WO1994009773A1

WO1994009773A1 - New antiviral agents

Info

Publication number: WO1994009773A1
Application number: PCT/US1993/010199
Authority: WO
Inventors: Darryl C. Rideout
Original assignee: The Scripps Research Institute
Priority date: 1992-10-26
Filing date: 1993-10-26
Publication date: 1994-05-11

Abstract

A method for treating or preventing retroviral infection in cells include contacting the cells with novel compounds. Lucifer Yellow and its analogs are coupled to compounds having carbonyl functional groups for reaction with the semicarbazide moiety of Lucifer Yellow to obtain the semicarbazone; alternatively, the naphthyl succinimide moiety of Lucifer Yellow or its analog is bound directly or through an amide linker to at least one aryl residue. Also, a method for inhibiting proliferation of retroviruses in cells includes contacting the cells with precursors to toxic conjugates; the precursors are used in therapeutic compositions. In preferred embodiments, the individual components are Lucifer Yellow and either aldehydes or dialdehydes or derivatives of Lucifer Yellow reacted on an equimolar basis with dialdehydes.

Description

NEW ANTIVIRAL AGENTS

Background of the Disclosure

Technical Field The invention relates to materials which are useful in treating or inhibiting retroviral infection in cells. Particularly, the invention features, in one general aspect, self-assembling naphthyl disulfonates which are effective in inhibiting the progress of human immunodeficiency virus ("HIV") infection; in another general aspect the invention features semicarbazones and other conjugates of Lucifer Yellow ("LY") and its analogs, or conjugates of the succinimide residue of LY and LY analogs, which are effective in inhibiting HIV and other retroviral infections.

Background Art

There are a number of viral diseases recognized as retroviral in origin in both humans and other animals. The most publicized such disease among humans is that caused by the human immunodeficiency virus ("HIV") as either acquired immunodeficiency syndrome ("AIDS") or AIDS-related complex ("ARC") . Other diseases of retroviral origin include hepatitis B and hepatitis delta. Among cats, retroviral diseases include those caused by the feline immunodeficiency virus ("FIV") and the feline leukemia virus ("FeLV") . A number of other animal species also contract retroviral-caused infections, such as the Visna virus infections of ungulates.

There have been a number of approaches to the treatment of such diseases, none of them entirely successful. Various active compounds have been suggested for the treatment of HIV infection, the best known being AZT and dextran sulfate.

Over a million Americans and several million people worldwide are infected with HIV, and treatment with the experimental drug AZT, produced by Burroughs Wellcome, appears to be of some help although it is clear that AZT is unable to cure AIDS and is extremely toxic. Nevertheless, AZT has been a considerable commercial success. There is no doubt that there is a continuing need for more effective antiviral agents which are effective against retroviral infections in general, and against HIV in particular.

The compound most closely related to the invention conjugates described herein is suramin sodium, which is a complex conjugate of trisodiu sulfonate naphthyl derivatives and aromatic nuclei linked through amide linkages. The structure of suramin is shown in Fig. 1A. The effects of suramin in treatment for AIDS and its mechanism of action have been studied by a number of workers (Balzarini, J., et al, Int J Cancer (1986) 32:451-457; Levine, A.M., et al. Annals Int Med (1986) 105:32-37; Broder, S., et al. Lancet (1985) 1:627-630; Cheson, B.D., et al, JAMA (1987)

258:1347-1351) . In general, this compound has been found helpful in treatment, although its toxicity precludes its use as a single agent in treatment. Side effects include ophthalmic damage, hypoadrenalism, nausea, thrombocytopenia and vomiting, and some deaths have been attributed to these side effects.

It has been suggested that the antiviral mechanism of suramin is due to binding of the polyanionic molecule to reverse transcriptase

(DeClercq, E. , Cancer Lett (1979) 8_:9-22) and a large number of suramin analogs have been prepared. A total of 90 suramin analogs, of which 57 showed the ability to inhibit HIV-I reverse transcriptase activity, and 24 of which were superior to suramin in this inhibition, were prepared by Jentsch, K. D., et al, J Gen Virol (1987) j5J3:2183-2192. A representative group of suramin analogs is shown in Figure 1. All of these are bis(naphthalene polysulfonic) acids, show ID50 values in the range of 5-42 μg/ml and, like suramin, eliminate the cytopathic effect of HIV-I against MT4 cells at 50 μg/ml. As stated above, suramin sodium is highly toxic; it is too toxic be used clinically, at least as a single agent, for treatment of HIV infection. The known analogs have similar drawbacks.

Certain additional publications discuss compounds that are related to those described and claimed herein. International PCT patent publication W088/00047, published 14 January 1988 and incorporated herein by reference, describes various materials which can be administered separately to a microenvironment and there assembled to create an active conjugate.

It appears that monocytes and macrophages are an appropriate target for an antiviral agent directed to HIV or other retroviral infection. In AIDS patients, evidence of HIV-I infection has been observed in monocytes from a variety of tissues, including brain, peripheral blood, lymph nodes, skin, and lung (Pauza, CD., et al, J Virol (1988) _62: (in press); Pauza, CD., cell Trmnunol (1988) 112:414-424) and human peripheral blood monocytes and onocyte-derived cell lines support viral replication in vitro. In addition, two other immunosuppressive retroviruses, MLDV and the Visna virus of ungulates, cause immunosuppression primarily by infecting monocytes. Others have shown that monocytes act as a reservoir for virus in infection (Ho, D. , et al. New Enσ J Med (1987) 317:278-286; Klatz ann, D., et al, Immunol Today (1986) 2:291-296). Since few T-cells are actually infected with HIV-I in AIDS patients, and since it is known that infected monocytes can shed HIV-I envelope protein in soluble form, it is inferred that infected monocytes may mediate the loss of helper T-cells in these patients (Siliciano, R.F., et al. Cell (1988) 54:561-575) .

It is recognized that there are many advantages to assembling a biologically active substance at the site of its intended activity, especially when the active substance can be formed from innocuous and nontoxic precursors. International PCT patent publication W088/00047, published 14 January 1988 and incorporated herein by reference, describes various materials that can be administered separately to a microenvironment and there assembled to create an active conjugate. One microenvironment which is of considerable interest is any of the loci of infection by HIV, and the possibility to administer nontoxic precursors of a drug which would self- assemble at the site of the HIV infection would clearly be advantageous in treating the disease and symptomology caused by this virus.

While WO88/00047 describes generically a concept of active conjugate self-assembly at a desired locus, and provides certain exemplification of this concept, no specific compounds useful in the treatment of HIV infection are disclosed.

Neither suramin nor its analogs offer the opportunity for localized formation through in situ self-assembly, as they are conjugated through amide or urea linkages which are not formed readily under in vivo conditions.

In a first aspect, the invention is focussed upon the microenvironment of retroviral infections, specifically the loci of infection by HIV, and to administering nontoxic precursors of a drug which would self-assemble at the site of the HIV infection as advantageous in treating the disease and symptomology caused by this virus. In this aspect, the compounds of the invention offer the capability of self-assembly in vivo and provide effective reagents to interrupt the course of HIV infection. In a second aspect, rather than relying on self-assembly at the site of infection, the invention is directed to administering compounds which are preassembled, and which are related to those used as components in the self-assembly aspect of the invention.

The conjugates according to the invention appear to home to monocytes.

The compounds of the present invention offer the capability to interfere with the life cycle of retroviruses in general, probably through interference with the activity of reverse transcriptase, and are thus useful as prophylactic and therapeutic agents in the treatment of retroviral infections.

Summary of the Invention

In a first aspect, the invention provides nontoxic precursors for conjugates capable of inhibiting HIV or other retroviral proliferation in infected or susceptible cells. The precursors are capable of self-assembly after contact with the cells to form conjugates which are toxic to the virus. This permits reduction of toxicity and, when administered in vivo, permits treatment with materials which are biologically safe to the subject, which nevertheless become toxic to the target infection when assembled at the appropriate site. Because the assembly is largely restricted to the locus of the target virus, any toxic side effects of the assembled conjugate to any subject being treated are minimized. In a second aspect, the invention provides relatively nontoxic conjugates capable of inhibiting retroviral infection in human and animal subjects. The conjugates are toxic to the retrovirus, and appear to inhibit reverse-transcriptase.

Disclosure of the Invention Thus, in one aspect, the invention features a method to inhibit proliferation of retroviruses in cells infected with or susceptible to said virus, which method comprises contacting the subject cells with amounts of precursors which will result in the assembly of an effective amount of conjugate in situ , which conjugate is effective in interrupting the virus infection.

In another aspect, the invention is directed to the conjugates obtained. These conjugates are derivatives of the commercially available nontoxic dye, Lucifer Yellow CH (abbreviated herein as LY) , LY and its analogs. In general, the conjugates are of the formula:

R-NHCONHN=CH-Y-CH=NNHCONH-R (1)

wherein R is of the formula:

wherein Z represents one or two noninterfering substituents, including the corresponding pharmaceutically acceptable salts, and Y is selected from the group consisting of the linking moieties shown in Fig. 2.

The conjugates are formed by the in situ reaction of the precursors of the formula: R-NHCONHN-CH-Y-CHO ( 2 ) and R-NHCONHNH₂ ( 3 )

wherein R and Y are as above defined.

In additional embodiments, the invention is directed to corresponding methods and compositions which involve formation of a conjugate of the formula:

R-

R-NHCONHN=C tT (6)

wherein R is as above defined; and wherein R¹ is H or alkyl (1-6C) ; and R² is alkyl (1-6C) or aryl (6-10C) or linked aryl (6-10C) containing 2-5 such aryl groups; said alkyl or aryl substituents being unsubstituted or substituted by 1-2 noninterfering groups.

The conjugates of Formula (6) are readily formed by the in situ reaction of the precursors of the formulas:

R-NHCONHNH₂ (3)

and

wherein R, R¹ and R² are as above defined.

In another aspect, as described above, the invention is directed to a method to inhibit proliferation of retroviruses in cells infected with or susceptible to said virus, wherein the method comprises contacting the subject cell with precursors of antiviral conjugate of Formula (1) or of Formula (6) . The preferred precursors of the compound of Formula (1) have the Formulas (2) and (3) . Preferred precursors of the compound of Formula (6) have the Formulas (3) and (7) . These precursors are capable of self-assembly in the presence of virus- susceptible or virus-infected cells.

In another aspect, the invention is directed to compositions containing the precursors of Formulas (2) and (3) or of Formulas (3) and (7) and to certain novel substances useful in preparing the compositions and in the method of the invention.

In another general aspect, the invention is directed to a method to inhibit proliferation of retrovirus in cells that are infected or susceptible to the virus, which method comprises contacting the cells with amounts of compounds shown below which are effective in interrupting or preventing the virus infection. In another aspect, the invention is directed to the compounds of the invention and the compositions containing them. These compounds are derivatives of the commercially available nontoxic dye, Lucifer Yellow (LY) , and its analogs, or of just the tricyclic succinimide residue thereof. In general, one group of the compounds are of the formula:

R-NHCONHN=C (6)

wherein R is as defined above. Formula (4) , and wherein Z represents one or two noninterfering substituents, Rl is H or alkyl(1-6C); and

R2 is alkyl(1-6C) or aryl(6-10C) or "linked" aryl(6-l0C) containing 2-5 such aryl groups, said alkyl or aryl substituents being unsubstituted or substituted by 1-2 non-interfering groups, and the pharmaceutically acceptable salts thereof.

Some typical embodiments of R2 are of the formula -Y-CHO wherein preferred forms of Y are shown in Fig. 2.

In still another aspect, the invention is directed to a method to prepare the compounds of Formula (1) which comprises contacting a compound of the formula RNHCONHNH₂ with a compound of the formula R1R2CO, and recovering the resulting conjugate.

An additional group of compounds effective in the methods and compositions of the invention is of the formula:

R-R3 (8)

wherein R is as defined above and R3 is an aryl-containing moiety of 6-10C or linked aryl moieties of 6-10C. The aryl groups in moiety R3 may be heterocycles or carbocycles and may be unsubstituted or substituted with 1-2 substituents that are noninterfering with the cytotoxic nature of the compounds of Formula (8) . R3 may contain 1-5 such aryl groups. Typical substitutions on such aryl groups include acidic moieties such as -COOH, P0₃ ^~2, B(0H)₂ and the like, alkyl(l-4C), alkoxy(l-4C) , halo, and the like wherein said such alkyl or alkoxy substituents may be further substituted with one or two additional substituents such as hydroxy, alkyl, alkoxy and the like. R may be linked directly to an aryl of R3 or may be linked through a suitable linker, such as, preferably, an amide of the formula -NHC0-.

The compounds of Formula (8) may be used in a manner analogous to those of Formula (6) and are themselves novel compounds. Description of Preferred Embodiments

Brief Description of the Drawings

Fig. 1A shows the structure of the compound sodium suramin.

Figs. 1-B through 1-J show the structures of representative analogs of suramin sodium.

Fig. 2 shows the structures of embodiments of the moiety Y in the precursors and conjugates of the invention. In their dialdehyde forms the Y moieties generate embodiments of R2 in the compounds of the invention.

Fig. 3 is an outline of the synthesis of the conjugate A4. Fig. 4 is a graph showing the effect of A4 and suramin on inhibition of reverse-transcriptase isolated from HIV-1.

Fig. 5 is a graph showing the effect of suramin and A4 on inhibition of reverse transcriptase isolated from feline immunodeficiency virus ("FIV") . Fig. 6 is a graph showing the inhibition of growth of FIV in feline kidney cells by compound A4 of the invention.

Fig. 7 is a graph showing the effect of A4 on the viability of infected feline cells containing FIV.

Fig. 8 is a graph showing the results of fluorescence activated-cell sorting ("FACS"), demonstrating that A4 is taken up and retained by a human macrophage-like cell line U937.

Fig. 9 is an outline of the synthesis of compounds designated Al to A9.

Fig. 9 is an outline of the synthesis of precursors A1-A9 and conjugates H1-H9 according to the invention.

Fig. 10 shows the structures of various compounds R-R3 of Formula (8) . Figs. 11-1 through 11-4 show summaries of the structures of compounds Al to A9 and HI to H9. Fig. 12 shows the structures of several compounds of Formula (6) .

Modes of Carrying Out the Invention

As noted above, the invention in one general aspect takes advantage of the self assembly of nontoxic precursors to form conjugates which are capable of inhibiting the proliferation of retroviruses in infected cells. The ultimate conjugates of Formula (1) are assembled in situ from precursors which are included in the set of ultimate conjugates of Formula (6) . The compounds of Formula (6) are assembled in situ from compounds of Formulas (3) and (7).

And, as noted above, in another general aspect, the invention provides compounds of Formula (6) or Formula (8), which are useful in inhibiting retroviral infection.

A. Nature of the Precursors and Conjugates

The conjugates formed in situ and the precursors thereof are derived from the nontoxic commercially available dye Lucifer Yellow (LY) or its analogs. LY has the formula:

and is thus a semicarbazide. The commercially available material is in the form of the lithium salt; however, other salts of the sulfonates, or even the free acid forms, can be used in the formulation. Of course, the ionization status of the sulfonate will depend on the pH of the medium which surrounds it. Suitable salts for use when the compound is administered therapeutically include any nontoxic, inorganic ions, such as sodium, potassium, ammonium, calcium, magnesium, and so forth, or may also be organic cations, such as quaternary amines, e.g., diethylamine, triethylamine or ethanolamine. In general, inorganic ions are preferred, as these confer greater solubility on the compounds and are less expensive. The free acid form can also be used in the manufacture of a suitable formulation which will adjust the pH to a physiologically compatible value.

By "analogs" of the Lucifer Yellow moiety is meant compounds other than LY of the formula:

NHCONHNH,

wherein Z represents one or two innocuous substituents in either ring such as lower alkyl, lower alkoxy, hydroxy, amino, thio, alkylamino, alkylthio, or halo. The nature of Z is not important in these analogs so long as it does not interfere with the self-assembly, with the efficacy of the conjugate, or with the non-toxicity of the precursor. Thus, by "analogs" of Lucifer Yellow, is meant those naphthalene disulfonate derivatives related to Lucifer Yellow which have different substituents in the naphthalene nucleus other than the single amino group present in Lucifer Yellow itself. As is the case with Lucifer Yellow, the analog sulfonates may be in the form the free acid or of any pharmaceutically acceptable salt.

Preferred forms of the compounds of Formula (1) include those wherein R represents the tricyclic residue of Lucifer Yellow-i.e., Z is NH₂ at the position shown.

In the compounds of formula 1, preferred embodiments of Rl are H and lower alkyl(1-4), most preferably H. By "alkyl" is meant a saturated hydrocarbyl of the indicated number of carbon atoms which is in a straight or branch chain form, such as methyl, ethyl, n-propyl, i-propyl, t-butyl, n-butyl, i-hexyl, n-hexyl, and 3-methylpentyl, provided sufficient C are included in the designation. With respect to R2, R2 can be alkyl, especially if Rl is not H. Compounds of Formula (1) in which R2 is aryl are preferred, however. By "aryl" is meant a 6-10 membered monocyclic or fused bicyclic ring system which has aromatic character. The ring may contain only carbons as ring members, or which may contain one or more heteroatoms such as N or O. The aryl substituents are, thus, typically, moieties such as phenyl, pyridyl, naphthyl, quinolyl, pyrimidyl, and the like. The aryl substituents may contain 1-2 nonhydrogen substitutions which do not interfere with the pharmacological effect of the conjugate. Typical substituents include, but are not limited to, lower alkyl, lower alkoxy, hydroxy, amino, thio, alkyl amino, alkyl thio, halo, carboxy, carbalkoxy, or CHO. The substituent R2 may also include multiple "linked" aryl (or, perhaps, more properly, "arylene" substituents wherein a total of 1-5 aryl moieties of 6-10C are linked through nonaromatic substituents or through a covalent bond to form an extended chain of aryl groups. Perhaps the simplest example of this is the embodiment of R2 is biphenyl wherein two phenyl groups are linked through a single covalent bond. Other examples of this type of "necklace" of aryl moieties are found in Figure 2, wherein in some instances phenyl groups are linked through -CONH- or -NHCONH-. Other types of linkage include hydrocarbylene linkages and conjugation-type linkages through -CH=CH-. As is the case for a single aryl substituent, the aryl groups forming the extended chain may be substituted with 1-2 noninterfering substituents. A particularly preferred substituent is -CHO. Particularly preferred also are those embodiments wherein at least two linked aryl groups are present in the substituent R2, and most preferred is the substituent which is a substituted biphenyl shown as Formula Y-4 in Fig. 2. A particularly preferred embodiment of R is that wherein R represents the tricyclic residue of Lucifer Yellow-i.e., Z is NΗ₂ at the position shown in formula (5').

Preferred embodiments of Y in formulas (1) and (2) are those represented by the formulas shown in Fig. 2; and in particular, more preferred are those shown as Y-l to Y-4, and most preferred is that represented by formula Y-2.

A series of preferred precursors and conjugates are constructed wherein the compound of formula (3) is a pharmaceutically acceptable salt of Lucifer Yellow and the compound of formula (2) is derived from Lucifer Yellow and Y dialdehydes of the various formulas shown in Fig. 2. These compounds of formula (2) are designated A1-A9 in Fig. 9. Further condensation in situ with LY results in conjugates of the corresponding hydrazones (semicarbazones) of formulas HI through H9.

The synthesis of all of these embodiments is outlined in Fig. 9. As shown in Fig. 9, the dialdehyde of the embodiments of Y shown as formulas Y-l to Y-9 in Fig. 2 is first prepared and reacted with an equimolar amount of Lucifer Yellow to obtain the precursors A1-A9; subsequent reaction with a second mole of LY in situ results in the toxic and antiviral conjugates H1-H9.

A series of preferred precursors and conjugates are constructed wherein the compound of formula (3) is a pharmaceutically acceptable salt of Lucifer Yellow and the compound of formula (2) is derived from Lucifer Yellow and Y dialdehydes of the various formulas shown in Fig. 2. These compounds of formula (2) are designated A1-A9 in Fig. 9. Further condensation in situ with LY results in conjugates of the corresponding hydrazones (se icarbazones) of formulas HI through H9.

Particularly preferred as precursors to the compounds of formula (1) are Lucifer Yellow itself, in the form of any pharmaceutically acceptable salt, as the compound of formula (3) , and the compound designated A2 herein, of the formula:

(A2)

which is a condensation production of LY with benzene- 1,3-dialdehyde.

When A2 is conjugated with LY, the conjugate has the formula designated H2:

LY-NHCONHN-HC —~(^~θ)

CH=NNHCONH-LY ^(H2) It is seen that some of the "precursors" to the compounds of formula (1) , which have the formula (2) , fall within the scope of the generic conjugate of formula (6) . The compounds of formula (6) have the formula:

wherein R, R¹ and R² are as above defined.

As in the previously described compounds, the preferred form of R is represented by the residue of Lucifer yellow containing an amino substitution in the naphthalene system. A preferred embodiment for R¹ is H. Preferred embodiments for R² include the residues represented by the reaction of the dialdehydes of the embodiments of Y represented in formula (2) on an equimolar basis with the Lucifer yellow or its analogs; these products of formula (6) represent the subset which are of formula (2), i.e., compounds A1-A9, discussed above.

However, other compounds of formula (6) are obtainable from alternative embodiments of the compound of formula (7) wherein R² is not derived from a dicarbonyl compound such as the dialdehydes which result in the compounds of formula A1-A9. In particular, preferred embodiments of R² include unsubstituted or mono- or disubstituted phenyl wherein the substitution is halo or is alkyl(1-4C) or alkoxy(1-4C) optionally further substituted by -OH or halo, unsubstituted, mono- or disubstituted biphenyl wherein the substituents are as set forth above, or wherein the substituent R² is derived from a more complex aldehyde or ketone such as that illustrated by CBDl in the examples below. Particularly preferred are those embodiments wherein R² is phenyl, either unsubstituted or having a single substituent which is a lower alkyl(1-4C), preferably methyl, and wherein R² is biphenyl, which is unsubstituted or has this preferred monosubstitution.

Thus, the production of the compounds of formula (6) results from the condensation of the compounds of formula (3) with those of formula (7) wherein the compounds of formula (6) include, as a subset, the compounds of formula (2) . In a preferred embodiment, Lucifer yellow itself is supplied as the precursor of formula (3) along with the preferred embodiments of the compounds of formula (7) .

Particularly preferred as conjugates are those derived from Lucifer Yellow itself, in the form of any pharmaceutically acceptable salt, and the compound of the formula:

i. e. , those of the formula

The synthesis of A4 from commercially available reagents is outlined in Fig. 3. As shown in Fig. 3, the commercially available compound 4,4'-dicyanobiphenyl is reduced in DIBAL and hydrolyzed to obtain the dialdehyde. The dialdehyde is then reacted with the lithium salt of Lucifer Yellow in a controlled reaction to obtain the single conjugate A4, which is then isolated using standard chromatographic methods. The lithium salt, is, if desired, converted to alternate pharmaceutically acceptable nontoxic salts. In addition. Fig. 9 shows a multiplicity of compounds analogous to A4 which are synthesized from the dialdehydes of the moieties in Fig. 2 in combination with Lucifer Yellow or an analog thereof. These compounds, Al through A9, are also useful as anti-viral agents.

In addition to the compounds of Formula (1) for use as conjugates, compounds which embody only the "R" portion of the Lucifer Yellow dye or its analog may be obtained and are useful antivirals. These compounds are of the Formula R-R3 wherein R is of the formula:

or its pharmaceutically acceptable salts. The embodiment of R which represents that found in LY per se is represented by R'; the generic formula representing this residue of Lucifer Yellow or its analog may be represented by R.

In the compounds of Formula (8) , R3 is an aryl-containing moiety analogous to those embodied in R2 but linked directly to the naphthyl succinimide residue in LY or its analog through the ring nitrogen of the succinimide. Some preferred embodiments of the compounds of Formula (8) are shown in Fig. 10.

Compounds of Formula (8) wherein the R substituent is linked directly to an aryl moiety of R3 are obtained by reacting the amino derivatized aryl moiety with the succinimide as exemplified hereinbelow. The nitrogen bound to the aryl group replaces the succinimide nitrogen resulting in the coupled compound of Formula (8) . Embodiments wherein the substituent R is coupled to the aryl portion of R3 through the nitrogen of an amide linkage are obtained by reaction of the carbocyclic acid hydrazide derivatives of the aryl substituent with the succinimide. Preferred embodiments of R3 include phenyl or biphenyl unsubstituted or substituted with one or two substituents which may be alkyl(1-4C) , halo, or alkoxy(1-4C) . The substituents may also be acidic substituents such as COOH, phosphonic acid or boronic acid residues.

Preferred compounds of the invention include:

wherein A is alkyl(1-8C) or -(CH₂)_nO(CH₂)_nH wherein each n is independently 1-4, and preferably is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, methoxymethyl, methoxyethy1, methoxy-n-propyl, methoxy-i-propyl, methoxy-n-butyl, ethoxy-i-butyl; ethoxymethy1, ethoxyethyl, ethoxy-n-propyl, ethoxy-i- propyl, ethoxy-n-butyl, ethoxy-i-butyl, n- propyloxymethyl, n-propyloxy ethyl, n-propyloxy-n- propyl, n-propyloxy-i-propyl, n-propyloxy-n-butyl, or n-propyloxy-i-butyl.

wherein Z is -(CH₂)_B-0-B wherein n is 1-4 and

B is a phenyl or a fused polyphenyl aromatic ring system substituted by 1-4 sulfonic acid residues. Preferred embodiments of B include 2,8-naphth-l-yl disulfonic acid, - , - ,-phenanthr-1-yl trisulfonic acid, and -,-,-, (tetraphenyl?) trisulfonic acid (TSP) .

wherein D is -P0₃"², -B(OH)₂, -(CH₂)_n-OH wherein n is 1-4, -COO(CH₂)„H wherein n is 1-4,

wherein E is alkyl(1-4C), or is

A or Z.

wherein D is as defined above.

B. Self-Assembly

From parameters describing the known uptake characteristics of LY, the relative volumes of endosomes and lysosomes inside cells, and the various kinetic parameters known to describe formation of hydrazones, it can be calculated that antiviral concentrations of the product hydrazone can be obtained inside monocyte lysosomes using sub-cytotoxic concentrations of the precursors, and without forming appreciable conjugate outside of the monocyte. These calculations are done manually or using computer simulation. In addition, verification of the various parameters can be obtained using a model system in cats involving feline im unosuppressive virus (FIV) which is a T-lymphytropic lentivirus, wherein the virus can infect feline T-lymphocytes, feline peripheral monocytes derived macrophages, and CCL94 feline kidney-derived fibroblasts in vitro. As the symptoms of FIV are similar to those of AIDS infections in humans, this provides a suitable model system (Pederson, N.C., et al Science (1987) 235:790- 793; Elder, J. , (1988, personal communication)).

The cytotoxicity of Lucifer Yellow and the analogs within the scope of the invention is low. The LD^ for LY injected intravenously in mice is greater than 1 g/kg (Silverstein, S., (1987, personal communication)) and LY exhibits no observable cytostatic activity in vitro against murine thiol- macrophages at 600 μM concentrations (Swanson, J.A. , et al, J Cell Biol (1985) 100:851-859).

c. Selectivity for Monocytes and Derivatives

Verification of the various parameters affecting dosage and administration can be obtained using a model system in cats involving feline immunosuppressive virus ("FIV") which is a T-lymphotropic lentivirus, wherein the virus can infect feline T-lymphocytes, feline peripheral monocytes derived macrophages and CCL94 feline kidney-derived fibroblasts in vitro. As the symptoms of FIV are similar to those of AIDS infections in humans, this provides a suitable model system for this particular indication, as well as for retroviral infection in general (Pederson, N. C. , et al. Science (1987) 235:790-793: Elder, J., (1988, personal communication)) .

The cytotoxicity of Lucifer Yellow and the analogs within the scope of the invention is low. The LD50 for LY-CH injected intravenously in mice is greater than 1 g/kg (Silverstein, S., (1987, personal communication) ) and LY exhibits no observable cytostatic activity in vitro against murine thiol-macrophages at 600 μM concentrations (Swanson, J.A., et al, J Cell Biol (1985) 100:851-859) . Lucifer Yellow itself is a nontoxic, fluorescent, membrane-impermeable dye (Stewart, W.W., Cell (1978) 14.:741) which is taken up by monocyte/macrophage endosomes through pinocytosis and is concentrated in the lysosomes. The rate of pinocytosis and accumulation in lysosomes is higher for macrophages, especially activated macrophages, than for other cell types such as fibroblasts (Besterman, J.M. , et al, J Cell Biol (1981) 11:916-917; Steinman, R.M. , et al, J Cell Biol (1976) .68:665-687) and does not bind to proteins to a substantial degree. The analogs of Lucifer Yellow which are within the scope of this invention exhibit similar properties. Because no specific receptor interaction is involved in the uptake of Lucifer

Yellow by monocytes, these analogs which retain the membrane impermeable nature of LY are similarly taken up by the lysosomes. In addition, unlike suramin, the LY and analog-derived compounds of the invention are not bound to serum proteins. Thus, their bioavailability is not diminished in this way.

Thus, the compounds derived from Lucifer Yellow and its analogs are expected to home to the virus-tropic cells, such as monocytes, rather than to the non-tropic cells, for example neutrophils. In addition, as the Lucifer Yellow-derived compounds are concentrated in the lysosomes. Concentration of the precursors in the desired target cells results, of course, in magnified selectivity.

Thus, the selectivity of precursors derived from Lucifer Yellow and its analogs are expected to home to the virus-tropic cells, such as monocytes, rather than to the non-tropic cells, for example neutrophils. In addition, as the Lucifer Yellow- derived materials are concentrated in the lysosomes, where the pH is relatively low (4-5) reaction to form the hydrazone (semicarbazone) is favored.

As described in PCT application WO88/00047, incorporated herein by reference, concentration of the precursors in the desired target cells results in a magnified selectively when the toxic material is formed by a second order reaction from the precursors. Thus, for example, a three-fold higher concentration of each precursor in the target cells as compared to other destinations would result in a nine-fold concentration differential between the target and the remaining tissues. In the present application, this is enhanced by the favorable conditions found in the targets for formation of the conjugate—i.e., the conditions are such that the rate constant for the forward reaction is increased.

D. Formulation and Administration

As illustrated hereinbelow, the compounds of the invention are successful in generating conjugates in situ which inhibit the proliferation of retroviruses in susceptible or infected cells. In conducting this inhibition of proliferation, the cells are contacted with the precursors which self-assemble in the cells or in the vicinity thereof to generate the effective conjugates. Contact of the cells can be conducted in culture or in vivo.

In the instances where the components are to be administered as pharmaceuticals, they are formulated in a conventional manner and administered either localized at the targeted site or systemically. As is generally the case for self-assembling, pharmaceutically active products, administration at the intended site of activity enhances the assembly of the biologically active conjugate by the high local concentrations of the components, and diffusion of the components away from the target site automatically diminishes their tendency to combine. As the compounds are to be administered as pharmaceuticals, they are formulated in a conventional manner and administered either localized at the targeted site or systemically. Generally known methods of formulation are employed, including sustained-release matrices, conventional excipients, and simple solutions, as described, for example, in Remington's Pharmaceutical Sciences (latest edition) , Mack Publishing Company, Easton, Pennsylvania.

Generally known methods of formulation are employed, including sustained-release matrices, conventional excipients, and simple solutions, as described, for example, in Remington's Pharmaceutical Sciences (latest edition) , Mack Publishing Company, Easton, Pennsylvania. In some instances, the conjugate formed by the precursors of the invention can be administered in a form in which conjugation has already taken place. Thus, the resulting pharmaceutically active conjugate can itself be formulated and administered to the patient. However, it is preferable to administer the precursors separately, either simultaneously and in the same location, or in different locations at the same time, or in the same location at different times.

Systemic administration is feasible in using the precursors as treatment for HIV infection, since it is known that LY is taken up and concentrated in the endosomes and lysosomes of macrophages. This has been reported for murine thiol-elicited macrophages. These organelles have lower Ph (about 5) , thus enhancing the production of the conjugated semicarbazone. This results in a concentration of the conjugate in T-cells which contact macrophages, as opposed to kidney or other parts of the body. The compound thus tends to accumulate and react in locations where the virus infection is likely to be found.

The dosage level required depends, of course, on the choice of specific end-products and/or components. For most embodiments, a dosage of the conjugate on the order of 0.1-5 g/week appears suitable; for LY and the complementary preferred compounds set forth above, i.e., the precursors administration of 100mg-3g/week of each component is appropriate.

In a preferred approach, by derivatizing the precursors so as to administer them in particulate form, for example, by conjugating them to a carbohydrate, selective killing of the macrophage cells which harbor the virus can be accomplished. Because the drugs are perceived as particles, they will be readily attacked by the macrophages.

Systemic administration is feasible in using the compounds as treatment for retroviral infection, since it is known that LY is taken up and concentrated in the endosomes and lysosomes of macrophages. This has been reported for murine thiol-elicited macrophages. This results in a concentration of the compound in T-cells which contact macrophages, as opposed to kidney or other parts of the body. The compound thus tends to accumulate and react in locations where the virus infection is likely to be found. The compounds can be administered intravenously, intramuscularly, orally, or intraperitoneally, at about 1-1000 g/kg body weight. The dosage level required depends, of course, on the choice of specific compound, as well as the nature of the subject being treated for retroviral infection and the magnitude of the infection or the potential therefor. Administration may have either therapeutic or prophylactic effects or both. For most embodiments, a dosage of the compound on the order of 0.1-5 g/week appears suitable.

In a preferred approach, by derivatizing the compounds so as to administer them in particulate form, for example, by conjugating them to a carbohydrate, selective killing of the macrophage cells which harbor the virus can be accomplished. Because the drugs are perceived as particles, they will be readily attacked by the macrophages.

The following examples are intended to il¬ lustrate, but not to limit, the invention.

Preparation A Preparation of Biohenyl-4.4 '- dicarboxaldehvde rθHC-fY-4)-CHO

Biphenyl-4,4^,-dicarbonitrile (0.679 mg, 3.33 mmol) was dissolved in 100 ml dry tetrahydrofuran (THF) and cooled to -78°C under nitrogen. DIBAL (7 ml of a 1.5 molar solution in toluene, 10.5 mmol) was added dropwise via syringe, and the mixture was allowed to warm to ambient temperature and was stirred for 18 hr. The reaction progress was followed by treating small aliquots with 10% aqueous H₂S0₄ and then saturated aqueous NaHC0₃. Aliquots were reduced with excess NaBH₄, and thin layer silica gel chromatography with 40:60 EtOAc/hexanes was used to detect the 4,4'- bis(hydroxymethyl)biphenyl and unreacted dinitrile 5 (the dialdehyde and dinitrile are almost inseparable on silica gel) . After the reaction was complete, the mixture was cooled to 0°C, and 10% aqueous H₂S0₄ was added cautiously with vigorous stirring until all the salts were dissolved. The organic layer was 10 separated, ad the aqueous layer was washed with CHC1₃ several times. The combined organic extracts were washed with saturated aqueous NaHC0₃, and then brine and finally dried over MgS0₄ and stripped of solvent to yield 85% of the desired product. ^XH NMR (CDC1₃) as I⁵ expected. HRMS: m/e=211.0761 (observed), m/e=211.0759 (expected for [Cι₄Hι₀O₂]₊) .

Preparation B Preparation of 4-Amino-1.8-naphthalic anhvdride-3.6-disulfonic acid. disodiuTn gait-- ⁰ This molecule is also an intermediate in the synthesis of Lucifer Yellow. The following is a modification of an existing procedure (Sealer and Forster, 1948). A 25-ml flask was charged with 3.3 ml of fuming H₂S0₄ (13 mmol S0₃) and cooled to 0°C. ⁵ 4-Amino-l,8-naphthalic anhydride (1 g, 4.7 mmol) was added in portions with stirring. The dark brown mixture was heated to 95^βC for 1.8 hr. Then the mixture was cooled to ambient temperature, and added carefully to 170 ml of doubly distilled water at 5^βC. ⁰ NaCl was added until the solution became saturated (about 75 g) . The mixture was filtered, and the residue was dried in vacuo, yielding 91% of the desired product. *H NMR (D₂0) : as expected. HRMS: 371.9484 (found); 371.9488 (expected for [C₁₂H₅NS₂0₉]-) . ⁵ Preparation C

Preparation of LY Conversion of the anhydride of Preparation B to Lucifer Yellow dilithium salt was carried out as described by Stewart (1981) . HRMS for the synthetic LY: m/e=443 .9929 (found) ; m/e=443 .9920 (calculated for

[C₁₃H₁₀N₅S₂O₉]-) .

Exam le _t Preparation of A2

Metaphthalaldehyde (190 g, 1.42 mmol) was dissolved in a mixture of 15 ml water and 8 ml methanol. A solution of LY dilithium salt (25 mg, 54 μmol) dissolved in 10 ml water was added dropwise over 90 min at 25°C. The reaction mixture was stirred for 16 hr at 25°C, and then extracted repeatedly with chloroform to remove the metaphthalaldehyde. The water was removed in vacuo, and the product was purified by using semipreparative Cι₈-silica high- pressure liquid chromatography (HPLC) and a gradient from 100% water to 12% acetonitrile/88% water (25 min, flow rate 2 ml/min) . Yield of the compound of Formula A2: 43%. ^l NMR as expected. δ 8.44 (S, 1H) , δ 7.52 (m, 1H) , δ 7.33 (m+S, 3H) , δ 7.00 (m, 1H) . HRMS: m/e=560.0165 (found), 560.0182 (expected for [C_MHM^SJO_ΪO)-).

Example 2 Preparation of A4 Biphenyl-4,4'-dicarboxaldehyde (1.55 g, 7.4 mmol) was dissolved in 300 ml DMF and heated to 40°C. A solution of LY (disodium salt, 1.7 g. , 4.1 mmol) in 60 ml dilute aqueous HC1 (pH adjusted to 5.0) was added dropwise over 18 hr at 40°C. After the solvent was evaporated, the product was dissolved in water and extracted with chloroform to remove excess dialdehyde. The aqueous layer was stripped of solvent, redissolved in 800 ml doubly distilled water, treated with 20 milliequivalents of Dowex-50W cation exchange resin (Li⁺ form) , and filtered through Celite. After evaporation, 2.2 g of crude product was obtained. The product was purified by using a full preparative reverse-phase diphenylsilica HPLC column (2.5 cm outside diameter, Vydac #219TP1022) with water as a solvent under isocratic conditions. After drying in vacua, the yield of the purified product was 0.57 g (21% based on LY) . *H NMR (D²0) as expected and ¹³C NMR (D₂0/DMSO) . HRMS: 636.0482 (found), 636.0495 (calculated for

.

Example 3 Preparation of H4 A solution of A4 (5.5 mg, 8.6 μmol) and LY (8 mg, 17 μmol) in dilute aqueous HCl (1.5 ml, pH 5.3) was stirred overnight at 35^βC. The product was purified by using a semipreparative reverse-phase C₁₈ silica column with H₂0 as the solvent (isocratic conditions) . The produce H4 elutes later than LY and earlier than A4_* The yield of H4 was 52% based on A4_* ^JH NMR in D₂0 as expected. HRMS: 1063.0356 (found), 1063.03887 (calculated for [QuH^Nu u]') .

Example 4 Preparation of Bl LY (10 mg, 22 μmol) was dissolved in dilute aqueous HCl (pH 5.4), and benzaldehyde was added until the solution was more than saturated (molar excess of benzaldehyde) . The mixture was stirred at 39°C for 18 hr and extracted repeatedly with chloroform to remove the benzaldehyde. After the water was removed in vacuo, the residue was purified by using semipreparative C₁₈-silica reverse-phase HPLC (see the A2 preparation procedure for details) . The yield of Bl, based on LY, was 76%. *H NMR as expected. HRMS: 532.0249 (found), 532.0233 (calculated for

Example 5 Preparation of B2 Lucifer yellow lithium salt (0.3 g) and 4- biphenylcarboxaldehyde (0.36 g) were added in a solution containing 30 ml THF and 20 ml distilled water. The solution was left at room temperature with stirring overnight. THF was removed from the solution filtered. Solid KC1 was added (10%) and the crude product was precipitated. B2 was then further purified by HPLC.

^XH NMR(DMSO-d_<j) as expected. HRMS: 610.0682 (observed); 610.0702 (expected). Example 6

Preparation of B3 Lucifer yellow lithium salt (0.36 g) and m-tolualdehyde (0.12 g) was mixed in a solution containing (30 ml THF and 20 ml distilled water) overnight. THF was removed from the solution by rotary evaporation under vacuum. The product was precipitated from the solution by adding solid NaCl (final concentration = 10%) *H NMR (DMSO-d as expected. Example 7

Preparation of CBD2 A. Trisodium 8-methoxy-l,3,6- pyrenetrisulfonate was prepared according to the published procedure (J Phvs Chem (1982) 86:4826) . This compound was reacted with 3-bromomethylbenzalde¬ hyde in methanol. The desired product, crystallized from ethanol and ether, was called CBD1. LRMS: 619, 597, and 575 found (expected for m³+2Na⁺, m³+H⁺+Na⁺, m"³+2H⁺ respectively) . B. One equivalent CBD1 (64 mg) and 3 equivalent LY (137 mg) were dissolved in distilled water (10 ml) . Solid NaCl was added to this solution (20% NaCl) . CBD2 was slowly precipitated from the solution. The precipitate (crude CBD2) was washed with ethanol and ether, and dried under vacuum, then purified by HPLC.

Example 8 Preparation of LD1 Lucifer yellow lithium salt (0.36 g) and biphenyl-4, 4'-biscarboxylic acid hydrazide (0.54 g) was reacted at 50°C in DMF aqueous solution (40 ml, 50% DMF) . The LD1 was purified from the solution by HPLC. *H NMR (DMSO-d as expected. gxample 9 Preparation of LD2 4-Amino-l,8-naphthalic anhydride-3,6- disulfonic acid, disodium salt (0.42 g) and ethyl 4- aminobenzoate (0.5 g) were added in distilled water (50 ml) . The mixture was heated to reflux for 18 hours, and filtered after cooling. Solid KC1 was added to the filtered solution. The precipitate was collected by filtration, washed with ethanol and ether, and dried to yield LD2 at 65%. ^XH NMR (DMSO- d₆) : as expected. LRMS: 597 (expected for m+H⁺) and 635 (expected for m+K⁺) (found) .

Example 10 Preparation of LD3 4-Amino-l,8-naphthalic anhydride-3,6- disulfonic acid, disodium salt (0.42 g, 1 equivalent) and 4-aminophenylphosphonic acid (0.35 g, 2 equivalents) were added in distilled water (50 ml) . The mixture was heated to reflux for 18 hours. The conversion was checked by HPLC and the reaction was not complete. Additional 4-aminophenylphosphonic acid (0.35 g, 2 equivalents) was added in the mixture and the mixture was heated to reflux for another 18 hours, and filtered after cooling. Solid KCl was added to the filtered solution (10% KCl) and the desired product was slowly precipitated from the solution. The precipitate was collected by filtration, washed with ethanol and ether, and dried under vacuum to yield LD3 at 57%. *HNMR (DMSO-d^: as expected. LRMS: 525 (m+H⁺) and 563 (m+K⁺) found.

Example 11 Preparation of LD4 4-Amino-l,8-naphthalic anhydride-3,6- disulfonic acid, disodium salt (0.11 g) and 3- aminophenylboronic acid monohydrate (0.16 g, 4 equivalents) were added in distilled water (40 ml) . The mixture was heated to reflux for 18 hours. Additional 3-amino phenylboronic acid (0.16 g) was added and the mixture was refluxed for another 18 hours. The mixture was filtered after cooling. Solid KCl was added to the filtered solution (10% KCl) and the desired product was precipitated from the solution. The precipitate was washed with ethanol and ether, and dried under vacuum to yield LD4 (150 mg) . Η NMR (DMSO-d6) : as expected. LRMS: 683 found (expected for [C₁₈H₉N₂Oι₀S₂BK_j ⁺] .

Example 12 Preparation of LD40H

4-Amino-l,8-naphthalic anhydride-3,6- disulfonic acid, disodium salt (0.42 g, 1 equivalents) and 4-aminophenethyl alcohol (Aldrich Chemical Co.) (0.40 g, 3 equivalents) were combined in distilled water (50 ml) . The mixture was heated to reflux for

18 hours and filtered over zeolite when cooled. Solid NaCl was added to the filtered solution (20% NaCl) and the desired product was slowly precipitated from the solution. The precipitate was collected by filtration, washed with ethanol and ether, and dried to yield LD40H at 54% yield. ^XH NMR (D₂0) : consistent with structure . LRMS : 513 (expected for [ C₂₀H₁₄O₉N₂S₂NaH₂ ⁺ ] ) and 537 found (expected for [C₂₀H₁₄O₉N₂S₂Na₂H⁺] .

Example 13 Preparation of LD5 4-Amino-1,8-naphthalic anhydride-3,6- disulfonic acid, disodium salt (0.11 g, 1 equivalent) and 2-(4-aminophenyl)-6-methyl-benzothiazole (0.30 g, 5 equivalent, Aldrich Chemical Co.) were added in the solution (DMF 30 ml, distilled water 10 ml) . The mixture was heated to reflux for 120 hours. The desired product was purified from the mixture by HPLC and C18 reverse phase column. LRMS: 710 and 672 found (expected for m"²+3K⁺ and m'²+2 K⁺+H⁺) . Example 14 Preparation of LD6 4-amino-l,8-naphthalic anhydride-3,6- disulfonic acid, disodium salt (0.42 g) of Preparation B and 4-biphenylcarboxylic acid hydrazide (MTM

Research Chemical) (0.42 g) were added in distilled water (50 ml) . The mixtures were heated to reflux for 18 hours, and filtered when cooled. Solid KCl was added to the filtered solution (10% KCl) and the desired product was slowly Precipitated from the solution. The precipitate was collected by filtration, washed with ethanol and ether, and dried to yield LD6 at 70%. *H NMR (DMSO-d : consistent with structure. LRMS: 565 found (expected for [C₂₅H₁₅0₉ϊJ₃K₃ ⁺ .

Example 15 Effect of A4 on HIV and FIV Compound A4 was first tested for its ability to inhibit reverse transcriptase isolated either from HIV-1 or from FIV using a standard protocol. The results are shown in Figures 4 and 5 for HIV-1 and FIV respectively. In each case, A4 was more potent than suramin in inhibiting the enzyme. The approximate IC50 values are 200 μM against HIV-1 and 20 μM against FIV.

A4 was also tested for its ability to inhibit the growth of FIV in a culture containing feline kidney cells using standard assays. As shown in Figure 6, using a concentration of 30 μg/ml A4, FIV growth and proliferation were virtually completely inhibited within 20 days following infection; administration of 20 μg/ml A4 resulted in corresponding inhibition 40 days thereafter. Smaller concentrations had similar effects but were less dramatic.

The results in Fig. 7 show the effect of A4 (20 μg/ml) on the viability of infected cells. This concentration of A4 was able to reestablish 100% viability of the infected cells within 50 days of infection.

Finally, Fig. 8 shows that the macrophage cell line U937 takes up and retains A4. In an in vivo test, compound A4 was injected into two mice intraperitoneally at a dose of 60 mg/kg. No apparent toxicity was evident 15 days later, showing that the compound is relatively non-toxic.

Example 16 Effect of Precursors and Conjugates on HIV The test culture system uses MT-2 cells, which are a T-cell-derived leukemic cell line derived from a person infected with HTLV-1 (Richman, D.D., et al., J EXP Med (1987) 166:1144-1149). When these cells are cultured and then infected with HIV-1, they form multinucleated macrocells called syncytia by self-infusion. These syncytia can be observed microscopically and quantitated.

Infected cell stock is prepared by adding HIV to 10 ml MT-2 cell suspension containing 6 x 10⁵ cells/ml in RPMI=1640 medium supplemented with 2mM glutamine, polybrene, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal calf serum. To 10 ml of MT-2 suspension is added 60 ul of HIV virus concentrate (10⁷ "tissue 50% infectious disease units" (TCID₅₀)/ml, strain 5735).

Solutions of the precursor or conjugate to be tested at 2x their final concentration were diluted with the infected cell stock in 24 well tissue culture plates. 100 ul each of the drug stock and infected cell stock were used. After 3-4 days incubation at 37°C, 5% C0₂, 100% humidity, the cells were checked for syncytia formation and rated on a scale from zero (no syncytia) to four (a maximum viral cytopathic effect— i.e., no antiviral activity).

Lucifer Yellow concentrations of 10 μM, 40 μM and 200 μM were tested; all showed no antiviral effect and received a rating of 4; LY was not antiviral even at 800 μM. A2 was tested at 10 μM, 40 μM and 200 μM; there was no antiviral effect at the two lower concentrations, but a slight effect (a rating of 3) at 200 μM; at 400 μM and 800 Ml A2 completely inhibited syncytia formation. When the drug contained a combination of 100 μM LY plus 100 μM A2, a marked antiviral effect (0 and 1 in two separate determinations) was found. Lower concentration levels of the combined precursors (20 μM and 5 μM of each gave no antiviral effect.

In the foregoing results, stock solutions used to prepare the combination drugs at concentrations of 10-fold the final concentration of the drug solution added to the wells were incubated together at 25°C for an hour before dilution to begin the incubation with the cell cultures.

In a subsequent experiment, conducted similarly, this recombination was not done, so no conjugation prior to addition to the culture could occur. Under these conditions, higher concentrations of the combinations were required. Concentrations of 100 μM A2 plus μM LY in this assay, without precombination, did not show an antiviral effect. However, when the concentration of both components was raised to 200 μM, a moderate (2-3) antiviral effect was exhibited, and combinations where both concentrations were 400 μM were strongly antiviral (0) . However, 400 μM A2 alone and 800 μM A2 also show pronounced antiviral effects, and 200 μM A2 alone, a moderate one (3) . At all concentrations of LY tested (100-800 μM) no antiviral effect was found.

Similar assays, but using HIV viral antigen expression in MDM as criterion for antiviral activity, were conducted. In these assays, the expression was rated from 0 (complete inhibition) to 180 (no drug effect) . In this assay, the following results were obtained, as shown in Table 1:

100 μM LY + 100 μM A2 83

As shown in the table, according to this assay, the effect of LY can be dramatically enhanced by the addition of A2. None of the compounds used was cytotoxic at the concentrations used hereinabove to either cat kidney epithelial cells after 4 days or to human monocyte-derived macrophages after 5 days. However, preincubated 150μM A2+250μM LY resulted in 50% inhibition of all growth over periods where unreacted components showed no effect.

Claims

Claims 1. A method to inhibit proliferation of retrovirus in cells infected with or susceptible to said virus which method comprises contacting said cells with an antiviral compound of the formula:

R2

R-NHCONHN=C

(1)

\

Rl

wherein R has the formula:

in which Z represents one or two non-interfering substituents; and wherein Rl is H or alkyl(1-6C); and

R2 is alkyl (1-6C) , or aryl(6-10C) or linked aryl(6-10C) containing 2-5 such aryl groups said alkyl or aryl substituents being unsubstituted or substituted by 1-2 non-interfering groups; or the pharmaceutically acceptable salts thereof.

2. The method of claim 1 wherein Rl is H.

3. The method of claim 1 wherein R2 is aryl(6-10C) or linked aryl(6-10C) containing 2-5 such aryl groups.

4. The method of claim 3 wherein the substituent

in formula (1) is of the formula =HC-Y-CH0 wherein Y is selected from the group consisting of Y-l to Y-9 in Fig. 2.

5. The method of claim 4 wherein the compound of formula (1) is of the formula:

wherein R is as defined in claim 1, or the pharmaceutically acceptable salts thereof.

6. The method of claim 5 wherein the compound is of the formula

or the pharmaceutically acceptable salts thereof.

7. A method to synthesize a compound of the formula

which method comprises contacting a compound of the formula

R-NHCONHNH₂ wherein R is as defined in claim 1, with a compound of the formula

wherein Rl and R2 are as defined in claim 1; and recovering the compound of Formula (1) .

8. A method to inhibit proliferation of a retrovirus in cells infected or susceptible to said virus which method comprises contacting said cells with an anti-viral compound of the formula

R-R3 (3)

wherein R has the formula:

in which Z represents one or two non-interfering substituents; and wherein R3 comprises aryl(6-10C) or linked aryl(6-10C) containing 2-5 such aryl groups, said aryl substituents being unsubstituted or substituted by 1-2 non-interfering groups; or the pharmaceutically acceptable salts thereof.

9. The method of claim 8 wherein the compound of Formula 8 is of the formula:

H3

10. The method of claim 8 wherein at least one aryl of R3 is bound directly to R.

11. The method of claim 8 wherein at least one aryl group of R3 is linked to R through a linker of the formula -NHCO- .

12. The method of claim 8 wherein the compound is selected from the group consisting of the compounds of Fig. 10.

13. A compound of the formula R-R3.

14. The compound of claim 13 which is of the formula

15. The compound of claim 13 wherein at least one aryl of R3 is bound directly to R.

16. The compound of claim 13 wherein at least one aryl group of R3 is linked to R through a linker of the formula -NHCO-.

17. The compound of claim 13 which is selected from the group consisting of the compounds of Fig. 10.

18. A method to synthesize a compound of the formula R-R3, wherein R3 is defined as in claim 8; and wherein R is coupled to R3 directly to an aryl residue, which method comprises treating a compound of the formula

or a salt thereof with a compound of the formula R3NH₂, under conditions wherein R-R3 is produced.

19. A method to synthesize a compound of the formula R-R3, wherein R3 is defined as in claim 8; and wherein R is coupled to R3 through a linkage of the formula -NHCO-, which method comprises treating a compound of the formula

or a salt thereof with a compound of the formula R3'- CON₃, wherein R3' represents the remainder of R3 other than -CO-NH- under conditions wherein R-R3 is produced.

20. A method to inhibit proliferation of retrovirus in cells infected with or susceptible to said virus which method comprises contacting said cells with the precursors of an antiviral conjugate, wherein said conjugate is of the formula:

R-NHCONHN=CH-Y-CH=NNHCONH-R (1)

wherein R has the formula:

in which Z represents one or two non-interfering substituents, including the pharmaceutically acceptable salts thereof,

Y is selected from the group consisting of the moieties shown as Y-l to Y-9 in Fig. 2; and wherein said precursors are capable of self- assembly into the conjugate in the presence of virus- infected or virus-susceptible cells.

21. The method of claim 20 wherein the precursors are of the formulas:

R-NHCONHN=CH-Y-CHO (2) and R-NHCONHNH₂. (3) 22. The method of claim 20 wherein R is

including the pharmaceutically acceptable salts thereof.

23. The method of claim 22 wherein the precursors of formulas (3) and (2) respectively are Lucifer yellow and A2, and the conjugate is H2.

24. A compound of the formula:

R-NHCONHN=CH-Y-CH=NNHCONH-R

wherein R has the formula:

in which Z represents one or two non-interfering substituents, including the pharmaceutically acceptable salts thereof, and

Y is selected from the group consisting of the moieties shown as Y-l to Y-9 in Fig. 2.

25. The compound of claim 24 wherein R is of the formula:

26. The compound of claim 25 which is selected from the group consisting of HI to H9 as shown in Figs. 11-1 through 11-4.

27. A composition of retrovirus to inhibit proliferation in cells infected with or susceptible to said virus which contains, as active ingredients, precursors of a compound of the formula:

R-NHCONHN=CH-Y-CH=NNHCONH-R

wherein R has the formula:

Y is selected from the group consisting of the moieties shown as Y-l to Y-9 in Fig. 2; wherein said precursors are capable of self- assembly into the conjugate in the presence of said cells.

28. The composition of claim 27 wherein said precursors are compounds of the formula:

R-NHCONHN=CH-Y-CHO (2) and

R-NHC0NHNH₂ (3 )

wherein R has the formula:

29. The composition of claim 28 wherein R has the formula:

including the pharmaceutically acceptable salts.

30. The composition of claim 29 wherein the compound of formula (2) is selected from the group consisting of A1-A9 as shown in Figs. 11-1 through 11-4.

31. A method to inhibit proliferation of retrovirus in cells infected with or susceptible to said virus which method comprises contacting said cells with the precursors of an antiviral conjugate wherein said conjugate is of the formula:

^•R

R-NHCONHN=C (6)

wherein R has the formula:

in which Z represents 1 or 2 noninterfering substituents, including the pharmaceutically acceptable salts thereof; and wherein R¹ is H or alkyl (1-6C) ; and R² is alkyl (1-6C) or aryl (6-10C) or linked aryl (6-10C) containing 2-5 such aryl groups; said alkyl or aryl substituents being unsubstituted or substituted by 1-2 noninterfering groups; and wherein said precursors are capable of self- assembly into the conjugate in the presence of said virus-infected or virus-susceptible cells.

32. The method of claim 31 wherein the precursors are of the formulas:

R-NHCONHNHj (3)

and

C=0 (7)

R¹

wherein R, R¹ and R² are as defined in claim 31.

33. The method of claim 32 wherein R is of the formula:

including its pharmaceutically acceptable salts.

34. The method of claim 31 wherein R¹ is H.

35. The method of claim 32 wherein the compound of formula (7) is selected from the group consisting of the dialdehydes of Y-l through Y-9 in Fig. 2.

36. The method of claim 32 wherein the compound of formula (7) is selected from the group consisting of benzaldehyde, 4-biphenylcarboxaldehyde and ortho-, meta- or paratolualdehyde.

37. The method of claim 32 wherein the compound of formula (7) is the condensation product of 2-, 3-, or 4-bromomethylbenzaldehyde with a salt of 8-methoxy-l,3,6-pyrenetrisulfonic acid.

38. A composition useful to inhibit the proliferation of retrovirus in cells infected with or susceptible to said virus wherein said composition contains as active ingredients, precursors of the compound of the formula:

^-R² R-NHCONHN=C (6)

wherein R has the formula:

in which Z represents 1 or 2 noninterfering substituents, including the pharmaceutically acceptable salts thereof; and wherein R¹ is H or alkyl (1-6C) ; and

R² is alkyl (1-6C) or aryl (6-10C) or linked aryl (6-IOC) containing 2-5 such aryl groups; said alkyl or aryl substituents being unsubstituted or substituted by 1-2 noninterfering groups; and wherein said precursors are capable of self- assembly into the conjugate in the presence of said virus-infected or virus-susceptible cells.

39. The composition of claim 38 wherein said precursors are of the formula:

R-NHC0NHNH₂ (3 )

and

R² _*

C=0 (7)

wherein R, R¹ and R² are as defined in claim 19.

40. The composition of claim 39 wherein R is of the formula:

including its pharmaceutically acceptable salts.

41. The composition of claim 38 wherein R¹ is H.

42. The composition of claim 39 wherein the compound of formula (7) is selected from the group consisting of Y-l through Y-9 in Fig. 2.

43. The composition of claim 39 wherein the compound of formula (7) is selected from the group consisting of benzaldehyde, 4-biphenylcarboxaldehyde and ortho, meta or peritolualdehyde.

44. The composition of claim 39 wherein the compound of formula (7) is the condensation product of

2-, 3-, or 4-bromomethylbenzaldehyde with a salt of

8-methoxy-l,3,6-pyrenetrisulfonic acid.

45. A compound of the formula:

R-NHCONHN=C: (6)

wherein R has the formula:

(4 )

in which Z represents 1 or 2 noninterfering substituents, including the pharmaceutically acceptable salts thereof; and wherein R¹ is H or alkyl (1-6C) ; and R² is alkyl (1-6C) or aryl (6-10C) or linked aryl (6-10C) containing 2-5 such aryl groups; said alkyl or aryl substituents being unsubstituted or substituted by 1-2 noninterfering groups.

46. The compound of claim 45 wherein R is of the formula:

including its pharmaceutically acceptable salts.

47. The compound of claim 45 wherein R¹ is H.

48. The compound of claim 45 wherein R*R²C= is the residue of the dialdehyde of any of Y-l to Y-9 of Fig. 2 upon equimolar reaction with a semicarbazide.

49. The compound of claim 45 wherein R*RC= is the residue of benzaldehyde, 4- biphenylcarboxaldehyde and ortho-, meta- or paratolualdehyde upon equimolar reaction with a semicarbazide.

50. The compound of claim 45 wherein R^XR²C= is the residue of the condensation product of 2-, 3-, or 4-bromomethylbenzaldehyde with a salt of 8-methoxy- 1,3,6-pyrenetrisulfonic acid upon equimolar reaction with a semicarbazide.