EP0609211A1 - Adenosine diphosphoribose polymerase binding nitroso aromatic compounds useful as anti-tumor and anti-retroviral agents - Google Patents

Abstract

The invention describes novel anti-tumor and anti-viral compounds. Said compounds include 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide, 5-nitroso-1 (2H) -isoquinolinone, 7-nitroso-1 (2H) -isoquinolinone, 8-nitroso-1 (2H) -isoquinolinone. The invention also describes compositions containing one or more of said compounds, as well as methods of treating viral infections and cancer using said compounds and said compositions.

Description

ADENOISE DIPHOSPHORIBOSE POLYMERASE BINDING NITROSO AROMATIC COMPOUNDS USEFUL AS ANTI-TUMOR AND ANTI-RETROVIRAL AGENTS

Field of the Invention

The present invention relates generally to the field of retroviral therapeutic agents and their use in treating viral infections and cancers. More specifically it relates to those therapeutic agents which inhibit ADP-ribose transferase, and in particular various nitroso- benzopyrones, nitroso-isoquinolinones and nitroso-benzamides.

Background of the Invention

The enzyme ADP-ribose transferase (ADPRT) (E.C.4.2.30) is a chromatin-bound enzyme located in the nucleus of most eukaryotic cells. The enzyme catalyzes the polymerization of the ADP- ribose moiety of nicotinamide adenine dinucleotide (NAD⁺) to form poly (ADP-ribose). The polymer is covalently attached to various nuclear proteins, including the polymerase itself.

The many varied roles that ADP-ribosylation plays in cellular metabolism have made ADPRT a target for drugs essentially useful for combating neoplasia and viral infections. Numerous physiological activities have been detected for compounds that inhibit the polymerase activity of ADPRT. Such activities include a cell cycle dependent prevention of carcinogen-induced malignant transformation of human fibroblasts (Kun, E., Kirsten, E., Milo, G.E. Kurian, P. and Kumari, H. L. (1983) Proc. Natl. Acad. Sci. USA 80:7219-7223), conferring also carcinogen resistance (Milo, G.E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Lett. 179:332-336), inhibition of malignant transformation in hamster embryo and mouse C3H10T1/2 cell cultures (Borek, C., Morgan, W.F., Ong, A. and Cleaver, J.E. (1984) Proc. Natl. Acad. Sci. USA 81:243-247), deletion of transfected oncogenes from NIH 3T3 cells (Nakayashu, M., Shima, H., Aonuma, S., Nakagama, H., Nagao. M. and Sugimara, T. (1988) Proc. Natl. Acad. Sci. USA 85:9066-9070), suppression of the mitogenic stimulation of tumor promoters (Romano, F., Menapace, L. and Armato, V. (1983) Carcinogenesis 9: 2147-2154), inhibition of illegitimate DNA recombinations (Waldman, B.C. and Waldman, A. (1990) Nucl. Acids Res. 18:5981- 5988) and integration (Farzaneh, F., Panayotou, G.N., Bowler, L.D., Hardas, B.D., Broom, T., Walther, C. and Shall, S. (1988) Nucl. Acids Res. 16: 11319-11326), induction of sister chromatid exchange (Ikushima, T. (1990) Chromosoma 99: 360-364) and the loss of certain amplified oncogenes (Grosso, L.E. and Pitot, H.C. (1984) Biochem. Biophys . Res. Commun. 119:473-480: Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci. USA 86:7442-7445). Compounds known to inhibit ADPRT polymerase activity include benzamide (Kun, E., Kirsten, E., Milo, G.E. Kurian, P. and Kumari, H. L. (1983) Proc. Natl. Acad. Sci. USA 80: 7219-7223), substituted benzamides (Borek, C, Morgan, W.F., Ong, A. and Cleaver, J.E. (1984) Proc. Natl. Acad. Sci. USA 81:243-247; Romano, F., Menapace, L. and Armato, V. (1983) Carcinoσenesis 9: 2147-2154; Farzaneh, F., Panayotou, G.N., Bowler, L.D., Hardas, B.D., Broom, T., Walther, C. and Shall, S. (1988) Nucl. Acids Res. 16: 11319-11326.; Grosso, L.E. and Pitot, H.C. (1984) Biochem. Biophys. Res. Commun. 119:473-480; Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci. USA 86:7442-7445), 3-aminonaphthylhydrazide (Waldman, B.C. and Waldman, A. (1990) Nucl. Acids Res. 18 : 5981-5988), isoquinoline, quercetin, and coumarin ( 1,2-benzopyrone) (Milo, G.E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Lett. 179: 332-336). The anti-transforming and anti-neoplastic effect of 1,2 benzopyrone were demonstrated in vitro and in vivo (Tseng, et al., (1987) Proc. Natl. Acad. Sci. USA 84:1107-1111).

Other known ADPRT polymerase activity inhibitors include 5-iodo-6-amino-1,2-benzopyrone as described in U.S. Patent application Serial No. 600,593, filed October 19, 1990 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and their Metabolites Useful as Cystostatic and Anti-Viral Agents" for use as anti-tumor and anti-viral agents. The cited patent discusses the possibility of using 5-iodo-6-nitroso-1,2-benzopyrone as an anti-tumor or anti-viral agent. The 6-nitroso-benzopyrones have not been hitherto known or described. The only remotely related compounds found in the literature are 6-nitro-1,2-benzopyrone and 6-amino-1,2-benzopyrone (6-ABP) (J. Pharm. Soc. Jap., 498 : 615 (1923)) for which, only scarce medicinal evaluation has been reported. In particular, testing was done for sedative and hypnotic effects (J. Pharm. Soc. Japan, 73:351 (1953); Ibid, 74:271 (1954)), hypothermal action (Yakugaku Zasshi, 78:491 (1958)), and antipyretic, hypnotic, hypotensive and adrenolytic action (Ibid, 83:1124 (1963)). No significant application for any of these compounds has been described except for 6-ABP. 2-nitrosobenzamide (Irne-Rasa, K.M. and Koubek, E. (1963) J. Org. Chem. 28:3240-3241), and 4-nitrosobenzamide (Wubbels, G.G., Kalhorn, T.F., Johnson, D.E. and Campbell, D. (1982) J. Org. Chem. 47:4664-4670), have been reported in the chemical literature, but no commercial use of these isomers is known. Neither of these articles suggest the use of nitrosobenzamides as ADPRT inhibitors. The anti-viral and anti-tumorigenic actions of substituted and unsubstituted 6-amino-1,2- benzopyrone and 5-iodo-6-amino-1,2-benzopyrone is the subject of copending U.S. patent applications Serial No. 585,231 filed on September 21, 1990 entitled "6-Amino-1,2- Benzopyrones Useful for Treatment of Viral Diseases" and Serial No. 600,593 filed on October 19, 1990 entitled "Novel 5-Iodo-6-Amino1,2-Benzopyrones and Their Metabolites Useful as Cytostatic and Antiviral Agents", which are incorporated herein by reference.

The precursor molecule, 1,2-benzopyrone

(coumarin), was shown to be an inhibitory ligand of adenosinediphosphoribosyl transferase

(ADPRT), a DNA-binding nuclear protein present in all mammalian cells (Tseng, et al. , (1987)

Proc. Nat. Acad, Sci. USA, 84:1107-1111). Hakam, et al ., FEBS Lett., 212:73 (1987) has shown that 6-amino-1,2-benzopyrone (6-ABP) binds specifically to ADRPT at the site that also binds to DNA, indicating that both 6-ABP and DNA compete for the same site on ADPRT. Synthetic ligands of ADPRT inhibit DNA proliferation, particularly in tumorigenic cells, (Kirsten, et al., (1991) Exp. Cell. Res. 193:1-4).

Subsequently, these ligands were found to inhibit viral replication and are the subject of the copending U.S. patent application entitled "6-Amino-1-2-Benzopyrones useful for Treatment of Viral Diseases," Serial No. 585,231, filed on September 21, 1990 which is hereby incorporated by reference.

Thus it is of interest to provide ADPRT polymerase activity inhibitors for use as anti- viral and anti-tumor agents. The subject invention provides for novel nitroso-1,2-benzopyrone, nitroso-benzamide and nitroso- isoquinolinone compounds, and various structurally related other nitroso compounds for use as anti-viral and anti-tumor therapeutic agents. The compounds taught for use in the subj ect invention are believed to be significantly less toxic and far more (500 to 1000 fold) potent than structurally analogous amino compounds.

Summary of the Invention

The subject invention provides for novel anti-tumor and anti-viral compounds. These compounds include 6-nitroso-1, 2-benzopyrone, 3-nitrosobenzamide, 5-nitroso-1(2H)-isoquinolinone, 7-nitroso-1(2H)-isoquinolinone, 8-nitroso-1(2H)-isoquinolinone. The invention also provides for compositions containing one or more of the compounds, and for methods of treating viral infections and cancer with these compounds and compositions.

Also provided for are methods of treating cancer and viral infections with 2- nitrosobenzamide and 4-nitrosobenzamide. Composition containing one or more of these compounds is also provided for.

Description of Figures

Figure 1 is a graph comparing the degree of ADRPT polymerase activity (ADPRP) inactivation exhibited by different concentrations of 6- nitroso-1,2-benzopyrone, 3-nitroso-benzamide, and nitroso-1(2H)-isoquinolinones (NOQ) (a mixture of the 5 and 7 nitroso isomers).

Figure 2 is a composite of graphs displaying the inhibitory effects of the ADRPT ligands on (A) 855-2 cells (a cell line of human B-cell lineage acute lymphoblastic leukemia), (B) H9 cells (a cell line of human T-cell lineage acute lymphoblastic leukemia), (C) HL-60 cells (a cell line of human acute nonlymphoblastic leukemia) and (D) K562 cells (a cell line of human chronic myelogenous leukemia). These cells were cultured while under the influence of the growth factors in 10% fetal bovine serum (FCS), whereas in (E) and (F) the 855-2 cells were cultured in the presence of autocrine growth factor activity (AGF) or low molecular weight-B-cell growth factor (BCGF, a T-cell derived lymphokine), respectively.

Figure 3 is a graph showing the inhibition of increasing levels of leukemic cell growth (in response to increasing concentrations of FCS) of 855-2 cells by 6-nitroso-1,2-benzopyrone (NOBP) and 3-nitrosobenzamide (NOBA).

Figure 4 is a graph showing that NOBP and NOBA inhibit the ability of human leukemic cells (855-2 and HL-60) to form colonies (CFU) from single cells in a semi-solid medium.

Figure 5 shows graph of the relative inhibitory effects of anti-leukemic doses of ADRPT ligands on the ability of (A) normal rhesus bone marrow stem cells or (B) human peripheral blood stem cells to form colonies in soft agar. Note that the NOBP and NOBA had minimal effect on normal cells.

Figure 6 shows graphs displaying the inhibitory effects of NOBP, NOBA and NOQ on four human brain tumor cell lines. Figure 7 is a graph comparing the effectiveness of NOBP with vincristine.

Figure 8 is a graph displaying the effects of NOBP, NOBA and NOQ on human breast tumor cell line MDA 468.

Figure 9 is a graph displaying the effects of NOBP, NOBA and NOQ on murine leukema cell line L 1210. Description of Specific Embodiments

The subject invention provides for several nitroso compounds that are ADPRT polymerase activity inhibitors. These compounds find use as anti-tumor and anti-viral compounds. Compound (I) has the following formula:

wherein R₁, R_2, R₃, R₄, R₅, and R₆ are selected from the group consisting of hydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, R₅, and R₅ is a nitroso group.

A preferred embodiment of compound I is where R₄ is the nitroso group, i.e., the molecule 6- nitroso-1,2-benzopyrone.

Compound II has the formula:

wherein R₁, R₂, and R₃ are selected from the group consisting of hydrogen and nitroso, and only one of R₁, R₂, and R₃ is a nitroso group.

Compound III has the formula:

wherein R₁, R₂, R₃, R₄, and R₅ are selected from the group consisting of hydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, and R₅ is a nitroso group.

Preferred embodiments of compound III are where either R₄ or R₂,is the nitroso group, i.e., 5-nitroso-1(2H)-isoquinolinone and 7-nitroso- 1(2H)-isoquinolinone, respectively.

The disclosed synthesis for 5-nitroso-l (2H)-isoquinolinone may produce 2 closely related structural isomers, 7-nitroso-1(2H)-isoquinolinone and 8-nitroso-1(2H)-isoquinolinone. Although experiments testing the biological activity of 5-nitroso-1(2H)-isoquinolinone may have contained significant quantities of 8-nitroso-1(2H)-isoquinolinone or 7-nitroso-1(2H)-isoquinolinone, all three isomers are believed to possess similar anti-tumor and anti-viral activity on the basis of their close structural similarity. This hypothesis may be conveniently tested by separating the isomers by thin layer chromatography or similar methods, and comparing the anti-tumor and anti-viral activities of the separated compounds. Detailed synthesis of 6-nitroso-1,2-benzopyrone, 3-nitroso-benzamide, 5-nitroso-1(2H)-isoquinolinone, 7-nitroso-1(2H)-isoquinolinone, and 8-nitroso-1(2H)-isoquinolinone, are provided in the example section below. In general, the nitroso compounds of the subject of invention may be synthesized by oxidizing a corresponding amino compound to a compound of the subject invention by oxidation with 3-chloroperoxybenzoic acid (or other peroxyacids) in ethyl acetate or a halocarbon solvent. Syntheses of these precursor amino compounds are described in the chemical literature and some of the compounds are commercially available. Some precursor amino compounds for oxidation to nitroso compounds of the subject invention are as follows: 3-amino- 1,2-benzopyrone (Spectrum Chemical Mfg. Corp., Gardena, CA 90248); 4-amino-1,2-benzopyrone (Aldrich, Rare Chemical Catalog); 5-amino-1,2- benzopyrone (by reduction of 5-nitro-1,2- benzopyrone, Chem. Abst. 57 16536d (1962)); 7- amino-1,2-benzopyrone (Gottlieb, et al., J.

Chem. Soc. Perkin. Trans. II 435 (1979)); 8- amino-1,2-benzopyrone (by reduction of 8-amino- 1,2-benzopyrone, Abdel-Megid, et al ., Egypt J.

Chem. 20:453-462 (1977)), and 4-amino-1(2H)- isoquinolinone, by reduction of the corresponding 4-nitro analog (Horning, et al. , (1971) Can. J. Chem. 49:2785-2796).

In addition to compounds (I) to (III), the subject invention contemplates various structurally related compounds that have similar carcinostatic and/or anti-viral activities. These structurally related compounds could be conveniently screened on the basis of their highly potent inhibitory effect on ADPRT polymerase activity. Structurally related compounds of interest include derivatives substituted by additional nitroso groups and small, e.g., C₁-C₃ alkyl groups. Also of Depending on the intended mode, the compositions may be in the solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, preferably in unit dosages. The compositions will include an effective amount of at least one of compounds (I) to (III), or pharmaceutically acceptable salts thereof, and in addition it may include any conventional pharmaceutical excipients and other medicinal or pharmaceutical drugs or agents, carriers, adjuvants, diluents, etc., as customary in the pharmaceutical sciences.

For solid compositions, in addition to the compounds (I) to (III), such excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The compounds of the subject invention may be also formulated as suppositories using, for example, polyalkylene glycols, for example, propylene glycol, as the carrier.

Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc., at least one of active compounds (I) to (III) in a pharmaceutical solution such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, DMSO and the like, to thereby form the injectable solution or suspension. interest are various nitroso substituted structurally related heterocyclic rings such as 3,4-dihydro-1(2H)-isoquinolinones, nicotinamides, pthalhydrazides, and 1,3- benzoxazine-2,4-diones.

Another aspect of the compounds of the subject invention are the ease with which they permeate cell membranes and their relative absence of non-specific binding to proteins and nucleic acid.

In practice, the ADPRT polymerase inhibitors of this invention, namely compounds (I) to (III), and any of their pharmaceutically acceptable salts, may be administered in amounts, either alone or in combination with each other, and in the pharmaceutical form which will be sufficient and effective to inhibit neoplastic growth or viral replication or prevent the development of the cancerous growth or viral infection in the mammalian host.

Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration such as oral, parenteral, transdermal, subcutaneous, or topical administration modes. The preferred method of administration of these drugs is intravenous, except in those cases where the subject has topical tumors or lesions, where the topical administration may be proper. In other instances, it may be necessary to administer the composition in other parenteral or even oral forms. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as, for example, sodium acetate, triethanolamine oleate, etc.

Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection. A more recently devised approach for parenteral administration employs the implantation of a slow-release or sustained-release systems, which assures that a constant level of dosage is maintained, according to U.S. Patent No. 3,710,795, which is incorporated herein by reference.

Any of the above pharmaceutical compositions may contain 0.1-99%, preferably 1-70% of the active ingredient.

Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in this art, and are described in detail in Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pennsylvania, 17th Edition, 1985. The composition or formulation to be administered will, in any event, contain such quantity of the active compound(s) that will assure that a therapeutically effective amount will be delivered to a patient. A therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. The amount of active compound administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. However, an effective dosage may be in the range of 1 to 12 mg/kg/day, preferably 1 to 5 mg/kg/day, given only for 1- 2 days at one treatment cycle. Generally, the upper limit for the drug dose determination is its efficacy balanced with its possible toxicity.

The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.

EXAMPLES

I. Synthesis and Characterization of 6- Nitroso-1,2-Benzopyrone

An example of a method for the preparation of 6-nitroso-1,2-benzopyrones is provided as follows: To a stirred solution of 6-amino-1,2-benzopyrone hydrochloride (4.00 g, 20 mmol) in water (40 ml) at 22ºC was added a solution of sodium tungstate (5.93 g, 20 mmol) in water (20 ml) followed by 30% aqueous hydrogen peroxide (5 ml) and stirring was continued for 1.5 hours. The oxidation product was extracted from the green-colored mixture with two 100 ml volumes of ethyl acetate, the combined extracts washed with 0.1 N HCl (50 ml) and then water (100 ml). The ethyl acetate was removed by rotary evaporation and the residue recrystallized from warm ethanol (250 ml).

Analysis of Reaction Product

The green crystals obtained from the recrystallization step (1.48 g, 42% yield) displayed light absorption at 750 nm characteristic of monomeric arylnitroso compounds. Mass spectrum: m/z (relative intensity): 175 (M⁺, 100), 161 (16.88), 145 (33.77), 133 (10.38), 117 (56.09), 89 (79.71), 63 (57.13). High resolution data for the M⁺ peak: calculated for C₉H₅NO₃: 175.0268; found: 175.0271 (deviation - 1.1 ppm). ¹H-NMR (CDCl₃, 300 MHz) δ (ppm) from TMS: doublet (6.572 and 6.604) H-4 split by H-3; doublet (7.472 and 7.501) H-8 split by H-7; doublet of doublets (7.860/7.866 and 7.889/7.798) H-7 split by H-8 and finely split by H-5; doublet (7.910 and 7.942) H-3 split by H-4; doublet (8.308 and 8.315) H-5 finely split by H-7. UV/VIS spectrum in ethanol, λ max (∈): 750 nm (46), 316 nm (8.96 × 10³), 274 nm (2.24 × 10⁴). Melting Point: The compound polymerizes above 160ºC, blackens and melts in the range of 325-340ºC. This nitroso-compound may also be prepared by reacting 6-amino-1,2-benzopyrone (as the free base) with 3-chloroperoxybenzoic acid in ethyl acetate or halocarbon solvents. II. Synthesis of 3-nitrosobenzamide

To a stirred solution of 3-aminobenzamide (Aldrich Chemical Co.) (0.476 g, 3.50 mmol) in ethyl acetate (50 mL) at ambient temperature was added 1.208 g of 3-chloroperoxybenzoic acid (commercial grade, 50-60% purity, Aldrich), whereupon the solution turned green. After 10 minutes the mixture was extracted with 0.14M aqueous sodium bicarbonate (58 mL), washed with three successive 40-mL portions of water, dried over sodium sulfate, then reduced in volume to 20 mL by rotary evaporation and placed in the freezer (-20ºC), whereupon the product slowly deposited as a light yellow solid during a period of 72 hours (0.180 g, 34% yield).

The 2-nitrosobenzamide and 4-nitrosobenzamide isomers may be similarly prepared by performing the above oxidation on 2-aminobenzamide and 4- aminobenzamide, respectively.

Analysis of Reaction Product

Melting point: The substance darkens above 135ºC, softens and apparently polymerizes in the range 150-160ºC, and melts at 240-250ºC (with decomposition). In solution the compound is green-blue. Mass spectrum: m/z (relative intensity): 150 (M⁺,100), 136 (10.9),_. 120 (77.2), 103 (31.6), 92 (46.5), 85 (22.8), 71 (33.3). High resolution data for the M⁺ peak: calculated for C₇H₆N₂O₂: 150.042928; found: 150.042900 (deviation =0.2 ppm). NMR spectrum: ¹H-NMR (DMSO-d₆, 300 MHz) δ (ppm) from TMS : broad singlet (7.737) N-H; t (7.824, 7.850, 7.875) H-5 split by H-4 and H-6; d (8.059 and 8.086) H-6 split by H-5; d (8.357 and 8.383) H-4 split by H-5; s (8.472) H-2. The singlet at 7.737 corresponds to 1 proton; the second N-H proton, spectrally non-equivalent in this compound, is overlaid by the doublet of H-4. This doublet integrates to 2 protons and can be resolved by addition of D₂O to the DMSO solution. UV-VIS absorption spectrum in absolute ethanol, λmax (£): 750nm (37.6), 304nm (5.35 × 10³) and 218nm (1.50 × 10⁴). An absorption maximum at 750nm is characteristic of monomeric arylnitroso compounds.

III. Synthesis of Nitroso-1 (2H)- isoquinolinones (a mixture of 5-nitroso and 7-nitroso-isomers)

1(2H)-Isoquinolinone (isocarbostyril) (Aldrich) was nitrated using a general method for isoquinoline compounds (C.G. LeFevre and R.J.W. LeFevre, J. Chem. Soc. 1470 (1935)). The nitration product (a mixture of the 5-nitro and 7-nitro isomers, as assigned by Y. Kawazoe and Y. Yoshioka, Chem. Pharm. Bull. (Tokyo) 16: 715-720 (1968), although one of the isomers could be the 8-nitro isomer) was then reduced to the corresponding amino-1(2H)-isoquinolinones using a combination of potassium borohydride and palladium-on-carbon catalyst in aqueous methanol. To the resultant amino-1(2H)-isoquinolinones (as free bases) (0.560 g, 3.50 mmol) in ethyl acetate (175 mL) at 30ºC was added 1.208 g of 3-chloroperoxybenzoic acid (Aldrich). The mixture became cloudy and after 20 minutes it was filtered, extracted with 0.14M sodium bicarbonate (58 mL), washed with two 50-mL portions of water, and dried over sodium sulfate. The volume of the solution was reduced to 50 mL by rotary evaporation and then placed in the freezer (-20ºC), whereupon an orange solid product was deposited (0.102 g). Analysis of Reaction Product

Melting point: substance darkens above 175ºC, softens, blackens and apparently polymerizes above 195ºC, and finally melts in the range 310- 335ºC. NMR analysis: ¹H-NMR (DMSO-d₆/D₂O, 300 MH_Z) δ (ppm) from TMS: m (6.723, 6.741, 6.752); m (7.511, 7.518, 7.533, 7.539, 7.547, 7.559, 7.577, 7.585); m (7.663, 7.674, 7.686. 7.698, 7.707); d (7.818, 7.846). In the absence of D₂O, the compound also displays a broad singlet at 11.90 ppm. The isomeric components were analytically resolved by thin-layer chromatography (silica gel plates, ethyl acetate solvent), giving two bands, R_f 0.82 and _Rf 0.72. Mass spectrum for R_f 0.82i m/z (relative intensity): 174 (M⁺, 100), 160 (26.8), 144 (93.0), 117 (90.8), 97 (21.9), 89 (96.1), 71 (24.1). High resolution data for the M⁺ peak: calculated for C₉H₆N₂O₂: 174.042928; found: 174.043200 (deviation = -0.3 ppm). For the component having R_f 0.72, M⁺, calculated for C₉H₆N₂O₂: 174.042928; Found: 174.043200 (deviation = -1.6 ppm). These data confirm that the compounds are mono-nitroso isomers.

IV. ADPRT Inactive Studies

The compounds of the subject invention were tested for their ability to inactivate the polymerase activity of adenosinediphosphoribosyl transferase (ADPRT). Assays were performed according to the method of Buki and Kun, Biochem. 27:5990-5995 (1988), using calf thymus ADPRT. The assay results as given in Table I provide the I₅₀ (the concentration of the compound that inhibits enzyme activity 50%) values for ADPRT of the nitroso precursor (6- amino-1,2-benzopyrone) and the more potent 5- iodo-derivative (Table I, compounds 1 and 2, respectively). The nitroso compounds (3,4,5 in Table I) are all highly active as anti-tumor and anti-HIV molecules (as shown in later sections) and are effective even after exposure of cells for a period as short as 30 minutes. 5-I-6- nitroso-1,2-benzopyrone (compound 6) in these studies has been shown to be a relatively poor inhibitor of ADRPT (It is believed that the iodo substitution deactivates the NO group as an electrophile) and its biological action is 10 times weaker than that of 6-NO-1,2-benzopyrone. For these reasons, the compositions of the present invention are believed to be superior to 5-I-6-nitroso-1,2 benzopyrone, which has been shown to be a poor permeant molecule.

TABLE I

I₅₀ data for aromatic inhibitors of ADPRT

No. Inhibitor I₅₀,μM

1 6-NH₂-1,2-benzopyrone* 370 2 5-I-6-NH₂-1,2-benzopyrone* 41

3 3-NO-benzamide 15

4 5(7)-nitroso-(2H)-isoquinolinone** 13 5 6-NO-1,2-benzopyrone 40

6 5-I-6-NO-1,2-benzopyrone 400

*biochemical precursor of nitroso compounds 5 and 6

**a mixture of the 5- and 7-nitroso compounds

Assay conditions: ADPRT, 0.4 μq; coDNA, 4 μq; inhibitor diluted between 0.8 and 600 μM, in 50 μl of 50 mM Tris-HC-l, 50 mM KCl, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.1 mM NAD ([32-P]-labelled), pH 7.5. Polymerization at 25ºC for 4 minutes. Figure 1 illustrates the % inactivation of ADPRT polymerase activity observed after 2 hours of incubation with the nitroso-compound inhibitors at several concentrations. Additional experiments involving the equilibration between ⁶⁵Zn⁺² and ADPRT-bound Zn⁺² suggest that the ADPRT inhibition activity of the nitroso compounds appears to act by destabilizing the protein through the ejecting of Zn⁺². (Buki K.G., Bauer P. T., Mendeleyev, F.; Hakam, H. and Kun E. (1991) FEBS Lett. 290:181- 185). The above mechanism of action for ADPRT inhibitors is speculative and does not constitute any limitation on claimed subject matter.

V. Biological Anti-Cancer Activities of

Nitrosobenzopyrones, Nitrosobenzamides and Nitroso-isoquinolinones

Experiments were perfomed in which various human leukemia cell lines were exposed to increasing concentrations of 6-amino-1,2- benzopyrone (ABP), 5-iodo-6-amino-1,2- benzopyrone (IABP), 6-nitro-1,2-benzopyrone (NO₂BP), 6-nitroso-1,2-benzopyrone (NOBP), 3-nitrosobenzamide (NOBA) or 5(7)-nitroso-l(2H)-isoquinolinone (NOQ) (a mixture of the 5-nitroso and 7 nitroso isomers), and the level of [³H] thymidine uptake was determined as a measure of cellular proliferation. As shown in Figure 2, for each of the cell lines tested (855-2 cells, Fig. 2A; H9 cells, Fig. 2B; HL-60 cells, Fig. 2C; K562 cells, Fig. 2D) the nitroso-containing ligands (NOBP, NOBA, NOQ) were able to inhibit ³H-thymidine uptake in lower molar concentrations than the other compounds. NOBP, NOBA and NOQ powerfully inhibited ³H-thymidine uptake at a concentration of 10 μM, a concentration at which the other compounds exhibited comparatively slight inhibitory effects. Experiments with H9 cells grown in 10% fetal bovine serum (FCS) (Fig. 2B) found NOQ to be the most potent inhibitor, demonstrating almost complete inhibition at 10 μM levels. NOBP demonstrated about a 30% decrease in thymidine uptake at 10 μM, and an almost complete inhibition of uptake at 100 μM. NOBA demonstrated about 75% level of inhibition at 10 μM, about 85% inhibition at 100 μM, and almost complete inhibition at 250 μM. The remaining amino and nitro compounds were significantly less potent and did not display complete inhibition until concentrations of 1000 μM were reached. Experiments with K562 cells grown in 10% fetal bovine serum (Fig. 2D) found NOQ and NOBP to be the most potent inhibitors of cell growth. Both NOQ and NOBP resulted in the almost complete inhibition at concentrations of 10 μM. NOBP was almost as potent as NOQ and produced about 90% inhibition at a concentration of 10 μM, and almost complete inhibition at a concentration of 100 μM . The other 3 compounds tested were significantly less potent. Experiments with 855-2 cells grown in 10% fetal bovine serum (Fig. 2A) found that NOQ, and NOBP produced almost complete inhibition at a concentration of 10 μM . At a concentration of 1μM, NOQ produced somewhat more inhibition than NOBP, and NOBP produced somewhat more inhibition than NOBA. Experiments using HL-60 cells (Fig. 2C) provided similar conclusions. The other 3 compounds tested were significantly less potent. The effect of different growth factors on the growth inhibitory effects of NOBP was tested. 855-2 cells that were grown in media with (1) 10% fetal bovine serum, (2) autocrine growth factor (AGF) and (3) low molecular weight-BCGF (a T cell derived lymphokine) were exposed to increasing concentrations of the ADRPT ligands. The results are provided in Figure 2 (A, E, F). Cells grown in each of the growth factors were all potently inhibited by the nitroso-containing compounds, with concentrations of 5 to 10 μM resulting in 100% inhibition, Thus, NOBP, NOBA and NOQ exert potent inhibitory effects regardless of the source of growth factor activity. In order to exclude the possibility that NOBP and NOBA manifest their growth inhibitory effects through inactivation of growth factors, the effects of 10 μM NOBP or NOBA (constant concentration) on 855-2 cells in the presence of increasing concentrations of fetal bovine serum (FCS) were tested (FCS) contains growth factors for 855-2 cells). The data are provided in Figure 3. Growth arrest occurs irrespective of the concentration of FCS. Thus, the mode of action of NOBP, does not appear to be by antagonism of growth factors but at ADPRT sites related to DNA-replication.

Tumor cell inhibitory concentrations of NOBP and NOBA were shown not to affect adversely the viability of normal cells. Experiments were performed in which the functions of various cancer cells (855-2 and HL-60 leukemia cells, D32, D37 and CRL 7712 glioblastoma cell lines, 186 medulla tumor cell line, L1210 murine leukemia cell line, MDA-468 human breast tumor cell line) and normal cells (neutrophil leukocytes and bone marrow or peripheal blood stream cells) were assessed in the absence or presence of the compounds. The results are shown in Figures 4-9. Together, the data indicate that a concentration of 10 μM of the nitroso-containing ligands effectively suppressed cancer cell growth but demonstrated only modest effects the functions on normal cells.

VI. Toxicity of NOBP

The cytotoxicity of 0, 2 μM, 4 MM, 8 μM and 10 μM NOBP was measured by examining the effect of the compound on the colony formation (CFU-GM) of normal human stem cells (PBSC). The results of the experiments are provided in figure 5B. Toxicity was not detected, even though levels of NOBP sufficient to block 855-2 cell proliferation completely were tested.

A similar CFU stem cell toxicity assay was performed in which comparisons were made between (ABP) 6-amino-l,2-benzopyrone 1 mM, (IABP) 5-I-6-amino-1,2-benzopyrone 250 μM, (NO₂BP) 6-nitro-1,2-benzopyrone (weakly active) 250 μM, NOBP 10 μM, and NOBA 10 μM . The results of the experiments are provided in figure 5A. Whereas the 6-amino-1,2-benzopyrone, 5-I-6-amino-1,2-benzopyrone and the 6-nitro derivative were toxic at the tested given doses, the almost ineffective (against tumor cells) 6-nitro derivative and the highly effective (against tumor cells) NOBP and NOBA were non-toxic.

The effects of 10μm NOBP and NOBA on superoxide generation by normal human peripheral blood neutrophil leukocytes was tested. The results are provided in table II. Only minor reductions in superoxide generation were observed. Table II

Effects of 10 μM NOBP and NOBA on the Generation of Superoxide by Human Neutrophils

nmol O₂-/hr/10⁵ cells (mean ± S.D.,, n=11)

10⁵ PMN + PMA: 55.9 ± 7.7

+10 μM NOBP 34.1 ± 14.1

+10 μM NOBA 44.4 ± 10.0 VII. Comparative Efficacy Studies

Vincristine, a highly toxic chemetherapeutic compound, is the currently used in the treatment of leukemia and other malignancies. Studies were performed in order to determine the concentration of vincristine that produces the same level of growth inhibition as 10 μM NOBP, when assayed on 855-2 leukemia cells grown in vitro. Vincristine was tested in doses of 0.1, 1, 10 and 100 μM. As shown in Figure 7. 100 μM of vincristine (a highly toxic concentration) was required to produce the same level of inhibition as 10 μM of NOBP, thus NOBP is about 10 times more potent than an equal concentration of vincristine, and is not toxic to normal cells.

Thus certain aromatic nitroso molecules that are also inhibitors of ADPRT polymerase activity may be useful chemotherapeutic cytostatic agents because of their effectiveness combined with low toxicity. VIII. Anti-HIV action of NOBP, NOBA and NOP on stimulated human lymphoblasts.

The ability of NOBP ( 6-nitroso-1,2- benzopyrone) and NOBA (3-nitrosobenzamide) to inhibit HIV infections were tested using the methods described in the Journal of Immunological Methods 76:171-183 (1985). Exposure to the two drugs was only for 30 minutes at the commencement of viral infection, and drugs were never re-added. The results given in Table III provide the ID₅₀ of HIV titer 10 days after infection of cell cultures with HIV. The data in Table III demonstrate that 10 μM of the nitroso-containing ligands causes a three log decrease in the HIV-1 infectivity titer.

TABLE III Test Sample Virus Titer (log ID ₅₀) 10 days

Virus Alone 5.25

+500 μM ABP 4.50

+250 μM IABP 4.66

+250 μM NO₂BP 4.93

+10 μM NOBP 2.01

+10 μM NOBA 1.05

+10 μM NOQ 1.73 IX. Cytocidal Activity of ADRPT ligands - MTT

Assay

Experimetns were performed to determine if the inhibition of proliferation of 855-2 cells seen in culture and in soft agar is due to the cytostatic or cytocidal effect of the nitroso compounds NOBP, NOBA, and NOQ. Cells at 1x10⁵/ml (concentration used in bone marrow assay) were treated with NOBP, NOBA and NOQ at

1, 2.5, 5 and 10μm for 2 hours then stimulated with 10% fetal calf serum and incubated for 24 hours. MTT (3-[4,5-Dimethyl-2-yl]-2,5- diphenyltetrazolium bromide) at 1 mg/ml was then added for 16 hours. The absorbance of the pelleted cell was then measured at 550nm after adding DMSO to solubilze the cells. Results: With 10μM NOBP, NOBA and NOQ, complete killing was observed in 855-2 cells at 100,000/ml.

All publications, patents, and patent applications cited above are herein incorporated by reference.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of pharmaceutical formulation or related fields are intended to be within the scope of the following claims.

Claims

Claims : What is claimed is:

1. A compound having the formula:

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are selected from the group consisting of hydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group.

2. A compound according to claim 1, wherein R₄ is a nitroso group.

3. A compound having the formula:

4. A compound having the formula:

wherein R₁, R₂, R₃, R₄, and R₅ are selected from the group consisting of hydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, and R₅ is a nitroso group.

5. A compound according to claim 4, wherein R₂ or R₄ is a nitroso group.

6. A composition for the treatment of cancer and retroviral diseases, said composition comprising a compound according to Claim 1.

7. A composition for the treatment of cancer and retroviral diseases, said composition comprising a compound having the formula:

wherein R₁, R₂, and R₃ are selected from the group consisting of hydrogen and nitroso, and only one of R₁, R₂, and R₃ is a nitroso group.

8. A composition for the treatment of cancer and retroviral diseases, said composition comprising a compound according to Claim 4.

9. A method for the treatment of cancer and retroviral diseases said method comprising the step of administering an effective amount of the compound according to Claim 1.

10. A method for the treatment of cancer and retroviral diseases said method comprising the step of administering an effective amount of the compound according to Claim 2 having the formula:

wherein R₁, R₂, and R₃ are selected from the group consisting of hydrogen and nitroso, and only one of R₁ , R₂, and R₃ is a nitroso group.

11. A method for the treatment of cancer and retroviral diseases said method comprising the step of administering an effective amount of thecompound according to Claim 4.

AMENDED CLAIMS

[received by the International Bureau on 26 March 1993 (26.03.93) ; original claims 1-11 replaced by amended claims 1-26 (10 pages) ]

1. A compound having the formula:

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group.

2. A compound according to claim 1, wherein R₄ is a nitroso group.

3. A compound having the formula:

4. A compound having the formula:

wherein R₁, R₂, R₃, R₄ and R₅ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄ and R₅ is a nitroso group.

5. A compound according to claim 4, wherein R₂ or R₄ is a nitroso group.

6. An anti-viral composition comprising an anti-viral effective amount of the compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂, R₃, R₄, and R₆ are selected from the group consisting of hydrogen and nitroso and only one R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group.

7. An anti-cancer composition comprising an anti-cancer effective amount of the compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂, R₃, R₄, R₅ and R₆ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group.

8. An anti-viral composition comprising an anti-viral effective amount of the compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂ and R₃ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂ and R₃ is a nitroso group.

9. An anti-cancer composition comprising an anti-cancer effective amount of the compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁ , R₂ and R₃ are selected from the group consisting of hydrogen and nitroso and only one of R₁ , R₂ and R₃ is a nitroso group.

10. An anti-viral composition comprising an anti-viral effective amount of the compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂, R₃, R₄ and R₅ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄ and R₅ is a nitroso group.

11. An anti-cancer composition comprising an anti-cancer effective amount of the compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂ , R₃, R₄ and R₅ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄ and R₅ is a nitroso group.

12. A method for the treatment of cancer said method comprising the step of administering an anti-cancer effective amount of a composition of a

compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁ , R₂ , R₃, R₄, R₅ and R₆ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group.

13. The method of claim 12 wherein R₄ is a nitroso group.

14. A method of inhibiting viral growth and replication within a cell in the substantial absence of cellular toxicity comprising contacting the cell with an anti-viral effective amount of a composition of a compound of the formula or pharmaceutically acceptable salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁ , R₂ , R₃, R₄, R₅ and R₆ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group.

15. The method of claim 14 wherein R₄ is a nitroso group.

16. The method of claim 14 wherein the virus is human immunodeficiency virus.

17. A method for the treatment of cancer said method comprising the step of administering an anti- cancer effective amount of a composition of a

compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂ and R₃ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂ and R₃ is a nitroso group.

18. The method of claim 17 wherein R₂ is a nitroso group.

19. A method of inhibiting viral growth and replication within a cell in the substantial absence of cellular toxicity comprising contacting the cell with an anti-viral effective amount of a composition of a compound of the formula or pharmaceutically acceptable salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂ and R₃ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂ and R₃ is a nitroso group.

20. The method of claim 19 wherein R₂ is a nitroso group.

21. The method of claim 19 wherein the virus is human immunodeficiency virus.

22. A method for the treatment of cancer said method comprising the step of administering an anti-cancer effective amount of a composition of a

compound of the chemical formula or a pharmaceutical salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂, R₃, R₄ and R₅ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄ and R₅ is a nitroso group.

23. The method of claim 22 wherein R₂ or R₄ is a nitroso group.

24. A method of inhibiting viral growth and replication within a cell in the substantial absence of cellular toxicity comprising contacting the cell with an anti-viral effective amount of a composition of a compound of the formula or pharmaceutically acceptable salt thereof:

in combination with a pharmaceutically acceptable amount of an inert carrier wherein R₁, R₂, R₃, R₄ and R₅ are selected from the group consisting of hydrogen and nitroso and only one of R₁, R₂, R₃, R₄ and R₅ is a nitroso group.

25. The method of claim 24 wherein R₂ or R₄ is a nitroso group.

26. The method of claim 24 wherein the virus is human immunodeficiency virus.

STATEMENT UNDER ARTICLE 19

The cancelation of claims 1-11 and the addition of new claims 1-26 were made in order to more clearly define the invention. The amendment does not go beyond the disclosure of the international

application as filed since full support for these claims can be found in the specification. In

particular, support for new claims 1-26 can be found at page 1, lines 4-7 and page 8 lines 1-30.