ANTI-VIRAL COMPOSITIONS

Field of the Invention

This invention is in the field of treatment of immune disorders associated with infection by Immunodeficiency Viruses, such as the Human Immunodeficiency Virus (HIV) .

Background of the Invention

There is, at present, intense effort in the private and public sector to discover pharmaceutical agents useful in treating and preventing AIDS and related immune disorders. The etiologic agent of AIDS is the

Human Immunodeficiency Virus (HIV) . Several isolates and strains of HIV have now been reported. See, e.g. , Barre-Sinousi, Science (1983); Vilmer et ai., Lancet i.:753 (1984); Gallo et al. , U.S. Patent 4,652,599; Levy et al. , U.S. Patent 4,716,102; and Vahlne et al., U.S. Patent

4,812,556. In addition, several other HIV-like viruses, collectively referred to as Immunodeficiency Viruses, have been identified as etiologic agents in immune dysfunctions in other animals. These include the Simian Immuno- deficiency Virus (SIV). See, e.g., Hirsh et al., Cell 49: 307 (1987) and Kestler et al. , Nature 331: 619 (1988).

These viruses have been classified in the family, Retroviridae. These are RNA-containing enveloped, icosohedral viruses of about 150 nm diameter and have a coiled nucleocapsid within a core structure. In the search for anti-HIV agents, much of the effort has been devoted to evaluating the anti-HIV activity of natural products and other compounds known to have tumoricidal, viricidal, bactericidal or other cytocidal activity. Some of such agents have shown promise, but many have not. In any case, a pharmaceutical agent effective in treating immune disorders associated with HIV infection has yet to be developed.

Phloroglucinols are a class of trihydroxylic phenols which were identified as natural products having anthelminthic activity. The naturally occuring members of this class can be extracted from plants of the species Medicosma sessiliflora (formerly, Melicope sessiliflora) in the family Rustaceae, which are indigenous to Australia. Bowden et al. , Brit. J. Pharmacol. 24: 714 (1965), report on the relative anthelminthic (Hymenolepis nana) activity of a variety of phloroglucinols and related compounds.

Bowden et al., J. Pharm. Pharmacol. 17: 239 (1965), report on the relative antibacterial (St phylococcus aureus) activity of a variety of phloroglucinols and related compounds. The authors report that "although antibacterial acitivity is often accompanied by high in vitro anthelminthic activity, this is not always so. " Chan et al., J. Org. Chem. 54: 2098 (1989), report anti-Herpes virus activity of phloroglucinols newly isolated from M. sessiliflora.

Broadbent et al., U.S. Patents 3,467,715 and 3,377,241, disclose anthelminthic activity of diacyl phloroglucinol derivatives.

1 Mizobuchi et al., Agric. Biol. Chem. - _9(3) :719(1985), report anti-fungal activity for 2,4,6,-trihydroxyacylphenones and related compounds.

Hartley, Aust. J. Bot. 33: 27 (1985), reports on 5 the reclassification of Medicosma and describe the genus and its species, including M^ sessiliflora.

Summary of the Invention

10 This invention is of a method for treating infection in an animal by an Immunodeficiency Virus, especially human immune disorders associated with infection by HIV and related human immunodeficiency viruses, by internally administering to the animal an

15.effective amount of phloroglucinol or a derivative of phloroglucinol which has the core trihydroxylic structure of phloroglucinol but has one or more of the protons in the hydroxyl groups or in the non-substitututed positions replaced. 0 This invention is also of certain novel phloro¬ glucinols and of use of such novel phloroglucinols to treat infection by an Immunodeficiency Virus.

This invention is also of a pharmaceutical composition for the treatment of infection in an animal by 5 an Immunodeficiency Virus comprising-phloroglucinol or a derivative thereof as described above and hereinbelow.

Detailed Description of the Invention

0 The method of the invention is useful in alleviating immune disorders associated with infection by Immunodeficiency Viruses, several of which have now been isolated. These include the isolates which have been variously identified as HIV, HTLV-III, HIV-1, HIV-2, LAV and ARV and which are herein collectively referred to as HIV. See, references cited above. It is postulated that alleviation of the immune disorders is achieved by

inhibition of direct infection of cells, especially lymphocytes, and by inhibition of syncytia formation. Alleviation of the immune disorders is manifested by normalization of T4 and T8 lymphocytes counts and of the T4:T8 lymphocyte ratio.

The compounds which are useful in the method of the present invention are phloroglucinol and derivatives of phloroglucinol which have the core trihydroxyl phenolic structure of phloroglucinol or in which the proton in one or more of the three hydroxyl groups is replaced, preferably by a lipophilic substituent such, i.e., a non-polar uncharged group such as an alkyl, alkenyl, alkyl aryl or alkenyl aryl group having 1 to 6 in-chain carbon atoms and one or more of the protons in the non-substituted positions are replaced by other moieties, preferably lipophilic moieties such as alkyl, alkenyl, alkyl aryl, alkenyl aryl, acyl or acyl aryl groups having 1 to 18 in-chain carbon atoms.

Illustrative of such compounds are those illustrated by Formula I, below.

in hich: FORMULA I

R 1, R3 and R5 are the same or different and are -H, -R ⁷ H, R ⁷ Ar, -C(0)R ⁷ H or -C(0)R ⁷ Ar; A a

R , R and R are the same or different and are -H, halogen, nitro, -R 8H, -R8Ar, -C(0)R8H or

-C(0)R ⁸ Ar;

7 _j _ R is C ₁ _ ₆ alkylene or alkenylene, optionally substituted with one or more halogen, -OH or =0 groups;

R is C. 1— ₁ o ₀ alkylene or alkenylene, optionally substituted with one or more halogen, -OH or =0 5 groups; and

Ar is -toluyl, naphthyl, phenyl or pyridyl

7 optionally substituted by -OH, halogen, -R H or

-C(0)R ⁷ H; or

T ^ A. -~ any two adjacent R , R , R , R , R and 0 R groups are together C ₂ _ ₄ alkylene, thereby forming a 5-, 6- or 7- member ring fused to the core phenolic ring, and one or more of such members are optionally substituted with -R ⁷ H, R ⁷ Ar, -C(0)R ⁷ H or -C(0)R ⁷ Ar.

The halogens are -Br, -F, -Cl and -I of which -F ις is the least preferred.

In the preferred method of the invention, the compounds which are employed are those of Formula I in which R 1, R3 and R5 are independently selected o from -H or -CH ₃ ;

R ² is -R ⁸ H; o is C. - _o alkylene or alkenylene, optionally mono- or di-substituted with -OH or = O; R ⁴ and R ⁶ are -C(0)R ⁸ H or -C(0)R ⁸ Ar; and 5 Ar is phenyl; or

1 and R2, or 2 and R3, are C2 alkylene, thereby forming a five member ring fused to the core phenolic ring and one of the two carbon atoms is 0 covalently bound to the R 2 position of a second compound which second compound otherwise has the structure of

Formula I, i.e., has the structure of Formula I

FORMULA I'

in which R ¹ ' , R ³ ' , R ' , R ⁵ ' and R ⁶ ' are as defined above for R , R , R , R and R , respectively.

As used throughout this specification and claims, terms such as "alkyl, "alkenyl," alkylene," alkenylene, "alkanol," "acyl" and other like terms encompass branched as well as linear carbon chains.

Phloroglucinol and the phloroglucinol derivatives which can be used in the method of the invention are synthesized by known technicrues. For example, preparation of phloroglucinol from trinitrobenzene or trinitrobenzoic acid is described by Pascoe, Che . Products 18: 454 (1955). Briefly, trinitrobenzene or trinitrobenzoic acid is reduced in the presence of tin and hydrochloric acid and the solution is neutralized and boiled for 15-20 hours. See, The Merck Index of Chemicals and Drugs, Merck & Co., Inc., Rahway, New Jersey.

Synthesis of derivatives of phloroglucinol is disclosed by Bowden et al., Brit. J. Pharmacol. 24: 714 (1965); Broadbent et. al. , U.S. Patent 3,467,715; and

Broadbent et al., U.S. Patent 3,377,241, all of which are incorporated herein by reference as though fully set forth

Exemplary of the compounds which can be prepared by the methods disclosed by Broadbent et al., U.S. 3,467,715 and 3,377,241 and which have now been demonstrated to have anti-HIV activity are diacylated compounds of Formula I, e.g., compounds of Formula I in

which R 3 and R5 are the same and are -H or

R and are the same and are -C(0)R H; is -H or -CH

,8 3' and,

R~ is C,_ ₁₈ alkylene. Specific examples of such compounds include the compounds listed in Table I.

TABLE I

Compound # Compound Name

90569 2,4-Diisobutyrylphloroglucinol

90578 2,4-Dipropionyl-6-methylphloroglucinol

90592 2,4-Divaleryl-6-methylphloroglucinol

90616 2,4-Dihexanoylphloroglucinol

90620 2,4-Di-heptanoylphloroglucinol

90621 ^' 2,4-Di-(4-methylvaleryl)phloroglucinol

90625 2,4-Dihexanoyl-6-methyl phloroglucinol

90629 2,4-Diheptanoyl-6-methylphloroglucinol

90649 2,4-Diisovaleryl-6-methylphloroglucino

90655 2,4-Dioctanoyl-6-methylphloroglucinol

90656 2,4-Dioctanoylphloroglucinol

90666 2,4-Dinonanoyl-6-methylphloroglucinol

90770 2,4-Di-(4-methylvaleryl)-6-methylphlor glucinol

Among such diacyl compounds, the compounds which are preferred for use in the method of the invention are the compounds of Formula I in which

R ¹ , R ³ and R ⁵ are -H;

Specifically preferred among such compounds are Compounds 90592, 90621, 90649, 90655, 90666 and 90770.

Compounds of Formula I which can be prepared by the methods disclosed by Bowden et al. , Brit. J. Pharmacol. 24: 714 (1965), include the compounds listed in Table II.

TABLE II

Compound # Compound Name 90574 2,4-Dibutyryl-6-methylphloroglucinol 90531 Acetylphloroglucinol 90532 Phloroglucinol triacetate 90525 2,4-Diacetylphloroglucinol 90533 Triacetylphloroglucinol 90548 2,4-Diacetylphloroglucinol 1-methyl ether

90543 2,4-Diacetylphloroglucinol 1,5-dimethy ether

90547 2,4-Diacetylphloroglucinol 1,3,5-trimethyl ether

2,4-Diacetylphloroglucinol triacetate

2,4-Diacetylphloroglucinol-l,3,5- tribenzoate

Phlorobutyrophenone 4-methyl ether

2-(4-Methylvaleryl)phloroglucinol

Monovalerylphloroglucinol

Phlorobutyrophenone-2,4-dimethyl ether

2,4-Diacetyl-6-methylphloroglucinol

2,4-Diacetyl-6-methylphloroglucinol 1-methyl ether 2,4-Diacetyl-6-methylphloroglucinol trimethyl ether

TABLE II (cont'd)

Compound tt Compound Name

90545 2,4,6-Tributyrylphloroglucinol 90700 2,4-Dipropionylphloroglucinol- triacetate

90590 2,4-Divalerylphloroglucinol 90590 2,4-Divalerylphloroglucinol 90575 2,4-Dipropionylphloroglucinol 1,3,5-trimethyl ether

90568 2,4-Dibutyrylphloroglucinol 1,3,5-trimethyl ether

90597 2,4-Diisobutyrylphloroglucinol 1,3,5-trimethyl ether

90644 2-Acetyl-4-valerylphloroglucinol 90717 2-Bromo-4,6-dibutyrylphloroglucinol 90771 2-Bromo-4,6-divalerylphloroglucinol 90694 2-Phenacetyl-6-methylphloroglucinol 90530 5,5'-Methylene bis-phlorobutyrophenone 2,4-dimethyl ether

Alternatively, phloroglucinol derivatives can be isolated from extracts of plants of the genus, Medicosma, especially, M__ sessiliflora. This plant can be found in northeastern Queensland, Australia from Gap Creek south to the Boonjie logging area, in rain forest to gallery forest and from sea level to about 750 m above sea level. The plant and its habitat are described by Hartley, Austrl. J. Bot. 33: 27 (1985), which is incorporated herein by reference. The plant is described as follows:

Shrub or small tree to 10 high. Branchlets subpersistently puberulent to glabrous, 2-4 mm wide at the internodes, not

1 corky; terminal bud pubescent to glabrous.

Leaves (subalternate to) opposite or (sub)opposite and (sub)verticillate in threes,6-27 cm long; petiole subpersiεtently 5 puberulent to glabrous, convex to canaliculate adaxially, swollen apically and often articulated with the blade, 5-45 mm long, 1-2 mm wide at the middle; blade subcoriaceous, sparsely and subpersistently puberulent on the midrib below to 10 glabrous, more or less conspicuously oil-dotted, elliptic or elliptic-oblong to oblanceolate, 5.5-22.5 cm long, 2-8.5 cm wide, about 2-4 times as long as wide, the base cuneate to rounded or narrowly and shallowly cordate, the main veins 25 (9-)12-16 on each side of the midrib, diverging at an angle of 70-80°, the apex obtuse to acuminate, often retuse. Inflorescences axillary, one- or few-flowered, in 'younger branchlets subsessile or with herbaceous axis to 20 1.5 mm long, in older branchlets additional flowers often produced on woody processes to about 2mm high; pedicel(s) obsolete to 0.5(-l) mm long. Flowers bisexual, 6-8 mm long, tetragonously ovoid in bud; sepals densely 25 pubescent to glabrous, imbricate for about 1/2 their length, ovate or suborbicular, 1-2 mm long; petals distinct of coherent into a tube for about 1/6 their length, white or cream-colored, densely pubescent abaxially, narrowly elliptic, 5-6.5 mm 30 long; staminal filaments distinct or connate into a tube for up to 1/5 their length, woolly at the margin, the antesepalous ones glandularmuricate near the apex, the antepetalous ones eglandular; anthers transiently pilose at the apex, 0.6-1 mm 35 long; disc cupular, 8-lobed with alternate deep and shallow sinuses, glabrous, 0.8-1.5 mm high; ovary pubescent toward the apex, 0.8-1 mm high;

l style pilose in the basal 1/3-1/2; stigma subclavate, 4-cleft, in a position about level with the antesepalous anthers. Follicles sparsely pubescent toward the apex or glabrous,

5 6-10 mm long; placental endocarp subfleshy; seed(s) brownish black, sparsely tuberculate and minutely papillate.

See, also, Berti et al., "Constituents of Ferns," 0 in Progress in Phytochemistry; Tomas-Lorente et al. , Phytochenistry 28: 1613 (1989); Arisawa et al., J. Natural Products 48: 455 (1985); Arisawa et al. , J. Natural Products 49: 298 (1986); and Fujita et al. , J. Natural Products 51: 708 (1988), all of which report 5 phloroglucinols from plants and all of which are incorporated herein by reference,

A typical extraction procedure comprises soaking dried, pulverized leaves in an organic solvent and fractionating the extract, such as on a hydrophobic and/or o hydrophilic column(s) and eluting with organic solvents, such as is described in the Examples, below.

Chan et al., J. Org. Chem. 54: 2098 (1989), report analysis of certain novel phloroglucinol derivatives from M. sessiliflora and is incorporated herein as though fully set forth.

Exemplary of the phloroglucinol derivatives which can be isolated from M. sessiliflora hexane or methyl ethyl ketone extracts are the compounds illustrated in Table III.

TABLE III

104402 108189

107925 107924

104553 104552

107923 107921

108190

As indicated in the Examples, below, not all of the natural product phloroglucinol derivatives have been demonstrated to have activity in an assay for inhibition of HIV infection, although all have been shown to inhibit syncytia formation. On the basis of -these assay results, among the single ring natural product-derived compounds, . the most preferred compound is Compound 104402; among the fused ring compounds, the compounds which are preferred for use in the method of the invention are 107923 and 107921. Additional phloroglucinol derivatives can be prepared from the natural derivatives of phloroglucinol by standard chemical procedures. It appears to be desirable in preparing such derivatives, to retain hydroxyl or lower alkoxy, especially methoxy, groups in the hydroxyl positions of phlorogucinol, i.e., in the -OR 1, -OR3 and -OR 5 positions; to replace the hydrogens i-n the R4 and R 6 positions with acyl groups, e.g., -C(0)R8H or

-C(0)R 8Ar; and to replace the hydrogen in the R2 o position with an alkyl or alkenyl group, e.g., -R H; or, in preparing fused ring derivatives, it is desirable to have the ring formed between the R 1 and R2 positions or the R 2 and R3 positions and to have such ring substituted by a phloroglucinol or a phloroglucinol derivative, e.g., a compound of Formula I'. It is to be understood that the formulae presented herein above and below are intended to encompass tautomers and isomers. In addition, the compounds of Formula I can form salts with inorganic and organic cations which salts are non-toxic and otherwise pharmaceutically acceptable, which salts are included within the scope of the invention. Exemplary of such cations are magnesium, calcium, sodium, potassium and piperazine. Also encompassed within the scope of the invention are pro-drugs of phloroglucinol or phloroglucinol derivatives, i.e., phloroglucinol or derivatives of phloroglucinol bound, usually covalently,

to another chemical group, which may impart enhanced stability, reduced toxicity or targeted delivery and which may or may not be active in the prodrug form, and which is cleaved off in the body of the treated animal to produce the active phloroglucinol or phloroglucinol derivative. In the method of the invention, compounds of Formula I are administered internally, e.g. , parenteraliy, such as by intravenous, subcutaneous or intramuscular injection or infusion, orally, rectally, buccally, transdermally or by inhalation. The compounds are typically administered in a pharmaceutically acceptable carrier or diluent selected on the basis of the route of administration. For solid formulations such as pills, powders, tablets, capsules and caplets, useful carriers include, among others, lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. Liquid carriers such as for oral, intranasal or ^" parenteral administration include, among others, glycerin, syrup, peanut oil, olive oil, saline, dextrose and water, optionally bufferred with organic or inorganic salts, for example, acetates, adipates, succinates or citrates of ammonium, potassium or sodium. Additional excipients may be added to adjust tonicity or, especially in the case of a formulation to be lyophilized, stabilizers such as, for example, gelatin, polyvinylpyrrolidine, cellulose, acacia, polyethylene glycol, pyrrolidone or mannitol. For rectal or vaginal administration, the compounds can be combined in powder form with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycol and molded into a suppository. For transdermal delivery, the compounds can be combined with an oily preparation, gel, cream or emulsion and administered via a transdermal patch.

In the method of the invention, compounds of

Formula- 1 are administered to an animal, especially a human, who has been or is suspected of having been infected with an Immunodeficiency Virus, especially HIV, in an amount effective to inhibit progression of immune dysfunction and, preferably, to stabilize or improve the immune dysfunction. The amount administered will vary depending upon a variety of factors including the stage of immune dysfunction progression and the age, weight and' general health of the animal being treated. In general, it is expected that the method of the invention will be carried out by administering from about 1 to 500 mg/Kg/day, although in particular cases a physician may elect to administer even higher doses. The effects of the method of the invention on immune disorders associated with infection by an Immunodeficiency Virus can be monitored by evaluating T4 and T8 cell counts and the T4/T8 ratio and the dosage rate and dose can be modified accordingly. Alternatively, the presence of virus in the bodily fluids of the animal can be monitored by evaluation of presence of virus antigens, or of antibodies to virus antigens, in sera. It is expected that the method of the invention will be carried out over a lengthy period of time, e.g., several weeks to months, or for the treatment to be repeated periodically, due to the ability of Immunodeficiency Viruses to remain latent within the infected animal.

Encompassed within the method of the invention is co-administration or simultaneous administration of phloroglucionol or of derivatives of phloroglucinol 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 such as AZT and ddC, HIV protease inhibitors and sCD4 and truncates thereof.

The Examples which follow are illustrative and not limiting of the invention. All compounds are referred to by the Compound Numbers assigned in Tables I and III, above. "EtOAc" means ethyl acetate and "HOAc" means acetic acid.

Examples

Example 1. Diacyl Phloroglucinol Derivatives. The compounds listed in Table IV, below, were tested in a HIV Infectivity Inhibition Assay substantially as follows. 100 uL of T4+ lymphocyte (AA5 or M0LT4 cells) stocks containing about 3 X 10 cells per mL were seeded on a 24 or 96 well plate. Then, to each well was added 100 uL of an HIV stock containing about 10 Infectious Units (which units are approximately equivalent to TCID D _c U _n units) . Virus was allowed to adsorb to the cells for about an hour at 37 C in a C0 ₂ incubator. Then, appropriate dilutions of a phloroglucinol derivative were added to each well. Control wells were prepared in the same manner except that no phloroglucinol derivative was added.

After incubating the cells for 7 to 10 days, HIV in' the conditioned medium was concentrated by precipitation with polyethylene glycol. The relative amounts of virus in the experimental and control wells was determined by assaying for reverse transcriptase activity in viral lysates and by an ELISA for p24 antigen. Results are reported in Table IV in which IC _5£) means the concentration of the phloroglucinol derivative which resulted in a reduction of 50 % of reverse transcriptase activity and of p24 concentration relative to controls. "N.A." means that no significant inhibition of infectivity was observed at the concentrations tested. Results reported in parentheses are the results of a second experiment.

The compounds listed in Table V, below, were tested in an HIV Syncytia Inhibition Assay substantially as described by Putney et al. , Science 234: 1392 (1986) and Matthews et al., Proc. Natl. Acad Sci. USA 8 : 5424 (1987). Briefly, various dilutions of phloroglucinol derivatives were added to 96 well plates. Then, CEM T4+ lymphocytes stably infected with HIV were mixed with unifected CEM or M0LT4 cells and seeded in the wells in amounts of approximately 70000 uninfected cells and 5000 infected cells per well. The plates were incubated for about 20 to 24 hours at 37 C in a C0 ₂ incubator. Giant cells, i.e., cells of at least 5-fold greater diameter than normal cells, were enumerated using an inverted microscope at 40X magnification. Control wells typically ave about 60 to 70 giant cells per well. Results are reported in Table V in which IC _5Q is the concentration of each phloroglucinol derivative which resulted in a 50 ' reduction in the number of giant cells, or syncytia, per well.

"N.A." indicates no detectable activity. "AZT" is 5-azidothymidine; and "ddC" is dideoxycytosine.

Example 2. Natural Phloroglucinol Derivatives.

Medicosma sessiliflora was collected in Australia i September, 1979 voucher specimen VKM 2666 is preserved in National Herbarium, Washington, D.C. Dry pulverized leaves from M _. sessiliflora (lOOg) were soaked in n-hexane for at least 24h. The n-hexane was removed by filtration and leave were then stirred in methyl ethyl ketone for at least 24 h. Filtration and concentration of filtrate under vacuum yielde 38 g of crude extract.

The methyl ethyl ketone extract (20 g) was chromatographed on silica gel 60 (230-400 mesh) in a Whatman Magnum 40 stainless steel column (4i7x47cm) (Whatman Chemica Separations, Inc., Clifton, New Jersey) in two lOg batches. The column was eluted sequentially first with CH_C1 ₂ (Solvent A), then with 0.2% H ₂ 0/1% CH ₃ OH/CH ₂ Cl ₂ (Solvent B) finally with 0.2% H ₂ 0/2% CH ₃ 0H/CH ₂ C1 ₂ (Solvent C) at a flow rate of 50 ml/min while monitoring at 254 nm. The first bioactive fraction containing compound

104402 (1.25 g) was obtained from the Solvent A eluate. Recrystallization from diethyl ether (ET ₂ 0) CH ₂ C1 ₂ gave 730 mg of white crystalline compound, 104402.

A second fraction (183 mg) was eluted from the silica column with Solvent B and crystallized from ET ₂ 0/CH ₂ C1 ₂ to give 38 mg. of compound 107924. The mother liquor was separated by multiple injections on a Mer Lichrosorb silica gel 60 column (7μm, 10x250mm) (EM Science, Cherry Hill, New Jersey) eluting with 0.1% HOAc/30 EtOAc/n-hexane at 4ml/min (800 psi) while monitoring at 335 nm. Compound 107922 (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 a Whatman ODS-3(40 μm) in a Whatman Magnum 40 stainless steel column (4.7x47 cm) (80 ml/min) while monitoring at

1 340 nm. Compound 107925 (860 mg) eluted with 70% CH ₃ 0H/H ₂ 0 and crystallized from CH ₂ C1 ₂ - The major peak eluted with 70 to 75% CH ₃ 0H/H ₂ 0 and gave 2 g of crude mixture. From this 2 g mixture, a 100 mg sample was fractionated by multiple injections on a Merck Lichrosorb silica gel column (7 μm, 10x250mm) and eluted with 0.1% HOAc/20% ETOAc/n-hexane at 4ml/ in (800 psi) while monitoring at 335 nm. Compound 108191 (6.8 mg) eluted at 10.2 ^" min. Compound 104553(43 mg) eluted at 10.7 min, and 0 compound 104552 (47 mg) eluted at 16 minutes.

The Solvent C elution from the silica gel column gave at least three additional bioactive fractions. The first 220 mg fraction which on trituration with 30% ETOAc/n-hexane gave 55 mg of insoluble material which was 5 compound 107921. The next major fraction wε a 480 mg mixture which gave 84 mg of 107923 on crystallization from ethyl ether. Fractionation of the mother liquor on a 60 ml silica Bond-Elut (3x4cm) (Analytichem International, Harbor City, California) with 0.1% HOAc/30%EtOAc/n-hexane gave first a fraction which was rich in compound 107923 and then a 63 mg mixture which was further purified using multiple injections on a Merck Lichrosorb silica gel column (7 μm, 10x250mm) with 0. l%HOAc/30%EtOAc/n-hexane at- 4ml/min (800 psi) while monitoring 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 silica Bond-Elut (60 ml) with 0.1% HOAc/30% ETOAc/n-hexane gave a mixture containing compounds 107923 and 108189. A second elution with 0.1% HOAc/70% EtOAc/n-hexane gave another mixture which was further purified on a Merck Lichrosorb silica gel 60(10x250 mm) column eluting with 0.1% HOAc/70%EtOAc/n-hexane to give compound 108190 (7 mg) . Provided below are analytical data for the above compounds.

104402:IR(CH,C1,)fi 1 ⁾ 3600-3100 ⁽ OH) ,3300-2400

-1 (hydrogen bonded hydroxyl), 1615cm (hydrogen-bonded carbonyl);

UV (MeOH)λmax(ε)274 CH ₃ ) ₂ CHCH ₂ ) , 25.2(d,

(CH ₃ ) ₂ CHCH ₂ ), 32.7(q, CH ₃ C0) 95.4(d, aromatic C-H), 105.0(bs, aromatic C) , 172.0 (bs, aromatic C-0), 204.0, 206.6 ppm

(2xs, C=0).

107921: Clcd for C ₃₁ H ₃₈ O _n 586.2414 found M ⁺ m/z

586.2407. ^] H NMR(CDC1 ₃ ) δ 0.94, 0.97 (4x3H, 2xd, 0=6.7Hz,

(CH ₃ ) ₂ CHCH ₂ ), 1.37, 1.49 (4x3H, 2xs, (CH^CHOH), 2.20 (2xlH, m,(CH ₃ ) ₂ CHCH ₂ ), 2.66,2.70 (2x3H, s, bs, CH ₃ C0),

3.06, 3.10 (2x2H, 2xdd, 0=7.1, 16.8Hz, (CH^CHCH^) , 4.80

(IH, d 0=5.6Hz, CHOH), 5.10 (IH, d, 0=5.7Hz, CHCH ) , 16.6, 15.9, 15.6 ppm (s, phenolic OH). ¹³ C N R ⁽ CDCl ₃ )δ, 22.8, 22.9 (q, (CH ₃ ) ₂ CHCH _£ ), 26.0 (d, , 32.6, 35.6 (q, CH ₃ C0), 33.1 (d, CHCH), 52.7, 53.2 (t, (CH^CHCH^ 73.4

(s, (CH ₃ ) ₂ C), 96.9(d, CHOH), 100.2 104.6, 105.1, 105.8, (s, aromatic C) , 165.3, 167.6, 170.4, 170.8, (s, aromatic C-0), 202.2

204.8, 207.2 207.5.ppm (s, C=0).

107923: H NMR(CDCl ₃ )δ0.98,l .04 (4x3H,d,bd,

(CH ₃ ) ₂ CHCH ₂ ), 1.38, 1.51 (4x3H, 2xs, (CH ₃ > ₂ CHOH), 2.20 (2xH, m, (CH ₃ ) ₂ CHCH ₂ ), 2.60, 2.70 (2x3H,s,bs, CH ₃ C0), 2.70, 3.10 (2x2H, 2xdd, 0=6.4,15.0Hz, (CH^CHCj^) , 4.80C1H, d, 0=5.7Hz, CHCH), 5.05 (IH, d, 0=5.7Hz, CHOH ⁾ , 15.6, 15.9, 16.6 ppm (s, phenolic OH). ¹³ C NMR (CDC1 ₃ ), δ, 22.7, 22.8, 22.9 (q, (CH ₃ ) ₂ CGCG ₂ ), 24.5, 25.5 (d, (CH) ₃ CHCH ₂ ), 32.6, 35.1 (q, CH ₃ C0), 33.1 (d, CHCH), 51.5 (t, (CH^CHCH^ , 73.5 (s, (CH ₃ ) ₂ C), 96.9(d, CHOH), 100.1, 104.8, 105.1 106.0(s, aromatic-C), 164.7, 167.7, 171.5 (s, aromatic C-0 ⁾ , 204.8, 205.1, 207.1 (s, C=0).

107922: Calcd for ^^^337.1561 , found M+H m/z 337.1643. ^] H NMR(CDC1 ₃ ) δ 0.97 (2x3H, d, 0=6.8Hz, (CH ₃ ) ₂ CHCH £, 1.45, 1.42 (2x3H, s, (CH ^ ) , 2.25 (IH, m, (CH ₃ ) ₂ CHCH ₂ ), 2.66(3H, s, CH ₃ C0) , 2.66, 2.87 (2H, dd, 0=5.3, 11.4Hz, (CH ₃ ) ₂ CHCH ₂ ), 2.89 (2H, AMX, 0x-5.)Hz, CH ₂ 0H), 3.86 (IH, t, 0=5.4Hz, CHOH), 15.3, 16.1 ppm (2xlH, s, phenolic OH), ¹³ C NMR (CDC1 ₃ δ 22.0 (d, (CH^CHCH^, 22.7 (q, (CH ₃ ) ₂ CHCH ₂ ), 25.1(q, (CH ₃ > ₂ C), 25.3 (t,

SUBSTITUTE SHEET

CH ₂ CH0H), 33.1(q, CH-.C0), 52.9(t, (CH ₃ ) ₂ CHCH ₂ ) , 68.3 (d,

CHOH), 79.9 (s, (CH ₃ ) ₂ C), 97.9, 104.1, 104.6 (s, aromatic C) , 160.3, 170.0, 170.1 (s, aromatic C-0) , 203.3, 206.8 ppm(s, C=0) 108191: Calcd for ^^^337.1651 , found M+H m/z 337.1644. ¹³ C NMR (CDC1.-) δ 22.0 (d, (CH^CHCH^ , 22.7 (q, (CH ₃ ) ₂ CHCH ₂ ) 25.1, 25.6(q, (CH^C), 25.3(t, CH ₂ CH0H), 33.0 (q, CH.-C0), 53.2(t, (CH ₃₂ CHCH ₂ ), 68.4 (d, CHOH, 80.1 (s, (CH ₃ ) _£ C), 169.7, 170.7 ppm (s, aromatic-C-O) , aromatic C and carbonyl peaks not detectable. 108189: Calcd for C _lg H ₂₀ 0 ₇ 354.1673, found M ⁺ m/z 354.1679. ^] H NMR(CDC1 ₃ + several drops of CD ₃ 0H), ό 0.97 (2.3H, d, 0=6.60Hz, (CH^CHCH^ , 1.25, 1.30 (2x3H, s, (CH ₃ C0H), 2.24 (IH, m, , 2.35, 3.15 (2H, AMX, 0=9.6, 15.0Hz, CHCH0H), 2.70(3H, s, CH- _j CO) , 3.00(2H, d, 0=6.8Hz,(CH ₃ ) _£ CHCH ₂ ), 3.53 ppm (IH, bd, 0-9.5Hz, CH ₂ CH0H), ¹³ C NMR (CDCl ₃ +severa1 drops of CD ₃ OH), δ, 22.2, 22.6 (q, (CH ₃ ) ₂ CHCH ₂ ), 25.1 (t, CH ₂ CH0H), 25.2 (q, (CH ₃ ) ₂ COH), 25.7 (d, (CH ₃ ) ₂ CHCH ₂ ), 32.9 (q, CH ₃ C0), 53.0 (t, (CH ₃ ) ₂ CHCH _£ ), 72.8(5, (CH^COH), 80.3 (d, CH _£ CH0H), 104.3, 104.4, 105.1 (s, aromatic C) , 169.6, 170.1 (s, aromatic C-0), 204.5, 207.0 ppm (s, C=0).

107924; Calcd for C ₁₅ H ₁₈ 0 ₆ 295.1181, found M+H m z 295.1184. Mixture of two isomers. ^] H NMR(CDC1.-), δ, 0.94, 0.95 (2x6H, d, (CH^CHCH^ , 2.20 (2xlH, , (CH ₃ ) ₂ CHCH ₂ ), 2.62, 2.67 (2x3H, s, CH.-C0) , 2.90, 2.94 (2x2H, AMX, 0=6.8Hz, (CH^CHCH^, 3.18, 3.20, 3.44(2x2H, AMX, 0-6.80, 17.8Hz, CH ₂ CH0H, 6.23, 6.24ppm (2x1H, d, CH=_0H), ¹³ C NMR (CDC1 ₃ ) δ 22.6, 22.7 (q, CH^CHCH^ , 24.9,

25.3 (d, (CH ₃₂ CHCH ₂ ), 31.2 (q, CH ₃ C0), 32.9, 33.1 (t,

CH ₂ CH0H), 51.3, 53.1 (t, , 31.2(q, CH ₃ C0),

32.9, 33.1 (t, CH ₂ CH0H), 51.3, 53.1 (t, ,

100.6, 102.7, 102.8 (s, aromatic C) , 104.4, 104.5 (d, CHOH ⁾ , 165.2

169.9 (s, aromatic C-0), 202.4 204.4, 204.9, 206.8 ppm (s, C=0) .

108190: Molecular weight = 352; ^] H NMR ⁽ CDCl- _j ⁾ : δ, 0.94, 0.95 (2x3H, d, 0=6.7Hz, (CH^CH), 1.27 (3H, s, CH ₃ (0H)C), 2.10 (IH, m, (CH ₃ ) ₂ CH), 2.64 (3H, s, CH ₃ C0) , 2.75 (2H, d, CH _£ CH0H), 2.96 (2H, d, COCH ₂ CH), 3.87, 4.01

SUBSTITUTE SHEET

(37,529),375(4941); UV (MeOH-0.1 N NaOH ⁾ 295 (39,059), 375 nm (infl): calcd for C _1g H ₂₄ 0 ₆ )336.1572, found M ⁺ m/z 336.1557; mp 117-120°C. Anal. Calcd for _1g H ₂ 40g: C, 64.27; H, 7.19. Found: C, 64.52; H, 7.33; ^] H NMR (CDC1 ₃ ⁾ 50.98 (6H,d,0=6.7Hz, (Cϋ ₃ ) ₂ CH), 1.86 (3H, s, CH ₃ C=CH ₂ ⁾ , 2.25

(!H,m,0-=6.7Hz,CH ₂ CH(CH3)2), 2.72(3H,s,br,CH ₃ C=0) , 2.75 (IH, dd, 0-15.5,7.3 Hz, CH ₂ CH0H), 3.05 (2H, AMX, C0CH_ ₂ CH), 3.11 (IH, dd, 0-15.5Hz, CH_ ₂ CH0H), 4.37 (IH, d, 0-7.3Hz, CHOH), 4.90, 5.00 (2x1H,s,br, Cji ₂ =), 10.1, 15.3, 16.2 ppm (3xaH, s, phenol ic-OH) . ¹³ C NMR (CDC1 ₃ > δ 18.5 (q, CH ₃ C=CH ₂ ), 22.8 ⁽ q,

(CH ₃ ) ₂ CH), 25.3 (d, CH(CH J £, 28.4(t, CH HOH), 33.0(q, CH ₃ CO), 53.0(t,CH ₂ CH), 77.7 (d.CHOH), 104.3 (s, aromatic C) , 112.9(t-CH ₂ ), 145.3(s, C=CH ₂ ), 165.5, 169.6, 170.5 (s, aromatic C-0), 204.4, 206.9 ppm (s,C=0). 104522: IR(CH ₂ C1 ₂ film) 3600-3100 (OH ⁾ , 3300-2400

(hydrogen-bonded hydroxyl), 1633 cm ^" (hydrogen-bonded carbonyl); UV (MeOH)(ε)273 (65,999), 341nm(8148 ⁾ ; λmax ⁽ ε ⁾ UV ⁽ MeOH-0.1 N NaOH) 294 (48,451), 320 nm (infl); calcd for C-gH^Og

336.1572, found M ⁺ m/z 336.1571; 'H NMR (CDC1 ₃ ⁾ δ 0.97

(2.3H,d,0=6.7Hz, (CH^CH), 1.27, 1.38 (2x3H, s,H0C(CH-.) ₂ ) ,

2.25 (lH,m,0=6.6Hz, CH ₂ CH(CH ₃ ) ₂ ) , 2.64 (3H,s, CH_ ₃ C0), 2.97

(2H, AMX, 0-6.9, 21.1Hz, C0CH ₂ CH), 3.07(2H, AMX, 0-9.3, 15.1 Hz,

CH ₂ CH0H), 4.84 (IH, t, 0-9.3Hz, CH0HCH ₂ ), 14.9, 15.6 ppm (2xlH, s, phenolic OH). ¹³ C NMR (CDC1 ₃ > δ 22.7 (q, (CH^CH),

24.7, 24.9 (q, (CH^C), 26.0 (d, (CH^CH ⁾ , 26.3 ⁽ t,

CH ₂ CH0H), 31. l(q, CH ₃ CO) , 51.4 (t, CH ₂ cO) , 71.7 (s,

C(CH ₃ ) _£ ) 92.9 (d, CH ₂ CH0H), 100.4, 104.6, 105.2 (3xs, aromatic C) , 166.8, 170.0, 170.7 (3xs, aromatic C-0), 202.0, 207.0 ppm (2xs, C=0). 104523: IR(CH,C1 ₉ film) 3600-3100(OH ⁾ , 3300-2400

-1 (hydrogen-bonded hydroxyl), 1636 cm (hydrogen-bonded carbonyl);

UV(MeOH) λ max (ε) 273(39,969), 341 (3737); UV(Me0H-0.1 N NaOH)

293(36,969), 320 nm (Infl); calcd for C ₁₈ H ₂₄ 0 ₆ 366.1572, found M ⁺ m/z 366.1564; mp 105-107°C; H NMR (CDC1 ₃ ) δ 0.99 (2x3H, d,0=6.7Hz, (CH ₃ ) ₂ CH, 1.27, 1.39 (2x3H,s, H0C(CH) _£ ) 2.20 (IH, rπ, 0=6.6, Hz, CH ₂ CH(CH ₃ ) ₂ ) , 2.70(3H, s, CH ₃ C0) ,

SUBSTITUTE SHEET

-24/ 1 - 2.81, 2.93 (2x1H, dd, 0=6.9, 14.5 Hz, COCH ₂ CH), 3.08 (2H, AMX, 0=9.0, 14.1 Hz, CH ₂ CHOH), 4.83 (IH, t, 0=9.OHz, CH0HCH ₂ ), 14.7, 15.8 ppm (2xlH,s,phenolic OH). ¹³ C NMR (CDC1 ₃ ) δ 22.7 (q, (CH ₃ )2CH), 24.7, 25.5 (q, (CH^C), 26.1 (d, (CH^CH), 26.2 (t, CH _£ CHOH), 33.0 (q, CH ₃ C0), 52.9 (t, CH ₂ CO) , 71.5 (s, C(CH ₃ ) ₂ ), 92.8(d, CH _£ CHOH), 100.3 104.4, 105.3 (3xs, aromatic C), 166.3, 166.7, 171.3 (3xs, aromatic C-0), 204.4, 204.5 (2xs, C=0) . Single-crystal X-ray diffraction analysis was carried out from three-dimensional intensity data collected on an Enraf-Nonius CAD-4 diffractometer [λ(Mo α)=0.71073 A ^' J equipped with a graphite monochromator. Data were collected by a variable speed 2Θ scan technique at 248 . A total of 5583 data (+h, +k, +1) were collected 2Θ 60°), of which 3623 were considered observed (l.>3δ(I) after correction for Lorentz and polarization effects and after averaging symmetry equivalent reflections Rj _t =0.013) . The molecule crystallizes as a hydrate from MeOH. Crystal data: triclinic, P 1, Z=2, a=9.852(2), b = 11.563(2), and c = 8.931(2) A, = 107.51 (2)°, β = 110.54 (2)° Y=73.36(2)° The structure was solved by direct methods. Non-hydrogen atoms were refined with anisotropic vibrational parameters; all hydrogens were located from difference Fourier synthesis and were refined with isotropic temperature factors. Full matrix least-square refinement (on F) converged to values of the standard crystal lographic residuals, R=0.043 and R =0.055. An extinction coefficient refined to 1.24(l)xl0~ . The final difference Fourier map was featureless.

107925: IR(KBr) 3600-3011 (OH) , 3100-2800 ⁽ hydrogen-bonded OH), 1626 cm (hydrogen-bonded OH), 1626 cm ^{" (} hydrogen-bonded carbonyl); Calcd for C ₁₃ H ₁₇ 0 ₅ 253.1076, found M+H m/z 253.1083; ^] H NMR <CDC1 ₃ > δθ.91 (2x3H, d, 0=6.4Hz

(CH ₃ ) ₂ CHCH £, 2.20 (IH, m, (CH ^ C ^, 2.60 (3H, s,

CH ₃ C0), 2.90 (2H,bd, 0=6.3Hz, CHCH _? ), 5.80 (IH, s, aromatic H),

16.2 ppm (3xH, bs, phenolic OH). ³ C NMR (CDC13 ⁾ δ 22.7 (q,

(2H, dd, 0-llHz, CH ₂ 0) , 4.16ppm (IH, m, CH ₂ CH0H ⁾ . ¹³ C NMR (CDC1 ₃ ): δ 22.7 (q, (CH ₃ ) _∑ CHCH ₂ ) , 25.0 (d, (CH ₃ ) ₂ CHCH ₂ ), 33.2 (q, CH ₃ C0), 52.3 (t, (CH^CHCH^ , 67.2 (d, CHOH), 67.8 (t, CH ₂ 0) , 79.8 (s, CH ₃ (OH)C(CH ₂ ) ₂ ) , 97.5, 104.0, 104.7 (s, aromatic C), 160.0, 170.0, 170.3 (s, aromatic C-0), 203.2, 206.9 ppm (s, C=0) .

-25- Example 3. Semi-synthetic Phloroglucinols.

Compound 104402 (40 mg) was heated with p-toluenesulfonic acid (40 mg) in toluene (6 mL) at 90-100"..under a nitrogen atmosphere. After 2.5 hours, 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 carbonare (1% w/v; 4 x 20 mL) and was dried over magnesium sulfate. Evaporation of the solvent under reduced pressure gave a mixture (40 mg) . This mixture was separated by multiple injections (4 mg each) on an HPLC semi-preparative scale reverse phase column (Magnum 9, 250 mm x 4.6 mm, Partisil 10 μm, ODS-3) with 0.1% trifluoroacetic acid/10% water/methanol as eluant at 5 mL per minute (3.4 Kpsi) . A mixture (6.4 mg) of 107922 and 108191 eluted as the major peak at 6.6 minutes. Separation of 107922 and- 108191 was carried out on the same Magnum 9 column (above) eluting with 0.1% trifluoroacetic acid/35% H ₂ 0/ methanol at 5 mL per minute (3.3 Kpsi). 107922 (1.9 mg) eluted at about 32 minutes and 108191 (2.8 mg) eluted at about 36 minutes. The above description and examples illustrate particular embodiments of the invention including the preferred embodiments thereof. However, the invention is not limited to the precise embodiments disclosed herein but, rather, encompasses all variations and modifications thereof encompassed within the scope of the following claims.