PT96331A - PREPARATION PROCESS OF PHARMACEUTICAL COMPOSITIONS, BASED ON FLOROGLUCINOL AND ITS DERIVATIVES - Google Patents

Description

72 011 14476 -2 I Ç & 0

Invention DESCR Field of

This invention pertains to the field of treatment of immunological disorders associated with immunodeficiency virus infection, such as the Human Immunodeficiency Virus.

Background of the Invention

There is presently an intense effort in the private and public sectors to discover agents - Pharmaceuticals useful in the treatment and prevention of AIDS and correlated immune disorders. The etiological agent of AIDS is the Human Immunodeficiency Virus (HIV). Several isolates and strains of HIV have been described. See, e.g., Barre-Sinousi, Science (1983); Vilmer et al., Lancet i .: 753 (1984); Gallo et al., U.S. Patent 4,652,599; Levy et al., J.S. Patent 4,716,102; and Vahlne et al., U.S. Patent 4,812,566. In addition, several other HIV-like viruses, collectively referred to as Immunodeficiency Virus, have been identified as eticological agents in immune dysfunctions in other animals. These include the Simian Immunodeficiency Virus (SIV). See, e.g., Hirsh et al., Cell 49; 307 (1987) and Kestler et al., Nature 531; 619 (1988). These viruses were classified in the family Retroviridae. They are icosahedral, envelope, RNA-containing viruses of about 150 nm in diameter and have in the core structure a spiral nucleocapsid.

In researching for anti-HIV agents, much of the effort has been devoted to assessing the anti-HIV activity of natural products and other compounds known to have tumoricidal, viricidal, bactericidal or other cytocidal activity. Many of these agents look promising, many do not. In any case, an effective pharmaceutical agent is still to be developed in the treatment of immunophysical disorders associated with HIV infection.

Floroglycinols are a class of trihydroxyl fissures which have been identified as natural products having antihelmintic activity. Members of this naturally occurring class 72 011 14476 -τ.- may be traits of plants of the species Medicosma sessiliflo-ra (formerly Melicope sessilíflora) of the Rustacea family, which are indigenous to Australia.

Sowden et al., Brit. J. Pharmacol. 24: 714 (1965), disclose the relative anthelmintic activity (HvmenQlepis nana) of various -Floroglucindis and correlated compounds.

Bowden et al., J. Pharm. Pharmacol. 17: 239 (1965), disclose the relative antibacterial (Staphylococcus areus) activity of various floroglucinds and correlated compounds. The authors note that " although antibacterial activity is sometimes accompanied by high anthelmintic activity in vitro. this is not always so ".

Chan et al., J. Ora. Chem. 54: 2093 (1939), report anti-virus activity of freshly isolated floroglucindis of M. sessiliflora.

Broadbent et al., U.S. Patents 3,467,715 and 3,377,241, disclose the anthelmintic activity of diacylglycine derivatives.

Mizobuchi et al., Aaric. Biol. Chem. 49 (3): 719 (1935), disclose the antifungal activity of 2,4, β-trihydroxyacylphenones and correlated compounds.

Hartley, Aust 3. Bot. 33: 27 (1935) deal with the reclassification of Medicosma and describe the genus of its species, including the species.

Summary of the Invention

This invention is a method of treating infestation in an animal by the Immunodeficiency Virus, especially the human immunological disorders associated with HIV infection and human immunodeficiency related viruses, by administering to the animal an effective amount of phloroglucinol or a phloroglucinol derivative having the trihydric structure of the phloroglucinol in the nucleus but having substituted one or more protons of the hydroxy groups or unsubstituted or substituted positions.

This invention also relates to the process of preparing certain novel phloroglucinols and the use of such novel phloroglycans to treat infection by the Immunodeficiency Virus.

By this invention a pharmaceutical composition for treating the infection is prepared in an animal by the Immunodeficiency Virus comprising flacoglucinol or a derivative thereof as described above.

Detailed Description of the Invention The present invention is useful for alleviating immunological disorders associated with infection by Immunodeficiency Virus, some of which have now been isolated. These include several isolates identified as HIV, HTLV-III, HIV-1, HIV-2, LAV and ARV, referred to herein as HIV. See the references cited above. It is postulated that the alleviation of immunological disorders is achieved by inhibiting the direct infection of cells, especially lymphocytes, and by inhibiting the formation of syncytia. □ Relief of immune disorders is manifested by normalization of T4 and TS lymphocyte counts and T4: TB lymphocyte ratio.

Compounds useful in the process of the present invention are flaroglucinol and floroglycinol derivatives having the trihydroxyl-phenolic core structure of flaroglucinol or in which the proton of one or more of the three hydroxyl groups is substituted for preferably a non-polar non-charged group, such as the alkyl, alkenyl, alkylaryl or acylaryl groups having 6 to 6 carbon atoms, and one or more protons of the unsubstituted positions are substituted by other radicals, preferably lipophilic radicals such as alkyl, alkenyl, alkylaryl, alkenylaryl, acyl or acylaryl, with the carbon atoms in the chain. Examples of such compounds are those shown by Formula I, below: (SEGUE FORMULA) 72 011 14476

OR3

where: R1, R2, R3 and R5 are the same or different and are -H, -R7H, -R7Ar, -C (O) R7H or -C (O) R7ftr; R2, R4 and R6 are the same or different and are -H, halogen, nitro, -RSH, -RsAr, -C (Q) RSH or -C (O) RsAr; R7 is C1 -C4 alkylene or alkenylene, optionally substituted with one or more halogens, -OH or = R8 is C1 -C4 alkylene or alkenylene, optionally substituted with one or more halogens, -OH or = 0.

Ar is toluyl, naphthyl, -phenyl or pyridyl, optionally substituted with -OH, halogen, -R7H or -C (O) R7H; or any two adjacent groups, R 1, R 2, R 3, R 4, and R 6 together form C 2 -C 4 alkylene, thus constituting a 5-, 6- or 7-membered ring fused to the phenolic ring of the nucleus, and one or more of such members is optionally substituted with -R 7 H, -R 7 Ar, -C <G) R 7 H or - (O) R 7 ftr.

Halogens can be -Br, -F, -Cl and -I, of which -F is or less preferred.

In the preferred process of the invention, the compounds which are employed are those of Formula I wherein R 1, R 2 and R 5 are, independently of one another, selected from -Η and -CH 3; R2 is -R8H; R8 is C1 -C4 alkylene or alkenylene, optionally substituted with -OH or = O; R4 and R6 are -C10) RaH or -C (O) RsAr; and Ar is phenyl; or R 1 and R 2 and R 3 and R 3 are C 2 alkylene thus forming a 5-membered ring fused to the phenyl ring of the nucleus and one of the two carbon atoms is covalently attached to the RA position of a second compound, second compound which has, on the other hand, the structure of Formula I, i.e., has the structure of formula I ': OR3'

R1, R2, R3, R4, R3 and are as defined above for R3, R3, R4, R4 and R6 respectively.

Throughout this description and claims, the terms "alkyl", "alkenyl", "alkylene", "alkenylene", "alkanol", &quot; acyl &quot; and other like terms, encompass both the straight and branched carbon chains. 0 floroglucino! and the phloroglucinol derivatives which may be used in the process of the invention are synthesized by techniques known per se. For example, the preparation of floroglucinol from trinitrobenzene or trinitrobenzoic acid is described by Pascoe, Chem. Products IS; 454 (b) &quot; 55 &gt;. Briefly, trinitrobenzene or trinitrobenzoic acid is reduced in the presence of tin and hydrochloric acid, the solution is neutralized and boiled for 15-20 h. See, The Merck Index of Chemicals and Druids. Merck Si Co., Inc., Rahway, New Jersay. Synthesis of derivatives of floroglucinol is described by Bowden et al., Brit. 3-Pharmacoi. 24: 714 (1965); Broadbent et al., U.S. Patents 3,467,715; and Broadbent et al., U.3. Patent 3,377,241, all of which are incorporated herein by reference only, but fully described.

Examples of compounds which can be prepared by the methods described by Broadbent et al., US 3,477,715 and 3,377,241 and which are now shown to have anti-HIV activity are the diacylated compounds of Formula I, such as the compounds of Formula I in which: R1, R3 and R5 are the same and are -H or -CH3; r4 3 R &amp; are the same and are -C (O) R 2, R 2, -H or -CH 3; and is C--C alquil alkylene. Specific examples of these compounds are those listed in Table I.

TABLE I

Compound tt 90569 9057B 90592 90616 90620 90621 90625 90629 90649 90655 90656 90666 90770

Name of Compound 2,4-diisobutyrifluoroglucinol 2,4-dipropionyl-6-methylphloroglucinol 2,4-divaleri-6-methylphloroglucinol 2,4-dihexanoylphloroglucinol 2,4-diheptanoylchloroglucinol 2,4-di- (4-methylvaleryl) florogluinol 2,4-dihexanoyl- -methylphloroglucinol 2,4-d i-heptanediyl-6-methoxyphloroglucinol 2,4-di isovaleryl-6-methylphloroglucinol 2,4-dioctane l-6-methoxyphloroglucine 2,4-dioctanoylflloroglucinol 2,4-di nonane i-6-methoxyphloroglucine 2,4-dihydro-4-methoxybenzoyl) -1-methoxyphenuloline

Among these diacylic compounds, the compounds which are preferred by the process of the invention are the compounds of Formula Σ wherein: R 1, R 3 and R 5 are -H; R2 is -H or -CH3; and R é is C--C alquil alquil alquilalkylene. Especially preferred are these compounds. the compounds 90592, 90621, 90649, 90655, 90666 and 90770.

Compounds of Formula I which may be prepared by the methods described by Bowden et al., Brit. J. Phartnacol, 24; 714 (1965), include the compounds listed in Table II.

Compound # 90574 90531 90532 90525 90533 90548 90543 90547 90651 90673 90540 90681 90774 90524 90536 90552 90546 90545 90700 90590 90575 90568 90597 90644 90717 90771 90694 TABLE II Name of Compound 2,4-dibutyl-1-6 -methylphloroglucinol acetylhydroglucyl triacetate Floroglucinol 2,4-diacetylchloroglucinol triacetylchloroglucinol ether 2,4-diacetyl-chloro-guinol-1-methyl ether 2,4-diacetyl-chloro-guinol-1,5-dimethyl ether 2,4-diacetylchloroquinazol-1,3,5-trimethyl ether triacetate 2,4-diacetylchlorphlorinol 1.3. 5-tri-benzoate of 2,4-diacetylchloroquinazol ether, florobutyrophenone 4-methyl 2- (4-methylvaleryl) floroglucinol, mannitol fluorophenrophenone 2,4-dimethyl 2,4-diacetyl-6-methoxyphenyl ether, 4-diacetyl-6-methylphloroglucinol-1-methyl ether 2,4-diacetyl-6-methylphloroglucinol tririmidine 2,4,6-tributyrylchloroglucinol triacetate 2,4-dipropionoglloroglucinol 2,4-divalerylchloroglucinol ether 2,4-dipropionylflurogluc 1,2,3-triisopropylethyl ether 2,4-dibutyrylphloroglucinol-1,3,5-trimethyl ether 2,4-diisobutyrylchloroglucinol-3,5-trimethyl-2-acetyl- 2-bromo-4,6-dibutyrylphloroglucinol 2-bromo-4,6-divalerylylchloroglucinol 2-naphthylthio-6-methylphloroglucino1,2-

90530 S.methylmethybis-Florobutyrophenones-2,4-dimethylacetamide

Alternatively, the phloroglucinol derivatives may be isolated from extracts of plants of the genus Medicosma, in particular H. ssssiliflora. This plant can be found in northeastern Oueensland, Australia, south of Sap Craek, up to the wood-cutting area of Boonjie and sea level up to 750 μm at sea level. The plant and its habitat are described by Hartley, Austr. J. Bot. 33: 27 (1985), hereby cited by reference. The plant is described as follows:

Shrub or small tree up to 10 m in height. Branch sub-persistently puberulent to glabrous, 2-4 mm wide internodes, non-carticiforme; terminal buttons pubescent to giabros. Leaves (subaltern to a> opposite or (sub) opposite and (sub) verticillated in the trees, 6 to 27 cm long; petiole subper- sistently puberulent to glabrous, convex to canaliculate adaxially, increased apically and sometimes articulated with the blade, 5 to 45 mm long, 1-2 mm wide in the sock, subcoriaceous, scarsa and subpersistant puberulent blade in the same central below glabrous, plus conspicuously oil - spattered, elliptical or elliptical - oblong hands up to oblabeled, 5.5-22.5 cm long, 2-8.5 cm wide, about 2 to 4 times longer than wide, with a rounded or narrow base and superficially cordiform, C9-) 12-16 major veins on either side of the central vein, diverging at an angle of 70-80®, from obtuse to acuminate apex, often retinal. Inflarescincias axillary, of one or some flowers, in younger sub-sessile branches or with herbaceous axes up to 1,5 mm in length, older branches other flowers frequently produced in woody processes up to 2 mm in height; obsolete) up to 0,5 (-1) mm in length. Bisexual flowers, 6-8 mm long, istragonalmerrfcs ovoid button; sepals from densely pubescent to glabrous, imbricate to about half their length, evaded or sub-orthocytic, 1-2 mm in length; petals of different thicknesses in a tube about 1/6 of the length, white or cream, densely pubescent

A narrow elliptical shape, 5-6.5 mm long; filaments of distinct stamens or cones in a tube, up to about one - fifth of their lengths, woolly on the margin, the tapirs are gyularularized near the apex, the ante - petals are eglandular; the anthers are transiently pilose ησ apex, 0.6-1 mm long; cupular disc with B lobes with alternately deep sinuses and glabrous surfaces, 0.8-1.5 mm in height; ovary pubescents in the felt of the apex, from 0.8-1 mm in height; hair stylus on base 1/3-I / 2; subgenerated stigma, with 4 slits, at a position approximately at the ante-sepals anthers. Finases slightly pubescent towards the apex or glabrous, 6-10 mm long; endocarpo placéntá-rio subcarnudo; blackish brown seed (s), sparsely tubercular and poorly papillary.

See also Berti et al., "Constituents of Ferns &quot;, in Progress in Phytochemistry; Tomas-Lorente et al., Phytchechemistry 28: 1613 (1989); Arisawa et al., J. Natural Products 45: 455 (1985); Arisawa et al., J. Natural Products 49; 298 (1986); and

Fujita et al., J. Natural Products 51: 708 (1989), all refer to plant floroglucines and are cited herein for reference only.

A typical extraction procedure comprises soaking, dried leaves sprayed in an organic solvent, fractionating the extract, for example on a hydrophilic and / or hydrophilic column, and eluting organic solvents as described in the Examples below.

Chan et al., J. Org. Chem. 54: 2098 (1989), describes the analysis of certain novel derivatives of flaroglucinol, from M. sessiiiflora. here quoted for reference even though integrally. Examples of phloroglucinol derivatives which may be isolated from extracts, hexane or methyl ethyl ketone from H. sessile are the compounds depicted in Example III. 72 011 14476 -11-

η3 &lt; γ ch3

TABLE III

104402

OH O

OH O

OH O

72 011 14476 -12-

OH

107921

108190

72 Οϋ 14476 -13-

As indicated in the Examples below, not all natural derivatives of floroglucinol have been shown to have activities in HIV infection inhibition assays, although all have been shown to inhibit syncytium formation. Based on the results of these assays, among the single ring compounds derived from natural products, the most preferred compound is G4402; among the fused ring compounds, compounds which are preferred for the process of the invention are 107923 and 107921. Other floroglucinol derivatives may be prepared from natural floroglucinol derivatives by classical chemical methods. In preparing these derivatives it appears desirable to retain the hydroxyl or lower alkoxy groups, especially the methoxy, at the hydroxyl positions of the phloroglucinol, i.e., at the -OR, -ORO, and -OR4 positions; replacing the hydrogens at the R 1 and R 1 positions by acyl groups, eg, -C (O) R 1 H or -C (Q) R 2 Ar; and replacing the hydrogen at the position with an alkyl or alkenyl group, e.g., -R 2 H; or, when preparing fused ring derivatives, it is desirable to have the ring formed between the R 1 and R 2 positions or between R 1 and R 2, and having this ring substituted with a floroglucinol or a floroglucinol derivative, e.g. , a compound of Formula I '.

It is to be understood that the formulas set forth hereinbefore and hereinafter are intended to encompass the tautomers and isomers. In addition, the compounds of Formula I may form salts with inorganic or organic cations, which salts are non-toxic and pharmaceutically acceptable which are included within the scope of the invention. Examples of these cations are magnesium, calcium, sodium, potassium and piparazine. Also encompassed by the scope of the invention are prodrugs of floroglucinol or derivatives of floroglucinol, i.e., floroglucinol and floroglucinol derivatives, usually covalently linked to another chemical group which may confer greater stability, reduced toxicity or action controlled and which may or may not be active in the prodrug form and which are cleaved in the body of the treated animal producing floroglucinol or active phloroglucinol derivatives.

In the process of the invention, the compounds of Formula I are administered internally, e.g., parenterally as an injection,

or intravenous, subcutaneous or intramuscular infusion, oral, rectal, buccal, transdermal or inhalation infusion. The compounds are usually administered in a pharmaceutically acceptable carrier or diluent, chosen based on the route of administration. For solid formulations such as pills, powders, tablets, capsules and dragées, useful carriers include, but are not limited to, lactose, terra alba, sucrose, talc, gelatin, agar, peciin, gum arabic, magnesium stearate and stearic acid. The carrier may also include a delayed action material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

Liquid carriers for oral, intranasal or parenteral administration include, but are not limited to, glycerol, syrup, peanut oil, olive oil, saline, dextrose and water, optionally buffered with organic or inorganic salts, eg acetates, adipates, succinates or ammonium, potassium or sodium citrates. Other excipients may be added to adjust the tonicity or, especially in the case of the formulation to be lyophilized, stabilizers such as, for example, gelatin, polyvinylpyrrolidine, cellulose, gum arabic, polyethylene glycol, pyrrolidone and mannitol.

For rectal or vaginal administration, the compounds may be combined in the form of powder with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycol, and shaped into a suppository. For transdermal administration, the compounds may be combined with an oily preparation, gel, cream or emulsion and administered by a transdermal patch.

In the process of the invention, the compounds of Formula I are administered to an animal, especially man, which has been or is suspected to have been infected by an Immunodeficiency Virus, especially HIv, in an amount effective to inhibit progression of immune dysfunction and preferably to stabilize or ameliorate immune dysfunction. The amount administered will vary depending on a variety of factors including the stage of progression of immunological dysfunction and the age, weight and general health status of the animal to be treated.

72 01 4446

It is generally believed that the process of the invention is carried out by administering from about 1 to 500 mg / kg / day, although in particular cases the physician may decide to administer even higher doses. The effects of the process of the invention on the immune disorders associated with infection by an Immunodeficiency Virus can be controlled by evaluating the T4 and TS cell counts and the T4 / TS ratio, and thus the dosage frequency and dose can be modified. Alternatively, the presence of virus in animal carpal fluids can be controlled by evaluating the presence of virus antigens or virus antigen antibodies in the serum. It is believed that the process of the invention should be conducted over a lengthy period of time, e.g. from several weeks to months, or that the treatment should be repeated periodically, because the ability of Immunodeficiency Virus to remain latent in the infected animal.

Covered by the process of the invention is co-administration or simultaneous administration of floroglucinol or of phloroglucinol derivatives with other pharmaceutically active agents, e.g., other agents which are also effective in inhibiting virus replication and syncytia formation, such as other nucleoside analogs, e.g., AZT and ddC, HIV protease inhibitors e = CD4 and their truncated moieties.

The following Examples illustrate and do not limit the invention. All compounds are referred to by the Numbers of Compounds assigned in Tables I and III above. &quot; EtOAc &quot; means ethyl acetate and &quot; HGAc &quot; means acetic acid.

EXAMPLES

Example 1. Diacylfloraolucinol derivatives

The compounds listed in Table IV, below, were checked by an HIV Infection Power Inhibition Trial, as follows. 100 μl of a &quot; stock &quot; of T4 + lymphocytes (AA5 or M0LT4 cells) containing about 3 x 10 6 cells per ml were seeded into a 4-well or 96-well plate. Then, to each well were added 100μ of a &quot; stock &quot; containing about 10 72 72 11 14476 1 1 ·

Infectious Units (approximately equivalent to TCID5, -, units). The virus was allowed to adsorb cells to 37Â ° C in a CO2 incubator. Appropriate dilutions of a floroglucinol derivative were then added to each well. Control wells were prepared in the same manner but without the floro- glucinol.

After incubating the cells for 7 to 10 days, the HIV from the conditioned medium was concentrated by precipitation with polyethylene glycol. The relative amounts of viruses in the experimental and control wells were determined by assessing reverse transcriptase activity in viral lysates and by ELISA for the p24 antigen. The results are given in Quadra IV where IC 50 means the concentration of the phloroglucinol derivative which results in a 50% reduction of reverse transcriptase activity and p24 concentration relative to the controls. "N.A. &quot; means that no significant inhibition of infectious power was observed at the concentrations tested. The results given in parentheses are the results of a second experiment.

5.0 3.0 0.8 (6, 6) 3.9 N.A. N.A. N.A. N.A. A. A. 1.0 8.5 9.5 V, below, were verified

Compound 90666 90770 104402 107924 797922 108189 104553 SS191 104552 90649 90575 90678 The compounds set forth in Table 72 OR 14476

In an HIV Syncytial Inhibition Trial Essay substantially as described in Putney et al., Science 254; 1392 (1986) to Matthews et al., Proc. Natl. Acad Sci. USA 84: 5424 (1987). Briefly, various dilutions of floroglucinal derivatives were added to 96-well plates. Lymphocytes, T4 + cells, which were previously HIV-infected, were mixed with uninfected CEM or M0LT4 cells and seeded into wells in amounts of about 70,000 uninfected cells and 5000 infected cells per well. The plates were incubated for about 20 to 24 hrs at 37 ° C in an incubator at 40 ° C. Giant cells, i.e., cells of diameter at least 5 times greater than that of normal cells, were counted using a 40X magnification microscope. Control wells had on average about 60 to 70 giant cells per well. The results are shown in Table V where IC 50 is the concentration of each floro-glucinol derivative which resulted in a 50% reduction in the number of giant or syncytial cells per well. &quot; MA &quot; indicates non-detectable activity, "AZT &quot; is 5-azido-thymidine and &quot; ddC &quot; is dideoxycytosine. -18-72 011 14476

TABLE V

Compound IC50 90569 7.5 90575 NA 90578 10.4 90582 NA 90592 1.5 90616 10.0 90620 5.8 90621 4.5 90625 10.1 90629 9.5 90649 1.5 90655 4.5 90656 6.7 90666 2.1 90770 1.4 90574 2.4 104402 5.6 107921 2.9 107923 5.0 107925 9.2 107924 17.0 107922 24.0 108189 24.0 104553 24.4 108191 41.2 104552 45.0

ΑΖΤ ddC 18.0 1.0 72 011 14476 -19-

Example 2. Natural derivatives of florogiucinol

Medicosma sessiliflora was harvested in Australia in September 1979; specimen voucher VKY 2666 is stored in the National Herbarium, Washington, D.C. Powdered leaves of M. ses-siflora (100 g) were macerated in n-hexane for at least 24 h. The n-hexane was removed by filtration and the sheets were then stirred in methyl ethyl ketone for at least 24 h. Filtration and concentration of the filtrate under vacuum yielded 38 g of crude extract. The methyl ethyl ketone extract (20 g) was chromatographed on silica gel 60 (230-400 mesh) on a Whatman Magnum 40 (4.7 x 47 cm) stainless steel column (Whatman Chemical Separatiores, Inc., Clifton, New Jersey) in two batches of 10 g. The column was eluted sequentially first with CH 2 Cl 2 (Solvent A), then with 0.27. H20 / 17. CH 3 OH / CH 2 Cl 2 (Solvent B), finally with 0.27. H 2 O / 2% CH 3 OH / CH 2 Cl 2 (Solvent C) at a flow rate of 50 ml / min while controlling at 254 nm. The first bioactive fraction containing compound 104.402 (1.25 g) was obtained from the eluate with Solvent A. Recrystallization from diethyl ether (Et2 O) / CH2 Cl2 provided 730 mg of the white crystalline compound, 104 402.

A second fraction (13 mg) was eluted from the silica column with Solvent B and crystallized from Et2 O / CH2 Cl2 to give 38 mg of compound 107,924. The mother liquor was separated by multiple injections on a Merck Lichrosarb silica gel 60 (7 pm, 10 x 250 mm) column (EM Science, Cherry Hili, New Jersey) eluting with 0.1% HCOA / 30% EtOAc / n -hexane at 4 ml / min (800 psi), controlling at 335 nm. Compound 107.922 (50 mg) eluted at 7.5 min.

A third major fraction (6.3 g), eluted from the initial silica gel column with Solvent B, was fractionated on a Whatman 0DS-ο (40 μm) on a Whacman Magnuim 40 (4.7 × 47 cm) stainless steel column ( 80 ml / min) while controlling at 340 nm. Compound 107925 (60 mg) was eluted with 70% CH3 OH / H2 O and crystallized in CH2 Cl2. The major peak eluted with 70 to 75% CH3 OH / H2 O and gave 2 g 72 011 14476

Of crude mixture. Of these 2 g of mixture, a 100 mg sample was fractionated by multiple injections on a silica gel (7 tm, 10 × 250 mm) column. Merck Lichrosorb eluted with 0.1% HDAc / 207. EtOAc / n-hexane at 4 ml / min, (SOO psi) while controlling at 335 nm. Compound 108191 (6.8 mg) eluted at 10.2 min. Compound 104553 (43 mg) eluted at 10.7 min. and compound 104552 (47 mg) eluted at 16 minutes. Elution with Solvent C on the silica gel column gave at least three other bioactive fractions. The first fraction, 220 mg, which after trituration with 30% EtOAc / n-hexane gave 55 mg of insoluble material which was compound 107.921. The next major fraction was a mixture, 480 mg, which gave 84 mg of 107923 after crystallization from ethyl ether. Fractionation of the mother liquor in a 60 ml silica Bcnd-Elut (3 x 4 cm) (Analytichem International, Harbor City, California with 0.1% H2 O / 30% EtOAc / n-hexane gave first a fraction which was rich in compound 1, 7923 and then a mixture, 63 mg, which was then purified using multiple injections on a Merck Lichrosorb silica gel column (7th, 10x250 mm) cam 0.1% H2Oc / 30% EtOAc / n-hexane at 4 ml / min (800 psi) while controlling at 335 nm to give compound 108189 (26 mg) The third major bioactive fraction, eluted from the silica gel column with solvent C, consisted of 225 mg of a crude mixture. fractionation of the crude mixture on Bond-Elut silica (60 ml) with 0.1% HCO3 / 30% EtOAc / n-hexane gave a mixture containing compounds 107923 and 108189. A second dilution with 0.1% HOOc / 70 Gave a further mixture which was further purified on a Merck Lichrosorb 60 silica gel column &lt; 10x250 mm) eluting with 0.1% HGAc / 70% EtOAc / n-hexane, obtained The compound 108190 (/ mg) was added

Analytical elements of the retained compounds are given below. 104402: I.V. (CH 2 Cl 2) 3600-3100 (DH), 3300-2400 (hydrogen bonded hydroxyl), 1615 cm -1 (hydrogen bonded carbonyl); UV CMeOH) λmax (O 274 (CH3) 2CHCH2), 25.2 d, (883) 288882). 32.7 (q, 88388), 95.4 (d, 8-8 72 011 14476 -21-

aromatic), 105.0 Ibs, aromatic C), 172.0 (os, aromatic C-Q), 204.0 206.6 ppm (2> g, C = O). ____ . [Î ±] Î ±] β [Î ±] β [3H], 2xd, J = 6.7Hz, (CH3) 2 ', CH3 / 2CHDH) , 2520 (ch3) 2 107921: Calc'd. for C; 586.2407. 1 H NMR (CDCl3)? .0, &lt; CHCH 2 &gt;, 1.37, 1.49 &lt; 4x3H, 2x CHCH2 &gt;, 2.66, 2.70 &lt; 3H, 5b, CH3CO), 3.06, 3.10 &lt; 2x2H, 2xdd , 3 = 7.1, 16.8Hz, (CH 3) 2 Ci-CH 2), 4.80 (1H, d 3 = 5.6 Hz, CHGH), 5.10 (1H, d, J = 5.7 Hz, CHCH) , 16.6, 15.9, 15.6 ppm (br, 2H). 13 C NMR (CDCl 3, δ, 22, B, 22.9 <q, (H 3) 2 CHCH 2), 26, C (d, CH 3) 2 H -CH 2 &gt;, 32.6, 35.6 &lt; q , CH 3 CO), 33.1 (1H, CHCH), 52.7, 53.2 (CH 3) 2 CHCH 2), 73.4 (CH 3), 2 CH 3, 96.9 (d, CHOH), 100.2, 104.6, 105.1, 105, S, δ, aromatic C), 165.3, 167.6, 170.4, 170.8, (s, C-aromatic), 202.2 , 204, B, 207.2, 207.5, ppm (cis, C = O). 107923: 1 H NMR (CDCl3): 0.98, 1.04, 1.38, 1.51 (4x3H, 2xs, (CH3) 2 CHOH), 2. 2.60, 2.70 (3H, s, bs, CH3 CO), 2.70, 15.0 Hz, (CH3) 2 CHCH2), 4.80 (1H, d, J = 5.7 Hz, CHCH), 5.05 (1H, d, J = 5.7 Hz, CHOH), 15.6, 15.9, 16.6 ppm 1, OH-Phenolic) .13 C NMR (CDCl3), 22.7, 22.8, 22.9 Iq, (CH3) 2CHCH2), 24.5, 25.5 &lt; d, (CH) 3 - CHCH2), 32.6, 35.1 (q, CH3 CO), 33.1 (d, CHCH), 51 , 5 (t, &lt; CH3) 2 CHCH2), 73.5 ee, (CH3) 2 &gt; 96.9 (d, 2H), 100.1, 104.8, 105.1, 106.0 (s, C aromatic), 164.7, 167.7, 171.5, C-aromatic), 204.8, 205.1, 207.1, C = O). 107922: Calc. Calc'd for C 25 H 25 N 3, 337.1561, there was obtained M + H m / z 1643. 1 H NMR (CDCl3)? £ 0.97 / iwTU h 1 i | (CH 3), CH 2 (CH 3) 2 CHCH 2), 1.42 (t, CH 3), 2 (CH 2) , 66.3 (3H, CH3 CO), 2.66, 2.87 (2H, dd, J = 5.3, 11.4Hz, CH3 CH2CH2), 2.89 AMX, J1 = 5.0Hz, CH2OH), 3.86 (1H, , t. 3 = 5.4 Hz, CHOH), 15.3, 16.1 ppm (2 H, s, OH-phenol), â € ƒâ € ƒâ € ƒ1 H NMR (CDCl3): Î'22.0 (CH3) , CH 3) 2 CHCH 2), 25.1 (q), (CH 3) 2 C), 25.3 (t, CH 2 CHOH), 33, CH 3), 52.9 (CH 3) 2 CH CH 2, 68.3 (d, CHOH) 79.9 (s, CH 3) 2 C), 97.9, 104.1, 104.6, aromatic C), 160.3, 170.0, 170.1 cis, C-aromatic), 203.3 , 206.8 ppmis, C = O). 10B19: Calc. for C19 H31 N3 337.1651, M + H m / z 337.1644. 13 C NMR (CDCl3): Î'22.0 (d, 3H)

(Q, <H3) 2 C>, 25.3 (t, H2 H2 O), 33.0 (q, CH3 CO), 53.2 (CH3) 2 CHCH2) t, (CH 3) 2 CH 2 H 2, 68.4 (d, CHOH, 30.1 Hz, (CH 3) 2 C), 169.7, 170.7 ppm (s, C- aromatic), aromatic C The title compound was prepared according to the procedure described in Example 2, except that the title compound was obtained as a white solid, mp 198 DEG-228 DEG C. Calc'd for C 20 H 25 N 3 O 3: 354.1673, H + m / z 354.1679. , 2.24 (1H, m, (CH 3 &gt; 2 CHCH 2), 2.35 (1H, m, CH 3) CH 3 CH 2 CH 2), 3.00 (28, d, J = 6.8Hz, <CH 3) 2 CHCH 2, (CDCl3 + several drops of CD30H), Î', 22.2, 22.6 <q, <lt; CH3) 2 CHCH2), 253 (1H, bd, J = 9.5Hz, CH2CHOH) ,  € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ (1, (CH 3 &gt; 2 Hg), 80.3 (d, CH 2 CHOH), 104.3, 104.4, 105.1 (s, aromatic C), 169.6, 170.1 cis , C-O-aromatic), 204.5, 207.0 ppm, C = O> 107924: Calc'd for MS: m / z 295.1118. Mixture of 2 isomers.  € ƒâ € ƒâ € ƒ1 H NMR (CDCl3), 0.94, 0.95 (2x68, d, (085) 2880), 2.20 (2â € ², 1H, m, (CHâ,ƒ) (2x3H, s, CH3C0), 2.90, 2.94 <2x2H, AMX, J = 6.8Hz, (CH3) 2CHCH2), 3.18, 3.20, 3.44 <2x28, AMX, (CDCl 3): 22.6, 22.7 (q, (H 3) 2 CHCH 2), 7.83 (d, , 24.9, 25.3 (d, CH 3) 2 HCH 2, 31.2 (q, C 83 CO), 32.9, 33.1 &lt; t, C 12 H 2 CH 2), 51.3, 53.1 (t, CH 3 &gt; 2CH 2 H 2), 100.6, 102.7, 102.8 (s, aromatic), 104.4, 104.5 (d, CHOH), 165.2, 169.9 , C-O-aromatic), 202.4, 204.4, 204.9, 206.8 ppm ís, 0 = 0). 108190: Molecular weight = 352; 1 H NMR (CDCl3): Î'0.94, 0.95 (2x38, d, J = 6.7Hz, (883.8), 1.27 (3H, s, CH3 (CH3) C), 2.10 , 2.76 (2H, d, CH 2 CHOH), 2.96 (28, d, CGCH 2 CH 3), 3.37, 4.01 (37.529), 375 (1H, 4941), UV (MeOH-G, IN NaOH) 295 (39,059), 375 nm (in -F): calc'd for C 18 H 226 O) 336.1572, there was obtained M + m / z 336.1557; mp 117-120 ° C. Anal. Calc. Calc'd for C 24 H 21 O 4: C, 64.47; H, 7.19. It was obtained; C, 64.52; H, 7.33; 1 H NMR (CDCl 3): 0.98 (68, d, J = 6.7 Hz, (CH 3) 2 CH), 1.86 (38, s, CH 3 C = CH 2), 2.25 (1H, 2.72 (38, s, br, CH3 C = O), 2.75 (18, dd, J = 15.5, 7.3Hz, CH2CHO8), 3.05 (28, AMX, CO2 CH3) ), 3.41 (1H, dd, 3.72, 1.14,

J = 15.5Hz, CH 2 CHOH), 4.37 (1H, d, J = 7.3Hz CHDH), 4.90, 5.00 (2x1H, s, br, 3, 16.2 ppm (3x H, s, OH-Phenol). 1 H-NMR (DMSO-d 6): δ (CH 3) = 22.5 (q, CH 3 C = CH 2) CH3 OH), 53.0 (t, CH2 CH3), 77.7 (CH3 OH), 104.3 (s, aromatic C), 112.9 (H2 H2), 145.3 s, CH = CH 2), 165.5, 169.6, 170.5 (s, C-aromatic), 204.4, 206.9 ppm (s, C = O). 104522: I.V. Film &lt; CH 2 Cl 2 &gt; 3600-3100 &lt; OH), 3300-2400 hydrogen bonded hydroxyl), 1633 cm-1 hydrogen bonded carbonyl); UV (MeOH) &lt; &gt; 273 (65,999), 341 nm (8143); λmax (i) UV (MeOH-0.1 N NaOH) 294 (48.451), 320 nm (Îμ-Fl); calc. for C 24 H 24 O 336.1572, M + m / z 336.1571; 1 H NMR &lt; CBCL3 &gt; t, 0.97 &gt; 2x3H, d, -J = 6.7Hz, CCH2 CH), 1.27, 1.38 (2x3H, s, HOC- (CH2) 2), 2.25 (1H, J = 6.6Hz, CH 2 CH (CH 3) 2), 2.64 (3H, 3, CH 3 CO), 2.97 (2H, AMX, J = 6.9, 21.1Hz, COOCH 2 CH), 3.07 (1H, t, J = 9.3Hz, CHOCH2), 14.9, 15.6 ppm (2x1H, 3, OH) -phenol ). 13 C NMR (CDCl 3): 22.7 (q, (CH 3) 2 CH), 24.7, 24.9 (q, <CH 3) 2 C), 26.0. (d, CH 3 CH 2 CH), 26.3 (t, CH 2 CHOH), 31.1 (q, CH 3 CD), 51.4 (t, CH 2 CO), 71.7 (s, CH (CH 3) 2), 92.9 , Aromatic C), 166.0, 170.0, 170.7 (3xs, C-O-aromatic), 202.0, 207.0 ppm &lt; , 2xs, 0 = 0). 104523: Films IV (CH3 Cl2) 3600-3100 (OH), 3300-2400 (hydrogen bonded hydroxyl), 1636 cm @ -1 (H bond attached); UV (MeOH) λmax (f) 273 39.969), 341 (3737); UV (MeOH-0.1 N NaOH) 293 (36.969), 32U nm CInFi); calc. for C 336.1572, M + m / z 336.1564; Federal Police. 105-1073 ° C; 1 H NMR (CDCl3) δ 0.99 (2x3H, d, J = 6.7Hz, (CH3 &gt; 2CH, 1.27, 1.39 &lt; 2x3H, s, HG CH3) 2 &gt;, 2.20 (1H, m , J = 6.6Hz, CH 2 CH (CH 3) 2), 2.70 (3H, s, CH 3 CO), 2.81, 2.93 <2x1H, dd, J = 0.9, 14.5Hz, ), 3.08 (2H, AMX, J = 9.0, 14.1 Hz, CH 2 CHOH), 4.83 (1H, t, J = 9.0 Hz, CHOCH2), 14.7, 15.8, ppm 2XiH, s, OH-Phenolol). NMR (CDCl 3): 22.7 (q, CH 3) 2 CH), 24.7, 25.5 (q, (CH 3) 2 C), 26.1 (d, CH 3) 2 CH), 26.2 (t, CH 2 CHOH) , 33.0 (q, CH3 CO), 52.9 (t, CH2 CH2), 71.5 (s, CH3) 2, 92.3 d, CH2 CH2 OH), 100.3, 3 (3: 3, aromatic C), 166.3, 166.7, 17.3, 31.3, 31.0, aromatic), 204.4, 204.5 (2xs, C = O). 72 011 14476 -24-

Single-crystal X-ray diffraction analysis - Was performed from three-dimensional intensity data obtained with a diffractometer Enraf-Nonius CAD-4 (ΜαΚα) = 0.71073 A3 equipped with graphite monochromator. The data were obtained by a variable speed search technique 2, at 248 K. A total of 5583 results were obtained (± h, + k, +1) (2 160 °) of which 3623 were considered (I 2: 3σ (I) ) after Lcrsntz correction, from the effects of polarization to the determination of means of the equivalent reflection of symmetry (Rint = 0.013). The molalate crystallizes as a hydrate in MeOH. Crystallization elements: trichloric, P 1, Z = 2, a = 9,852 (2), b = 1, 563 (2), and = 8.931 (2) A = 107.51 (2) 54 (2) * τ = 73.36 (2) °. The structure was determined by direct methods. Atoms other than hydrogens were &quot; refined &quot; with anisotropic vibrational parameters; all hydrogens were localized by Fourier-difference synthesis and were "refined &quot; with isotropic temperature factors. The complete least squares matrix of "refining * 1 (in F) converged to values of classical crystallographic residues. R = 0.043 and R w = 0.055. An extinction coefficient "refined" to 1.24 (1) x 10-è. The map of final Fourier differences was uncharacteristic. m, 107925: IR (KBr) 3600-3100 (OH), 3100-2800 hydrogen bonded C0H), 1626 cm-1 (hydrogen bonded carbonyl); Calc. for C 31 H 275 253.1076, M + H m / z 253.1033; 1 H NMR (CDCl 3) δ 0.91 (2 H, d, J = 6.4 Hz CCH 3) 2 CHCH 2}, 2.20 (1H, m, (CH 3) 2 CHCH 2), 2.60 (3H, s, CH 3 C 90 (2H, bd, J = 6.3Hz, CHCH2), 5.30 (1H, s, aromatic H), 16.2 ppm &lt; 3xH, bs, phenolic OH).  € ƒâ € ƒâ € ƒ1 H-NMR (CDCl3) Î'22.7 &quot; (2H, dd, J = 11 Hz, CHâ,ƒ), 4.16 ppm (1H, CHâ, Hâ, Hâ, ...). 1 H-NMR &lt; CHCl3): 6.227 (qq. (CH 3), 52.3 (t, CH 3) 2 CH CH 2), 67.2 (CH CH), 67.3 (t-CH 3) (S, C (aromatic), 160.0, 170.0, 170.3 (s) 1, aromatic C-O), 203.2, 206.9 ppm (s, C = O).

Example 3. Semi-synthetic Floroalucinols.

Compound 104,402 (40 mg) was treated with p-toluenesulfonic acid (40 mg) in toluene (6 ml) at 90-100 ° under a nitrogen atmosphere. After 2.5 h, the mixture was concentrated, taken up in ether (25 ml) and washed with aqueous sodium carbonate (1% w / v, 4 x 20 ml). The ether layer was washed with aqueous sodium carbonate (1% w / v, 4 x 20 mL) and dried over magnesium sulfate. Evaporation of the solvent under reduced pressure provided a mixture (40 mg). This mixture was separated by multiple injections (4 mg each) into a reversed-phase, semi-preparative HPLC scale (Magnum 9, 250 mm x 4.6 mm, Partisil 10 μm, 0DS-3) 1% trifluoroacetic acid / 10% water / methanol as eluent at 5 ml / min (3.4 Kpsi). A mixture (6.4 mg) of 107922 and 108191 was eluted as the major peak. Separation of 107922 and 08191 was performed on the same Magnum 9 column (above) eluting with 0.1% trifluoroacetic acid / 35% H2 0 / methanol at 5 ml / min. (3.3 Kpsi). 107922 (1.9 mg) eluted at about 32 min and 108191 (2.8 mg) elute at about 36 min. The foregoing description and examples illustrate particular arrangements of the invention, including their preferred arrangements. However, the invention is not limited to these precise arrangements described herein but, on the contrary, include all variations and modifications falling within the scope of the following claims.

Claims (3)

A process for the preparation of a pharmaceutical composition for treating immune dysfunction associated with infection in an animal with an immunodeficiency virus, characterized in that it comprises associating floroglucinol or a derivative of floroglucinol having the trihydroxyl structure of the fluoroglucinol but which has substituted one or more protons of the hydrazyl groups or the unsubstituted positions with a pharmaceutically acceptable carrier. 2. A compound according to claim 1, wherein the phloroglucinol derivative is a compound of formula I OR 3 Wherein R 1 and R 2 are the same or different and are -H, -R 7 H, -R 7 Ar, -C (O) R 7 H or -C <Q) R 7 Ar; Ra and Ra are the same or different and are -H, halogen, nitro, -RSH, -RaAr, -CiQ) RsH or -C (O) RsAr; R7 is C1 -C4 alkylene or alkenylene, optionally substituted by one or more halogen groups, = O or -OH; R 1 is C 1-6 alkylene or alkenylene, optionally substituted by one or more halogen groups, = O or -OH; and Ar is -toluyl, naphthyl, phenyl or pyridyl, optionally substituted by -OH, halogen, -R7H or -C1-6alkyl; or any two R 3 groups together form a 5-, 6- or 7-membered alkylene, fused to one or more of these members are R 2, R 3, R 4 and R 5 are adjacent C 2 -C 4, of the nucleus, and one substituted with -R7H, -R / Ar, -C <0) R7H or -C (O) R7Ar. 14476 A process according to claim 2, wherein the compound of formula I is 107923 or 107921 Lisbon, 2'i. TEN. nineteen ninety By SMITHKLINE BEECHAM CORPORATION - 0 OFFICIAL AGENT -