WO1990004968A1

WO1990004968A1 - Inhibition of human retroviruses

Info

Publication number: WO1990004968A1
Application number: PCT/US1989/004807
Authority: WO
Inventors: Gen-Ichiro Nonaka; Kuo-Hsiung Lee; Yung-Chi Cheng; Robert E. Kilkuskie
Original assignee: University Of North Carolina At Chapel Hill; Biotech Research Laboratories, Inc.
Priority date: 1988-10-31
Filing date: 1989-10-31
Publication date: 1990-05-17
Also published as: CA2001898A1

Abstract

Tannin derivatives are a new class of HIV reverse transcriptase inhibitor. Among these compounds are new galloylquinic acids having at least one depside galloyl group. These compounds inhibit the growth of HIV in cells with low cytotoxicity.

Description

INHIBITION OF HUMAN RE ROV RUSES

BACKGROUND OF THE INVENTION

The present application is a continuation-in- part of application Serial No. 07/264,558 filed October 31, 1988, which application is hereby incorporated by reference.

Mention of Governmental Support

This invention was funded in part by grant AI- 25697 from the National Institute of Allergies and Infectious Diseases, awarded to Y.C. Cheng, K.H. Lee, and A.J. Bodner.

Field of the Invention

This invention relates to new drugs for the treatment of AIDS (acquired immunodeficiency syndrome) .

Invention Disclosure Statement ^"

Since the discovery of human immunodeficiency virus [HIV (HTLV-III LAV) ] , much progress has been made in elucidating the genomic structure of HIV as well as the mechanisms of HIV infection, and this has been reviewed.^{1 *3} Since reverse transcriptase (RT) plays a very important role in controlling the replication of the HIV, RT is one of the most attractive targets in the development of anti-AIDS drugs. Among the many drugs tested, suramin, phosphonoformate (PFA) , 3'-azido-2* ,3 '- dideoxythymidine triphosphate (AZTTP) , 2' ,3'-dideoxy- cytidine triphosphate (DDCTP) , ribavirin and HPA-23 have been demonstrated to be useful in the treatment of AIDS patients.²*⁴ The selective antiviral action of some of these drugs was suggested to be associated with the unique behavior of RT².

Tannins are water-soluble polyphenolic compounds, usually having molecular weights between 500 and 3,000, which, besides giving the usual phenolic reactions, have the ability to precipitate alkaloids, gelatine and proteins. For this reason, crude extracts have been used since ancient times in tanning leather. Hasla , Ch. 18, "Vegetable Tannins", in the Biochemistry of Plants, Vol. 7, pages 527-556 (1981) . Some sources of vegetable tannins of commercial importance are listed in Haslam Table 1.

Tannins are conventionally classified as either hydrolyzable or condensed tannins, .the hydrolyzable tannins, which may be hydrolyzed by acids, alkali and tannase into alcohols and phenolic acids, are further classified into gallotannins (yielding gallic acid) , the ellagitannins (yielding ellagic acid) , and other hydrolyzable tannins (yielding other acids) . In the case of the gallotannins, the possible alcohol components include D-Glucose, D- Hamamelose, D-Xylose, proto-Querσitol, 1,5-anhydro-D-Glucitol, Quinic acid, methyl-beta-D-Glucoside, and Salidroside. Gallotannins were first discovered in galls but are also found in normal plant tissues. Both mono- and polygalloyl tannins are known. Nishioka, Chemical and Biological Activities of Tannin, Oriental Healing Arts International Bull., 11:9-27 (January 1986). Nishioka notes that "the greatest obstacle to chemical research on tannins is the difficulty of separating pure compounds, even with modern techniques." Nonetheless, a number of have been isolated. These include the gallotannins 3-0-, 4-0-, 5-0-, 3,4-di- 0-, 3,5-di-0-, 4,5-di-0- and 3,4,5-tri-0-galloylquinic acids. Busgunyram et al., Ogtticgenustrtm 23:2621-23 (1984) . Polygalloyl glucoses have also been separated. Nishizawa, et al., J. Chem. Soc. , Perkin Tans. 1: 2963- 68 (1982) . The latter researchers succeeded in separating the gallotannins of Chinese galls according to their degree of galloylation (G5-G12) by a combination of Sephadex LH-20 chromatography and normal- phase HPLC. See also Nishizawa, et al., Chem. Pharm. Bull., 28(9) :2850-52 (1980); Nishizawa, et al., J. Chem. Soc. Perkin Trans., 1: 961-965 (1983). Elagitannins have also been isolated.

A number of tannins have been examined by Kakiuchi for inhibitory activity against the reverse transcriptase of avian RNA myeloblastosis virus. It was found that hydrolyzable tannins inhibited the polymerization catalyzed by this virus' reverse transcriptase. The ellagitannins tested were generally more effective than the gallotannins tested. The only gallotannins examined were derivatives of glucose; pentagalloylglucose and hexagalloyl glucose were the most active species tested. It will be noted from our Table 1 that the galloylglucoses (compounds 1-51) were among the least potent inhibitors of HIV among the compounds we tested. No galloylquinic acids were tested by Kakiuchi. See Kakiuchi, et al., J. Natural Products. 48: 614-21 (1985).

However, Kakiuchi did not determine whether any tannins inhibit the reverse transcriptase of a human retrovirus, and particularly of HIV. Moreover, Inouye et al., in J. Antibiotics. 4_0:100-104 (1987) observed that while janiemycin and colistin strongly inhibited the reverse transcriptase of avian myeloblastosis virus, they failed to suppress the replication of HIV in MT-4 cells at the tested concentrations. Thus inhibition of AEV RT is not a reliable guide to whether a compound inhibits HIV RT. Cellular components may adversely affect the activity of the inhibitory compound.

Balzarini et al. , in Biochemical Pharmacology. vol. 37, No. 12, 2395-2403 (1988) disclose a number of compounds which are much more effective in inhibiting Moloney murine leukemia virus than in inhibiting human HIV. Thus, inhibition of one type of retrovirus at a reasonable dosage does not necessarily imply inhibition human HIV at a comparable dosage. At this point, there is no way of ascertaining how the host cells process the cytotoxic agent, so that one skilled in the art cannot predict from inhibition of one retrovirus the inhibition of any other retroviruses.

Nor does Kakiuchi demonstrate that any tannin inhibits any retrovirus in culture, merely that certain tannins inhibit avian myeloblastosis virus. It has been reported that 4-methyl-2- amino-pyridine-palladium chloride (MAP) inhibits reverse transcriptase in disrupted retrovirus (Rauscher murine leukemia virus) but not in infected cells (except in such high concentrations as are toxic to the cells per se) . Consequently, the is vivo inhibitory activity of the tannin compounds of the present invention towards HIV would have been unpredictable prior to applicants* discovery even if the .in vitro inhibitory action against HIV RT had been known.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the aforementioned deficiencies in the prior art.

It is another object of the present invention to provide new compounds for treating AIDS.

It is a further object of the present invention to provide methods for inhibiting the reverse transcriptase activity of the AIDS virus. It is yet another object of the present invention to provide a method for inhibiting the growth of HIV-infected cells in a patient suffering from an HIV infection.

According to the present invention, compounds are provided for inhibiting the growth of human retroviruses, particularly HIV. The compounds used in the present invention include gallotannins, ellagitannins, condensed tannins, complex tannins, and other related compound.

The gallotannins are classified into four groups: gallotannins with a glucose core (compounds 1- 5) , gallotannins with a quinic acid core (compounds 6-8 and compounds 52-56) , gallotannins with a shikimic acid core (compounds 9-12) , and gallotannins with a hamamerose core (compounds 36-37) .

The ellagitannins used in the present invention are ellagitannins with a glucose core (compounds 13-19) , ellagitannins with a modified hexahydrox^'y-diphenoyl moiety and/or^'related acyl group (compounds 25-31 and 33) , ellagitannin dimers (compounds 20 and 2) , ellagitannin tetramer (compound 22) , ellagitannins with an open-chain glucose core (compounds 23, 24, 32, and 38), and ellagitannins with a triterpenoid moiety (compounds 34 and 35) .

The condensed tannins include monomers (compounds 39 and 43), dimers (compounds 40 and 44), trimers (compounds 41 and 45) , tetramers (compounds 42 and 47) , and a pentamer (compound 48) .

Compounds 50 and 51 represent the complex tannins consisting of the component unit [ (--)-catechin] of condensed tannins and an ellagitannin moiety linked through a carbon-carbon linkage, while compound 49 is a caffeic acid tetramer.

Several ellagitannins were found to be particularly potent inhibitors of HIV-RT, including chebulinic acid (28) and chebulagic acid (29) . These compounds have a modified hexahydroxydiphenoyl group in the molecule. Punicalagin (31) , which has an additional hexahydroxydiphenoyl group, was shown to be less active than punicalin.

Since the more potent compounds, punicalin (30) and punicacortein-C (32) , have a gallagyl (tetraphenoyl) moiety in their molecule, it is postulated that the gallagyl moiety seems to play an essential role in HIV RT inhibition in this class of compounds.

The compounds may be provided in the form of a partially purified extract of tannic acid, such as a gallotannin, ellagitannin, condensed tannin, etc. , or a fraction thereof. However, the compounds are preferably provided in pure form. While these compounds have been isolated from natural sources, now that their structures are known, it is possible to devise means for their synthetic preparation. Pharmaceutically acceptable derivatives, such as salts, amides, and esters, may be substituted ^"for the acids themselves.

The ability of the compounds of the present invention to inhibit the growth of HIV-infected cells is believed to be at least partially attributable to their ability, observed in vitro. to inhibit the action of reverse transcriptase. Thus, in another aspect, this invention relates to the use of these compounds to inhibit reverse transcriptase, _in vitro or jLn vivo, whether for therapeutic, diagnostic, or drug screening purposes.^"

The activity of these compounds may also be attributable in part to their activity against DNA polymerase, and so another aspect of the present invention is their use in inhibiting DNA polymerase.

For the purposes of the present invention, the term "inhibition" refers to any ability to diminish activity or growth, and does not require the abolition thereof. The terms "tannins" and "tannic acids" are used interchangeably unless specifically indicated otherwise. The term "tannins" must be interpreted so as to cover all of the compounds of Table 1.

The appended claims are hereby incorporated by reference as an enumeration of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the anti-HIV effect of tannin compounds 12, 28, 29, 30, 31, and 32 as measured by p24 antigen capture.

Figure 2 shows the effect of tannin compounds

12, 28, 29, 30, 31, and 32 on H9 cell growth.

Figure 3A shows the effect of punicalin on HIV RT.

Figure 3B shows the time course in the presence of punicalin at 12.5 μM.

Figure 4 shows ethidium displacement by punicalin.

Figure 5 compares the inhibition of DNA polymerase alpha (1) , gamma (2) , and beta (3) , and of HIV RT (4), by compound 54.

Figure 6 (a) through (i) show the chemical structures of compounds 1-56.

DETAILED DESCRIPTION OF THE INVENTION In searching for natural products which can be used as anti-AIDS agents, it was found that the tannic acids showed potent inhibitory activity against HIV reverse transcriptase. The tannic acids were obtained either from a commercial source or extracts prepared from either Turkish or Chinese galls. As seen in Table 4, the extract from the commercial source showed the strongest inhibitory effect against HIV reverse transcriptase (74% at 100 μg/ml) . Subsequent bioassay- directed fractionation of this tannic acid mixture resulted in the isolation of at least four new tetragalloylquinic acids.

The inhibitory effect of the tannins according to the present invention against purified HIV-RT obtained from HIV infected H9 lymphocyte cells is shown in Table 1, at a dose of 20 μg/ml. Among the 51 tannins tested, twelve samples (8, 11, 12, 14, 18, 23, 24, 28, 29, 30, 31, and 32) inhibited HIV-RT more than 40% at the concentration used.

Trigalloylquinic acid, compound 8, as well as di- and tri-galloylshikimic acids, compounds 11 and 12, are among the more potent inhibitors of HIV. This observation is consistent with the findings disclosed in our prior application. Serial No. 07/264,558, that galloylquinic acids having at least three galloyl groups are potent inhibitors of HIV.

At a concentration of 20 μg/ml, punicalin, compound 30,^" and punicacortein-C, compound 32, showed greater than 80% inhibition of HIV RT. Chebulinic acid, compound 28, and chebulagic acid, compound 29, inhibited HIV RT more than 70%.

It is interesting to note that punicalagin, compound 31, which has an additional hexahydroxydiphenoyl group, was shown to be less active than punicalin, compound 30. Since the more potent compounds, 30 and 32, have a gallagyl (tetraphenoyl) moiety in their molecule, the gallagyl moiety seems to play an essential role in HIV RT inhibition.

The inhibition of reverse transcriptase by compound 30, punicalin, is concentration dependent, and the reaction catalyzed by reverse transcriptase in the presence of the drug is linear with time, as shown in Figures 3 and 4. The degree of inhibition by punicalin varied with the concentration of substrates in the reaction mixture, as shown in Table 3. This could be due to the interaction of punicalin with DNA, which is about 65-fold less than actinomycin-D, as demonstrated by ethidium bromide displacement assay, as shown in Figure 4.

In Figure 3, the effect of punicalin on HIV RT was demonstrated by incubating different amounts of punicalin in the standard reverse transcriptase mixture for 60 minutes. After washing and counting, the drug- treated samples were compared to drug-free controls. In Figure 3B, the time course in the presence of punicalin at 12.5 μM is shown. The drug was incubated in the standard assay mixture for various times. After washing and counting, samples were compared to drug-free controls. In Figure 4, punicalin was incubated in three reaction conditions as indicated in Table 3. The rest of the condition is the same as the regular DNA reaction mixture wherein each condition in a 50 μl volume contained 50 mM Tris HC1 pH 8.0, 8 mM MgCl₂ , 100 μg/ml bovine serum albumin, 100 μM of dATP, dGTP, and dCTP, and 1 mM DTT. The concentration of activated DNA and dTPP used in the assay is described in Table 3. The reactions were incubated for 30 minutes at 37^βC. The rest of the conditions are identical to the RT assay.

Among the twelve most active tannins mentioned above, compounds 12, 28, 29 30, 31, and 32 were selected for further evaluation against the growth of HIV infected H9 lymphocytes. As demonstrated in Figure 1, these six tannins showed significant anti-HIV activity. At concentrations of 20 and 5 μg/ml, HIV replication was inhibited more than 50% of the control HIV replication. Compounds 28, 29, and 32 showed anti-HIV activity even at 1 μg/ml.

Two of these tannins caused only mild cytotoxicity against the growth of uninfected H9 cells at 1 and 5 μg/ml (less than 25% inhibition) . Compound 30, especially, showed low cytotoxicity even at 20 μg/ml (less than 15% inhibition) . Further investigation was undertaken to determine whether the anti-HIV-RT activity and anti-HIV activity are independent events or whether these tannins inhibit HIV replication by acting on sites other than reverse transcriptase. Compound 30 was added at different steps in a standard virus inhibition assay. The antiviral effect was greater when compound 30 was present during the infection step than when it was present only during culture. Pretreatment of the virus with compound 30 had no effect on the virus. These results are very different from those of similar studies using 2' ,3'-dideoxycytidine (DDC) or dextran sulfate as the drugs, as shown in Table 2. These results suggest that compound 30 is likely to inhibit HIV propagation at the step of virus and cell interaction. Another possibility is that the virus could serve as a carrier of compound 30 into cells to inhibit HIV-RT. It is hypothesized that the anti-HIV action and the anti-HIV RT action of compound 30 are two independent events. The specific examples which follow are for purposes of illustration only, and not for limitation.

The tannin samples used in the present invention were isolated from the plant materials as reported previously: 1-0-galloyl-β-D-glucose (l) {6}, 1,6-di-O-galloyl-β-D-glucose (2) (6), 1,2,6-tri-O- galloyl-3-D-glucose (3) (7), l,2,3,6-tetra-0-galloyl-3- D-glucose (4) {8} l,2,3,4,6-penta-0-galloyl-/3-D-glucose (5) {9}, 4-0-galloylquinic acid (6) {10}, 1,4-di-O- galloylquinic acid (7) {11}, 1,3,4-tri-O-galloylquinic acid (8) {11}, 3-O-galloylshikimic acid (9) {12}, 3-0- digalloylshikimic acid (10) {12}, 3,5-di-O-galloyl- shikimic acid (11) {12}, 3,4,5-tri-0-galloylshikimic acid (12) {13} , 2,3-O-hexahydroxydiphenoyl-D-glucose (13) {14}, strictinin (14) {15}, pedunculagin (15) {16}, 1-β-O-galloylpedunculagin (16) {17}, eugeniin (17) {18}, corilagiin (18) {15}, punicafolin (19) {15}, phillyraeoidin A (20) {19}, sanguiin H-6 (21) {14}, sanguiin H-ll (22) {14}, castalagin (23) {20}, vescalagin (24) {20}, geraniin (25) {21}, elaeocarpusin (26) {22}, furosin (27) {23}, chebulinic acid (28) {24}, chebulagic acid (29) {25}, punicalin (30) {26}, punicalagin (31) {26}, punicacortein C (32) {27}, terchebin (33) {28), castanopsinin A (34) {29}, castanopsiniin A (35) {28}, hamamelitannin (36) {29}, 2'3,5-tri-0-galloylhamamelose (37) {30}, grandinin (38) {31}, (-)-epicatechin (39) {32}, procyanidin B-2 (40) {32}, procyanidin C-l (41) {32}, cinnamtannin A₂ (42) {32}, (-)-epicatechin 3-O-gallate (43) {33}, procyanidin B-2 3,3'-di-O-gallate (44) {33}, procyanidin C-l 3,3' ,3"-tri-0-gallate (45) {33}, procyanidin A-2 (46) {34}, cinnamtannin B*_| (47) {32}, cinnamtannin B₂ (48) {32}, lithospermic acid B (49) {35}, acutissimin A (50) {36}, and mongolicain A (51) {37}. Compound numbers are in parentheses ( ) and literature references in curly brackets { }.

HIV RT ASSAY

The HIV RT assay was performed according to the method described by Cheng et al. {4} . The immuno- affinity purified enzyme used was isolated from virions released by human T cells infected with HIV. Poly rA oligo d , ₀ from Pharmacia, Piscataway, N.J. , was used as the template to measure the incorporation of [³H]dTMP (20 μM) . The percentage of inhibition was determined by comparing the reverse transcriptase activity of tannin containing assays to that of the tannin of the drug free controls.

HIV GROWTH INHIBITION ASSAY

The standard HIV inhibition assay was per¬ formed by incubating H9 lymphocytes (3.5 x 10⁶cells/ml) in the presence or absence of HIV-1 (HTLV-IIIB) for one hour at 37°C. The cells were washed thoroughly to remove unabsorbed virions and were resuspended at 4 x 10⁵ cells/ml in culture medium. Aliguots of l ml were placed into wells of 24 well culture plates containing an equal volume of medium and equal concentration of test compound diluted in culture medium. After incubation for three days at 37^βC, the cell density of uninfected cultures was determined by cell counts to assess toxicity of the test compound.

A p24 antigen capture assay was used to determine the level of virus presence infection released into the medium of HIV cultures. The ability of compounds to inhibit HIV replication was measured at four different concentrations of test compound. Test compounds were considered to have anti-HIV activity if p24 levels were <70% of untreated culture.

Inhibition of virus replication by punicalin (compound 30) , DDC, and dextran sulfate, were further compared in a modified inhibition experiment which is summarized in Table 2. In this experiment, virions were incubated for one hour at 37*C in the presence or absence of the compounds. The preincubated virions were then diluted tenfold into cells, and the one hour infection step was carried out with or without fresh compound. Following the infection and washing steps, the three days of culture were done with and without compound.

ETHIDIUM DISPLACEMENT ASSAY This assay was performed using an Aminco fluoro colorimeter. Three ml of a solution containing 2.5 μM EDTA was mixed with 20 μl of 300 μM calf thymus DNA. This solution was placed into the fluorocolorimeter, and it was adjusted to 100% relative intensity. Then, additions of the drug in DMSO or DMSO alone were added to the tube. A relative intensity reading was taken after every addition. A change of one on this scale was considered one unit of ethidium displacement (cf. references 38 and 39) . The ethyl acetate soluble portion of tannic acid was fractionated using a column of Sephadex LH-20 to yield seven fractions. These fractions were separated according to the degree of galloylation⁵ , and were composed of mono (G-l) to octa- (G-8) galloylated compounds. Among these, fraction G-4, which contained tetragalloylated compounds, showed the strongest inhibitory effect against HIV RT (Table 2) . Subsequent repeated preparative high performance liquid chromatography on reverse phase columns yielded active compounds 53, 54, 55 and 56. Compound 52 was isolated from the G-3 fraction, which was less active than G-4.

A comparison of the physical constants and the ¹H and ¹³C-NMR spectra indicated that 52 is 3,4,5,-tri- O-galloylquinic acid.⁶'⁷ The 1H-NMR spectra of 53, 54 and 55 showed the signals from a depside galloyl group (doublets at S 7.34 and 7.36 for 53, 7.43 and 7.50 for

54 and 7.35 and 7.42 for 55) along with the singlets of galloyl groups and the signals of the 3,4,5-triacylated quinic acid moiety. The ¹³C-NMR spectra of 53, 54 and

55 also indicated the presence of a depside galloyl group (signals at δ 114.7, 117.6, 143.7 and 151.2 for 53, 114.7, 117.5, 143.2 and 151.2 for 54, and 114.6, 117.5, 143.6 and 151.2 for 55, see Table 7). After partial hydrolysis in boiling water, ⁸»⁹ 53, 54 and 55 gave gallic acid and 3,4,5-tri-O-gallylquinic acid (52), which were identified by HPLC. Therefore, 53, 54 and 55 have a core structure of 52 with a depside galloyl group. The position of the depside galloyl group on 53, 54 and 55 was determined by comparison of their chemical shifts of the hydroxymethine carbons of quinic acid moieties with those of 52. In the spectrum of 53, the signal due to C-4 of quinic acid moiety was observed to be 0.4 ppm downfield shifted (Table 7) while those due to C-3 and C-5 of the same moiety remained unchanged. This implies that the depside galloyl group of 53 is attached to the galloyl group at the C-4 position.⁸ _' ⁹ In the case of 54 and 56, the signals due to C-5 and C-3 positions were observed to be 0.4 and 0.3ppm downfield shifted compared to that of 52, respectively. Therefore, the structures of 53, 54, and 55 were characterized as the new 3,5-di- O-galloyl-4-0- digalloylquinic acid _τ 3,4-di-0-galloyl-5- digalloylquinic acid and 3-0-digallσyl-4,5-di-O- galloylquinic acid, respectively.

The ^'H-NMR spectrum of 56 showed three acylated hydroxymethine protons due to the quinic acid moiety and four singlets of galloyl groups (δ 7.02, 7.07, 7.13 and 7.17). The ¹³C-NMR spectrum also indicated that 56 is a tetragalloylquinic acid. There was no signal from a depside galloyl group in the ¹H and ¹³C-NMR spectra. Therefore 56 is characterized as 1,3,4,5,-tetra-O-galloylquinic acid.

Many galloylquinic acids are reported in the literature.⁶ _' ⁷ _' ¹⁰ However, 53, 54 and 55 are the first examples of galloylquinic acids with depside .galloyl groups.

Example 1: Isolation and Characterization of HIV RT Inhibitors from Tannin

¹H- and ¹³C-NMR spectra were measured on a JEOL JNM-GX400 spectrometer using TMS as an internal standard. Specific rotation was determined on a AUTOPOL (Rudolph Research) polari eter. High performance liquid chromatography (HPLC) was performed on a Waters model 600OA chromatograph equipped with a model 440 absorbance detector, a U6K injector and a TSK-ODS 80Tm (4.6 i.d. x 150mm, Toyo Soda) column for the reverse phase HPLC [(mobile phase, acetonitrile-water-formic acid(15:85:l) ] ; and a Porasil (3.9 i.d. x 300mm, Waters) for the normal phase HPLC [mobile phase, n-hexane- methanol-tetrahydrofuran-formic acid (55:33:11:1) containing oxalic acid lg/1]⁵ Preparative HPLC was performed on a Walters model 501 solvent delivery system using a micro-Bondapak column (19 i.d. x 150 mm) and a Waters differential refractometer, which utilized a solvent system of acetonitrile-water formic acid (20:80:0.5, 15:85:0.5 and 13:87:0.5). Low pressure chromatography was performed with a FMI pump utilizing a Fuji-gel RQ-3 column.

Tannic acid, purchased from Aldrich Chemical Company, Inc., Milwaukee, WI, (lot No. 05720KM, 50g) was partitioned with ethyl acetate and water. The ethyl acetate layer (47.8g) was chromatographed on a Sephadex LH- 20 (4.5 i.d. x 45 cm) column, eluted with ethanol, ethanol-water (increasing water content of 10%, 20%, 30% and 40%) and then ethanol-water-acetate. Each fraction (50-100ml) was analyzed by normal phase HPLC. Repeated chromatography yielded fractions containing mono to heptagalloylated compounds. The fraction which contained tetragalloyl compounds (G-4, 2.69g) was further fractionated by low pressure chromatography on Fuji gel RQ-3 column and a preparative HPLC to yield compounds 53(48mg), 54(35mg), 55(30mg) and 56(48mg) as amorphous powders. Compound 52 (186mg) was isolated from the fraction containing trigalloyl moieties as amorphous powders by the same methods described above.

3.4.5-Tri-O-σalloylquinic acid (52) An amorphous powder; [α]D²⁰ -124o(c=0.20, acetone-d₆ ) ; ¹ H- NMR (acetone) S 2.33 (IH, dd, J=6.5 and 14.3 Hz, C₆-H) , 2.44 (2H, , C₂-H and H'), 2.58 (IH, dd, J=3.9 and 14.3Hz, C₆H), 5.52 (IH, dd, J=3.3 and 8.3Hz, C₄ -H) , 5.81(1H, m, W_1/2=15HZ, C5-H) , 5.83(1H, m, w_1/2=25Hz, C₃ - H) , 7.06(2H, s, galloyl), 7.09(2H, s, galloyl), and 7.17 (2H, s, galloyl); and for ¹³C-NMR see Table 7.

3,5-di-O-σalloyl-4-0-diσalloylquinic acid (53) An amorphous powder [α]_D ²⁰ -91° (c=0.53, acetone); ¹ H- NMR(acetone-d₆) 52.34(IH, dd, J=6.2 and 14.4Hz, C₆- H) , 2.43(2H, , C₂-H and H'), 2.57(1H, br.d, C₆-H'), 5.54(IH, dd, J=3.0 and 8.4Hz, C₄-H) , 5.84(2H, m, C₃ - and C₅-H), 7.09(S)*, 7.17(S)*, 7.24(S)**, 7.34(d, J=2.0Hz) *, 7.36(d, J=2.0HZ)*, 7.10(S)**, 7.11(s)**, 7.18(s)** and 7.23(s)**; and for ¹³C-NMR see Table 7.

3,4-di-O-σalloyl-5-0-diσalloylαuinic acid (54) An amorphous powder; [α]₀ ²⁰ -92° (c=0.60, acetone); ^lHNMR(acetone-d₆) δ 2.33-2.45(3H, m, C₂-H, H¹ and C₆- H) , 2.58(IH, dd, J=3.5 and 14.5, C₆-H'), 5.52 (IH, dd, J=3.5 and 8.7Hz, C₄-H) , 5.83(IH, m, W_1/2=l5Hz, C₅-H) , 5.86(1H, m, W_1/2=25HZ, C₃-H) , 7.05(s)*, 7.07(s)*, 7.28(s)*, 7.43(d, J-2.0HZ)*, 7.50(d, J=2.0Hz)*, 7.05(s)**, 7.06(s)**, 7.09(s)** and 7.29(s)**; and for ¹³C- NMR see Table 7.

3-0-diσalloyl-4-5-di-O-σalloylquinic acid (55) An amorphous powder; [α]_D ²⁰ -106* (c=0.48, acetone); ^lH- NMR(acetone-d₆ ) δ 2.34(1H, dd, J=6.1 and 14.2Hz, C₆- H) , 2.40-2.48(3H, m, C₂-H, H¹ and C₆-H'), 5.52(IH, dd, J*=3.3 and 8.6Hz, C₄-H), 5.81(1H, m, W_1/2=l5Hz, C₅ -H) 5.86(1H, m, W_1/2=25HZ, C₅-H) , 7.04(s)*, 7.17(s)*, 7.26(s)*, 7.35(d, J=2.0HZ)*, 7.42(d, J=2.0Hz)*, 7.06(s)**, 7.15(s)**, 7.24(s)** and 7.28(s)**; and for ¹³C-NMR see Table 7.

1.3.4.5-Tetra-O-galloylquinic acid (56) An amorphous powder; [α]_D ²⁰ -72'(c=0.20, acetone); ¹ H- NMR(acetone-d₆ ) δ 2.47(IH, dd, J=7.8 and 13.7Hz, C₆- H) , 2.85-3.00(3H, m, C₂-H and C₆-H'), 5.61(1H, dd, J=3.5 and 8.6Hz, C₄-H) , 5.95(1H, m, W_1/2=l5Hz, C₅-H) , 5.88(1H, m, w_1/2=25Hz, C₃-H), 7.06(2H, s, galloyl), 7.07(2H, s, galloyl), 7.13(2H, s, galloyl), and 7.17(2H, s, galloyl) ; and for ^{τ 3}C-NMR see Table 7.

In the above characterization data, * denotes signals due to an m-digalloyl group and ** signals due to a p-digalloyl group. Example 2: Bioloσical Activity

The effects of 52 (3,4,5-GQA), 53(3,5,-G-4- diGQA) , 54(3,4-G-5-diGQA) , 55(3-diG-4,5-GQA) and 56(1,3,4,5-GQA) against HIV RT isolated from infected cells and HIV infected H9 lymphocytes are summarized in Table 6. Compounds 53, 54, 55 and 56 showed 90, 89, 84 and 94% inhibitory activity against HIV RT at lOOμM, respectively. Compounds 53, 54 and 55 exhibited more than 50% inhibitory activity at 10μM, and there was no significant difference in inhibitory activities among these three tetragalloylquinic acids. The inhibitory activities of 56 and 52 were shown to be less than those of 53, 54 and 55. This indicated that the depside galloyl group in the molecules of 53, 54 and 55 plays an important role in their inhibitory effects.

These compounds (52-56) also exhibited the inhibitory activity against the growth of HIV in infected H9 lymphocytes (61- 70% inhibition at 25 μM) . At this level, these galloylquinic acids showed little cytotoxicity against the uninfected H9 cells (0-25%) , and no cytotoxicity was observed at 6.25 and 1.25μM. The growth of HIV was inhibited even at 6.25μM (44-59%) and 1.25μM (13-34%). It is interesting to note that the inhibition of HIV RT and 30μM and HIV growth at 25μM by 53, 54^* and 55 was we^'ll correlated. This correlation was also found between HIV RT inhibition at lOμM and HIV growth at 6.25μM.

The inhibitory effect of 54 against DNA polymerase alpha, beta and gamma, as well as HIV-RT, is shown in Figure 5. The sensitivity of these DNA polymerases against 54 was found to be quite different. DNA polymerase alpha is most sensitive to 54. The ID₅₀ of 54 for DNA polymerase alpha(ID_{S 0}=0.065 ± 0.019μM) is about 1/450 of that for HIV RT (ID₅₀=29 ± 18μM) , and the sensitivities are in the order of DNA polymerase alpha>gamma-(ID₅₀=2.5+O.9μM)>beta(21±llμM)=HIV RT. As mentioned above, 54 inhibits HIV RT in vitro and HIV growth in H9 cells, and the percent inhibitions in these assays are well correlated. Therefore, the HIV growth inhibition by 54 might be due to its inhibitory effect against HIV RT. However, Compound 54 was much more potent against DNA polymerase alpha, which was not well correlated with inhibition of cell growth. These results imply that there are other factors in uninfected cells which render them resistant to these compounds.

Compounds 53, 54 and 55 appear to be the first examples of plant products that demonstrate potent inhibition of both HIV RT activity and HIV growth in culture. The structures of these compounds are unique compared to those of the other known HIV RT inhibitors. These compounds are intended for use as anti-AIDS drugs. Compounds 52 and 56, though less effective, may also be useful. Combination of these compounds with each other and with other anti-AIDS drugs may also be of value.

HIV RT assay: The HIV RT assay was performed according to the method described by Cheng et al. , incorporated by reference herein to the extent pertinent. The enzyme was immunopurified from virion- containing extracts of human T cells infected with HIV lymphotropic virus. Poly rA oligo dT_{t 0} from Pharmacia, Piscataway, N.J. was used as the template to measure the incorporation of [³H] dTMP (20μM) . The percentage of inhibition was determined by comparing the RT activity of drug containing assay to that of the drug- free control. Results are shown in Table 4.

HIV growth inhibition assay: This assay was performed by incubation of H9 lymphocytes (IxlO⁷ cells/ml) in the presence or absence of HIV-1 (HTLVIIIB) for 1 hour at 37^βC. Cells were washed thoroughly to remove unadsorbed virions and resuspended at 4 x 10⁵ cells/ml in culture medium. Aliquots (1ml) were placed in wells of 24 well culture plates containing an equal volume of test compound (diluted in test medium) . After incubation for three days at 37"C, cell density of uninfected cultures was determined to assess toxicity of the test compound. A p24 antigen capture assay was used to determine the level of HIV infection in HIV treated cultures. The ability of test compounds to inhibit HIV replication was measured at four different concentrations of test compound relative to infected or uninfected cultures. Test compounds were considered to be active if p24 levels were <70% of infected, untreated cultures. The results are shown in Table 5.

DNA polymerase assay: Human DNA polymerase alpha¹¹ , beta¹² and gamma¹ - were purified as described previously. DNA polymerase alpha activity was assayed at 50μl reaction mixture containing 50mM Tris buffer (pH 8.0), lmg/ml of bovine serum albumin, 6mM MgCl₂ , ImM dithiothreitol, lOOμg/ml of gapped duplex DNA¹³ , 10μM[³H]TTP (ICi/mmol) , 50μM each of dATP, dCTP and dGTP, and approximately 0.01 units of DNA polymerase activity. After incubation at 37°C for 1 hour, the DNA was precipitated onto glass fiber filters with a 5% trichloroacetic acid, lOmM pyrophosphate solution and counted for radioactivity as previously described¹¹. DNA polymerase beta, gamma and HIV RT were assayed in the same manner except that lOOmM KCl was included in the reaction mixture. The effect of the galloylquinic acids (52-56) in HIV cell growth is shown in Table 6.

The tannins described hereinabove can be administered as a partially purified extract of tannic acid, such as a polygalloyl, elagalloyl, condensed tannin, complex tannin, and the like. However, the compounds are preferably administered in pure form. Pharmaceutically acceptable derivatives, such as salts, amides, and esters, may be substituted for the acids themselves.

Compositions within the scope of the present invention include compositions wherein the tannin derivative is contained in an effective amount to achieve inhibition of the reverse transcriptase activity of HIV. Determination of the effective amounts is within the skill of the art.

In addition to the tannic acid derivatives, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically Preferably, the preparations, particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.1 to 99 percent, preferably from about 25-85 percent by weight, of active compound, together with the excipient.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.

Suitable excipients include, in particular, fillers such as sugars, including lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellu- lose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxy methyl starch, crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum Arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses.

Other pharmaceutical preparations -which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plastiσizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. in addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffine hydrocarbons, polyethylene glycols or higher alkanols. In addition, it is also possible to use gelatin rectal capsules which consists of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffine hydrocarbons.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds, as well as emulsions in appropriate pharmaceutically acceptable oils.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation.

TABLE 1: Inhibitory Effect Of Tannins Against HIV RT

Percent Inhibition

No, Compound Class+ 0μg/ ml 50 μg/ml * S. D.

1 l-0-Galloy-/9-D-glucose

2 1,6-Di-0-galloy-/3-D-glucose

3 1,2,6-Tri-0-galloyl-,β-D-glucose

4 1,2,3,6-Tetra-0-galloyl-,9-D-glucose

5 1,2,3,4,6-Penta-0-galloyl-0-D-glucose

6 4-0-Galloylquinic acid

7 1,4-Di-O-galloylquinic acid

8 1,3,4-Tri-O-galloylquinic acid

9 3-O-Galloylshikimic acid

10 3-0-Digalloylshikimic acid

11 3,5-Di-0-galloylshikimic acid

12 3,4,5-Tri-O-galloylshikimic acid 46+ 1

13 2,3-Hexahydroxydiphenoyl-glucose

14 Strictinin

15 Pedunculagin

16 l(/3)-0-Galloylpedunculagin

17 Eugeniin

18 Corilagin

19 Punicafolin

20 Phillyraeoidin A

21 Sanguiin H-6

22 Sanguiin H-ll

23 Castalagin

24 Vescalagin

25 Geraniin

26 Elaeocarpusin

27 Furosin

28 Chebulinic acid

29 Chebulagic acid

30 Punicalin

31 Punicalagin

32 Punicacortein C

33 Terchebin

34 Castanopsinin A

35 Castanopsiniin A

36 Hamamelitannin

37 2• ,3,5-Tri-O-galloylhamamelose

38 Grandinin

39 ( - ) -Epicatechin

40 Procyanidin B-2

41 Procyanidin C-l

42 CinnamtanninA₂

43 (-)-Epicatechin 3-0-gallate

44 Procyanidin B-2 3,3•-di-O-gallate

45 Procyanidin C-l 3,3•,3"-tri-0-gallate

46 Procyanidin A-2

47 CinnamtanninB

48 CinnamtanninB₂

49 Lithospermicacid B Mg salt

50 Acutissimin A t

51 Mongolicain A

+ G: gallotannin, E: ellagitannin, C: condensed tannin,

EC: complex tannin # Caffeic acid tetramer

Positive control: AZT triphosphate O.lμM 70+ 6%

Phosphonoformate 0.6μM 71+ 6%

Data represent means of percent inhibition plus a standard deviation rounded to whole numbers.

TABLE 2. Inhibitor Present

Virus Infection Per Cent

Inhibitor Preinfection Step Culture Virus Inhibition

GN-30 51 (10 μg/ml)

+ 0 + + 97

+ + 93

+ 39 to

DDC + 72 (0.01 μg/ml)

+ 2 + + 78 + + 3

+ 77

Dextran sulfate + 100 (1 μM)

+ 94 + + 100

+ + 98 80

TABLE 3: Effect of Punacalin on HIV-RT

Varied Assay Mixtures Components B

Activated DNA (μg/ml) 75 75 375 dTTP (μM) 5 25 5

Percent Activity (50μg/ml) mean 11.4+2 34.4+7 45.3+8

TABLE 4. Inhibitory Effect of the Fractions from Tannic Acid Against HIV RT

Fractions Inhibition at lOOμg/ml

(%)

G-l 0

G-2 10+15

G-3 49+4 to oo

G-4 83+2

G-5 68+5

G-6 56+11

G-7 49+17

G-8 48+14

positive control: AZT triphosphate lOOnm 64+6%

Phosphonoformate 0.6 M 63+9%

TABLE 5. Inhibitory Effect of Tannic Acids Against HIV RT

Inhibition at lOOμg/ml

Tannic acid (from a commercial 74+ 7 source)

Tannic acid (Turkish galls 7+ 8 extract)

Tannic acid (Chinese galls extract) 10+14

positive control:

AZT triphosphate at lOOμM 64+6% Phosphonoformate 0.6μM 63+9%

TABLE 6. Effect of Galloylquinic Acids on HIV Cell Growth and HIV RT Activity

HIV RT-Activity (% Inhibition)

Compounds lOOμm 30μM lOμM

3 , 4 , 5-TriGQA (52) 58+6 48+11 39+10

3,5-G-4-diGQA(53) 90+3 73+5 53+3

3,4-G-5-diGQA(54) 89+5 70+9 55+11 > o

3-DiG-4,5-GQA(55) 84+7 63+6 50+11

l,3,4,5-TetraGQA(56) 94+2 84+7 36+6

Indicates percent inhibition of uninfected H-9 cell growth in the presence of drug.

The μM concentration of 1 was 30.8, 7.7 and 1.5μM, respectively.

TABLE 7. ³C-NMR Data 52, 53, 54, 55 and 56

52 53 54 55 56 3,4,5-G 3,5-G-4-diG 3,4-G-5-diG 3-diG-4,5-G 1, 3,4,5

Quinic acid moiety 0-0 110.0(4C) 72.7(+0.4) 70.1(+0.4) 69.2(+0.3) 121.3(2

C-l 74.4 74.3 74, 74.3 79.8 C-2 38.6 38.6 38. 38.6 37.3 C-3 68.9 68.9 68, 69.2 (+0.3) 68.6 C-4 72.3 72.7(+0.4) 72, 72.4 71.9 C-5 69.1 69.7 70.1(+0.4) 69.6 69.4 C-6 36.5 36.5 36.4 36.5 33.3 COOH 175.9 176.0 176.0 175.8 172.2

Gallic acid moiety

C-l 120.5 120.6 12l.2(2 121.0 121.1 121.3(2 121.3 121.3 121.8 121.9

C-2,6 109.8 109.9 110.1(6 110.0 110.0 110.3(2 110.1 110.6 114.6

117.5

C-3,5

C-4

-COO-

All spectra were measured in acetone-d₆ using TMS as an internal standard.

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Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting the reverse transcriptase (RT) activity of a reverse transcriptase of a human retrovirus which comprises contacting a living or nonliving material comprising such a reverse transcriptase with an inhibitory amount of a tannin having human retrovirus RT- inhibiting activity or an inhibitory derivative thereof.

2. The method according to claim 1 wherein the tannin is selected from the group consisting of gallotannins, ellagitannins, condensed tannins, and complex tannins.

3. The method according to claim 2 wherein the tannin is a gallotannin.

4. The method according to claim 3 wherein the gallotannin is a galloylquinic acid having at least three galloyl groups.

5. The method according to claim 4 wherein the galloylquinic acid is a tetragalloylquinic acid.

6. The method according to claim 5 wherein the acid is 3,5,-di-O-galloyl-4-O-digalloylquinic acid.

7. The method according to claim 5 wherein the acid is 3,4,-di-O-galloyl-5-O-digalloylquinic acid.

8. The method according to claim 5 wherein the acid is 3-O-digalloyl-4,5-di-O-gallylquinic acid.

9. The method according to claim 5 wherein the acid is 1,3,4,5,-tetra-O-galloylquinic acid.

10. The method according to claim 4 wherein the galloylquinic acid is 1,3,4-tri-O-galloylquinic acid.

11. The method according to claim 3 wherein the gallotannin is 3,5-di-0-galloylshikimic acid.

12. The method according to claim 3 wherein the gallotannin is 3,4,5-tri-O-galloylshikimic acid.

13. The method according to claim 2 wherein the tannin is an ellagitannin.

14. The method according to claim 13 wherein the ellagitannin is strictinin.

15. The method according to claim 13 wherein the ellagitannin is corilagin.

16. The method according to claim 13 wherein the ellagitannin is castalagin.

17. The method according to claim 13 wherein the ellagitannin is vescalagin.

18. The method according to claim 13 wherein the ellagitannin is chebuliniσ acid.

19. The method according to claim 13 wherein the ellagitannin is chebulagic acid.

20. The method according to claim 13 wherein the ellagitannin is punicalin.

21. The method according to claim 13 wherein the ellagitannin is punicalagin'.

22. The method according to claim 13 wherein the ellagitannin is punicacortein C.

23. The method according to claim 2 wherein the tannin is a condensed tannin.

24. The method according to claim 23 wherein the condensed tannin is cinnamtannin B₂ •

25. The method according to claim 1 wherein the reverse transcriptase essentially corresponds to the reverse transcriptase of a human retrovirus.

26. The method according to claim 25 wherein the reverse transcriptase essentially corresponds to a reverse transcriptase of HIV.

27. A method for inhibiting the growth of HIV-infected cells in a patient suffering from an HIV infection which comprises administering to the patient an effective amount of a tannic acid derivative having HIV-infected cell growth inhibitory activity or an active derivative thereof.

28. The method according to claim 27 wherein the tannin is selected from the group consisting of gallotannins, ellagitannins, condensed tannins, and complex tannins.

29. The method according to claim 27 wherein the tannin is a gallotannin.

30. The method according to claim 29 wherein the gallotannin is a galloylquinic acid having at least three galloyl groups.

31. The method according to claim 30 wherein the galloylquinic acid is 1,3,4-tri-O-galloylquinic acid.

32. The method according to claim 28 wherein the gallotannin is 3,5-di-O-galloylshikimic acid.

33. The method according to claim 29 wherein the galklotannin is 3,4,5-tri-O-galloylshikimic acid.

34. The method according to claim 28 wherein the tannin is an ellagitannin.

35. The method according to claim 34 wherein the ellagitannin is strictinin.

36. The method according to claim 34 wherein the ellagitannin is corilagin.

37. The method according to claim 34 wherein the ellagitannin is castalagin.

38. The method according to claim 34 wherein the ellagitannin is vescalagin.

39. The method according to claim 34 wherein the ellagitannin is chebulinic acid.

40. The method according to claim 34 wherein the ellagitannin is chebulagic acid.

41. The method according to claim 34 wherein the ellagitannin is punicalin.

42. The method according to claim 34 wherein the ellagitannin is punicalagin.

43. The method according to claim 34 wherein the ellagitannin is punicaσortein C.

44. The method according to claim 28 wherein the tannin is a condensed tannin.

45. The method according to claim 44 wherein the condensed tannin is cannamtannin B₂.

46. A method for inhibiting the propagation in human cells of a human retrovirus which comprises exposing infected cells to punicalin or an inhibitory derivative thereof.

47. Use of a tannin or derivative thereof having HIV-infected cell growth inhibitory activity in the manufacture of a means for the treatment of an HIV infection.