu +, 1 / 2Cu2 +. It is also possible to mention the addition salts obtained by reaction of the acid of formula (I) with an organic base, such as alkylamines and dialkylamines (where each alkyl moiety is a linear or branched C1-C8 radical) N-hydroxyalkylamines wherein the alkyl moiety is a straight or branched chain C1-C8 divalent radical (e.g., 2-hydroxyethylamine), saturated or unsaturated mononuclear cyclic amines (e.g., pyridine, 3-methylpyridine, pyrrolidine, piperidine, 4-methylpiperidine, morpholine, thiomorpholine, piperazine, 4-methylpiperazine, 4-phenylpiperazine, 4- (4-chlorophenyl) piperazine, 4- (2-hydroxyethyl) piperazine and hexamethyleneimine, as well as amino acids (eg Arg, His, Orn,
Lys, Gly, Ala, Phe, Glu, Leu, Ile, Nle, Nva, Megly, Pro, 4Hyp) where each acid function is capable of being blocked according to the methods used in peptide synthesis.

The esters of the compounds used according to the invention correspond to the formula (I) in which
R = COOZ or A-COOZ, where Z comprises a linear or branched C1-C8 aliphatic hydrocarbon residue, and the amino group that it may contain is NH2, or one of the monoalkylamino, dialkylamino, N-hydroxyalkylamino and cyclic amino groups such as as defined in the context of the addition salts above.

 In general, acids are preferred over their salts and esters.

 The compounds of formula (I) can be prepared according to known methods, by application of conventional reaction mechanisms.

dans laquelle Y est halogène, de préférence Br ou Cl,
avec un composé de formule
H-R
(III), où R est COOH, A-COOH,
en présence d'un métal alcalin, d'un hydroxyde de métal alcalin ou d'un hydrure alcalin.A preferred process is that of reacting a 2,4-di-tert-butyl-4-halophenol of formula

in which Y is halogen, preferably Br or Cl,
with a compound of formula
HR
(III), where R is COOH, A-COOH,
in the presence of an alkali metal, an alkali metal hydroxide or an alkaline hydride.

 In carrying out this process, the OH group of the phenolic function of the compound of formula (II) and the COOH group of the compound of formula (III) can be protected according to known methods.

Details, variants and improvements to the previous method are described in particular in the
International Patent Application PCT / FR91 / 00882.

dans laquelle R représente un groupe COOH ou un groupe
A-COOH, où A est un reste hydrocarboné aliphatique, ou un sel ou un ester de ce composé, en association avec un véhicule pharmaceutiquement acceptable.The invention also proposes to provide pharmaceutical compositions intended to combat an infection by the viruses of the HIV type, and more particularly of the HIV-1 and HIV-2 type, containing as active ingredient at least one compound of formula

wherein R represents a COOH group or a group
A-COOH, where A is an aliphatic hydrocarbon residue, or a salt or an ester thereof, in combination with a pharmaceutically acceptable carrier.

 Advantageously, in formula (I), A is a C 1 -C 11 aliphatic hydrocarbon radical.

 Among the active ingredients used in the pharmaceutical compositions of the invention, mention should be made more particularly of the compounds of formula (I) in which R represents a COOH group, as well as their salt and esters.

 The salts and esters of the compounds used in the formulation of the pharmaceutical compositions of the invention are those previously described, but acids are generally preferred rather than their salts and esters.

 The invention specifically envisages a pharmaceutical composition intended to fight against an infection by the viruses of the HIV type, and more particularly of the HIV-1 and HIV-2 type, containing as active principle 3,5-di acid. t-butyl-4-hydroxybenzoic acid, or a salt or an ester thereof, in combination with a pharmaceutically acceptable carrier.

 The pharmaceutical compositions according to the invention can be administered in the usual manner to man enterally or parenterally, especially by oral see. They can be in the form of solid, semi-solid or semi-liquid and liquid preparations. Examples include tablets, capsules and dragees, suppositories, gels, ointments and creams, injectable solutions or suspensions, drops and syrups.

 Given the low solubility of the compounds of formula (I) in the usual physiological solvents, solid preparations, semisolids and in particular gels and ointments which are particularly suitable for local application at the level of the anogenital organs more particularly concerned, are preferred. in the process of transmitting AIDS.

These formulations may for example be associated with a condom. Injectable preparations, such as alcohol suspensions, may also be considered, for example for intravaginal injections.

 In these compositions the active ingredient is generally mixed with one or more usual pharmaceutically acceptable excipients well known to those skilled in the art.

 The pharmaceutical compositions may contain in particular from 0.1 to 20% by weight of active principle consisting of at least one compound of formula (I). The amount of active ingredient that is administered obviously depends on the patient being treated, the route of administration and possibly the progress of the disease.

I - EXAMPLE OF FORMULATION OIL-CREAM
WATER FOR LOCAL APPLICATION.

 The following is a non-limiting example of a formulation of 3,5-di-t-butyl-4-hydroxybenzoic acid (BG4) as an oil-water cream for topical application.

A first mixture of CBS CUTINA: 6,000 is heated to 75 ° C.
(marketed by the Henkel Company and consisting of
glycerol stearate, cetostearyl alcohol,
cetyl palmitate, coconut triglycerides).

- TEFOSE 1500: 9,000
(marketed by the Gattefosse Company and constituted
polyoxyethylene glycol monodipalmitostearate).

- Isocetyl stearate: 10,000 - Cg and C10 triglycerides (55/45): 5,000
Poured into said first mixture, a second mixture also heated to 750C consisting of - demineralized water: 66,200 - PHENONIP which is a preservative paraben: 0.300
The emulsion thus prepared is cooled, then to 350C, the following mixture is incorporated - 3,5-di-t-butyl-4-hydroxybenzoic acid
finely pulverized: 0,500 - ISOSTEARIC LABRAFIL: 3,000
(marketed by the Gattefosse Company and constituted
of polyoxyethylenated isostearic glycerides).

 Cooling and stirring are stopped at 300C.

It - ACTIVITY AGAINST VIRUSES OF THE TYPE
HIV RESPONSIBLE FOR AIDS.

 The following description relates to the results of the study of the virucidal action of 3,5-di-t-butyl-4-hydroxybenzoic acid (BG4) on the infectivity of the HIV-1 virus.

 A) PRINCIPLE OF THE STUDY.

 This study evaluated the inhibition of the infectivity of the HIV-1 virus for target cells, after treatment of said virus with 3,5-di-tert-butyl-4-hydroxybenzoic acid (BG4) to determine the conditions necessary to reduce by at least 5 log the infectivity of the HIV-1 virus on the cellular model.

 The target cells used are MT4 cells (S. Harada et al., Science, 1986, 229, 563566, F. Rey et al., J. of Virol, Meth., 1987, 16, 239249). The MT4 test allows a fast response; ten days after infection of the MT4 cells, it is possible to observe a cytopathogenic effect and to detect a reverse transcriptase activity in the supernatants of the cultures infected with control virus samples.

 B) MATERIAL.

 1) 2,6-Di-tert-butyl-4-hydroxybenzoic acid.

The study was carried out with micronized crystals of 3,5-di-t-butyl-4-hydroxybenzoic acid
(crystals of the order of 0.5 to 5 pm) in suspension.

 A difficulty of the study to reside in the low solubility of BG4. Its solubility in DMSO is acceptable but has not been used because of the toxic effect of DMSO on cells; similarly the esters or salts of BG4 have not been used because of the increase in pH, harmful to the cells they cause.

 2) Virus.

 The LAI strain of the HIV-1 virus is produced on chronically infected CD4 + T lymphoblastoid cells (CEM-HIV-1 / LAI line). Every three or four days, the cells are centrifuged, resuspended in culture medium (RPMI 1640 containing 10% fetal calf serum, 1% antibiotics and 1% glutamine). They are then mixed with 50% uninfected CEM cells at the final concentration of 0.56 cells per milliliter to maintain viral production. The virus present in the culture supernatant is concentrated by ultracentrifugation at 17 ° C rpm for 3 hours. The pellet is resuspended in the NTE buffer, aliquoted and stored at -800C.

 The titre in reverse transcriptase activity of the virus stock used for this study will be of the order of 107 cpm / ml, and its infectivity titre of the order of 106 IU / ml (Infectious Units / milliliter) on MT4 cells.

 3) MT4 cells.

 It is a human T cell line transformed by HTLV-1. During superinfection of these cells by HIV-1, a cytopathogenic effect and the death of more than 807 of the cells are observed 8 or 10 days after infection.

 This cytopathic effect is directly correlated to the amount of virus used for infection, its replication and the expression of viral antigens by the cells. All the inhibition of this effect therefore corresponds to an inhibition of the multiplication of the LAI strain of the HIV-1 virus, followed by measurement of the reverse transcriptase activity in the culture supernatants.

 The MT4 cells are cultured in RPMI medium (Wittaker) supplemented with 103 fetal calf serum (Labsystem), 18 antibiotics (PSN, Gibco) and 1% glutamine (Gibco).

 C) METHOD.

This study was conducted in two stages
the treatment of the HIV-1 virus with three concentrations of BG4;
the residual infectivity of the treated virus on MT4 cells.

 1) Treatment in kinetics of the HIV-1 virus with three doses of BG4.

 The virus used is diluted in BG4 solutions to a final concentration of 0.5%, 0.1% or 0.05. The contact times with these solutions are 15, 30 and 60 minutes.

 At the end of the treatment, each sample is immediately diluted 1 / 10th in NTE buffer and ultracentrifuged at 40,000 rpm for 35 minutes, in order firstly to eliminate the product, and secondly to concentrate the residual viral particles. The pellet is then resuspended in culture medium, filtered through 0.45 clam, aliquoted and stored at -800C before being tested in vitro infectivity on MT4 cells.

Several witnesses are made
- HIV-1 virus diluted in culture medium and frozen at -800C, as a positive control.

 - The HIV-1 virus diluted in culture medium, then diluted, ultracentrifuged and stored in the same way as the treated samples, to evaluate the loss of infectivity due to centrifugation and filtration.

 - The HIV-1 virus diluted in culture medium, left under the temperature conditions of the experiment for 60 minutes, then ultracentrifuged and stored in the same way as the treated samples, in order to evaluate the loss of infectivity due under experimental conditions.

 - Each concentration of BG4, virus-free, diluted 1 / 10th in NTE buffer and then ultracentrifuged under the conditions described for the virus treated. This sample serves as a check of a possible toxicity on the cells of the infectivity test, due to traces of residual product not eliminated during the centrifugation.

 - The cells of the infectivity test uninfected.

2) Deriving the infectious titles of the
HIV-1 treated or not. using an MT4 cell infectivity test.

 a) Monitoring of the cytopathic effect after inoculation of MT4 cells.

 3.105 MT4 cells are deposited in each 24-well COSTAR plate well. Two wells are inoculated with 0.25 ml of treated virus and two others with 0.1 ml. The wells are supplemented to 1 ml with culture medium and the cells are incubated for 1 hour at 370C. After viral absorption, the residual inoculum is removed and the cultures are reincubated with 1 ml of medium of sequential dilutions per well (2 wells per dilution).

 After 3 days at 37 ° C., 750 μl of supernatant are removed in each well, stored at -800 ° C. and replaced with culture medium The cells are diluted 3 to 4 times to adjust them to 3 × 10 5 cells per ml.

 At day 6, the cells and the cytopathic effect are observed under the microscope and the cell concentration adjusted to 3.105 cells per ml after taking 750 μl of supernatant. At day 10, cytopathic effect and cell death were assessed by microscopic observation after trypan blue staining.

 Each sample of control or treated virus is tested in duplicate.

 b) Measurement of reverse transcriptase acivite (F. Barre et al., Science, 1983, 220, 868-871).

 Assays of the reverse transcriptase activity are carried out from the supernatants taken from each well and kept at each passage (days 3, 6 and 10).

The reverse transcriptase activity is assayed from 0.5 ml of culture supernatant concentrated 50 times by ultracentrifugation for 5 min at 95 ccm rpm on the
Centrifuge TL 100, Rotor 100.2 from Beckman Company.

The pellet is resuspended in 10 μl of buffer
NTE (0.1M NaCl, Tris C, CIM, 0.001M EDTA, pH 7.8) containing 0.1% Triton X100. The enzymatic activity is revealed by the addition of 40 l of the following reaction mixture: 50 mM Tris pH 7.9; KCl 20 mM
50 mM MgCl 2; 1 mM dithiotreitol; polyA 0.05 OD / ml oligodT 12-18 0.05 OD / ml; 3HTTP 511Ci (specific activity = 30 Ci / mmol). After 1 hour at 37 ° C., the acid-insoluble products are precipitated with 20% trichloroacetic acid, filtered through Millipore 0.45 μm, and the radioactivity ss is measured using a BETAmatic type scanning counter. of the Kontron Company.

 D) RESULTS.

Viral expression by the target cells
MT4 inoculated with the treated virus samples is compared to the infectious titres of the control virus samples. This comparison allows to conclude when to the effectiveness of the molecule BG4 to inactivate the virus
HIV-1 in vitro.

 Table I below reports the titration results in dilutions of untreated control virus samples on MT4 cells.

Table I

<tb><SEP> Activity <SEP> reverse
<tb> Samples <SEP> Dilution <SEP> transcriptase <SEP> in <SEP> ECP
<tb><SEP> cpm / 0 <SEP> 5ml <SEP> at <SEP> our <SEP>
<tb><SEP> +3 <SEP>'<SEP> +6 <SEP> +9
<tb><SEP> Positive <SEP> 10-6 <SEP> 304 <SEP> 41 <SEP> 561 <SEP> 47 <SEP> 605 <SEP> ++
<tb> HIV-1 <SEP> 521 <SEP> 7 <SEP> 560 <SEP> 71231 <SEP> ++
<tb><SEP> 10-7 <SEP> 185 <SEP> 15 <SEP> 792 <SEP> 107 <SEP> 145 <SEP> ++
<tb><SEP> 521 <SEP> 14 <SEP> 077 <SEP> 86 <SEP> 152 <SEP> ++
<tb><SEP> 10-8 <SEP> 1 <SEP> 196 <SEP> 9 <SEP> 342 <SEP> 40 <SEP> 719 <SEP> ++
<tb><SEP> 482 <SEP> 32 <SEP> 683 <SEP> 156 <SEP> 879 <SEP> ++
<tb><SEP> control of <SEP> 10-5 <SEP> 512 <SEP> 7 <SEP> 843 <SEP> 47 <SEP> 816 <SEP> ++
<tb> centrifugation <SEP> 687 <SEP> 139 <SEP> 112 <SEP> 089 <SEP> ++ <SEP>
<tb><SEP> 673
<tb><SEP> -6 <SEP> 593 <SEP> 2 <SEP> 974 <SEP> 36 <SEP> 309 <SEP> ++
<tb><SEP> 399 <SEP> 1 <SEP> 195 <SEP> 60 <SEP> 951 <SEP> ++
<tb><SEP> 10-7 <SEP> 710 <SEP> 1 <SEP> 241 <SEP> 56 <SEP> 429 <SEP> ++
<tb><SEP> 504 <SEP> 574 <SEP> 1 <SEP> 217 <SEP> ++
<tb> Witness <SEP> 10-5 <SEP> 379 <SEP> 89 <SEP> 979 <SEP> 153 <SEP> 069 <SEP> ++
<tb> of <SEP> conditions <SEP> 271 <SEP> 7 <SEP> 884 <SEP> 128 <SEP> 821 <SEP> ++
<tb> experimental
<tb><SEP> 10-6 <SEP> 425 <SEP> 3 <SEP> 397 <SEP> 15 <SEP> 082 <SEP> ++
<tb><SEP> 400 <SEP> 1 <SEP> 327 <SEP> 99 <SEP> 082 <SEP> ++
<tb><SEP> 10-7 <SEP> 414 <SEP> 580 <SEP> 2 <SEP> 938 <SEP>
<tb><SEP> 852 <SEP> 370 <SEP> 8 <SEP> 988 <SEP> +
<tb> Cells <SEP> 1 <SEP> 074 <SEP> 643 <SEP> 1 <SEP> 130 <SEP>
<tb> witnesses <SEP> 356 <SEP> 826 <SEP> 655
infected <tb> no <SEP>
<Tb>
In Table I, ECP represents the cytopathic effect detected at day 9 after infection and after staining with Trypan blue (-: no cytopathic effect, +: about 70% mortality, ++: 100% mortality) . Viral expression was evaluated by measuring reverse transcriptase (RT) activity in culture supernatants; it is considered positive when the RT activity is greater than 5 CCC cpm / 0.5 ml of supernatant
Table II below reports the cytopathic effect induced by virus samples treated or not treated with 0.5 and 1% BG4 on MT4 cells at day 9 after inoculation.

Table II

<tb> Samples <SEP> Dilution <SEP> ECP
<tb><SEP> tested
<tb> Control <SEP> positive <SEP> HIV-1 <SEP> 10-8 <SEP> ++
<tb><SEP> control of <SEP> centrifugation <SEP> 10-6 <SEP> ++
<tb><SEP> LED of <SEP> 10-6 <SEP> ++
<tb> experimental <SEP> conditions
<tb> Control <SEP><SEP> Toxicity <SEP> BG4 <SEP> 0.5% <SEP> 1/10 <SEP> None
<tb><SEP> toxicity
<tb> Control <SEP><SEP> Toxicity <SEP> BG4 <SEP> 1% <SEP> 1/10 <SEP> None
<tb><SEP> toxicity
<tb> HIV-1 <SEP> Treated <SEP> 15 <SEP> minutes <SEP> 1/10 <SEP> ++
<tb> by <SEP> BG4 <SEP> to <SEP> 0.5% <SEP>
<tb> HIV-1 <SEP> Treated <SEP> 30 <SEP> minutes <SEP> 1/10 <SEP> ++
<tb><SEP> ar <SEP> BG4 <SEP> to <SEP> 0 <SEP> 5% <SEP>
<tb> HIV-1 <SEP> Treated <SEP> 60 <SEP> minutes <SEP> 1/10 <SEP> ++
<tb> by <SEP> BG4 <SEP> to <SEP> 0.58 <SEP>
<tb> HIV-1 <SEP> Treated <SEP> 15 <SEP> minutes <SEP> 1/10 <SEP> ++
<tb> by <SEP> BG4 <SEP> to <SEP> 1%
<tb> HIV-1 <SEP> Treated <SEP> 30 <SEP> minutes <SEP> 1/10 <SEP> ++
<tb> by <SEP> BG4 <SEP> to <SEP> 1%
<tb> HIV-1 <SEP> Treated <SEP> 60 <SEP> minutes <SEP> 1/10
<tb> by <SEP> BG4 <SEP> to <SEP> 1%
<tb> Control <SEP> infected <SEP> no <SEP> cells
<Tb>
In Table II, the cytopathic effect
(ECP) was detected on D + 9 day after inoculation of the cells and after staining with Trypan blue (-: no cytopathic effect, + about 70% dead cells, ++: 100% dead cells).

 Table III below reports the expression of HIV-1 virus (reverse transcriptase activity) by MT4 cells inoculated with 0.5% and 1% BG4 treated virus samples.

Table III

<tb><SEP> Activity <SEP> reverse
<tb> Samples <SEP> Dilution <SEP> transcriptase <SEP> in <SEP> ECP
<tb><SEP> cpm / 0.5ml <SEP> at <SEP> day
<tb><SEP> +3 <SEP> +6 <SEP> +9
<tb> HIV-1 <SEP> Treated <SEP> 10-1 <SEP> 9 <SEP> 969 <SEP> 98 <SEP> 760 <SEP> 135 <SEP> 396 <SEP> ++
<tb> 15 <SEP> minutes <SEP> 13 <SEP> 757 <SEP> 82 <SEP> 893 <SEP> 74 <SEP> 637 <SEP> ++
<tb> by <SEP> BG4 <SEP> to <SEP> 0.5% <SEP>
<tb> HIV-1 <SEP> Treated <SEP> 10-1 <SEP> 6 <SEP> 558 <SEP> 39 <SEP> 745 <SEP> 67 <SEP> 658 <SEP> ++
<tb> 30 <SEP> minutes <SEP> 3 <SEP> 968 <SEP> is considered positive when the RT activity is greater than 5000 cpm / 0.5 ml of supernatant
E) CONCLUSIONS.

Previous results show that
3,5-Di-t-butyl-4-hydroxybenzoic acid (BG4) is capable of inactivating the infectivity of HIV-1 for MT4 cells in vitro.

 Treatment of the virus with the suspended BG4 molecule at a theoretical concentration of 1% for 60 minutes results in a 5 log reduction in virus infectivity for this CD4 + lymphoid cell line.

Claims (8)

1) Use of Formula Compounds

HIV-2.  in which R represents a COOH group or an A-COOH group, where A is an aliphatic hydrocarbon residue, and their salts and esters, for obtaining a medicament for the treatment and prevention in humans of an infection with HIV-type viruses, and more particularly of the HIV-1 type and  2) Use according to claim 1, characterized in that in formula (I), A is a C 1 -C 11 aliphatic hydrocarbon residue.  3) Use according to claim 1, characterized in that in formula (I), R represents a COOH group.  4) Use of 3,5-di-tert-butyl-4-hydroxybenzoic acid and its salts and esters, according to one of claims 1 or 3, for obtaining a drug for treatment and prevention in humans of an infection by the viruses of the HIV type, and more particularly of the HIV-1 and HIV-2 type.  5) A pharmaceutical composition for combating an infection with HIV-type viruses, and more particularly of the HIV-1 and HIV-2 type, characterized in that it contains, as active ingredient, at least one compound of formula

 wherein R is COOH or A-COOH, where A is an aliphatic hydrocarbon residue, or a salt or an ester thereof, in combination with a pharmaceutically acceptable carrier.  6) Pharmaceutical composition according to claim 5, characterized in that in the formula  (I), A is a C 1 -C 11 aliphatic hydrocarbon radical.  7) Pharmaceutical composition according to claim 5, characterized in that in formula (I), R represents a COOH group.  8) A pharmaceutical composition for combating an infection with HIV-type viruses, and more particularly of the HIV-1 and HIV-2 type, according to one of claims 5 or 7, characterized in that it contains as a active ingredient 3,5di-t-butyl-4-hydroxybenzoic acid, or a salt or an ester thereof, in combination with a pharmaceutically acceptable carrier.