WO1993001193A1

WO1993001193A1 - Marine compounds as hiv inhibitors

Info

Publication number: WO1993001193A1
Application number: PCT/US1992/005517
Authority: WO
Inventors: Shing Huey Mai; Vasant Kumar Nagulapalli; Ashok D. Patil; Alemseged Truneh; John W. Westley
Original assignee: Smithkline Beecham Corporation
Priority date: 1991-07-09
Filing date: 1992-06-30
Publication date: 1993-01-21
Also published as: AU2307492A

Abstract

The present invention discloses novel marine compounds, and derivatives thereof, which inhibit HIV-1 infection.

Description

l_____J.fi

MARINE COMPOUNDS AS HIV INHIBITORS

Field of Invention

The present invention relates to novel marine alkaloids which inhibit HIV infectivity.

Background of the Invention

The hallmark of the Acquired Immunodeficiency Disease, AIDS, is a progressive decline in the number of CD4⁺ cells leading to the demise of immune function and consequent susceptibility to opportunistic infections, the primary cause of death. Human immunodeficiency virus type 1 (HIV-1) is the primary causative agent of AIDS. This highly variable virus shows selective tropism for CD4⁺ cells which is determined by recognition of the HIV envelope glycoprotein gpl20 with the CD4 cell-surface receptor protein. The manner in which HIV infection leads to the slow but progressive decline in CD4⁺ cells has not been established.

Therefore, numerous approaches are being investigated in the search of potential anti-HIV drugs and vaccines.

It has been shown that an agent which antagonizes HIV in vitro, azidothymidine (AZT) , also provides a therapeutic benefit in vivo. Thus antagonism of HIV is a therapeutic strategy for AIDS. One approach to inhibit HIV infectivity is to antagonize the HIV/host- cell interaction. That is, to antagonize the virus from binding and/or entering a host cell.

The process of viral infection is initiated by the attachment of HIV to cells through a high affinity interaction between gpl20 and the CD4 receptor protein, located on the cell surface. Subsequent to that, the virus enters the host cell by fusion of the viral and cellular membranes.

There is another process, virus-mediated cell fusion, which is also initiated by the interaction of gpl20 with the CD4 receptor protein. Cells infected with the HIV virus can express viral envelope proteins, ultimately detected on the infected-cell's surface. Thus gpl20 on the surface of infected cells can bind to CD4 on uninfected cells leading to the fusion and consequent formation of multinuclear giant cells (i.e., syncytiu ) . This process is envisioned as a cell-cell equivalent of the binding and fusion events between HIV and an uninfected cell.

Compounds, which antagonize HIV infection (i.e., which prevent viral binding and cell entry) , will ultimately inhibit HIV replication, since HIV cannot replicate without utilizing the biosynthetic apparatus of the host cell that it infects. It is thus an object of the present invention to isolate compounds which selectively inhibit HIV infection.

Summary of the Invention

In one aspect, the present invention is a compound represented by the structure:

wherein: n is an integer from 6-10; Rl is

wherein m is 0 or an integer from 5-10/ the dotted line represents an optional double bond; R2 is selected from the group consisting of hydrogen, hydroxy, lower alkyl (C1-C4₎ , lower alkenyl (C2-

C4) , alkoxy(C1-C ₎ , aryl, amino, guanidinyl, guanidinyl alkylene(Cι-C_{5) f} -0(CH₂)_pNH(C=NH)NH₂,

wherein p is an integer from 1-6; or a pharmaceutically acceptable salt thereof.

In related aspects, this invention is a compound represented by the structure:

"CH₃

wherein: q is 0 or an integer from 5-10; r is 0 or an integer from 1-3;

R3 is selected from the group consisting of hydrogen, hydroxy, lower alkyl(C1-C ₎ , lower alkenyl(C2-

C4) , alkoxy(C1-C4₎ , aryl, amino, guanidinyl, guanidinyl alkylene(C!-C5_{) f} -0(CH₂)pNH(C=NH)NH ,

In yet another related aspect, this invention is a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable^' carrier.

In further related aspects, this invention relates to a method of inhibiting HIV infection which comprises administering to a patient a pharmaceutically effective amount of a compound isolated from the marine sponge Batzella, wherein said compound inhibits HIV infectivity in vitro. This invention also relates to a process for preparing an HIV inhibitor which comprises extracting the marine organism Batzella with an organic solvent, separating the extract into fractions containing HIV antagonist activity, and purifying said extract to an essentially homogeneous compound which inhibits HIV infectivity in vitro.

Brief Description of the Drawings

FIG. 1 shows the mass spectral analysis of Compound

II of the present invention.

FIG. 2 shows the mass spectral analysis of Compound

III of the present invention. FIG.3 depicts the results of a competitive ELISA assay (i.e., "Bind") wherein the binding inhibition of sT4 (also referred to as CD4) to immobilized recombinant HIV gpl20 is determined in the presence of the compounds of the invention. FIG. 3 also depicts the cytotoxicity results (i.e., "Cyto") of the SupTl cell line (measured by the inhibition of ^H-thymidine incorporation) in the presence of the compounds of the invention.

"Native" refers to those compounds in the present invention that were not chemically modified after their isolation. "Dmp dvtv" refers to compounds of the present invention that were reacted with the compound 2,4-pentanedione. "Cph dvtv" refers to compounds of the present invention that were reacted with the compound 1,2-cyclohexanedione.

Detailed Description of the Invention

The present invention relates to compounds which inhibit the interaction between HIV and the human cell- surface protein, CD4. In particular the invention relates to a class of alkaloids derived from marine sponges which inhibit HIV binding and/or subsequent fusion of HIV-infected cells and uninfected cells. Preferably the compounds of this invention are isolated from the genus Batzella. More preferably, they are isolated from a newly discovered species of Batzella (herein referred to as Batzella sp.) identified by Dr. Rob Van Soest of the Institute of Taxonomic Zoology, University of Amsterdam.

The taxonomic description of the genus Batzella is in accordance with R. van Soest ("Marine Sponges from Curacao and Other Caribbean Localities. Part III.

Poecilosclerida", ££:1-167 (1984)). Other taxonomic descriptions are from P. Berquist (Sponges, p. 136-180, University of California Press, Berkeley and Los Angeles, CA (1978)), . Hartman (in Synopsis and Classification of Living Organisms, p. 640-666, S.P. Parker ed., MacGraw-Hill, New York (1982)) and R. van Soest .supra. :

Phylum Porif ra: A sedentary, filter-feeding metazoan which utilizes a single layer of flagellated cells (choanocytes) to pump a unidirectional water current through its body. The sponge body is isolated from the external environment by a perforated epithelium, one cell thick. Both the internal - t - flagellated epithelium (the choanoderm) and the external epithelium (the pinacoderm) differ from other etozoan epithelium in that they lack a stable basement membrane. Between these two thin layers is a third region, the mesohyl, which can vary in composition and extent, but which always includes some mobile cells and some skeletal material.

Class Demospongiae: Marine or freshwater sponges with a siliceous skeleton in which megascleres are usually either monaxons or tetraxons, a triaxon being present as a major spicule type in one subclass only. There are microscleres of diverse types. The spicule skeleton can be supplemented or replaced by a spongin skeleton which is utilized either as a cementing element for the mineral skeleton, or to form fibers. Some genera have lost all specialized skeletal components. At the subclass level primary emphasis is given to reproductive pattern, whether oviparous or viviparous, and to the type of larva produced. Orders are defined on the type of megascleres and microscleres present, on the organization and composition of the skeleton and on the detail of reproductive patterns.

Subclass Ceractinomorpha: Demospongiae in which the typical reproductive pattern is viviparous: most species for which reproductive sequences are known incubate parenchymella larvae. The megascleres in the group are always monaxonid, triaenes are never present. Microscleres are generally sigmoid or chelate never asterose. Spongin is an almost universal component of the skeleton.

Order Poecilosclerida: The largest and structurally most diverse order of the Demospongiae. Poecilosclerida are Ceractinomorpha with a skeleton which is always composed of a combination of spicule and spongin fiber. The megascleres are monactine or diactine with many curious structural variations. Spiny spicules are common. Both fiber and spicule skeletons can be complex and show a great regional differentiation. Microsclere types are varied. The larvae are parenchymelle with incomplete ciliation, the posterior pole is always bare, and anterior and posterior poles may show differential pigmentation. Family Esperiopsidae: Sponges in which megascleres are monactine or diactine, have a fasciculate arrangement, and are organized into reticulate or plumoreticulate tracts provided with more or less spongin. In general, the megascleres are uniform in size and shape throughout the sponge, but included in some genera with ectosomal megascleres smaller than those of the choanosome. Microscleres are sigmas and chelas of varied form

Genus Batzella: Esperiopsidae with a reduced, loosely plumose skeleton of strongyles (tornotes) ; no ectosomal skeleton; no microscleres.

The newly discovered organism Batzella sp. is very similar to the Great Barrier Reef Sponge Batzella frutex (see, Pulitzer-Finali, "Some new or little-known sponges from the Great Barrier Reef of Australia", Boll Mus 1st Biol Univ Genova_f ^:87-141 (1982)). It is found in the Caribbean Sea as further described in the Examples section. Moreover, once the location is known, Batzella sp. can be collected and used to isolate the compounds of the present invention.

By using phase extraction chromatography, the compounds of the present invention can be extracted into an organic solvent and then further purified. Such solvents are known to one skilled in the art. For example, the compounds of the invention can be extracted into methyl alcohol, ethyl alcohol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, acetone and the like. Preferred solvents are methyl alcohol or ethyl acetate. However, it is not to be construed that the extraction is limited to just one solvent. More preferably, it is a two-solvent extraction, using two miscible solvents, e.g., methyl alcohol and 1,2-dichloroethane to extract the compounds of the present invention. If necessary. the extract may be desalted by column chromatography.

Preferably, a nonionic polymeric resin, such as XAD-2 is used.

The chromatographic separation is carried out by employing conventional chromatography (e.g., gravity, flash, high pressure (i.e., HPLC) or thin layer chromatography (TLC) ) . Common materials used are alumina and silica gel as well as other materials known to one skilled in the art. For example, column chromatography with non-ionic resin or by high performance liquid chromatography employing a reverse phase resin may be used. The fractions containing HIV antagonist activity can be assayed by a gpl20/CD4 binding assay described more fully below. Generally, more than one chromatographic separation step is employed. In a preferred procedure, one or more separations are carried out employing column chromatography and a final separation is carried out employing preparative thin layer chromatography. When conventional column chromatography is employed, silica gel is the preferred adsorbent. When more than one chromatographic separation is required, silica gel may be used in all the separations, employing different eluting agents. In addition, chromatography using silica gel may be combined advantageously with a different adsorbent, e.g., Sephadex LH-20. Other adsorbents such as alumina, styrene-divinylbenzene copolymers (e.g., HP-20, HP-30, HP-40) and Amberlite

(e.g., XAD-2, XAD-4, XAD-16) may also be employed. A mixture of methanol, methylene chloride, water and formic acid has been found to be especially useful in the fractionation and recovery of the active compounds of the present invention on silica gel (Siθ2) .

The mixture may be employed in isocratic, step gradient or continuous gradient systems. Once isolated, the fractions containing the homogeneous compound(s) may be concentrated under reduced pressure.

The purified products can then be analyzed for purity, structure, etc. by such techniques as NMR (e.g.,

!H, ¹³C, DEPT, l-H-iH 2D COSY) , MS (fast atom bombardment mass spectrometry (FAB-MS) and Tandem) , IR, UV and chemical degradation. All the compounds of the present invention are alkaloids of marine origin. Preferably they are polycyclic guanidine alkaloids and derivatives thereof. Such compounds have the general formula:

-Ri

wherein: n is an integer from 6-10; Rl is

wherein m is 0 or an integer from 5-10; the dotted line represents an optional double bond; R2 is selected from the group consisting of hydrogen, hydroxy, lower alkyl (C -C4₎ , lower alkenyl(C2^_

C4) , alkoxy(C1-C4₎ , aryl, amino, guanidinyl, guanidinyl alkylene(C!-C5_{) f} -0(CH )pNH(C=NH)NH₂,

wherein p is an integer from 1-6. Preferably n is 7-9; m is 6-9; and p is 3-5. More preferably, n is 8-9; m is 7-9; and p is . It is further noted that the variables n, m and p are selected independently of each other. - to ¬

other compounds of the present invention are of the formula:

-CH₃

wherein: q is 0 or an integer from 5-10; r is 0 or an integer from 1-3;

R3 is selected from the group consisting of hydrogen, hydroxy, lower alkyl(C_.-C4₎ , lower alkenyl(C2-

C4) , alkoxy(C1-C4₎ , aryl, amino, guanidinyl, guanidinyl alkylene (Cχ-C5) _f -0 (CH2) _pNH (C=NH) NH₂,

wherein p is an integer from 1-6.

Preferably, q is 6 to 10; r is 1-3; and p is 3-5. More preferably q is 7 to 10; r is 1 or 3; and p is 4.

Furthermore the variables q, r and p are selected independently of each other.

Preferably, R2 and/or R3 are selected from the group consisting of hydroxy, lower alkyl (C1-C4) , lower alkenyl (C₂-C₄) , alkoxy (C!-C₄) , -0 (CH₂)_pNH(C=NH)NH₂,

More preferably R2 and/or R3 is selected from the group consisting of -0 (CH )pNH(C=NH)NH₂,

Aryl refers to phenyl or naphthyl, which may optionally be independently substituted by one or two alkyl(Cι_5), alkoxy(C1-.4) , hydroxy, carboxy, carboalkoxy, carboxamide, phenylalkylene (C1-4) carboxyalkylene(Cι_5) , carboalkoxyalkylene(Cι_5) , carboxamidealkylene(Cι_5) , carboxyalkyloxy(Cι_5) , carboalkoxyalkyloxy(Cι_5) , carboxamidealkyloxy (C1-5) , amino, mono-alkylamino(C]__5) , di-alkylamino(Cι_5) , aminoalkylene(Cι_5) , guanidinyl or guanidinylalkylene(Cι_5) groups.

The present invention further encompasses derivatives of compounds of the present invention which comprises chemical modifications known to those skilled in the art. Chemical modifications include, but are not limited to, hydrolysis, esterification, acetylation, and alkylation, which do not destroy the inhibitory function(s) of the present invention. For illustrative purposes, the guanidine residues may be blocked by a condensation reaction with a three-carbon unit. The three-carbon unit may be a β-dialdehyde, β-ketoaldehyde, β-keto ester, malonic ester, or other combinations of these functional groups as further exemplified in the examples section. Furthermore, said derivatives retain the ability to inhibit HIV infectivity at a comparable (i.e., within one log unit) or lower concentration than the unmodified compounds, but with reduced cytotoxicity.

That is, to inhibit HIV binding to uninfected cells and/or to inhibit subsequent fusion of HIV to uninfected cells and/or the fusion of HIV-infected cells to uninfected cells with an IC50 cytotoxicity less than the unmodified compounds.

Acid addition salts of the compounds of the present invention are prepared in a standard manner in suitable solvents. In brief, an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, or succinic is added to the parent compound, in particular, the acetate salt form is especially usef l. In addition, certain of the compounds may form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkylating reagent, such as hydroxide, carbonate or alkoxide containing the appropriate cation. Cations such as Na⁺, K⁺, Ca^⁺ and H ⁺ are examples of cations present in pharmaceutically acceptable salts. Further examples of derivatives include homologs and iso ers. Homologs shall mean compounds with structural formulas that differ from the compounds of the present invention by one or more carbon atoms and one or more pairs of hydrogen atoms. For example, higher homologs of compound V (see Examples) have a C o^H21 ^{or C}11^H23 side chain in place of the CgH^g side chain. Isomers include, without limitation, enatiomers, optical isomers and stereoisomers (e.g., cis and trans, + and -, d and 1) .

The compounds of the present invention can be assayed for their ability to inhibit HIV infection via binding assays and functional (e.g., fusion or infectivity) assays. One binding assay entails a competition ELISA, measuring the binding inhibition of sT4 to immobilized recombinant gpl20 in the presence and absence of the compounds of the invention. A similar competition RIA entails measuring the binding inhibition of labelled sT4 to (immobilized) recombinant gpl20. Another method entails the inhibition of HIV gpl20 binding to CD4+ cells. Bound gpl20 is detected by gamma counting when using ¹²^ι-iabelled gpl20 (e.g., by the Bolten-Hunter method) or by flow cytometry when using mouse anti-gpl20 antisera and FITC (fluorescein isothioc anate) labelled goat anti-mouse Ig antisera. These assays are performed essentially as described in Arthos et al. (Call, .52:469-81 (1989)) and Sattentau et al. ( Exp Med_r 1 ϋ:1319-34 (1989)), and are incorporated by reference herein.

One functional assay (i.e., syncytium assay) comprises the inhibition of cell fusion between chronically infected cells and uninfected CD4⁺ cells. In essence, HIV infected H9 cells (R. Gallo, National Institute of Health, Bethesda, MD, USA) are cocultivated overnight with uninfected cells at a ratio of 1:2, in the presence of an inhibitory compound. Such assays are performed essentially as described by Sleckman et al., (Nature. 328:351-3 (1987)) and are hereby incorporated by reference.

A non-viral syncytium assay can also be used; This assay measures the inhibition of fusion between cells expressing HIV env protein and CD4⁺ cells as disclosed in U.S. application serial number, 07/587,011, filed September 24, 1990 (Clark et al., "Human Lymphoid Cells Expressing HIV Envelope Protein gpl60") and incorporated by reference herein.

Another functional assay is the virus infectivity assay. This assay comprises infection of T-lymphocytes or macrocyte/macrophages with HIV. At six or more days post-infection measurement of particle-associated reverse transcriptase activity and/or p24 antigen levels can be determined (See, for example, Clapham et al., HalLiX_fi, 337: 368-370 (1990) or McDougal et al., J Iirnmin Meth_f 2£: 171-183 (1985)).

Pharmaceutical compositions of the compounds of the present invention may be formulated as solutions of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is - N - especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

Alternatively, the compounds of the present invention may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about lg per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly or filled into a soft gelatin capsule.

The dosage ranges for administration of the compounds of the present invention are those to produce the desired effect whereby symptoms of HIV or HIV infection are ameliorated. Effective inhibition may be achieved by using one or a combination of two or more compounds of the present invention. Effective inhibition of HIV is defined as at least one log reduction in the infectivity of free virus preparations.

As used herein, a pharmaceutically effective amount refers to the amount administered so as to maintain an amount which suppresses or inhibits secondary infection by syncytia formation or by circulating virus throughout the period during which HIV infection is evidenced such as by presence of anti-HIV antibodies, presence of culturable virus and presence of p24 antigen in patient sera. The presence of anti-HIV antibodies can be determined through use of standard ELISA or western assays for example, anti-gpl20, anti-gp41, anti-tat, anti-p55, anti-pl7 antibodies, etc. The dosage will generally vary with age, extent of the infection, and counterindications, if any, for example, immune tolerance. The dosage can vary from 0.001 mg/kg/day to 50 mg/kg/day, but preferably 0.01 to 1.0 mg/kg/day.

In addition, the compounds of the present invention are useful as tools and/or reagents to study protein- protein interactions. For example, the instant compounds selectively inhibit the interaction between HIV and the T-cell surface protein, CD4. Hence, the instant compounds are useful as an SAR (structure activity relationships) tool to study, select and/or design other molecules to inhibit HIV. The Examples which follow are illustrative, and not to be construed as limiting of the present invention.

EXAMPLES

Collection, extraction and isolation

The sponge Batzella sp. was collected by hand using SCUBA at a depth of 20-30 meters throughout the southern portion of the Berry Islands, Bahamas (ca. 25.5° N, 77.5° W) . A sample of the sponge was deposited in the Zoological Museum of Amsterdam, under a ZMA registry number POR.8788. It was identified by Dr. Rob Van Soest of the Institute of Taxonomic Zoology, University -_b- of Amsterdam, as a new species of the genus Batzella.

The freeze-dried sponge (114g) was extracted with EtOAc and eOH-CH2Cl2 (1:1) to give 5.7g and 25.2g extracts respectively. The MeOH-CH2Cl2 extract was then extracted with CH2CI2 to remove excess salt resulting in a dark red/brown residue (6.7g) . 2.1g of the residue was applied to a column of Sephadex® LH-20 and eluted with MeOH:H2θ (1:1). Fractions (15-20ml) were monitored by UV and pooled. Combined fractions (27-37) were active in a gpl20/CD4 binding assay and contained several compounds that were chromatographed over a column of silica (Siθ2) gel (Kieselgel-60, 230-400 mesh) eluting with a solvent gradient system from MeOH:CH₂Cl2:H2θ:HCOOH (10:90:1.5:2.5) to MeOH : CH₂Cl2 :H2θ :HCOOH (25 : 75 : 2 .5 : 3 .5) . Fractions with same TLC profile were combined to yield individual fractions which were further purified by extensive preparative TLC (PTLC) employing several different solvent systems to give 11 compounds in pure form. The initial fractions 58-95 were further purified via repeated PTLC on Siθ2 using MeOH:CH2Cl2:H2θ:HCOOH

(15:85:1.5:2.5) as elutent to yield compounds V (Rf 0.5,

57 mg) and IV (Rf 0.49, 11 mg) . Fractions 110-155 contained active compounds of identical Rf values and appeared homogeneous on Siθ2TLC using

MeOH:CH2Cl2:H₂0:HCOOH (20:80:2.5:3.5) as eluent. They were finally separated by repeated Siθ2 gel column chromatography and PTLC using Me2CO:MeOH:CH₂Cl2:H20:HCOOH (40:30:30:2.5:3.5) to get compounds III (Rf 0.44, 37 mg) and II (Rf 0.47, 31 mg) . Other fractions (197-221) were purified via Siθ2PTLC using Me₂CO:MeOH:CH2Cl2:H₂0:HCOOH (40:30:30:2.5:3.5) followed by another Siθ2 PTLC employing

MeOH:CH2Cl2'-H20:HCOOH (20:80:2.5:3.5) to yield compound I (Rf 0.41, 31 mg) . The polar fractions (241-330) had the most abundant component in the crude extract. Final purification of this fraction was effected using long (150 cm) and thin (1.5 cm) Siθ2 gel glass column eluted - π- with MeOH:CH₂Cl₂:H₂0:HCOOH (25:75:2.5:3.5) to yield VI

(Rf 0.29, 0.737 g) . The compounds II-IV represent a novel class of alkaloid natural products.

This procedure was then successfully scaled-up to isolate more material from 2,300g of freeze-dried sponge.

Structure Determination

The structure of compounds I to VI were determined by techniques such as NMR ^H, ¹³C, DEPT, 1H-¹H 2D COSY, HOHAHA, C-H 2D COSY, COLOC, HMBC) , MS (FAB & Tandem) , IR, UV, and finally chemical degradation.

Compound T This colorless, amorphous water soluble powder [MW 421 (M+H) , C22^H4l^N6°2' 6Dbe, 7 exchangable] showed bands at 3200-3120, 1695 and 1680-1650 cm^"1 in its IR spectrum

& the UV (MeOH) spectrum showed λ max at 206 (ε 9782) and

288 nm (ε 5889) . The ¹³C NMR and DEPT spectra confirmed the presence of 22 carbon atoms of which five are quaternary, one methine, fifteen methylenes and one methyl. An intense ion at m/z 114 (C5H12N3) observed in its FABMS is derived from the fragment m/z 132 (confirmed by MS-MS) by the loss of a molecule of water indicating a four methylene chain containing branched heteroatoms. NMR data (ID & ¹H-¹H 2D COSY) together with mass spectral fragmentation demonstrated the following system to be present:

NH₂ 0=C-0-CH2-CH2-CH2-CH2-NH-C=NH

In addition to the above fragment, two more moieties were established for I based entirely on NMR data.

CH- (CH₂) 8"CH₃ and N-CH₂~CH₂-CH₂-C= These three systems were linked as shown in structure I. The conclusive proof for the proposed structure was obtained from the analysis of HMBC spectrum of I. The most important correlations observed - \Z- were between H-13 (δ 4.37) and the ¹³C signals at 165.9

(C-6), 103.0 (C-7), 152.5 (C-8) , 153.3 (C-12) , 37.3 (C- 14), and 25.0 ppm (C-15) . All above spectral data is in excellent agreement with crambine A (Berlinck et al., > Te Lett_r .21:6531-6534 (1990)).

Compound I.

Compound II

Compound II is an amorphous, colorless, water soluble powder with IR (KBr) bands at 3600-3100 (N-H & O-H) , 3100-2800 (C-H) , 1733 (ester), 1696 and 1684

(unsaturated ester), 1653-1635 cm^-1(C=C or C=N) . The UV spectrum is very similar to that of I and has λ max 205 (ε 20098) and 288 nm (ε 6824) . The FABMS of II displays a molecular ion at 768 (M+H) and corresponds to the molecular formula C42H74N9O4 by HRFABMS. The FABMS also shows two more peaks at m/z 754 and 740 attributable to the molecular ions of lower homologs of II. The prominent ion at m/z 350 (C2o^H36^N3°2) indicated that II consisted of a molecule of I connected through an oxygen or nitrogen to a tricyclic guanidine system bearing a long alkyl side chain. The iH- MR (MeOH-d4) spectrum exhibited most of the signals associated with I. In addition, it also displayed a triplet at δθ.89 (CH3-42,

3H, J=6.1 Hz, δc 14.4), a doublet at δl.27 (CH₃-33, 3H, J=6.3 Hz, δc 18.4) and another triplet at δ4.14 ppm (CH2- 22, 2H,J=7.0 Hz, δc 65.09). Five methine protons appeared at δ3.93 (CH-25, , 1H, δc 58.2), 3.83 (CH-32, m, 1H, δc 49.4) 3.52 (CH-28, m, 1H, δc 57.0), 3.51 (CH-30, m,1H, δc 53.1) and 3.11 ppm (CH-24, m, 1H, δc 48.8) . The DEPT analysis of the ¹³C NMR spectrum of this alkaloid II showed that thirty-five of the forty-two carbon atoms bear protons and of these, six are primary, twenty-seven are secondary and two are tertiary. The proton-proton and proton-carbon connectivities in the right-hand portion of II were deduced from the ^-H-^-H COSY, HOHAHA, HMBC and particularly from the COLOC experiment. In addition to the correlations observed between H-13 and C-6, C-7, C-8, C-12, C-14 and C-15 in the HMBC experiment, the CH-32 multiplet at δ3.83 showed crosspeaks to carbon signals at d 170.7 (C-23) , 58.0 (CH-25) , 48.7 (CH-24) and 18.3 (33-CH₃) . In the COLOC spectrum the quaternary carbon signal of C-23 showed strong correlation between δ65.09 (CH-22) , 49.4 (CH-32) , 58.2 (CH-25) and 48.8 (CH-24) . Based on this evidence and fully supported by l-H-¹!! COSY as well as HOHAHA experiments, the major fragment in addition to those in I was established as:

X-CH-CH-CH-CH₂-CH₂-CH-CH-CH?-CH₂ (X = N or O) I I I I I

Me X X X X

Finally, the methanolysis reaction determined the point of connectivity of the two MS fragments. The correct structure and relative stereochemistry are indicated below. The lower homologs of II have C7H15 and C_QRI-J in place of the C9H19 side chain of compound II.

Compound II.

Compound TIT

With the structure of II in hand, the structures of the closely related alkaloids III & IV were readily assigned. Compound III, a white, amorphous, water soluble powder exhibited an IR spectrum (KBr) with bands at 3600-3100 (O-H & N-H) , 3100-2800 (C-H) 1691 and 1686 (unsaturated ester), 1632 (C=C, C=N) , and 1347-1080 cm^" ¹. The UV (MeOH) spectrum had λ max at 206 (εl6582) , 289 (ε9298), and 340 nm (ε4375) . The FABMS displayed a molecular ion at 738 (M+H) which corresponded to the molecular formula C θHg8^N9^{0 in its} HRFABMS. The FABMS also showed peaks at m/z 724 and two further molecular ions at m/z 752 & 766 attributable to higher homologs of compound III. An intense signal at m/z 625 (loss of 113 from M⁺) suggested the presence of the same n-butyl guanidine ester moiety present in I and subsequent fragmentation also showed similarity to the cyclic part of I, while a peak at m/z 320 indicated that III had the same ring system as II with one additional unsaturation and an alkyl chain two methylenes shorter. The ^H NMR spectrum of III, like II, had all the signals of I except for the terminal methyl group (22-CH3) which now appeared as a two hydrogen multiplet at δ4.15 (δc 65.5 ppm) . Absence of two methine protons at δ3.11 and 3.63 ppm, present in II, and the emergence of a singlet at δ2.31 ppm led to the conclusion that there was a double bond (δl02.4 & 144.7ppm) adjacent to the ester carbonyl carbon which is also supported by ^■'-H-^H COSY spectrum of III in which the 25-CH shows crosspeaks only to 26-CH2 and not 24-CH observed in case of II. The proton (CH- 25) next to the unsaturated ester underwent a considerable shift (1.1 ppm) and appeared as a doubled doublet (δ4.48 ppm) . The ¹³C NMR spectrum and DEPT analysis of II confirmed the presence of 2 methyls, 25 methylenes, 4 methines and 9 quaternary carbon atoms as shown in figure 3. The higher homologs of III have CgH 7 and C9H19 side chains in place of the C7H15 side chain as illustrated below. -2|-

Compound III

Compound IV

Another of the alkaloids from the Batzella, albeit isolated in small amounts but active in an Elisa assay, showed a band at 1731 cm^-1 ester (C=0 in its IR spectrum and displayed a molecular ion at 463 (M+H) in its FABMS. The elemental composition was derived as C2sH47Ngθ2

(HRFABMS) . The base peak at m/z 350, also seen in the MS of II, and another peak at m/z 114 together with NMR data suggested that the compound IV had the same n~butyl guanidine ester moiety attached to the right-hand portion of II through an ester. The ¹H NMR (MeOH-d4) spectrum of IV shows five methine proton signals associated with the tricyclic guanidine ring system, virtually identical with those of II. The ¹³C NMR and DEPT analysis indicated the presence of 3 quaternary, 2 methyl, 15 methylenes and 5 methine carbon atoms in IV.

Compound IV.

-^■2.2-

Compound V

This white, amorphous, inactive and highly toxic metabolite had IR and UV spectral properties similar to those of I. It showed peaks at 3600-3100, 3100-2800, 1701, 1688, 1652, 1219 and 1092 cm^"1 in IR spectrum and had UV (MeOH) λ max at 206 and 287 nm. The FABMS which shows a molecular ion at 489 (M+H) also had peaks at m/z 503 and 517, due to the higher homologs of V. An intense ion at m/z 114, similar to all other compounds suggested the presence of same n-butyl guanido ester moiety. The molecular weight of V, which had an extra 68 mμ in its MS accounted for a methyl, 2 methylenes and an additional unsaturation in the form of an additional ring. Careful analysis of the H- H & C-H 2D spectra suggested structure V. The higher homologs of V had Cιo^H21 ^anc ^11^H23 ^ln pia-ce of the CgH^g side chain of compound V.

Compound V.

Compound VI This abundant metabolite was readily identified as ptilomycalin A by the comparison of its spectral data with the literature (Kashman et al., &££., 111:8925-8926 (1989)) . Compound VI

Hydrolysis s udies

Acid hydrolysis of T

Compound I (5 mg) was dissolved in CHCI3 (3 ml) to which was added BBr3 (0.3 ml) under 2. The reaction mixture was then refluxed for 30 min. and the solvent evaporated to dryness. The residue was dissolved in H2O

(10 ml) and extracted with CHCI3. The CHCI3 layer after evaporation of the solvent yielded a colorless gum which was purified by (1) RP-18 PTLC (MeOH) and (2) Siθ2PTLC using MeOH:CH₂Cl2:H 0 (10:90:1.5) to get the TLC homogeneous acid la (2.3 mg, MW 308, C17H30N3O2) . The

!H NMR spectrum of la showed absence of the n-butyl guanido ester moiety while the remaining protons appeared at identical positions as seen in I.

Compound la.

Base methan^plysis of I

Excess of NaOMe (25 %wt. in MeOH, 1 ml) was added to solution of I (5 mg) in MeOH (3 ml) and the sealed tube was heated at 65°C for 16 hrs. The solvent was removed and the residue dissolved in H2O (15 ml) , neutralized with dilute CH3COOH and extracted with CHCI3 (3 xl5 ml) . The CHCI3 layer after evaporation gave a residue which was purified by Siθ2 gel PTLC using MeOH:CH2Cl2:H2θ (10:90:1) to obtain lb (lb, MW 322, ^C18^H32^N3°2) • ^{In lts IR} spectrum (KBr) it had bands at 3600, 3000, 2800, 1700, 16888, 1682, 1219 cm^"1 and UV (MeOH) has λ max 205, 287 nm. The l-HNMR spectrum displayed a sharp singlet at δ3.74 ppm (3H) for a methyl ester group formed after the removal of the n-butyl ester guanido moiety.

Compound lb.

Base hydrolysis of TI In a sealed glass tube was placed a mixture of II (12.5 mg) dissolved in MeOH (3 ml) and NaOMe (2 ml) and heated at 65°C for 16 hrs. The reaction mixture was worked up as described above to obtain a mixture of two major degradation products. Siθ2 gel PTLC using MeOH:CH₂Cl2:H₂0:HC0OH (15:85:0.5:0.5) furnished Ha and lib (see below), identified from their spectral data. The UV active left hand Ha (MW 337,

gave a blue color with vanillin/H2S0 and showed a sharp triplet (δ 3.53, 2H, J=7.1 Hz) and singlet (δ3.71ρpm, 3H) in its ¹HNMR (MeOH-d4) spectrum ascribed to the -CH2-O and -OCH3 groups respectively. On hydrolysis of II, the proton adjacent to the carboxylic ester group, in lib (MW 350, M+H, C20^H36^N3°2) ^is epimerised to give a signal at δ2.01 ppm (t, 1H, J=10.5 Hz) that is due to an axial hydrogen coupled to two other axial hydrogens. In II the corresponding signal is at δ3.11 ppm (t, 1H ,J=4Hz) . The large change in the chemical shifts is due o the effect of the two nitrogen atoms in the ring. -2£\- The same phenomenon can be seen in the chemical shift difference between the signals of the two methylene hydrogens on the other fused 6-membered ring at δ2.2 and 1.4 ppm. The five membered ring, which must be "cis- fused" to the 1,5, 9-triazadecalin ring system is clearly defined by the HOHAHA experiment. The three methine protons appeared as a complex multiplet (δ3.58-3.69 ppm), which was resolved when the spectrum was recorded in CDCI3 or CgDg. The ¹³C NMR and DEPT spectra are consistent with the structure lib. Attempts to cleave II using HC1, (CH3)3SiI or aq. NaOH were unsatisfactory.

Compound Ha. Compound lib.

Base hydrolysis of III

A mixture of NaOMe (25%, 1.5 ml) and III (8 mg) in

MeOH (3 ml) was heated in a sealed tube at 65°C for 16 hrs. Reaction was worked up in a similar way as described for II to yield a residue, which after Siθ2 gel PTLC using MeOH:CH2Cl2:H2θ (10:90:1) yielded the identical Compound Ha obtained from II and the methyl ester, Hlb, whose FABMS displayed molecular ion at 333 (C18H33N3O2) . In the ^XR NMR (MeOH-d4) of Hlb the singlet (3H) for the methyl was observed at δ3.75 ppm while the rest of the spectrum exhibited all the proton signals associated with Compound Hlb. -24- Base hydrolysis of IV

Compound IV (3 mg) was dissolved in MeOH (2 ml) to which was added NaOMe (0.75 ml) and the reaction carried out under identical conditions described above to yield Hb as identified by direct comparison with the sample obtained from the hydrolysis of II.

Base hydrolysis of V

NaOMe (3 ml) was added to a solution of V (20 mg) in MeOH (5 ml) and the reaction performed as described above for I-IV. The methyl ester Vb, shows IR and UV spectra identical to that of V plus a methyl ester singlet at δ3.75 ppm in its ¹H NMR spectrum.

Compound Vb.

Hydrolysis of V resulted in the cleavage of the butyl guanido ester group and the formation of methyl ester Vb. Detailed analysis of the ¹H-¹E COSY spectrum of Vb indicated that the methine proton at δ4.48 ppm (18-CH) is coupled only to methylene protons at δl.65 (19-CH2) .

Absence of any crosspeaks between methine proton and 9- CH2 (δ2.81 & 3.34 ppm) rules out the possibility of an alternate structure which would carry a carbon-carbon double bond in the 7-19 position rather than 7-8, as shown in Compound V.

Acid hydrolysis of V

Compound V (8 mg) was dissolved in CHCI3 (5ml) to which was added BBr3 (0.8 ml) and the reaction mixture refluxed for 30 minutes. The solvent was evaporated to dryness and residue dissolved in H2O (20ml) and extracted with CHCI3 (3 x 15ml) . Purification by Siθ2 PTLC using MeOH:CH2Cl2 :H₂0 (10:90:1.5) furnished acid Va

(4 mg) whose FABMS showed M+H at 376.

Compound Va.

Base hydrolysis of ptilomycalin A (VT.

Hydrolysis of ptilomycalin A with aq. NaOH and methanolic NaOH furnished acid Via & methyl ester VIb respectively, as identified from their spectral data.

Compound VI (20 mg) was dissolved in aq. NaOH (5 ml) and the reaction mixture heated to 65°C for 16 hrs.

Removal of the solvent yielded a residue which was dissolved in water (25 ml), neutralized with dil. CH3COOH, extracted with CHCI3 (3 x 25 ml) and purified by PTLC using MeOH:CH₂Cl2:H₂0 (15:85:2.5) to yield acid Via.

Compound Via,

Base methanolysis of ptilomycalin A .VI.

Compound VI (20 mg) was dissolved in MeOH (5ml) to which was added NaOMe (4 ml) and the reaction carried out under identical conditions described above to yield VIb. The ¹H NMR shows a methyl ester singlet at δ3.77.

Protection of the guanidine groups

Compound Ha (25 mg) was treated with IN NaOH (5ml) in dioxane (10 ml) with phenoxy acetyl chloride at 0°C for 1 hour to give phenoxy acetyl derivative VII.

Compound VII,

Under the same conditions, the acid lib (25 mg) was converted to its phenoxy acetyl derivative, compound VIII.

Compound VIII.

Esterification Condensation of acid lib and alcohol Ha: A mixture of the phenoxy acetyl alcohol, VII (lOmg) , and phenoxy acid, VIII (10 mg) was stirred at room temperature with dicyclohexyl carbodiimide (DCC) , N,N- dimethyl aminopyridine (DMAP) , in dry (i.e., molecular sieve) dimethyl formamide (DMF) and methylene chloride at room temperature for 24 hours. The solvent was then evaporated to dryness and the residue was treated with 10% Pd/C to remove the phenoxyacetyl group. The ester was then purified by preparative TLC to yield compound IX.

Compound IX.

Modification of the σnani ine residue using 2_r pentanedione Compound II (100 mg) was refluxed (100°C) in a mixture of 2,4 pentanedione (0.5 ml) and sodium bicarbonate (50 mg) in 95% ethanol (10 ml) for 18 hours. The majority of the ethanol was evaporated, diluted with water (20 ml) and then extracted with dichloromethane (3 x 20 ml) . The combined extracts were dried over anhydrous sodium sulfate and evaporated to give a crude product. The crude product was purified on silica gel to yield Compound Ilm^.

Compound Ilm^.

__3o-

Modif cation of the σuanidine residue using 1 _r 2- cyclohexanedione

Compound II (100 mg) was stirred at room temperature (20°C) in a mixture of 95% ethanol (3 ml) and 1,2-cyclohexanedione (200mg) in 0.5M NaOH (10 ml, pH

>12) overnight. The majority of the ethanol was evaporated, acidified with cold acetic acid (4°C) to pH of approximately 5.0, and then extracted with dichloromethane (3 x 100 ml) . The dichloromethane was evaporated to give a crude product. The crude product was then purified on silica gel (15:85:1.5:2.5 MeOH:CH2CH2:H 0:HCOOH) to yield Compound IIπ_2.

Compound IIm2,

Compound III was also modified using 1,2- cyclohexanedione (see structure A) . Both modified compounds II and HI were active in an Elisa assay and showed reduced cytotoxicity relative to the unmodified compounds.

Compounds I, HI, IV, and V were also modified using 1,2 cyclohexanedione to yield compounds H, A, G and B, respectively. Similarly, compounds I, III, IV and V were modified using 2,4 pentanedione to yield compounds D, C, F and E, respectively.

Compound A. - =sl-

Compound B,

Compound C.

Compound D,

Compound E,

Compound F,

Compound G,

Compound H.

Additional derivatives were prepared by refluxing with 1,2-Diketo cyclohexane, phenyl glyoxal or butanedione.

Table 1 summarizes eleven compounds tested for activity in a CD4 gpl20 binding assay and the IC50 cytotoxicity values.

(Ptilomycalin A)

* Not toxic at highest concentration tested.

The Whole cell binding assays were performed essentially as described by Arthos et al. .Cell, 57:469- 481 (1989)) and Sattentau et al. (J Exp Med, 1211:1319- 1334 (1989)), and are incorporated by reference herein.

In the ELISA assay, goat anti-mouse antibody was coated onto microtiter plates which in turn immobilizes a monoclonal antibody to HIV gpl20. gpl20 is then captured onto the plates and appropriate concentrations of compounds of the present invention are applied followed by the addition of soluble CD4. The bound CD4 is then quantitated by treatment with HRP-conjugated rabbit anti-CD4. In the Whole cell binding assay, gpl20 is incubated with the compound(s) of the present invention for 30 minutes followed by the addition of CD4⁺ T cells for 30 minutes. The cell associated gpl20 is then detected by treatment with a monoclonal antibody to gpl20 followed by FITC-conjugated goat anti-mouse antibody. The analysis is then performed by flow cytometry.

Cytotoxicity was tested with the CD4⁺ cell line, SupTl (J. Hoxie, Univ. of Pennsylvania, Philadelphia, PA, USA) (see also, Sattentau et al., J Exp Med, 170:1319-1334 (1989)). The compounds of the present invention were dissolved in DMSO in order to maximize solubility and provide a common solvent system. The final concentration of DMSO in the cytotoxicity assay did not exceed 2% of the total volume. Cytotoxicity was measured as the inhibition of H-thymidine incorporation into the host cell's genome after exposure to the compounds of the present invention. Alternatively, cell viability can be measured 18-20 hours after exposure to the compounds of the present invention by measuring the reductive capacity (e.g., MTT [3-(4,5-dimethylthiazol-2- yl)-2,5 diphenyl tetrazolium bromide] or XTT reagents), and thereby indirectly measure cell viability, of cells in a microtiter format. Other cell lines that are available to one skilled in the art include CEM, MOLT 4, AA5, MT-2 and H9 (see, Jacobs, J Na l Cancer Inst_r .21:231 (1965) ) .

Table 2 summarizes the results of a syncytial assay, i.e., the ability of compounds to inhibit HIV- induced fusion of CD4 positive T-cell lines.

TABLF, 2

(Note: Visual cell killing is only an approximate measure of cytotoxicity.)

The syncytial assay is performed essentially as follows.

The activity of test compounds was assessed for their ability to inhibit the HIV-induced fusion of CD4 positive T-cell lines. Serial 2 fold dilutions of standard positive control sT4 (soluble T4 or CD4) or test Batzella compound were made in RPMI 1640 containing 10% fbs and 20 μl of each dilution were added to duplicate wells of half-area 96 well tissue culture plates (COSTAR) . Cells from a CEM cell line persistently infected with HIVTUB ^anc 9^ro^wn ϊⁿ RPMI 1640 containing

10% fetal bovine serum were suspended in fresh medium at a concentration of 1.25 x 10⁵ cells/ml. Uninfected MOLT 4 cells (CD4 positive target cells) were suspended in fresh medium at a concentration of 1.75 x 10^ cells/ml. To each well of plates containing serial sT4 dilutions 40 μl of each cell suspension were added. Fusion of the infected CEM and uninfected MOLT 4 cells was determined microscopically 24 hours later, and the number of syncytia were counted (see, e.g., Matthews et al., PNAS :5424, 1987) .

In addition, the compounds of the present invention can be assayed in a virus neutralization assay (or HIV infectivity assay) as follows.

1. 100 μl of appropriate dilutions of "inhibitor" (i.e., purified compound, natural product extract, etc.) are added to wells of assay plate. Generally 20 mM is more than enough compound to cause complete inhibition of processing in HIV infected T-cell lines.

2. 100 μl containing 40 Infectious Units of virus

(IIIB Stock = 10^ infectious units/ml) mixed with

100 μl of AA5 or M0LT4 cells (3 x 10⁵/ml) and incubated 60 min at 37°C in CO2 incubator.

3. Add 100 μl of virus dilutions (4-fold serial) for titration of virus stock on the same or a separate plate.

4. Add 100 μl AA5 or MOLT4 cells at a concentration of 3 x 10⁵/ml.

5. Harvest 90 or 180 μl culture medium at 5-10 days post infection and add to 96-well plate containing 10 or 20 μl 5% Triton X-100. Samples can be stored at -80°C or immediately assayed for RT and p24 ELISA.

Alternative Infectivity Assays: Alternative assays can be set up by infecting cells in bulk for 1 hr, washing cells to remove unabsorbed virus, then adding 100 μl (3 x lO^/ml) of infected cells per well. Dilutions of inhibitors are then added to the infected cell cultures at 0 hr post infection. The above examples and description fully disclose the present invention, including preferred embodiments thereof. This invention, however, is not limited to the precise embodiments described herein, but encompasses all modifications within the scope of the art to the claims which follow.

Claims

What is claimed is:

1. A compound in substantially pure form represented by the structure:

wherein : n is an integer from 6-10; Rl is

wherein m is 0 or an integer from 5-10; the dotted line represents an optional double bond; R2 is selected from the group consisting of hydrogen, hydroxy, lower alkyl(C1-C4₎ , lower alkenyl(C2-

C4) , alkox (C1-C4₎ , aryl, amino, guanidinyl, guanidinyl alkylene(C!-C5_{) r} -0(CH₂)_pNH(C=NH)NH₂,

2. The compound of claim 1 wherein R^ is

-3<? -

3. The compound of claim 2 wherein n = 9 and m = 8.

. The compound of claim 3 wherein R2 is selected from the group consisting of -0

wherein p is an integer from 1-6.

5. The compound of claim 1 wherein R^ is

-CH₃

6. The compound of claim 5 wherein n = 9 and m 6.

7. The compound of claim 6 wherein R2 is selected from the group consisting of -0(CH )_pNH(C=NH) H₂,

wherein p is an integer from 1-6.

8. A compound in substantially pure form represented by the structure:

-CH₃

- no ¬ where in : q is 0 or an integer from 5-10; r is 0 or an integer from 1-3;

R3 is selected from the group consisting of

5 hydrogen, hydroxy, lower alkyl(C1-C4₎ , lower alkenyl(C2-

C4) , alkox (C1-C ₎ , aryl, amino, guanidinyl, guanidinyl alkylene tCi-Cs_{) r} -0 (CH₂) _pNH (C=NH) NH₂ ,

5 9. The compound of claim 8 wherein q is 7 to 10.

10. The compound of claim 8 wherein r is 1 or 3.

11. The compound of claim 9 wherein R3 is further 0 selected from the group consisting of

wherein p is an integer from 1-6,

12. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable 0 carrier.

13. A pharmaceutical composition comprising the compound of claim 8 and a pharmaceutically acceptable carrier. 5

14. A method of inhibiting HIV infection which comprises administering to a patient a pharmaceutically effective amount of a compound isolated from the marine sponge Batzella, wherein said compound inhibits HIV infectivity in vitro.

15. A method of inhibiting HIV infection which comprises administering to a patient a pharmaceutically effective amount of the compound of claim 1.

16. A process for preparing an HIV inhibitor which comprises: extracting the marine organism Batzella with an organic solvent; separating the extract into fractions containing HIV antagonist activity; and purifying said extract to an essentially homogeneous compound which inhibits HIV infectivity in vitro.

17. The process of claim 16 in which the HIV inhibitor is the compound of claim 1.