Method for the suppression of viral growth

Abstract

A method for suppressing undesired viral growth in a host which comprises administering to the host an effective amount of a compound of the formula: ##STR00001##
wherein R₁, R₂, R₃ and R₄ are independently selected from the group consisting of HO—, CH₃O— and CH₃(C═O) O—. The method is exemplified by inhibiting Tat transactivation of a lentivirus and in suppressing Herpes simplex virus.

Description

This is a continuation-in-part of U.S. application Ser. No. 08/627,588, filed Apr. 4, 1996 now U.S. Pat. No. 5,663,209, which is a division of U.S. application Ser. No. 08/316,341, filed Sep. 30, 1994.

The invention described and claimed herein was made in part with funds from Grant No. AI 32301 from the National Institutes of Health and in part with funds from U.S. Army Medical Research Grant DAMD 17-93-C3122. The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the isolation, purification and characterization of derivatives of 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (nordihydroquaiaretic
where:

C⁻: Control sample (no DNA, no drug)

CT⁻: Control sample (+DNA, no drug)

C⁺: Drug-treated sample (no DNA, +drug)

CT⁺: Drug-treated sample (+DNA, +drug)

Optimization of the Transfection Technique: Various techniques are utilized in the DNA transfection of eukaryotic cells. These procedures include DNA coprecipitation with calcium phosphate or cationic polymers, cell membrane weakening either by chemical means (detergents, solvents, enzymes, amphophilid polymers) or by physical means (thermic, osmotic or electric shocks, or particle bombardment). These techniques suffer, to some extent, variable efficiency and varying degrees of cytotoxicity.

Prerequisites for cells to be amenable to DNA uptake, i.e., to cross the intact cytoplasmic membrane, are “compaction and masking of DNA charges” (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). These requirements have been successfully met with the newly developed Transfectam® procedure. The Transfectam reagent (dioctadecylamidoglycyl spermine) is a synthetic cationic lipopolyamine which contains a positively charged spermine headgroup with a strong affinity for DNA (K_d=10⁵−10⁻⁷ M). This spermine headgroup is covalently attached to a lipid moiety by a peptide bond. The lipospermine molecules bind to DNA, coating it with a lipid layer. In the presence of excess lipospermine, cationic lipid-coated plasmid DNA vesicles are formed and the lipid portion of the complex fuses with cell membrane. DNA internalization is-believed to occur by endocytosis.

Transfectam-mediated transfection has been shown to offer greater efficiency than existing methods (Barthel et al., DNA and Cell Biology 12 (6): 553-560, 1993). In addition, Transfectam® is a stable and virtually non-cytotoxic reagent. However, factors for optimization of transfection in the specific COS cell line had to be addressed. These factors include the duration of transfection, the ratio of the Transfectam reagent to DNA, DNA concentration and other dilution factors such as NaCl volume and strength. The results of optimization of transfection conditions are shown below.

a) Duration of transfection: COS cells were incubated with a fixed plasmid DNA concentration in time course studies. These studies aimed at the selection of the suboptimal incubation time point for inhibition studies of SEAP expression by various test compounds. The results of the time-course induction of SEAP expression (results not shown) indicate a gradual time-dependent increase in SEAR expression. The onset of this induction began at less than 4 hours and reached a maximum at 24 hours. No significant difference was observed between the 10, 12 and 15-hour values. Therefore, the 12-15 hours endpoint was selected as the appropriate suboptimal incubation period for inhibition of SEAP expression in all subsequent drug screen studies.

b) DNA concentration: The optimal DNA concentration for transfection was determined based on previous studies with Transfectam reagent (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). For contransfection, the ratio 2:1 (nonselected gene/selected gene) was found to be the most appropriate as reported elsewhere (Hsu et al., Science 254: 1799-1802, 1991, the entire contents of which are hereby incorporated by reference and relied upon). The nonselected pBC12/HIV/SEAP plasmid was utilized at a concentration of 0.35 μg/well and pBC12/CMV/t2 plasmic coding for Tat function at a concentration of 0.75 μg/well in Linbro® 24 flat bottom well of 17-mm diameter plates.

c) Ratio of Transfectam to DNA and Determination of Ionic Strength: The optimal ratio of Transfectam® (DOGS) to plasmid DNA and the ionic strength of NaCl used were a modification of the previously reported values (Loeffler and Behr, Methods in Enzymology 217: 1799-1802, 1993) and determined as follows: From the original 1 mg/0.400 ml (2.38 mg/ml) stock solution of Transfectam® prepared in 10% (v/v) ethanol in distilled water, 6 times the volume (μl) of stock solution was required for each μg DNA used. The optimal ionic strength of the solution was provided by an appropriate volume of 150 mM NaCl determined by the relation:
Volume (μl) of NaCl=Volume (μl) Transfectam/0.6

The results of Tat-induced SEAP levels in the standard assay after optimization of these conditions, are illustrated in FIG. 2. Briefly, COS cells were maintained in Isocove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal calf serum (FCS) and antibiotics. Triplicate cell samples were seeded at a density of ≈1.5×10⁵ cells per well in Linbro® 24 flat bottom wells of 17-mm diameter and incubated in a humidified 95% O₂/5% CO₂ incubator at 37° C. until they reached 50% confluency. Subconfluent cells were transfected using the lipospermine procedure (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). The medium of the subconfluent cells was aspirated and replaced by 300 μl of fresh minimum medium (IMDM supplemented with 3% FCS). COS cells were transfected with either pBC12/HIV/SEAP alone (0.35 μg/well) or pBC12/CMV/t2 (coding for Tat function) at 0.175 μg/well+pBC12/HIV/SEAP (0.35 μg/well) or with buffer alone (no DNA control samples). The plates were incubated for 12 to 15 hours after which, 700 μl of complete medium (IMDM containing 10% FCS) were added. Cells were then incubated for 48 hours after which, a 250-μl aliquot was removed from COS cell culture supernatants and heated at 65° C. for 5 minutes to selectively inactivate endogenous phosphatases (SEAP is heat stable). The samples were then centrifuged in a microfuge for 2 minutes. One hundred μl of 2×SEAP assay buffer (1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl₂; 10 mM L-homoarginine) were added to 100-μl aliquot of the samples. The solution was mixed and transferred into a 96-well flat-bottom culture dish (Corning). Twenty μl of pre-warmed substrate solution (120 mM p-nitrophenylphosphate dissolved in 1×SEAP assay buffer) were dispensed with a multipipetter into each well containing the reaction mixture. A₄₀₅ of the reaction was read at 5-minute intervals at 37° C. for 60 minutes using an EL340i microplate reader (Bio-tek Instruments, Inc.) with 5-seconds automatic shaking before each reading. The change in absorbance was converted in mU of SEAP expression as previously described (Berger et al., Gene 66: 1-10, 1988) and plotted against time.

These results indicate a nearly 65-fold increase in SEAP induction after 1 hour relative to the control (no DNA) levels or the induction of nonselected gene (HIV/SEAP) alone.

Assay-Guided Isolation of Creosote Bush Extract Active Component(s) by Countercurrent Chromatography: As stated labove, the differential fractionation and purification by countercurrent chromatography (CCC) of the creosote bush extract constituents led to the isolation of two major components (Table 1). 6.8 mg of the component termed Gr was isolated from the Green fraction on SiO₂ TLC. The total percent yield was ≈0.051% based on the original plant powder. 9.3 mg of the component Lo was isolated from the Yellow fraction (Ye).

Inhibition of Tat-TRS Activity by Extracts from Creosote Bush Leaves and Flowers: In several plant extracts tested with the SEAP assay, only the extract from the creosote bush, Larrea tridentata, leaves and flowers showed significant inhibitory activity of HIV Tat protein. Creosote bush displayed a dose-response inhibition of SEAP expression as illustrated in FIG. 3. Briefly, triplicate samples of COS cells were transfected with a mixture of pBC12/HIV/SEAP and PBC12/CMV/t2 (coding for Tat function) in 2:1 ratio, using the lipospermine procedure as described above. Cells were incubated for 12-15 hours after transfection. Creosote bush extract stock solution (10 mg/ml) was made in calcium/magnesium-free PBS and 10% DMSO, and filter-sterilized using a Millex®-GS 22 μm filter (Millipore). The appropriate concentrations of creosote bush extract were added to the transfected cells at a final DMSO concentration of 0.2% and samples were incubated for 48 hours. For SEAP analysis, a 250-μl aliquot was removed from COS cell culture supernatants, heated at 65° C. for 5 minutes to selectively inactivate endogenous phosphatases (SEAP is heat stable) and centrifuged in a microfuge for 2 minutes. One hundred μl of 2×SEAP assay buffer (1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl₂; 10 mM L-homoarginine) were added to 100-μl aliquot of the samples. The solution was mixed and transferred into a 96-well flat-bottom culture dish (Corning). Twenty μl of pre-warmed substrate solution (120 mM p-nitrophenylphosphate dissolved in 1×SEAP assay buffer) were dispensed with a multipipetter into each well containing the reaction mixture. A₄₀₅ of the reaction was read at 5-minute intervals at 37° C. for 60 minutes using an EL340i microplate reader (Bio-tek Instruments, Inc.) with 5-second automatic shaking before each reading. The percent inhibition of SEAP expression was calculated at 30 minutes as follows:
% Inhibition=100−[(CT⁺−C⁺)/(CT⁻−C⁻)×100]
where:

C⁻: Control sample (no DNA, no drug)

CT⁻: Control sample (+DNA, no drug)

C⁺: Drug-treated sample (no DNA, +drug)

CT⁺: Drug-treated sample (+DNA, +drug)

As seen in FIG. 3, the onset of this inhibition started at a concentration of 20 μg/ml and reached a maximum inhibitory activity at 600 μg/ml. The estimated EC₅₀ (the. concentration exhibiting 50% of inhibition) for this crude material was 110 μg/ml. As the purification of the active ingredients progressed, there was a stepwise increase in the activity of the active ingredient(s) which tripled (68%) with the organic phase (OG) fraction compared to 21% from the original total crude extract.

Inhibition of HIV cytopathic Effects: A compound inhibiting Tat transactivation should in principle block HIV replication. Consequently, creosote bush extract was tested at the National Cancer Institute (NCI) for inhibition of HIV-1 cytopathic effects using the soluble-formazan assay (Weislow et al., JNCI 81: 577-586, 1989, the entire contents of which are hereby incorporated by reference and relied upon). In principle, CEM-SS cells (ATCC, Rockville, Md.) are cocultivated with HIV-producing H9 cells. Viruses infect the host CEM-SS cells, replicate and kill most of the CEM-SS cells in a week. If the drug inhibits HIV production, CEM-SS cells are protected from HIV-induced cell death. The tetrazolium (XTT) reagent is therefore metabolically reduced by the viable cells to yield a colored formazan product which is measurable by colorimetry at 450 nm.

In practice, triplicate samples of CEM-SS cells (5000) were plated in 96-well microtiter plate. Appropriate concentrations of test compounds were added in a final volume of 100 μl calcium/magnesium-free PBS in 5% DMSO. Control samples received the compound medium (PBS) alone. Five minutes later, 500 highly infectious HIV-1 producing II9 cells or normal H9 cells were added to the wells containing the appropriate drug concentrations. The microtiter plates were incubated at 37° C. in 95% O₂/5% CO₂ for 6 days after which a 50-μl mixture of XTT and N-methylphenazonium methosulfate (PMS) was added. The plates were reincubated for additional 4 hours for the color development (XTT formazan production). The plates were sealed, their contents were mixed by automatic shaking and the OD₄₅₀ of samples was determined in a microplate reader. Each value represents the average of 3 determinations. No significant difference was found between the means of the duplicate values of the uninfected cells and HIV-challenged cells, in presence of test compounds. In contrast, there was a significant difference (p<0.05) between HIV-challenged samples in the presence or absence of test compounds.

The results of these studies are summarized in Table 2. At a concentration of 0.75 μg/ml for component Gr, there was an average 58% protection (cell viability) against HIV as opposed to 15% viability in drug-free samples challenged with HIV. At a concentration as low as 0.187 μg/ml, component Lo exhibited even stronger inhibitory activity of HIV cytopathic effects. The cell viability was 87%, very close to that of not treated control cells (89%), in contrast to 14% viability for the drug-free samples challenged with HIV. These compounds were devoid of cytotoxicity at the concentrations used.

TABLE 2
INHIBITION OF HIV-1 CYTOPATHIC EFFECTS BY CREOSOTE
BUSH EXTRACT COMPOUNDS IN THE SOLUBLE-FORMAZAN
ASSAY.
	Concentration	Percent of live cells at
	of the test	day 6 as measured by XXT
	sample which	Formazan production
	yielded max	Un-	HIV	HIV
	protection	infected	infected	infected
	against HIV	plus	plus	minus
	without killing	test	test	test
Test Sample	the cells μ/ml	sample	sample	sample
Fraction Green		0.187	59	67	16
Component Gr		0.75	80	67	16
	-duplicates
Component Gr		0.75	70	48	14
Fraction		0.187	60	57	14
Yellow (Ye)
Component Lo		0.187	91	86	14
	-duplicates
Component Lo		0.187	89	87	14

Structure elucidation of the active components of creosote bush extract: The chemical characterization of the purified plant active constituents was achieved mainly by mass spectroscopy and by H- and C- nuclear magnetic resonance (NMR). Component Lo was found to be a mixture of four related compounds (L1, L2, L3 and L4). Resolution and characterization of each peak of the mixture was accomplished by gas chromatography (GC) using an analytical non-destructive capillary cross-linked 5% phenylmethylsiloxane (HP-5) column attached to a mass spectroscope (MS). The GC studies revealed that the first compound (L1) represented 6% of the mixture; the second (L2) was 76% of the mixture (MW=316); the third (L3) was isomeric with L2 and represented 9% of the total mixture (MW=316); and the fourth compound (L4) represented 9% of the mixture (MW=358). The time (minutes) of elution of these compounds is indicated on the peaks (FIG. 4).

Component Gr consisted of fifteen compounds. Resolution and characterization of each peak of the mixture was accomplished by gas chromatography (GC) using analytical non-destructive capillary cross-linked 5% phenylmethylsiloxane (HP-5) column attached to a mass spectroscope (MS). The GC studies revealed that four (G1, G2, G3 and G4) of the fifteen compounds. The time (minutes) of elution of these compounds is indicated on the peaks (FIG. 5).

The structures of these eight compounds (L1, L2, L3, L4, G1, G2, G3 and G4) are described as follows:

L1 has the composition C₁₈H₂₂O₄ and has been identified as a previously known chemical, 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (
where:

C⁻: Control sample (no DNA, no drug)

CT⁻: Control sample (+DNA, no drug)

C⁺: Drug-treated sample (no DNA, +drug)

CT⁺: Drug-treated sample (+DNA, +drug)

Each point represents the average of two determinations. No significant difference was apparent between the EC_50s of Mal 4 and NDGA which were 8 μg/ml (25 μM) and 6 μg/ml (20 μM), respectively. The EC_50s are defined as the inhibitory concentration of the compound at which the Tat regulated HIV transactivation is reduced to 50% of that in untreated control cells.

The inhibition of transactivation of HIV promoter activity by Mal 4 and NDGA were compared in Table 3.

TABLE 3
INHIBITION OF TRANSACTIVATION OF HIV PROMOTER
ACTIVITY BY NATURAL COMPOUNDS MAL 4 and NDGA.
	Inhibition of Tat-induced Scap Expression
Test	(% inhibition/concentration of test compound)
Compound	%	μm	%	μm	%	μm	%	μm
Mal 4	13.6	9.5	60.4	31.3	92	62.7	100	95
NDGA	17.0	9.9	73.8	32.6	88.1	65.2	92.9	99

The compounds NDGA and Mal 4 were assayed as described for FIG. 6. Control samples were run in quadruplicate. The percentage inhibition was determined after 30 minutes and the OD₄₀₅ values were:

C⁻: Control sample (no DNA, no drug): 0.091

CT⁻: Control sample (+DNA, no drug): 0.805.

The quantitation of XTT formazan production as a measurement of viable cells in Mal 4 treated cultures of CEM-SS cells in shown in FIG. 7. Each figure shows infected or uninfected CEM-SS target cells (10⁴/M well) with serial dilutions of Mal 4. EC₅₀ represents the concentration of Mal 4 (e.g. 13.4 μM) that increases (protects) XTT formazan production in infected culture to 50% of that in uninfected, untreated culture cells. IC₅₀ represents inhibitory or toxic concentration of Mal 4 (e.g., 325 μM, estimated) that reduces XTT formazan production in uninfected cultures to 50% of that in untreated, uninfected control cells. Levels of XTT formazan in untreated, infected control cells were 9% of those in untreated, uninfected control cells. The soluble-formazan assay for HIV-1 cytopathic effects was conducted according to the procedure described by Weislow et al., JNCI 81: 577-586, 1989, the entire contents of which are hereby incorporated by reference and relied upon.

It will be appreciated that the invention in its broadest method aspects contemplates the use of NDGA or any of its pharmaceutically acceptable derivatives for suppressing undesired viral growth in a host. These derivatives comprise modifications of NDGA where one or more, and preferably all four, of the hydroxy groups are replaced by, for example, other substituents such as CH₃O—, CH₃(C═O)O—, or the like. Preferably, however, these substituents are selected to provide water-solubility without affecting therapeutic properties. The active compounds to be used in the invention can, therefore, be structurally represented as follows: ##STR00019##
wherein R₁, R₂, R₃ and R₄ are each independent HO—, CH₃O—, CH₃(C═O)O— or other pharmaceutically acceptable equivalent thereof, with a preference for substituents which provide water-solubility. As representative water-soluble derivatives of NDGA suitable for use herein, there may be mentioned compounds of the foregoing formula wherein each R₁-R₄ substituent is the group. ##STR00020##
or
the group ##STR00021##

Alternatively, the R₁ and R₂ substituents and the R₃ and R₄ substituents may be joined together with the adjacent carbon atoms of each phenyl ring to form cyclic structures as follows: ##STR00022##

As indicated, the foregoing are given only for purposes of illustration as those in the art will appreciate that other alternative pharmaceutically acceptable R₁-R₄ substituents may be employed to provide water-solubility or to obtain functionally equivalent pharmacological results.

While the foregoing disclosure is directed primarily to the suppression of HIV Tat transactivation, the compounds disclosed herein are also applicable to suppress other undesired viral gene expression. In other words, the invention is not limited to use of the indicated compounds for the suppression of Tat transactivation of a lentivirus. The effectiveness of the compounds against other viral gene expression can be readily determined by testing the compounds in appropriate art-recognized test systems. Thus, for example, the compounds of the invention have been found to be effective for inhibition of the Herpes simplex virus. Without intending to be bound by any theory as to how the compounds function against this virus or other virus, it is believed that the compounds proceed by a common mechanism which includes inhibiting proviral transcription and transactivation essentially as earlier described herein.

This broader application of the invention is illustrated hereafter with data showing the suppression of Herpes simplex virus replication by inhibition of transcription and transactivation of HSV IE gene α₄ (ICP₄).

By way of explaining this further aspect of the invention, it is generally known that manifestation of Herpes simplex virus (HSV-1, HSV-2) infection has frequently been observed in HIV infected patients. Initial treatments with anti viral agents (such as acyclovir for HSV and reverse transcriptase and viral protease inhibitors for HIV-1) have been found to be quite effective. However, drug resistance and high sensitivity to multiple drugs often developed in these patients with time. Antiviral strategies aimed at controlling the replication of wild type and mutants of both viruses by drugs which can control both viruses thus are clinically important. Transcription of proviral HIV and HSV IE genes (α₀, α₄, α₂₂, α₂₇, and α₄₇) (Ref. Deluca, N. A. and Schaft, P., J. Virol. 14, 8, 1974) are essential for replication of these two respective viruses. The expressions are thoroughly host dependent, utilizing RNA polymerase II and cellular transcription factors. The promoter activities of proviral HIV and HSV α₄ (ICP₄) gene are regulated by host protein Spl in addition to other cellular factors (Ref. Jones, K. A. and Tjian, R., Nature 317, 179 1985).

The use of the invention for suppressing Herpes simplex virus (HSV-1, HSV-2) is illustrated hereinafter using tetramethoxyl nordihydroguaiaretic acid (CH₃—O)₄ NDGA, 4N of the formula: ##STR00023##
to inhibit HSV ICP₄ gene transactivation and HSV replication in vero cells at a dosage range that is not toxic to the host cells (see Table 4).

In addition, in comparison with acyclovir (ACV), a commercially available HSV drug, HSV virus showed no drug resistance toward tetramethoxyl NDGA (4N) following ten viral passages in vero cells. The IC₅₀ for 4N remained essentially the same from first passage to tenth passage (IC₅₀ app 10 μm) while the concentration of acyclovir required to inhibit HSV-1 increased from 7.5 μm at first passage to 440 μm at the 10th passage (see Table 5A) Consequently, tetramethoxyl NDGA, 4N, was able to also inhibit the acyclovir resistant strain (HSV-1 SM44-CV4r) very effectively (IC₅₀ of 6.9 μm) (see Table 5B).

It is also noted that tetramethoxyl NDGA 4N has been used in an animal system, i.e. to treat HSV infected skin of guinea pigs. At a concentration of 60 mg/ml, 4N eliminated HSV-1 growth completely in five days following daily topical treatment of the drug at the infected skin areas (FIG. 8).

Two male guinea pigs (350 gm body weight) were used in the guinea pig experiment. The back skin of the animal was shaved and equally divided into six patches. Each skin patch was pinched with seven-pin needles and HSV-1 suspension was applied topically to infect each punched area. Twenty-four hours after infection, three agents (ABPS, 2 mg/ml, ABPS 4 mg/ml, tetramethoxyl NDGA, 4N 60 mg/ml and acyclovir 60 mg/ml) were applied to the infected areas five times per day for five days. Picture (FIG. 8) was taken 96 hours following infection.

Comparing with the two positive controls (HSV-SC, HSV-C, i.e. infection without drug treatment), the result indicated that agent ABPS is ineffective while tetramethoxyl NDGA and acyclovir are effective in suppression of HSV-1 replication. Differing from acyclovir, however, tetramethoxyl NDGA has the advantage in that it is a mutation insensitive drug (Table 5A and Table 5B).

TABLE 4
Tetramethoxyl NDGA Effect
Concentration
of (CH3O)4 NDGA
μM	% Inhibition	IC50
I. Inhibition of HSV-1 Replication in Vero Cells
14	97.50	7.7	μM
7	40
3.5	20
1.75	<0
0.875	<0
0.437	<0
II. Inhibition of HSV-1 ICP4 Transactivation in Vero Cells
80	78	33	μM
70	68
60	64
50	52
40	35
30	30
20	8
III. Inhibition of Growth of Vero Cells
280	79.62	142	μM
140	43.95
70	9.55
35	7.64
17.5	7.64
8.75	3.18
4.37	1.27

TABLE 5
Comparison Between the Drug Sensitivities of Acyclovir and
Tetramethoxyl NDGA Against HSV-1 in Vero Cells
A. IC50 at different passages
	IC50 μM
Passage	ACV	[CH3]4 NDGA
1	7.5	11.7
2	37.8	4.4
3	>88.8	8.2
4	138.4	5.9
5	>220	9.9
10	440	10

Virus which recovered from medium of each passage at IC₅₀ drug concentration were used to infect the subsequent passage of the cells.

B. Drug sensitivities of tetramethoxyl NDGA against ACV sensitive or
ACV resistant strain of HSV-1
	HSV-Sm44-ACVg		HSV-18m44-CV4r
Drug	(IC50)	(μM)	(IC50)
ACV	<6.9		117.6
[CH3]4 NDGA	6.9		6.9

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Thus, it is to be understood that variations in the derivatives of NDGA and the method of suppression of Tat transactivation can be made without departing from the novel aspects of this invention as defined in the claims.

Claims

1. A method for suppressing viral growth in a host which consists essentially of administering to the host an effective viral growth suppressing amount of a composition consisting essentially of a compound of the formula: ##STR00024##

2. The method of claim 1 4, wherein said compound is the water-soluble substituent is —O(C═O)CH₂NH(CH₃)₂.Cl.

3. The method of claim 1 for suppressing 4, wherein the host is infected with Herpes simplex virus in the host .

4. A method for suppressing viral growth in a host infected with a virus comprising (a) providing a composition comprising a substantially purified compound and (b) administering said composition to the host in a dosage having an effective amount of the compound to suppress viral growth, wherein the compound is a derivative of nordihydroguaiaretic acid (NDGA) having the formula: ##STR00025##

5. The method of claim 4, wherein the water-soluble substituent is —O(C═O)CH₂NH₂.

6. The method of claim 4, wherein the compound inhibits viral transcription.

7. The method of claim 4, wherein the compound inhibits transactivation of viral gene.

8. The method of claim 4, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-2,3-dimethylbutane (4-O-methyl-NDGA).

9. The method of claim 4, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3-O-methyl-4-O-acetyl-NDGA).

10. The method of claim 4, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,3′,4-tri-O-methyl-NDGA).

11. The method of claim 4, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,4,4′-tri-O-methyl-NDGA).

12. The method of claim 4, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylbutane (3′,4-di-O-methyl-3-O-acetyl-NDGA).

13. The method of claim 4, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3,3′-di-O-methyl-4-O-acetyl-NDGA).

14. The method of claim 4, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylbutane (4,4′-di-O-methyl-3-O-acetyl-NDGA).

15. The method of claim 4, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3,4′-di-O-methyl-4-O-acetyl-NDGA).

16. The method of claim 4, wherein R₁, R₂, R₃ and R₄ are not each CH₃O— or CH₃(C═O)O— simultaneously.

17. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 95 μM.

18. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 62.7 μM.

19. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 31.3 μM.

20. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 25 μM.

21. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 9.5 μM.

22. A method of inhibiting replication of an acyclovir-resistant virus in a cell comprising the steps of:

(a) providing a substantially purified compound having a formula: ##STR00028##

23. A method of treatment of acyclovir-resistant viral infection in a subject comprising the steps of:

(a) providing a substantially purified compound having the formula: ##STR00031##

24. A method of treatment of a subject infected with a virus, wherein the virus is resistant to acyclovir comprising the steps of:

(a) providing a composition comprising a substantially purified compound; and

(b) administering said composition in a dosage having a therapeutically effective amount of the compound to the subject, wherein the compound has the formula: ##STR00034##

25. The method of claim 24, wherein the water-soluble substituent is —O(C═O)CH₂NH₂.

26. The method of claim 24, wherein the water-soluble substituent is —O(C═O)CH₂NH(CH₃)₂.Cl.

27. The method of claim 24, wherein the compound inhibits viral transcription.

28. The method of claim 24, wherein the compound inhibits transactivation of the viral gene.

29. The method of claim 24, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-2,3-dimethylbutane (4-O-methyl-NDGA).

30. The method of claim 24, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3-O-methyl-4-O-acetyl-NDGA).

31. The method of claim 24, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,3′,4-tri-O-methyl-NDGA).

32. The method of claim 24, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,4,4′-tri-O-methyl-NDGA).

33. The method of claim 24, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylbutane (3′,4-di-O-methyl-3-O-acetyl-NDGA).

34. The method of claim 24, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3,3′-di-O-methyl-4-O-acetyl-NDGA).

35. The method of claim 24, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylbutane (4,4′-di-O-methyl-3-O-acetyl-NDGA).

36. The method of claim 24, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3,4′-di-O-methyl-4-O-acetyl-NDGA).

37. A method of treatment of viral infection in a host comprising the steps of: (a) providing a composition comprising a compound; and (b) administering said composition in a dosage having a viral inhibitory amount of the compound to the host, wherein the compound has the formula selected from the group consisting of: ##STR00037## ##STR00038##