WO1991006589A1

WO1991006589A1 - New anti-hiv compounds belonging to aurintricarboxylic acid

Info

Publication number: WO1991006589A1
Application number: PCT/US1990/006408
Authority: WO
Inventors: Rudiger D. Haugwitz; Venkatachala Narayanan; Marc CUSHMAN
Original assignee: The United States Of America, Represented By The Secretary, United States Department Of Commerce; Purdue Research Foundation
Priority date: 1989-11-03
Filing date: 1990-11-05
Publication date: 1991-05-16
Also published as: AU6728290A

Abstract

The invention relates to the synthesis of compounds with anti-retroviral activity. The synthesis of substantially pure aurintricarboxylic acid monomer and structurally defined polymers, analogs and derivatives thereof, is described. The compounds are shown to have potent anti-retroviral activity, particularly against HIV-1.

Description

NEW ANTI-HIV COMPOUNDS BELONGING TO AURINTRICARBOXYLIC ACID

FIELD OF THE INVENTION The present invention is related to the chemical synthesis of a substantially pure monomer of aurintricar- boxylic acid (ATA), structurally defined polymers, analogs and/or derivatives thereof and to defining anti-viral activity of the new compounds, particularly against human immunodeficiency virus (HIV) . BACKGROUND OF THE INVENTION

Treatment of a mixture of salicylic acid and formaldehyde with sulfuric acid and sodium nitrite results in the formation of a solid substance known as aurintri- carboxylic acid (ATA) (Caro, Chem. Ber. 1892, 25, 939). This material was originally believed to have the tri- phenylmethane dye structure 1 shown in Figure 1, and this structure has persisted in the current literature even though studies reported by Gonzalez, Blackburn, and Schliech seem to indicate quite clearly that ATA is actually a heterogeneous mixture of polymers which was represented schematically as structure 2 (Gonzalez et al, T. Biochem. Biophys. Acta 1979, 562, 534), also shown in Figure 1.

ATA is a potent inhibitor of cellular processes that depend on the binding of nucleic acids to proteins. This inhibition of the binding of nucleic acids to pro¬ teins may be due to the fact that the polymeric and polyanionic nature of ATA resembles the structures of oligonucleotides. To the extent that ATA occupies the oligonucleotide binding sites of a wide variety of pro¬ teins that normally act on oligonucleotides, it can be regarded as an oligonucleotide mimetic. Examples include the inhibition of protein synthesis by blocking the attachment of mRNA to the ribosome (Grollman et al, Proc. Natl. Acad. Sci. U.S.A. 1968, 61, 719; Stewart et al, Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 97; and Kreamer et al. Can. J. Biochem. 1978, 56, 1162) and inhibition of aminoacyl-tRNA synthetase (Igarashi et al. Can♦ J. Biochem. 1975, 53, 120). ATA is also known to reduce the affinity of the DNA primer for reverse transcriptase (Givens et al. Nucleic Acids Res. 1976, 3, 405), and to interfere with the activities of DNA (Seki et al, Biochem. Bioohvs. Res. Commun. 1977, 79, 179; Tsutsui et al, Biochem Biophys. Acta 1978, 517, 14; and Nakane et al, Eur. J. Biochem. 1988, 177, 91) and RNA (Blumenthal et al, Biochem. Biophys. Res. Commun. 1973, 55, 680; Liao et al, J. Med. Chem 1975, 18, 117; and Iapalucci-Espinoza et al, Mol. Cell Biochem. 1983, 55, 41) polymerases. Ribonucleo- tide reductases from a variety of sources are inhibited uniformly by ATA (Baumann et al, Naturforsch. 1984, 39c, 276), as are an array of ribonucleases (Apirion et al. Antibiotics (NY) , 1975, 3, 327; Zuag et al. Cell, 1980, 19, 331; Gonzalez et al. Biochemistry 1980, 19, 4299; Schulz-Harder et al, Biochem. Biophys. Res. Commun. 1982, 104, 903; Ramon et al, FEMS Microbiol. Lett. 1986, 36, 9; Eslami et al, Bioche . Interna . 1986, 13, 163; and Kumar et al, Indian J. Exper. Biol. 1986, 24, 79). ATA has recently been shown to block DNA-cellulose binding to progesterone, estrogen, glucocorticoid, and 1.25-dihy- droxyvita ine D3 receptors, suggesting that steroid hormone receptors and nucleic acid polymerase may have similar polynucleotide binding sites (Moudgil et al. Life Sci. 1980, 27, 1159; Moudgil et al, Archs. Biochem. Biophys. 1982, 213, 98; Mellon, Biochem. Pharmacol. 1984, 33, 1047; Moudgil et al. Steroid Biochem. 1985, 23, 125; and Moudgil et al, J. Steroid Biochem. 1985, 22, 747).

It was recently demonstrated that ATA prevents the cytopathic effect of HIV-1 in ATH8 cell culture as well as the expression of. p24 in H9 cells infected with HIV-1, and it has been suggested that this effect may be due to the inhibition of HIV-1 reverse transcriptase (Balzarini et al, Biochem. Biophys. Res. Commun. 1986, 136, 64) and/or the blockade of the HIV/CD4 cell receptor (Schols et al, Proc. Natl. Acad. Sci. USA 1989, 86, 3322). In addition, it has been reported that the triphenylmethane dye fuchsin acid, as well as ATA, inhibit the cytopathic effect of HIV-1 in MT-4 cell culture (Baba et al, Biochem. Biophys. Res. Commun. 1988, 155, 1404). A variety of authentic triphenylmethane dyes sharing the same skeletal structure as fuchsin acid, along with ATA, have also been reported to inhibit Raucher leukemia virus reverse transcriptase, E. coli RNA polymerase, and protein biosynthesis (Liao et al, supra) . These observations seem to contradict the conclusions of other workers "that aurintricarboxylic acid in the form of the commonly accepted structure 1 is ineffective as an inhibitor of protein nucleic acid interactions, and that the inhibitory activity of an aurintricarboxylic acid preparation is proportional to the degree of polymerization" (Gonzalez et al, supr ) . In light of such conflicting reports, the question arises as to why do non-polymeric, authentic triphenylmethane dyes inhibit protein nucleic acid interactions (Liao et al, supra) . Assuming that the known inhibition of HIV-1 reverse transcriptase by both ATA structure 2 and fuchsin acid is responsible for the observed anti-HIV activity (Balzarini et al, supra; Schols et al, supra; and Baba et al, supra) , a related question is whether or not a com¬ pound having the commonly accepted but incorrect structure 1 of ATA would have any potential as an anti-AIDS agent. In order to provide answers to such outstanding problems, one of the steps taken was to synthesize substantially pure monomer of ATA. Such a compound was not heretofore known or described.

SUMMARY OF THE INVENTION It is, therefore, an object of the present inven- tion to provide a substantially pure monomer of ATA.

It is another object of the present invention to prepare structural analogs of the monomer of ATA, includ¬ ing a variety of substituted di- and tri-phenylmethanes.

It is a further object of the present invention to synthesize homogeneous, structurally defined polymers related to ATA and to prepare structural analogs of polymeric ATA.

It is yet another object of the present invention to fractionate polymeric ATA and polymeric ATA analogs on the basis of molecular weight and to identify the anti-HIV activity and cytotoxicity of the various fractions.

A still further object of the present invention is to manufacture compounds having greater potency and less cytotoxicity than ATA so as to design drugs that would be useful for the treatment of AIDS and other viral diseases.

Various other objects and advantages will become evident from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the originally conceived structure of triphenylmethane, and the actual polymeric form of ATA. Figure 2 outlines the syntheses of triphenyl carbinol.

Figure 3 outlines the syntheses of 4,4' ,4"- trihydroxy-5,5' ,5"-tricarboxytriphenyl ethane, 5,5',5"- tricarbomethoxy-4,4',4"trihydroxytriphenylmethane, 4,4'- dihydroxy-3,3'-dimethyl-5,5'-dicarboxydiphenylmethane, 5,5 ' ,5"-trimethyl4,4' ,4"-trihydroxy-3,3' ,3"-trimethyl- triphenylcarbinol, and 5,5' ,5"- ricarbomethoxy-4,4' ,4"- trihydroxy-3,3' ,3"-trimethyltriphenylcarbinol.

Figure 4 outlines the syntheses of 5,5'-dibromo-

3,3' ,3"-dicarboxy-2 , 2'-dihydroxydiphenyl-methane, 3,3'- dicarboxy-2,2'-dihydroxydiphenylmethane, 3,3'dicarbo- methoxy-2,2'-dihydroxydiphenylmethane, aurintriphosphoric acid, and aurintrisulfonic acid.

Figure 5 outlines the gel permeation chromatogra¬ phy fractionation of ATA. Figure 6 outlines the equilibrium dialylsis and ultrafiltration fractionation of ATA.

Figures 7-25 provide in-vitro anti-HIV drug screening results for compounds described herein. DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by the chemical synthesis of substantially pure monomer, structurally defined polymer, analogs, fractions and derivatives of ATA, preferably having anti-viral and more preferably having anti-HIV activity.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

The term "substantially pure" as used herein means the compound is as pure as can be obtained by conventional isolation and purification means.

EXAMPLE 1 Synthesis of the ATA monomer

A three step synthesis that gave the triphenylcar¬ binol 6 (ATA monomer) is depicted in Scheme I shown in Figure 2. This is the covalent hydrate of the hypotheti¬ cal structure 1, which could not be obtained despite various attempts. The successful route relied on the use of chlorine as a protecting group in order to control the regiochemistry during the first step and to prevent the further reaction of the diphenylmethane in the presence of formaldehyde and acid to form polymers of the phenol- formaldehyde type. Thus, 3-chlorosalicylic acid 3 was treated with formaldehyde and sulfuric acid to give the diphenylmethane 4. Both of the chlorine atoms present in 4 were then removed by hydrogenolysis over palladium on charcoal to afford methylene disalicylic acid 5. Reaction of intermediate 5 with salicylic acid in the presence of sulfuric acid and sodium nitrite then gave the triphenyl- carbinol 6, which^' is the covalent hydrate of structure 1. The triester 7 was also prepared by treatment of 6 with diazomethane.

EXAMPLE 2 3,3'-dichloro-5,5'-dicarboxy-4,4'-dihydroxydiphenylmethane 111 3-Chloro-salicylic acid (1.72 g, 10 mmol) was dissolved in methanol (10 mL) . Water (2.5 mL) was added and the flask was cooled to -5°C in an ice-salt bath. Concentrated sulfuric acid (30 mL) was added during 20 min. while the temperature was maintained at -5 to 0°C. The reaction mixture was then stirred at this temperature for 1 h while an aqueous solution of 37% formaldehyde (4 mL) was added. The temperature was then maintained at 0°C for 1 h before the reaction mixture was left at room temperature (about 22°-24°C) for 24 h. The mixture was poured into crushed ice (150 g) and the precipitate was filtered and dried to give the product 4 (1.7 g, 96%) as a solid. The analytical sample was recrystallized from chloroform-methanol (2:1): mp 302°C; UV (ethanol) 316 nm (8,300); IR (KBr) 3400-2850, 1680, 1610, 1460, 1290, 1235, 1170, 870, 780 cm"¹; -R NMR (200 MHz, acetone-d^) δ 7.68 (s, 2 H), 7.47 (s, 2 H), 3.87 (s, 2 H) ; ¹³C NMR (DMSO-dβ) δ 171.53, 155.23, 135.57, 132.29, 128.77, 120.64, 114.32, 37.89; CIMS m/e. (relative intensity) 357 (MH⁺, 93), 339 (100). Anal. Calcd for C₁₅H₁₀CL₂O₆: C, 50.42; H, 2.80. Found: C, 50.11; H, 2.72.

3,3'-dicarboxy-4,4'-dihydroxydiphenylmethane (5

Compound 4 (1.78 g, 5 mmol) was dissolved in ethanol (30 mL) and triethylamine (15 mL) . Pd/C (10%, 500 mg) was added to the solution and the mixture was stirred under an atmosphere of hydrogen for 48 h. The catalyst was filtered off, the solvent was evaporated, and water (100 mL) was added to the residue. The solution was cooled and acidified by addition of cone, hydrochloric acid (5 mL). The white precipitate was filtered and dried to give the product 5 (1.32 g, 92%) as a solid. The analytical sample was recrystallized from chloroform- methanol (2:1): 268-269°C; UV (ethanol) 312 nm (7,530); IR (KBr) 3400, 3250-2980, 1680, 1620, 1590, 1490, 1445, 1280, 1299 cm"¹; ^XH NMR (200 MHz, acetone-dg) δ 10.95 (s, 2 H, exchangeable with D₂0), 7.73 (d, 2 H, J = 2 Hz), 7.33 (dd, 2 H, J = 8 and 2 Hz), 6.88 (d 2 H, J = 8 Hz), 3.89 (s, 2 H); ¹³C NMR (DMSO-ds) δ 171.85, 159.55, 136.13, 132.05, 129.79, 117.28, 112.71, 38.72; CIMS m/e (relative intensity) 289 (MH⁺, 100), 271 (37). Anal. Calcd for C₁₅H₁₂0₆: C, 62.50; H, 4.16. Found: C, 62.67; H, 3.98.

EXAMPLE 4 3,3' ,3"-tricarboxy-4,4' ,4"-trihvdroxytriphenylcarbinol (6) Powdered sodium nitrite (0.276 g, 4 mmol) was added with vigorous stirring to concentrated sulfuric acid (3 mL) . A mixture of compound 5 (0.576 g, 2 mmol) and salicylic acid (0.276 g, 2 mmol) was added in portions. The mixture was stirred until it was homogeneous and was then poured into a solution of sodium nitrite (0.276 g, 4 mmol) in sulfuric acid (3 mL) . Stirring was then contin¬ ued at room temperature for an additional 18 h. The mixture was then poured into crushed ice (100 g) with stirring. The orange precipitate was filtered and dried to give the product 6 (0.686 g, 78%): mp 236-238°C (dec); UV (ethanol) 312 (9.935), 412 (3,425), 553 nm (10,460); IR (KBr) 3400, 3200-2840, 1670, 1610, 1575, 1480, 1430, 1350, 1290, 1200, 1130, 1070 cm^-1; ^:H NMR (200 MNz, acetone-dg) δ 11.12 (s, 1 H, exchangeable with D₂0), 7.88 (3 H, d, J = 2 Hz), 7.46 (3 H, dd, J = 9 and 2 Hz), 6.92 (d, 3 H, J = 9 Hz), 5.58 (brs, exchangeable with D₂0); ¹³C NMR (acetone-dβ) δ 172.65, 161.99, 139.16, 136.45, 130.17, 117,60, 112.28, 80.59; FABMS (negative ion mode) m/e (relative intensity) 439 (M - H⁺, 100). EXAMPLE 5

3,3' ,3"-trimethoxycarbonyl-4,4' ,4"-trihvdroxytriphenyl¬ carbinol (7)

A solution of diazomethane in ether (20 mL, prepared from 0.824 g of nitrosomethylurea) was added to a solution of 6 (0.440 g, 1 mmol) in ether (20 mL) at 5°C and the mixture was kept at this temperature for 48 h. A few drops of acetic acid were added to the solution, the solvent was evaporated, and the product was purified by flash chromatography over silica gel (60-200 mesh, 40g) . The analytical sample was recrystallized from hexane- methylene chloride (1:1): mp 189-190°C (dec); UV (ethanol) 312 nm (11,365); IR (KBr) 3460, 3180, 1680, 1610, 1590, 1490, 1445, 1340, 1295, 1210, 1080 cm"¹; ^XH NMR (200 MNz, acetone-dβ) δ 10.82 (s, 3 H, exchangeable with D₂0); 7.78 (d, 3 H, J = 2 Hz), 7.29 (dd, 3 H, J = 9 and 2 Hz), 6.93 (d, 3 H, J - 9 Hz), 3.89 (s, 9 H) ; ¹⁰C NMR δ 170.22, 160.58, 137.38, 135.15, 128.46, 117.18, 111.57, 80.17, 52.16 cm"¹; CIMS m/e (relative intensity) 483 (MH⁺, 54), 465 (100), 331 (16). Anal. Calcd for C₂₅H₂₂0₁₀: C, 62.24: H, 4.56. Found: C, 61.88; H, 4.60.

EXAMPLE 6 Synthesis of 4,4' ,4"-trihydroxy-5,5' ,5"-tricarboxytri- phenylmethane (8)

A solution of ATA monomer 6 (0.660 g., 1.5 mmols) in absolute ethanol (150 ml) was shaken under hydrogen atmosphere at 80 lbs/in² using 10% palladium on carbon as catalyst for 72 h. The catalyst was filtered off and the solvent was removed under reduced pressure using a rota- vapor, to give 0.625 g (98%) of 4,4'4:-trihydroxy-5,5' ,5"- tricarboxy triphenylmethane: m.p. 280-282°C (decomposes). FABMS (positive ion mode) 425 (MN⁺, 9%); IR (KBr) 3600- 2800 (br), 1670, 1610, 1590, 1485, 1440, 1285, 1180 (br), 1070 cm^-1; ^*H NMR (200 MHz, acetone-dβ) δ 7.65 (3 H, d, J = 2.1 Hz), 7.28 (3 H, dd, J = 8.5 and 2.3 Hz), 6.91 (3 H, d, J = 8.5 Hz), 5.51 (1 H, s); ¹³C-NMR (acetone-dβ) δ 172.30, 161.37, 137.47, 135.35, 131.21, 118.21, 112.84, 54.19. See Figure 3.

EXAMPLE 7 Synthesis of 5,5' ,5"-tricarbomethoxy-4,4' ,4"-trihydroxy- triphenylmethane (9)

To a solution of 8 (0.424 g, 1 mmol) in ether (20 ml), a solution of diazomethane (prepared from 0.618 g of nitrosomethyl urea in 20 ml ether) was added at 5C and kept 24 h at this temperature. A few drops of acetic acid were added to remove excess of diazomethane. The solvent was removed under reduced pressure and the residue was chromatographed over silica gel (60-200 mesh, 20 g) using 1% ethyl acetate in dichloromethane as eluent to give 5,5'5"-tricarbomethoxy-4,4'4"trihydroxytriphenylmethane (0.355 g, 76%). The product was recrystallized from hexanedichloromethane (about 2:1): m.p. 201-202°C; CIMS (isobutane ionizing gas) 467 (MH⁺, 100%); IR (KBr) 3400- 3060 (br), 2960, 1675, 1610, 1585, 1480, 1435, 1335, 1290, 1260, 1200 (br), 1180, 960, 825, 780, cm^-1; -K NMR (200 MHz, chloroform-d.) δ 10.72 (3H, exchanged with D₂0), 7.52 (3 H, d, J = 2.0 Hz), 7.18 (3 H, dd, J = 8.75 and 2.35 Hz), 6.92 (3 H, d, J = 8.5 HZ), 5.38 (1 H, s), 3.89 (9 H, s), ¹³C-NMR (chloroform-d) δ 170.33, 160.31, 136.41, 134.10, 129.88, 117.85, 112.18, 53.98, 52.27. See Figure 3.

EXAMPLE 8 Synthesis of 4,4'-dihvdroxy-3,3'-dimethyl-5,5'-dicarboxy diphenylmethane (11) A mixture of 3-methyl salicylic acid (7.6 g, 0.05 mole) dilute sulfuric acid (25%, 100 ml) and formaldehyde solution (37%, 4.05 mL) was heated at 90°C, with stirring. After 4 h, another 2.0 ml of formaldehyde solution was added to the reaction mixture, and the mixture was heated at 90°C for a total period of 12 h, cooled to room temper¬ ature and poured into crushed ice (100 g) . The white precipitate was filtered and dried to give 7.82 g (99%) of 11. The product was recrystallized from ethanol-chloro¬ form (1:1), m.p. 296-298°C; CIMS (isobutane ionizing gas) 317 (MH⁺, 56%); IR (KBr) 3500-2830 (br) , 1675, 1620, 1470, 1445, 1300 (br), 1230, 1200, 1145, 900, 790 cm^"1; *H NMR (200 MHz, DMSO-dβ) δ 7.52 (2 H, S), 7.14 2 H, s), 3.77 (2 H, s), 2.19 (6 H, s). See Figure 3. EXAMPLE 9 Synthesis of 5,5' ,5"-trimethv-4,4' ,4"-trihvdroxy-3,3' ,3"- trimethyltriphenylcarbinol (12)

Concentrated sulfuric acid (25 mL) was taken in a 100 mL round bottomed flask. Powdered sodium nitrite (2.07 g, 0.03 mole) was added slowly with stirring. An intimate mixture of 3-methyl salicylic acid (1.52 g, 0.01 mol) and 4,4'-dihydroxy-3,3'-dimethyl-5,5'-dicarboxy diphenylmethane (3.16 g, 0.01 mol) was added in small portions over a period of 20 minutes. The dark red reaction mixture was left at room temperature (~22-24C) for 16 h and then poured over crushed ice (100 g) . The precipitate was filtered and dried to give 4.32 g (90%) of (12) as a dark red powder, m.p. > 310°C: FABMS (positive ion mode) 465 (MH⁺-H₂0, 100%), FABMS (negative ion mode) 481 (M-H, 10%), 463 (M-H-H₂0, 100%); IR (KBr) 3400 (br) , 3100-2800 (br) 1670, 1585, 1420, 1375, 1300 (br) , 1190, 1025, 890, 785 cm"¹; ^XH NMR (200 MHz, DMSO-dg) δ 7.50 (3 H, s), 7.24 (3 H, s) 2.17 (9 H, s), ¹³C-NMR (DMSO-dβ) δ 171.35, 157.57, 136.09, 134.47, 125.40, 123.54, 109.39, 77.99, 14.34. See Figure 3.

EXAMPLE 10 Synthesis of 5,5' ,5"-tricarbomethoxy-4,4' ,4"-trihvdroxy- 3,3' ,3"-trimethyltriphenylcarbinol (13 To a solution of 12 (0.974 g, 2 mmol) in methanol

(20 ml) cooled to 5°C, a solution of diazomethane in ether (25 ml, prepared from 1.545 g of nitrosomethyl urea) was added. The mixture was kept at 5°C for 48h. A few drops of acetic acid were added to remove excess of diazo- methane, solvent was removed under reduced pressure, and the residue was purified by flash column chromatography over silica gel (230-400 mesh, 20g) using dichloromethane- hexane (2:1) as- eluent. The isolated yield of the tri- methyl ester (13) is 0.650 g (62%); CIMS (isobutane ionizing gas) 525 (MH⁺, 8%), 507 (MH⁺-H₂0, 100%); IR (KBr)

^" 3420 (br), 3180 (br), 2945, 1680, 1610, 1520, 1440, 1340,

1280, 1195, 1125, 780 cm^"1; ^XH NMR (200 MHz, chloroform-d) δ 11.04 (3 H, s, exchanged with D₂o), 7.74 (3 H, s), 7.28 (3 H, s) 3.90 (9 H, s), 2.22 (9 H, s). See Figure 3.

EXAMPLE 11 5,5'-dibromo-3,3' ,3"-dicarboxy-2,2'-dihydroxydiphenyl- methane (15)

A stirred solution of 5-bromosalicylic acid (4,34 g, 0.02 mmol) in water (2 mL) was cooled to -5°C and concentrated sulfuric acid (80 mL) was added dropwise while the temperature was maintained between -5 and 0°C. A mixture of 38% aq formaldehyde (10 mL) and methanol (10 mL) was added to the reaction mixture over a period of 20 min. Stirring was continued for 2 h at 0°C. The reaction mixture was left at room temperature for 14 h and then poured into crushed ice. The white precipitate was filtered, washed with water, and dried to give the product 2 (4.13 g, 93%). The analytical sample was recrystallized by dissolving the solid in a minimum amount of methanol and then adding chloroform: mp 290°C (dec); IR (KBr) 3400-2800, 1655, 1600, 1445, 1425, 1285, 1230, 1170, 870, 855, 790, 675 cm"¹; -H NMR (200 MHz, acetone-dg) δ 7.90 (d, 2 H, J = 2 Hz), 7.56 (d, 2 H, J=2 Hz), 4.02 (s, 2 H) ; CIMS m/e (relative intensity) 477 (MH⁺, 100), 429 (84), 249 (24), 231 (43), 217 (25). See Figure 4.

EXAMPLE 12 3.3'-dicarboxy-2,2'-dihydroxydiphenylmethane (16)

5,5'-Dibromo-3 ,3 '-dicarboxy-2 ,2 '-dihydroxy¬ diphenylmethane (15, 4,46 g, 0.01 mol) was dissolved in 10% alcoholic potassium hydroxide (150 mL). Palladium on activated carbon (10%, 0.5 g) was added and the solution was hydrogenated at room temperature and atmospheric pressure for 20 h. The catalyst was filtered off, the solvent evaporated, and water (100 mL) was added to the residue. The alkaline solution was cooled in an ice bath and neutralized with dilute hydrochloric acid. The precipitate was filtered, washed with water, and dried to

• give the product .4 (2.62 g, 91%). The analytical sample was recrystallized from methanol-chloroform: mp 284- 1230, 1175, 1145, 1065, 880, 740 cm"¹; -K NMR (200 MHz, acetone-dg) δ 7.70 (dd. 2 H, J = 7 and 2 Hz), 7.26 (dd. 2 H, J = 7 and 2 Hz), 6.72 (t, 2 H, J = 8 Hz); CIMS m/e (relative intensity) 289 (MH⁺, 80), 271 (100), 151 (8), 139 (7) . See Figure 4.

EXAMPLE 13 3,3'-dicarbomethoxy-2,2'-dihydroxydiphenylmethane (17

A cooled solution of diazomethane (prepared from 0.9 g of nitroso methyl urea) in ether (20 mL) was added to a solution of 3,3'-dicarboxy-2,2'-dihydroxydi¬ phenylmethane (0.5 g, 1.74 mmol) in methanol (15 mL) at 5°C. The solution was stored in a refrigerator for 4 h. The solvent was evaporated and the residue was crystal¬ lized from chloroform-hexane to give the product 4 (0.52 g, 96%): mp 125-126°C; IR (KBr) 3400, 3080, 1660, 1605, 1430, 1285, 1245, 1190, 1140, 990, 740 cm^"1; ^XH NMR (200 MHz, acetone-dβ) δ 7.72 (dd. 2 H, J = 8 and 2 Hz), 7.34 (dd. 2 H, J = 8 and 2 Hz), 6.84 (t, 2 H, J = 8 Hz), 4.00 (s, 2 H) , 3.94 (s, 6 H); CIMS m/e. (relative intensity) 317 (MH⁺, 100), 285 (35), 157 (93). See Figure 4.

EXAMPLE 14 Preparation of Ammonium Salt of Aurintriphosphonic Acid

(19)

Concentrated sulfuric acid (3.6 mL) was placed in a 25 mL two-necked flash equipped with a mechanical stirrer. The flask was cooled in a dry ice-acetone-water bath at 0-10°C and sodium nitrite (0.52 g, 7.47 mmol) was added in small portions with vigorous stirring. 2- Hydroxyphenylphosphonic acid (prepared according to: Naito et al., Shiritsu Daiσaku Yakaqakubu Kivo, 1957, 5, 43; Chem. Abstrl. 1958, 52, 6248; Freedman et al., J. Orq. Che . , I960, 25, 140) (1.30 g, 7.47 mmol) then was added in small portions with stirring. The reaction mixture became a brown solution. Formaldehyde (0.23 g of 37% solution, 2.8 mmol) was added dropwise to the flask with vigorous stirring, while the temperature of the bath was kept at -5°C. After the addition was complete, the reaction mixture was stirred to 0-5°C for 20 min, then room temperature for another 10 min. Crushed ice (10 g) was added to the reaction mixture. The resulting mixture was a dark red solution. Extraction with ether and methylene chloride failed to separate the product from the solution.

The solution was basified with concentrated ammonium hydroxide to pH 8 to Hydrion paper. This solu¬ tion then was placed in a dialysis tube (molecular weight cut-off = 3500). The dialysis was suspended in a water jar and the water was agitated with a magnetic stirrer. The dialysate was replaced with fresh water several times until there was only a very faint color present in the dialysate. The retentate was rotary evaporated. The residue was dried in an Abderhalden drying pistol to obtain 290 mg of the polymeric ammonium salt of aurintri¬ phosphonic acid as a dark red glass.

The 1H NMR spectrum (D₂0) with TMS as external reference) of the red glass displayed absorptions at δ 5.50-6.80 (m) . The ¹³C NMR spectrum displayed absorptions at δ 27.84, 74.65, and a complex multiplet in the region at 108-150. The infrared spectrum (KBr) of this salt displayed absorptions at 3160, 1569, 1393, 1232, 1124, 1073, 1018 and 878 cm-1. EXAMPLE 15

Preparation of Ammonium Salt of Polymeric Aurintrisulfonic Acid

In a 50 mL three-necked flask equipped with a mechanical stirrer, thermometer and nitrogen gas inlet, there was placed concentrated sulfuric acid (7.4 mL) . The flask was cooled in an ice bath while sodium nitrite (1.06 g, 15.3 mmol) was added with vigorous stirring. Sodium 2- hydroxyphenylsulfonate (prepared according to: Chase et al., J. Chem. Soc. , 1963, 50) (300 g. 15.3 mmol) was added to the solution in small portions with stirring. The flask then was cooled in a dry ice-acetone-water bath. Formaldehyde (0.60 g of 37% solution, 7.4 mmol) was added to the flask with vigorous stirring while the temperature of reaction mixture was kept below 5°C. After the addi¬ tion of formaldehyde was complete, the reaction mixture was stirred at room temp (~22-24C) for another 30 min and then poured into crushed ice (10 g) . The solution was neutralized with concentrate ammonium hydroxide. This solution then was placed in a dialysis tube (molecular weight cut-off = 3500). The dialysis tube was suspended in water and the water was agitated with a magnetic stirrer. The dialysate was replaced with fresh water several times until there was only a very faint color present in the dialysate. The retentate was rotary evaporated. The residue was dried in an Abderhalden drying pistol for 18 h to afford 400 mg of the ammonium salt of aurintrisulfonic acid (MW>3500) as a dark red glass.

The IR spectrum (KBr) of the ammonium salt of aurintrisulfonic acid (MW>3500) displaced absorptions at 3448, 3158, 1401, 1210, 1033 and 633 cm"¹.

All the dialysates were collected and subjected to ultrafiltration using an Amicon pressure cell fitted with a Diaflo YC05 membrane (molecular weight cutoff = 500). The retentate was rotary evaporated and the residue was dried in an Abderhalden drying pistol to afford 330 mg of red glassy solid. This solid was dissolved in water and subjected for another ultrafiltration with a Diaflo YM2 membrane (molecular weight cutoff = 1000). Virtually all of the solution passed through this membrane. The ultra- filtrate was rotary evaporated and the residue was dried in an Abderhalden drying pistol to afford 323 mg of red glassy solid.

EXAMPLE 16 Preparation of Polymeric Bromoaurintricarboxylic Acid (22) ATA (1.266 g) was added to glacial acetic acid (50 Ml) and the mixture was cooled in an ice bath. A mixture of bromine (0.8 Ml, density 3.11) in acetic acid (10 Ml) was added dropwise during 30 min. The reaction mixture was then left at room temperature for 22 h. The solvent was removed under reduced pressure and ice (30 g) was added. The precipitate was filtered and dried to yield bromoaurintricarboxylic acid (1.43 g) . Elemental analysis indicated that the brominated polymer contains 28.90% bromine.

EXAMPLE 17

12

3, 3 ' , 3"-tricarboxy-5,5' ,5"-trimethyl-4,4 ' ,4"- trihydroxytriphenylmethane. Triphenyl carbinol 12 (1.0 g) was dissolved in absolute ethanol (100 ML) and the cata¬ lyst Pd/C (10%, 0.3 g) was added. The contents were stirred under hydrogen atmosphere at 80 psi for 48 h. The catalyst was filtered off and the solvent was removed under reduced pressure to give 0.92 g (95%) of the triphe¬ nylmethane, which was recrystallized from chloroform- methanol: mp > 300°C (dec); IR (KBr) 3400 (br) , 3100 (br), 1660, 1605, 1470, 1430, 1265, 1180, 1125, 1005, 880, 785 cm^"1; ^*H NMR (200 MHz, Methanol-d₄) δ 7.44 (bs, 3H) , 7.13 (bs, 3H), 5.34 (s, 1H) , 2.19 (s, 9H) ; FABMS (negative ion mode) 465 (M+H, 47%), 275 (35%), 185 (100%).

EXAMPLE 18

4,4'-Methylenebis(l-hydroxy-2-naphthoic acid). A mixture of l-hydroxy-2-naphthoic acid (1,88 g, 10 mmol) sulfuric acid (25% aq., 100 mL) and formaldehyde (37% aq. , 1.0 mL) was heated at 90°C for 6 h, left at room tempera¬ ture for 12 h, and then poured over crushed ice and the precipitate (1.82 g, 94%); filtered and dried: mp 290- 293°C (dec); IR (KBr) 3420 (br), 3080 (br) , 1665, 1645, 1590, 1520, 1465, 1300, 1260, 1160, 1100, 9915, 770 cm^"1; CIMS mle (relative intensity) 389 (MH+, 9%), 345 (49%), 157 (100%); ^LH NMR (200 MHz, DMS0-d₆) δ 8.45-8.41 (m, 2H), 7.97-7.93 (m, 2H) , 7.66-7.51 (m, 4H) , 7.37 (s, 2H) , 4.61 (s, 2H) .

EXAMPLE 19

Note: This substance was prepared by a modifica- tion of a published procedure: Artis, J. D.; Thibert,

R.J.; Mclntosh, J.M.; Zak, B. Microchem. J. 1981, 26, 487.

5,5'-Dichloro-2,2'-dihydroxy-3,3'-disulfonyloxy- diphenylmethane disodium salt. 2,2'-Methylenebis (4- chlorophenol) (8.00 g, 29.7 mmol) was dissolved in chloro- form (300 mL) in 500 mL three-necked flask equipped with a mechanical stirrer, an addition funnel, and a nitrogen inlet. A solution of chlorosulfonic acid (20.00 mL, 301 mmol) in chloroform (30 mL) was added to the flask over a period of 1 h. The reaction mixture was then stirred under a nitrogen atmosphere at room temperature for 8 h. The white solid that precipitated out of solution was collected by filtration. This solid was dissolved in water (150 mL) and then neutralized with 10% NaOH. The solution was concentrated on a rotary evaporator. The solid residue was then recrystallized from a 40% (v/v) solution of ethanol in water. The solid was collected by filtration and dried in a drying pistol for 28 h to afford the product (3.99 g, 28%): mp > 300°C; ^XH NMR δ 3.54 (s. 2H) , 7 . 11 (d , 2H, J=2 Hz ) , 7 . 23 (d, 2H, J=3 Hz ) ; ¹³C NMR δ 32 . 61 , 119 . 38 , 125 . 67 , 130 . 61 , 132 . 65 , 133 . 78 , 156 . 50 .

EXAMPLE 20 GPC fractionation of ATA GPC separations were carried out using a Shodex

GPC HF-2003 semi-prep chromatography column. Solvent consisted of 100% THF, flow rate was 3.5 mL per minute, and elution was followed by U.V. detection at a wavelength setting of 254 nm. ATA (Aldrich, 1.5 g) was dissolved in 100 mL HPLC grade THF. This solution was filtered through a medium sintered glass funnel to remove residual solids. Following a second filtration through a .45 micron nylon filter 2.0 mL samples were placed on the column. The GPC traces of these samples indicated that ATA was a mixture of polymeric components with a variety of molecular weights. The elution time for this material was 14.0 minutes wide (20 mins 00 sees - 37 mins 36 sec). Frac¬ tionation was accomplished in a series of steps. The first of these involved separating the crude material into two components 1-067.005 and 1-067.004 (See Fig. 5). The second step involved taking these two fractions and dividing them into a second series of four components. This was accomplished by dividing the first two components into two nearly equal parts to yield compounds 1-076.001, 1-076.002, 1-076.003, and 1-076.004. The final step in the fractionation involved repeating this procedure one more time to give eight fractions 1-076.005 - 1-076.012. As is evident from the mean retention times of these eight compounds a separation of the original material based on molecular weight has been accomplished without loss of material. The sample material was converted to solid form by the following procedure. Following removal of vola- tiles the residual material was taken up in ~20 mL 1:2 NH₄OH:H₂0 and extracted three times to a total of 30 mL with methylene chloride. The aqueous layer was then acidified by the dropwise addition of 1:1 HCl (conc):H₂0 until solid ATA began to precipitate. After cooling in the refrigerator for several hours the ATA fraction was recovered by filtration of the suspension through a medium sintered glass funnel. After washing with a small amount of cold water the solid was dried by storage overnight in a desiccator.

ATA was also fractionated by standard equilibrium dialysis and ultrafiltration techniques, described in Figure 6. The results are presented in Tables 1 and 2. TABLE 1 0

5

0

5

GPC Retention Times and Weight Distributions of Fractions Obtained by Equilibrium Dialysis and Ultrafiltration 0 Dialysis

& Ultrafiltration- GPC Analysis¹- 5 Fraction MW range Weight Number Retention Area number (daltons) (%) of Peaks Time (min) (%)

0

5

<500 32.6

0

¹ Shodex HF-2003 semi-preparative column, THF solvent, flow rate 3.5 mL/min. Antiretroviral Activity

Antiretroviral activity of various compounds was determined by the standard assay employing CEM-V cell line and HIV-1 as the representative infecting agent and was conducted at the National Cancer Institute, Developmental Therapeutics Program, Bethesda, MD. The results are shown in various reports presented herein and made a part hereof. See Figures 7-25.

In summary, the results presented herein indicate that the synthetic routes and the compounds described herein are useful models for the preparation of a variety of di- and tri-phenylmethanes, ATA analogs and the like which have been exemplified herein by polymeric aurin¬ triphosphonic, aurintrisulfonic and bromoaurintricar- boxylic acid, etc., and which possess minimal cytotoxicity but high anti-retroviral activity, particularly against HIV. Of course, the methodology described herein can be suitably modified for the preparation of other homogeneous polymers or compounds related to ATA and for the commer- cial scale synthesis by one of ordinary skill in the art.

A pharmaceutical composition in accordance with the present invention comprises an effective amount of the compound of the present invention to inhibit cytopathic activity of a retrovirus and a pharmaceutically acceptable carrier. A method of treating retroviral infection comprises contacting retroviral-infected cells with anti¬ retroviral amount of the compound of the present inven¬ tion.

It is understood that the embodiments described herein are for illustrative purposes only and that various changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A substantially pure compound having anti¬ retroviral activity, selected from the group consisting of: (a) 3, 3 ' 3"-tricarboxy-4,4 ' , 4 " -trihydroxytri- phenylcarbinol ;

( b ) 5,5' -dicarboxy-3 , 3 ' dichloro-4 , 4 ' dihydroxydi¬ phenylmethane ;

(c) 3 , 3 ' -dicarboxy-4 , 4 ' -dihydroxy-5 , 5 ' - dimethyldiphenylmethane ;

(d) 3,3' -dicarboxy-4 , 4 ' -dihydroxydinaphthyl- methane ;

( e ) 5,5' -dibromo-3 , 3 ' -dicarboxy-2 , 2 ' -dihydroxydi¬ phenylmethane ; (f) 5,5'-dichloro-2,2'-dihydroxy-3,3'-disulfonyl- oxydiphenylmethane disodium or ammonium salt;

(g) 3 , 3 ' , 3 " - tricar boxy- 4 ,4,4" -trihydroxy- triphenylmethane ;

(h) 3 , 3 ' , 3 " -tricarboxy-4 , 4 ' , 4 " -trihydroxy- 5 , 5 ' , 5 " trimethyl triphenylmethane ;

(i) polymeric aurintriphosphonic acid; (j) polymeric aurintrisulfonic acid; and (k) polymeric haloaurintricarboxylic acid.

2. A composition of matter comprising an effec- tive amount of the compound of claim 1 to inhibit retro- viral cytopathic activity and a pharmaceutically accept¬ able carrier.

3. A method of inhibiting retroviral activity, comprising contacting retroviral infected cells with an effective amount of the compound of claim 1 to inhibit retroviral activity.

4. A fractionation product of aurintricarboxylic acid polymer, said fraction having anti-retroviral activi¬ ty and the following properties as determined by equilib- rium dialysis, ultrafiltration membrane molecular weight cutoff and GPC retention time on Shodex HF-2003 semi- preparative column using THF solvent and a flow rate of 3.5 ml/min, said fraction being selected from the group consisting of:

(a) MW > 12,000, GPC retention time 27.995 - 29.024 min;

(b) 12,000>MW>7,000, GPC retention time 29.025- 29.604 min;

(c) 7,000>MW>3,500, GPC retention time 29.605- 29.754 min;

(d) 3,500>MW>2,000, GPC retention time 29.755- 29.859 min; (e) 2,000>MW>1,000, GPC retention time 29.860-

30.929 min;

(f) 1,000>MW>500, GPC retention time 30.930 min;

(g) GPC retention time 25 min 48 sec - 26min 59 sec; (h) GPC retention time 27 min 0 sec - 27 min 59 sec;

(i) GPC retention time 28 min 0 sec - 29 min 11 sec; (j) GPC retention time 29 min 12 sec - 29 min 59 sec; (k) GPC retention time 30 min 0 sec - 30 min 47 sec; and

(1) GPC retention time 30 min 48 sec

5. A composition of matter comprising an effec- tive amount of the fraction of claim 4 to inhibit retro¬ viral cytopathic activity and a pharmaceutically accept¬ able carrier.

6. A method of inhibiting retroviral activity, comprising contacting retroviral infected cells with an effective amount of the fraction of claim 4 to inhibit retroviral activity.

7. A method for the synthesis of substantially pure monomeric form of di- and tri-methylmethanes, com¬ prising the steps of: (a) reacting a halogenated benzene derivative with formaldehyde and acid to produce halogenated diphenylmethane, the halogen serving as a protecting group for controlling the regiochemistry of the reaction and for preventing polymerization of diphenylmethane;

(b) dehalogenating the halogenated diphenylmethane by hydrogenolysis in the presence of a catalyst; and then (c) reacting the dehalogenated diphenylmethane with an oxidant and a benzene derivative to obtain the desired product.

8. Use of an effective amount of the compound of claim 1 to inhibit retroviral activity.

9. Use of an effective amount of the fraction of claim 4 to inhibit retroviral activity.