JP3070767B2 - Adenosine diphosphoribose polymerase binding nitrosoaromatic compounds useful as antitumor and anti-retroviral agents - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention generally relates to retroviral therapeutics and their use in treating viral infections and cancer. More particularly, it relates to therapeutic agents that 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-binding enzyme located in the nucleus of most eukaryotic cells. The enzyme catalyzes the polymerization of the ADP-ribose component of nicotinamide adenine dinucleotide (NAD ⁺ ) to form poly (ADP-ribose). The polymer is covalently linked to various nucleoproteins, including the polymerase itself.

The many different roles that ADP-ribosylation acts in cell metabolism has been to make ADPRT a target for drugs that are substantially useful in attacking neoplastic and viral infections. Many physiological activities have been detected for compounds that inhibit the polymerase activity of ADPRT. Such activity has been demonstrated by cell cycle-dependent prevention of carcinogen-induced malignant transformation of human fibroblasts (Kun, E., Kirsten, E., Milo, GEK).
urian, P. and Kumari, HL (1983) Proc. Natl. Acad. Sci.
USA 80 : 7219-7223) and conferring carcinogen resistance (Milo,
GE, Kurian, P., Kirsten, E. and Kun, E. (1985) FEBS Le
tt. 179 : 332-336), hamster embryo and mouse C3H10T1 / 2
Inhibition of malignant transformation in cell culture (Borek, C., Mor
gan, WF, Ong, A. and Cleaver, JE (1984) Proc. Natl. A
USA 81 : 243-247), deletion of an oncogene transfected from NIH 3T3 cells (Nakayashu, M., Shima,
H., Aonuma, S., Nakagama, H., Nagao, M. and Sugimura, T.
USA, 85 : 9066-9070), suppression of mitogen stimulation of tumor promoters (Romano, F., et al., Proc. Natl. Acad. Sci.
Menapace, L. and Armato, V. (1983) Carcinogenesis 9 :
2147-2154), inhibition of illegitimate DNA recombination (Waldman, B. et al.
C. and Waldman, A. (1990) Nucl. Acids Res. 18 : 5981-59
88) and integration (Farzaneh, F., Panayotou, GN, Bowler,
LD, Hardas, BD, Broom, T., Walther, C.and Shall, S.
(1988) Nacl. Acids Res. 16 : 11319-11326), Induction of sister chromatid exchange (Ikushima, T. (1990) Chromosoma 99 : 3).
60-364) and loss of certain amplified oncogenes (Gross
o, LEand Pitot, HC (1984) Biochem. Biophys. Res. Co
mmun. 119 : 473-480; Shima, H., Nakayasu, M., Aonuma, S., S
ugimura, J. and Nagao, M. (1989) Proc. Natl. Acad. Sci. U
SA 86 : 7442-7445).

Compounds known to inhibit ADPRT polymerase activity include benzamide (Kun, E., Kirsten, E., Milo,
GEKurian, P. and Kumari, HL (1983) Proc. Natl. Aca
d.Sci.USA 80 : 7219-7223), substituted benzamides (Bore)
k, C., Morgan, WF, Ong.A.and Cleaver, JE (1984) Pro
c. Natl. Acad. Sci. USA 81 : 243-247; Romano, F., Menapac
e, L. and Armato, V. (1983) Carcinogenesis 9 : 2147-2
154; Farzaneh, F., Panayotou, GN, Bowler, LD, Hardas,
BD, Broom, T., Walther. C. and Shall, S. (1988) Hacl. A
cids Res. 16 : 11319-11326; Grosso, LEand Pitot, HC
(1984) Biochem. Biophys. Res. Commun. 119 : 473-480; Sh
ima, H., Nakayasu, M., Aonuma, S., Sugimura, T.and Nagao,
M. (1989) Proc. Natl. Acad. Sci. USA 86 : 7442-7445), 3
-Aminonaphthyl hydrazide (Waldman, BCand Waldma
n, A. (1990) Nucl. Acids Res. 18 : 5981-5988), isoquinoline, quercitrin, and coumarin (1,2-benzopyrone) (Milo, GE, Kurian, P., Kirsten, E. and Kun, E.
(1985) FEBS Lett. 179 : 332-336). 1,2-
The anti-transformation and anti-malignant effects of benzopyrone have been demonstrated in vitro and in vivo (Tseng, et al., (1987).
(Proc. Natl. Acad. Sci. USA 84 : 1107-1111).

Other known ADPRT polymerase activity inhibitors are
“Novel 5-Indo-6-Amino-1,2-Bonzopyrones and
their Metabolites Useful as Crstostatic and Anti−
Viral Agents ", as described in U.S. Patent Application No. 600,593, filed October 19, 1990.
Includes 5-iodo-6-amino-1,2-pensopyrone for use as tumor and anti-viral agents. The cited patents disclose 5 anti-tumor or anti-viral agents.
Discuss the possibility of using iodo-6-nitroso-1,2-benzopyrone.

6-Nitroso-benzopyrone has not been previously known or described. The only slightly related compounds found in the literature are 6-nitro-1,2-benzopyrone and 6-amino-1,2-benzopyrone (6-AB
P) (J. Pharm. Soc. Jap., 498 : 615 (1923)), for which only a few medical evaluations have been reported. In particular, the test is based on sedative and hypnotic effects (J. Pharm. Soc. Ja
pan, 73 : 351 (1953); supra, 74 : 271 (1954)), hypothermic action (Yakugaku Zasshi, 78 : 471 (1958)) and antipyretic, hypnotic, hypotensive and adrenaline degrading actions (supra, 83 : 1124).
(1963)). No significant use for any of these compounds has been described except for 6-ABP.

n-Nitrosobenzamide (Irne-Rasa, KMand Kou
bek, E. (1963) J. Org. Chem. 28 : 3240-3241) and 4-nitrosobenzamide (Wabbels, GG, Kalhorn, TF, John).
son, DEand Campbell, D. (1982) J. Org. Chem. 47: 4664
44670) have been reported in the chemical literature, but the commercial use of their isomers is unknown. These documents do not suggest the use of nitrosobenzamide as an ADPRT inhibitor.

Substituted and unsubstituted 6-amino-1,2-
The anti-viral and anti-tumor effects of benzopyrone and 5-iodo-6-amino-1,2-benzopyrone are described in "6-Am
ino-1,2-Bonzopyrones Useful for Treatment of Vit
No. 585,231 and "Novel 5-Iodo-6," filed Sep. 21, 1990, entitled "Al Diseases".
−Amino−1,2-Bonzopyrones and Their Metabolites U
1 of the title of "seful as Cytostatic and Antiviral Agents"
This is the subject of U.S. Patent No. 600,593, filed October 19, 990, which is incorporated herein by reference.

The precursor molecule, 1,2-benzopyrone (coumarin), is adenosine diphosphoribosyltransferase (ADPR)
T), an inhibitory ligand for DNA-binding nucleoprotein present in all mammalian cells (Ts
eng, et al., (1987) Proc. Natl. Acad. Sci. USA, 84: 1107-
1111).

Hakam et al., FEBS Lett., 212 : 73 (1987) show that 6-amino-1,2-benzopyrone (6-ABP) also specifically binds to ADPRT at sites that bind to DNA. This suggests that 6-ABP and DNA compete with the same site on ADPRT. Synthetic ligands for ADPRT inhibit DNA growth, especially in neoplastic cells (Kirsten, et al., (199
1) Exp.Cell.Res. 193 : 1 to 4). Subsequently, these ligands were found to inhibit viral replication and were filed on September 21, 1990, entitled "6-Amino-1,2-Bo
nzopyrones useful for Treatment of Viral Diseases "
No. 585,231, which is incorporated herein by reference.

Thus, it is of interest to provide inhibitors of ADPRT polymerase activity for use as anti-viral and anti-tumor agents. The present invention relates to novel nitroso-1,1
Provided are 2-benzopyrone, nitroso-benzamide and nitrosoisoquinolinone compounds, as well as various other structurally related nitroso compounds. The compounds taught for use in the present invention are likely to have significantly lower toxicity (500-1000 fold) than structurally similar amino compounds.

SUMMARY OF THE INVENTION The present invention provides novel anti-tumor and anti-viral compounds. These compounds include 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide, 5-nitroso-
Includes 1 (2H) -isoquinolinone, 7-nitroso-1 (2H) -isoquinoline, 8-nitroso-1 (2H) -isoquinolinone. The invention also provides compositions comprising one or more compounds, and methods for treating viral infections and cancer with these compounds and compositions.

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

DESCRIPTION OF THE FIGURES FIG. 1 shows various concentrations of 6-nitroso-1,2-benzopyrone, 3-nitroso-benzamide, and nitroso-
FIG. 4 is a graph comparing the degree of inactivation of ADRPT polymerase activity (ADPRP) exhibited by 1 (2H) -isoquinolinone (NOQ) (mixture of 5 and 7 nitroso isomers).

FIG. 2 shows (A) 855-2 cells (human B-cell line acute lymphoblastic leukemia cell line), (B) H9 cells (human T
-Cell line acute lymphoblastic leukemia cell line), (C) HL
FIG. 4 is a graphical composition having the inhibitory effect of ADRPT ligand on -60 cells (human acute non-lymphoblastic leukemia cell line) and (D) K562 cells (human chronic myelogenous leukemia cell line). These cells are cultured under the influence of growth factors in 10% fetal calf serum (FCS), whereas (E)
In (F) and (F), 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.

FIG. 3 shows 6-nitroso-1,2-benzopyrone (NOB
855- with P) and 3-nitrosobenzamide (NOBA)
Figure 4 is a graph showing the inhibition of increasing levels of leukemic cell proliferation of two cells (in response to increasing concentrations of FCS).

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

FIG. 5 shows a graph of the relative inhibitory effects of anti-leukemic doses of ADRPT ligand on the ability of (A) normal bone marrow stem cells or (B) human peripheral vascular dry cells on soft agar. It is noted that NOBP and NOBA had minimal effect on normal cells.

FIG. 6 shows NOBP and NOB against four human brain tumor cell lines.
3 shows a graph showing the inhibitory effects of A and NOQ.

FIG. 7 is a graph comparing the efficacy of NOBP with vincristine.

FIG. 8 shows NOBP, NOB against human breast tumor cell line MDA468.
6 is a graph showing the effects of A and NOQ.

FIG. 9 shows NOBP, NO against murine leukemia cell line L1210.
6 is a graph showing the effects of BA and NOQ.

DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention provides certain nitroso compounds that are ADPRT polymerase activity inhibitors. These compounds are used as anti-tumor and anti-viral compounds.

Compound (I) has the following formula: Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are selected from the group consisting of hydrogen and nitroso, and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , And R
One has passed out of ₆ have a nitroso group].

A preferred embodiment of the compound I is where R ₄ is a nitroso group, i.e. the molecular 6- nitroso-1,2-benzopyrone.

Compound II has the following formula: Wherein R ₁ , R ₂ , and R ₃ are selected from the group consisting of hydrogen and nitroso, and only one of R ₁ , R ₂ , and R ₃ is a nitroso group.

Compound III has the following formula: Wherein R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are selected from the group consisting of hydrogen and nitroso, and only _one of R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ One is a nitroso group].

In a preferred embodiment of compound III, either R ₁ or R ₂ is a nitroso group, ie, 5-nitroso-1 (2H)
-Isoquinolinone and 7-nitroso-1 (2H) -isoquinolinone.

The disclosed synthesis for 5-nitroso-1 (2H) -isoquinolinone is based on two very related structural isomers, 7-nitroso-1 (2H) -isoquinolinone and 8-nitroso-1 (2H)-. Isoquinolinone can be produced. Experiments testing the biological activity of 5-nitroso-1 (2H) -isoquinolinone have shown significant amounts of 8-
Includes nitroso-1 (2H) -isoquinolinone or 7-nitroso-1 (2H) -isoquinolinone, but all three isomers have similar anti-tumor and anti-tumor based on their close structural similarities. -It appears to have viral activity. This hypothesis can 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.

6-nitroso-1,2-benzopyrone, 3-nitroso-
Benzamide, 5-nitroso-1 (2H) -isoquinolinone, 7-nitroso-1 (2H) -isoquinolinone and 8-
The detailed synthesis of nitroso-1 (2H) -isoquinolinone is
Provided in the example section below.

In general, the nitroso compounds of the present invention can be synthesized by oxidizing an amino compound corresponding to a compound of the present invention by oxidation with 3-chloroperoxybenzoic acid (or other peroxyacid) in ethyl acetate or a halocarbon solvent. . The synthesis of these precursor amino compounds
It is described in the chemical literature and some compounds are commercially available. Some precursor amino compounds for oxidation to the nitroso compounds of the present invention are as follows:
-Amino-1,2-benzopyrone (Spectrum Chemical Mf
g. Corp., Gardena, CA, 90248); 4-amino-1,2-benzopyrone (Aldrioh, Rare Chemical Catalog); 5-amino-1,2-benzopyrone (reduction of 5-nitro-1,2-benzopyrone) By Guttlieb, et al., J. Chem. Soc. Perkin. Tra
ns. II 435 (1979); 8-amino-1,2-benzopyrone (8
Abdel-Me by reduction of amino-1,2-benzopyrone
gid, et al., Egypt J. Chem. 20: 453-462 (1977)) and 4-amino-1 (2H) -isoquinolinone (by reduction of its corresponding 4-nitro analog, Horning, et al., ( 197
1) Can. J. Chem. 49: 2785-2796).

In addition to compounds (I)-(III), the present invention also relates to various structurally related compounds having similar anti-cancer and / or anti-viral activity. These structurally similar compounds are conveniently screened based on their high possible inhibitory effect on ADPRT polymerase activity. The structurally related compounds of interest include derivatives substituted with additional nitroso groups and small, eg, C ₁ -C ₃ , alkyl groups. Also, various nitroso-substituted structurally related heterocyclic rings, such as 3,4-dihydro-1 (2H) -isoquinolinone, nicotinamide, phthalhydrazide and 1,3-benzoxazine-2,4-dione. Are also the subject of the present invention.

Another aspect of the compounds of the present invention is their ease of entering cell membranes and the relative absence of non-specific binding to proteins and nucleic acids.

Indeed, the ADPRT polymerase inhibitors of the present invention, ie, compounds (I)-(III) and any of their pharmaceutically acceptable salts, alone or in combination with each other, and inhibit malignant growth or virus replication Alternatively, it can be administered in a pharmaceutical form that is sufficient and effective to prevent cancer growth or viral infection in a mammalian host.

Administration of the active compounds and salts described herein is 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. The preferred method of administration of these drugs is intravenous, except where the subject has a local tumor or injury, where local administration is appropriate. In other cases, it may be necessary to administer the composition in another parenteral or even oral form.

Depending on the intended embodiment, the composition may be a solid, semi-solid, or liquid dosage form, such as an injection, tablet, suppository, pill, special capsule, powder, liquid, suspension or the like, It is preferably present in unit dosage. The composition comprises an effective amount of at least one compound (I)-(III), or a pharmaceutically acceptable salt thereof, and further comprises any pharmaceutical excipient and other medical or pharmaceutical agent. Drug, or substance, carrier, adjuvant, diluent, and the like.

With respect to solid compositions, in addition to compounds (I)-(III), for example, pharmaceutical classes of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,
Excipients such as magnesium carbonate and the like can be used. The compounds of the present invention can also be formulated as suppositories, using, for example, polyalkylene glycols such as propylene glycol as carriers.

Liquid, especially injectable compositions, are pharmaceutical solutions such as water,
It can be prepared by dissolving or dispersing at least one active compound (I)-(III) in salt solution, aqueous dextrose, glycerol, ethanol, DMSO and the like, whereby an injectable solution or suspension A liquid can be formed.

If desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and other substances such as sodium acetate, triethanolamine oleate and the like.

Parenteral injection administration is commonly used for subcutaneous, intramuscular or intravenous injection or infusion. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for solution in liquid prior to injection.

A recently developed approach for parenteral administration is described in U.S. Pat. No. 3,710,795, which is incorporated herein by reference, which has a removable or sustained release that ensures that a constant level of administration is maintained. Use a system port.

Any of the above pharmaceutical compositions, wherein the active ingredient is 0.1-99%,
Preferably, it can contain 1 to 70%.

The actual methods for preparing such dosage forms are known, or will be apparent, to those skilled in this art and
ton's Pharmaceutical Sciences, Mack Publishing Comp
any, Easton, Pennsylvania, 17th edition, 1985. In any event, the composition or formulation to be administered will contain an amount of the active compound so as to ensure that a therapeutically effective amount will be administered to the patient. A therapeutically effective amount means an amount effective to prevent or alleviate the progress of the existing condition of the patient to be treated.

Of course, the amount of active compound to be administered will depend on the patient to be treated, the patient's weight, the severity, the mode of administration and the judgment of the prescribing physician. However, the effective dose can range from 1 to 12 days, 1 to 12 days in a single treatment cycle.
mg / kg / day, preferably in the range of 1-5 mg / kg / day. In general, the upper limit for drug administration is its efficiency balanced by its potential toxicity.

While the present invention has been described, the following examples are illustrative and not limiting.

Example I. Synthesis and characterization of 6-nitroso-1,2-benzopyrone An example of how to prepare 6-nitroso-1,2-benzopyrone is provided as follows: water (40 ml) at 22 ° C. To a stirred solution of 6-amino-1,2-benzopyrone hydrochloride (4.00 g, 20 mmol) in water (2.
0ml), sodium tungstate (5.93g, 20mmol)
Solution, followed by 30% aqueous hydrogen peroxide (5 ml),
Stirring was continued for 1.5 hours. The oxidation products are extracted from the green mixture with a volume of 100 ml of ethyl acetate and the combined extracts are washed with 0.1N HCl (50 ml) and then with water (100 ml).
And washed. Ethyl acetate was removed by rotary evaporation,
The residue was crystallized from hot ethanol (250 ml).

Analysis of the reaction product The green crystals obtained from the crystallization step (1.48 g, 42% yield) showed an absorbance at 750 mm characteristic of a monomeric aryl nitroso compound. Mass spectrum: m / z (relative intensity): 175 (M ⁺ , 100), 161 (16.88), 145 (33.7
7), 133 (10.38), 117 (56.09), 89 (79.71), 63 (57.1)
3). Strong analytical data for M ⁺ peak: calculated for _{C 9 H 5 NO 3: 175.0268} ; Found: 175.0271 (deviation -1.1pp
m). ¹ H-NMR from TMC (CDCl ₃ , 300 MHz) δ (ppm): H-4 split with tablet (6.572 and 6.604) H-3; H- with tablet (7.472 and 7.501) H-7.
8 split; tablet tablet (7.860 / 7.866)
And 7.889 / 7.798) H-7 split with H-8 and fine split with H-5; tablet (7.910 and 7.942) H-3 split with H-4; tablet (8.308 and 8.315) H-5 with H-7 Fine split. Λ _max for UV / VIS spectrum in ethanol
(Ε): 750 nm (46), 316 nm (8.96 × 10 ³ ), 274 nm (2.24
× 10 ⁴ ). Melting point: Compound polymerizes above 160 ° C, 325-340
Blackens in the range of ° C. and melts.

The nitroso-compound can also be prepared by reacting 6-amino-1,2-benzopyrone (as a free salt) with 3-chloroperoxybenzoic acid in an ethyl acetate or halocarbon solvent.

II. Synthesis of 3-nitrosobenzamide 1,208 g of a stirred solution of 3-aminobenzamide (Aldrich Chemical Co.) in ethyl acetate (50 ml) at ambient temperature.
3-chloroperoxybenzoic acid (commercially available, 50-60%
(Aldrich) was added, after which the solution turned green. After 10 minutes, the mixture was extracted with 0.14 M aqueous sodium bicarbonate (58 ml), washed three times with 40 ml of water, dried over sodium sulfate and then reduced to a volume of 20 ml by rotary evaporation and Freezer (-20
C), after which the product slowly precipitated as a pale yellow solid for 72 hours (0.180 g, 34% yield).

2-Nitrosobenzamide and 4-nitrosobenzamide isomers are converted to 2-aminobenzamide and 4
-Aminobenzamide can be similarly prepared by carrying out the above oxidation.

Synthesis of reaction product Melting point: The material darkens above 135 ° C., softens in the range of 150-160 ° C., and apparently polymerizes and melts (with decomposition) at 240-250 ° C. In solution, the compound is greenish 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). M ⁺ Strong analytical data for the _{peak: C 7 H 6 H 2 O} 2 Calculated for: 150.04292
8; Found: 150.042900 (deviation = 0.2 ppm). NMR Spectrum: ¹ H-NMR from _{TMS (DMSO-d 6, 300MHz} ) δ (ppm):
Broad singlet (7.737) NH; t (7.824, 7.850, 7.87)
5) H-5 split with H-4 and H-6; d (8.059
And 8.086) H-6 split with H-5; d (8.357 and 8.383) H-4 split with H-5; s (8.472) H
-2. The singlet at 7.737 corresponds to one proton;
Second NH which does not correspond spectroscopically in this compound
Protons are overlaid by the H-4 tablet. The tablet integrates with the two protons and can be broken down by the addition of D ₂ O to the DMSO solution. UV-VIS absorption spectrum in anhydrous ethanol, λ
_max (ε): 750 nm (37.6), 304 nm (5.35 × 10 ³ ) and 218n
m (1.50 × 10 ⁴ ). The maximum absorbance at 750 nm is characteristic of a monomeric aryl nitroso compound.

III. Synthesis of nitroso-1 (2H) -isoquinolinone (mixture of 5-nitroso and 7-nitroso-isomers) 1 (2H) -isoquinolinone (mesocarbostyril)
(Aldrich) was nitrated using the general procedure for isoquinoline compounds (CGLefevre and RJWLeFe).
vre, J. Chem. Soc. 1470 (1935)). The nitration products (Y. Kawazoe and Y. Yoshioka, Chem. Pharm. Ball. (Toky
o) a mixture of 5-nitro and 7-nitro isomers as selected by 16 : 715-720 (1968), one of which may be the 8-nitro isomer) Using the combination of potassium borohydride and palladium on carbon catalyst in aqueous methanol, the corresponding amino-1
Reduced to (2H) -isoquinolinone. To the resulting amino-1 (2H) -isoquinolinone (as free base) in ethyl acetate (175 ml) at 30 ° C. 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 (58ml), washed twice with 50ml water and dried over sodium sulfate. The volume of the solution was reduced to 50 ml by rotary evaporation and then placed on a freezer, after which an orange solid product precipitated (0.102 g).

Melting point: The material thickens, softens, darkens above 175 ° C. and apparently polymerizes above 195 ° C. and finally melts in the range 310-335 ° C. NMR analysis: ¹ from TMS
H-NMR (DMSO-d ₆ / O ₂ O, 300MH ₂ ) δ (ppm): m (6.723, 6.
741,6.752); m (7.511,7.518,7.533,7.539,7.547,7.55
9,7.577,7.585); m (7.603,7.674,7.686,7.698,7.74
7); d (7.818,7.846). In the presence of D ₂ O, the compound also shows a broad singlet at 11.90 ppm. The isomer components were analytically resolved by thin-layer chromatography (silica gel plates, ethyl acetate solvent) to give two bands, R _f 0.82 and R _f 0.72. Mass spectrum for R _f 0.82: m / z (relative intensity): 174 (M ⁺ , 100), 160 (2
6.8), 144 (93.0), 117 (90.8), 97 (21.9), 89 (96.
1), 71 (24.1). High analytical data for M ⁺ peaks: C
₉ calculated for _{H 6 N 2 O 2: 174.04298} ; found: 174.04320
0 (deviation = -0.3 ppm). R _f 0.72, with respect to components having M ^+, calcd for _{C 9 H 6 N 2 O 2} : 174.042928; Calculated: 17
4.043200 (deviation = -1.6 ppm). These data confirm that the compound is a mono-nitroso isomer.

IV. ADPRT Inactivation Studies Compounds of the invention were tested for their ability to inactivate the polymerase activity of adenosine diphosphoribosyltransferase (ADPRT). The assay was performed using bovine thymus ADPRT using Buki and kum, Biochem. 27: 5990.
5995995 (1988). Assay results as given in Table I show that the nitroso precursor (6-
Amino-1,2-benzopyrone) and more likely 5-
Of the iodo-derivatives (Table I, compounds 1 and 2 respectively)
Provides the I ₅₀ value (concentration of compound that inhibits enzyme activity by 50%) for ADPRT. Nitroso compounds (in Table I
3,4,5) have very high activity as anti-tumor and anti-HIV molecules (as shown in later sections)
And it is effective even after exposure of the cells for a short period, such as 30 minutes. 5-I-6 nitroso-1,2-benzopyrone (Compound 6) in these studies has been shown to be a relatively low inhibitor of ADPRT (iodine substitution appears to inactivate the NO group as an electrophile). And its biological effects are 10 times weaker than those of 6-NO-1,2-benzopyrone. For these reasons, the compositions of the present invention appear to be superior to SI-6-nitroso-1,2-benzopyrone, which has been shown to be a poor penetrating molecule.

Assay conditions: ADPRT, 0.4 μg; _CO DNA, 4 μg; 50 mM Tris-HCl, 50 mM KCl, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.1 mM NAD ([32-P] -labeled) 50 μl of inhibitor diluted between 0.8-600 μM in 50 μl of a pH 7.5 solution. Polymerization for 4 minutes at 25 ° C.

FIG. 1 shows the% inactivation of ADPRT polymerase activity observed after 2 hours of incubation with nitroso compound inhibitors at several concentrations.

Additional experiments involving the equilibrium between ⁶⁵ Zn2 ⁺ and Zn ^{+ 2} in ADPRT binding indicate that the ADPRT inhibitory activity of the nitroso compounds appears to act by destabilizing the protein by release of Zn ^{+ 2.} Suggest (Buki KG, Bauer P.
T., Mendeleyev, F .; Hakam, H. and kun E. (1991) FEBS Le
tt.290: 181-185). The mechanism of action described above for ADPRT inhibitors is speculative and does not limit the scope of the claims.

V. Biological anti-cancer activity of nitrosobenzopyrones, nitrosobenzamides and nitroso-isoquinolinones Various human leukemia cell lines have increased concentrations of 6-amino-1,2-benzopyrone (ABP), 5-iodo-6. - amino-1,2-benzopyrone (IABP), 6- nitro-1,2-benzopyrone (NO ₂ BP), 6- nitroso 1,2-benzopyrone (NOBP), 3- nitrosobenzamide (NOBA) or 5
(7) -Nitroso-1 (2H) -isoquinolinone (NOQ)
(Mixtures of 5-nitroso and 7-nitroso isomers) were exposed and experiments were performed where the level of [3H] thymidine uptake was determined as a measure of cell proliferation. As shown in FIG. 2, the individual cell lines tested (855-2 cells,
2A; H9 cells, FIG. 2B; HL-60 cells, FIG. 2C; K562 cells, FIG. 2D) with respect to the nitroso-containing ligand (NOB
P, NOBA, NOQ) is ³ H- at lower molar concentrations than the other compounds.
Thymidine intake could be inhibited. NOBP, NOBA and N
OQ potently inhibited ³ H-thymidine uptake at a concentration of 10 μM, at which concentration the other compounds showed relatively little inhibitory effect.

H grown in 10% fetal calf serum (FCS)
Experiments with 9 cells (FIG. 2B) found that NOQ was the most likely inhibitor, indicating that 10 μM
The level showed the most complete inhibition. NOBP is 10 μM
Showed about a 30% reduction in thymidine intake, and 10
0 μM showed almost complete inhibition of uptake. NOBA is 1
0 μM showed 75% level of inhibition, 100 μM showed about 85% level of inhibition, and 250 μM showed almost complete level of inhibition. The remaining amino and nitro compounds had a significantly lower potential and did not show complete inhibition until a concentration of 1000 μM was reached.

Experiments on K562 cells grown in 10% fetal calf serum (FIG. 2D) found that NOQ and NOBP were the most potential inhibitors of cell growth. Both NOQ and NOBP resulted in almost complete inhibition at a concentration of 10 μM. NOBP has almost the same potential 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 three tested compounds had significantly lower likelihood.

855-2 grown in 10% fetal calf serum
Experiments on cells (FIG. 2A) show that NOQ and NOBP
M concentrations were found to produce almost complete inhibition. At a concentration of 1 μM, NOQ produced a somewhat higher inhibition than NOB, and NOBP produced a somewhat higher inhibition than NOBA. The other three compounds tested had significantly lower likelihood.

The effect of various growth factors on the growth inhibitory effect of NOBP was tested. Elevate 855-2 cells grown in medium with (1) 10% fetal calf serum, (2) autocrine growth factor (AGF) and (3) low molecular weight-BCGF (T cell derived lymphokine). Exposure to concentrations of ADRPT ligand. The results are provided in FIG. 2 (A, E, F). Cells grown with individual growth factors were probably inhibited by the nitroso-containing compound and resulted in 100% inhibition at concentrations of 5-10 μM. Therefore, NOBP, NOBA and NOQ
Shows a potential inhibitory effect despite the source of growth factor activity.

To exclude the possibility that NOBP and NOBA may have their growth inhibitory effects through inactivation of growth factors, 10 μM NOBP or 10 μM against 855-2 cells in the presence of increasing concentrations of fetal calf serum (FCS). The effect of NOBA (constant concentration) was tested (FCS contains growth factors on 855-2 cells).
The data is provided in FIG. Growth inhibition occurs regardless of the concentration of FCS. Therefore, the mode of action of NOBP is
Not because of growth factor antagonism, but
It appears to be at an ADPRT site involved in DNA replication.

Tumor cell inhibitory concentrations of NOBP and NOBA have been shown to have no adverse effect on normal cell viability. Various cancer cells (855-2 and HL-60 leukemia cells, D32, D37 and CRL771
2 glioblastoma cell line, 186 medullary tumor cell line, L121
Experiments were performed in which the function of the murine leukemia cell line, the MDA-468 human breast tumor cell line) and the function of normal cells (neutrophil leukocytes and bone marrow or peripheral blood cells) were evaluated in the absence or presence of the compound. Was. The results are shown in FIGS. Furthermore, the data show that a nitroso-containing ligand at a concentration of 10 μM effectively inhibited cell proliferation and exhibited its function on normal cells with only modest effects.

VI. Toxicity of NOBP The cytotoxicity of 0.2 μM, 4 μM, 8 μM and 10 μM NOBP
Conine formation of normal human stem cells (PBSC) (CFU-GM)
It was determined by testing the effect of the compound on The results of that experiment are provided in FIG. 5B. Toxicity is
No level of NOBP was detected, even when levels of NOBP sufficient to inhibit 855-2 cell proliferation were tested.

The comparison is (ABP) 6-amino-1,2-benzopyrone 1 mM,
(IABP) 5-I-6-amino-1,2-benzopyrone 250μ
M, a similar CFU stem cytotoxicity assay performed between 250 μM (NO ₂ BP) 6-nitro-1,2-benzopyrone (weak activity), 10 μM NOBP, and 10 μM NOBA. The results of the experiment are shown in FIG. 5A. 6-amino-1,2-
Benzopyrone, 5-I-6-amino-1,2-benzopyrone and 6-nitro derivatives are toxic at the doses tested but are almost ineffective (on tumor cells).
The nitro derivatives and the very effective (on tumor cells) NOBP and NOBA were non-toxic.

The effect of 10 μM NOBP and NOBA on superoxide production by normal human peripheral blood neutrophil leukocytes was tested. The results are shown in Table II. Only a slight decrease in superoxide formation was observed.

VII. Comparative efficiency studies Vincristine, a highly toxic chemotherapeutic compound,
It is currently used for the treatment of leukemia and other malignancies. A study was performed 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 inhydro. Vincristine 0.1, 1,10 and
Tested at a dose of 100 μM. As shown in FIG. 7, 100 μM vincristine (high toxic concentration)
Required to produce the same level of inhibition as 0 μM NOBP, NOBP is therefore likely to be about 10-fold higher than an equal concentration of vincristine and is not toxic to normal cells.

Thus, certain aromatic nitroso molecules, which are also inhibitors of ADPRT polymerase activity, can be useful chemotherapeutic cytostatics because of their effectiveness combined with low toxicity.

VIII. NOBP, NOBA and Stimulated Human Lymphoblasts
Anti-HIV Effect of NOQ The ability of NOBP (6-nitroso-1,2-benzopyrone) and NOBA (3-nitrosobenzamide) to inhibit HIV infection was determined by the Journal of Immunological Methods 76: 171-183 (1
985). Those two
Exposure to the seed drug was only 30 minutes at the onset of viral infection, and the drug was never added again.
The results given in Table III provide an HIV titer of ID ₅₀ 10 days after infection of the cell culture with HIV. The data in Table III shows that 10 μM of the nitroso-containing ligand was HIV-1.
FIG. 7 shows that it causes a 3-log reduction in infectious titer.

IX. ADPRT ligand cytocidal activity-MTT assay To determine whether the inhibition of 855-2 cell growth found in culture and soft agar is due to the cytostatic or cytocidal effects of the nitroso compounds NOBP and NOQ. Was performed. Cells at 1 × 10 ⁵ / ml (concentration used for bone marrow assay) were treated with NOBP, NOBA and 1,2.5,5 and 10 μM.
Treated with NOQ for 2 hours, then stimulated with 10% fetal calf serum and incubated for 24 hours. Next, 1mg /
MTT (3- [4,5-dimethyl-2-yl] -2,5-
Diphenyltetrazolium bromide) was added for 16 hours. Next, the absorbance of the pelleted cells was measured at 550 nm after adding DMSO to lyse the cells.

Results With 10 μM NOBP, NOBA and NOQ, complete killing was observed in 855-2 cells at 100,000 / ml.

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

The foregoing specification is believed 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 can be made within the scope of the invention.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Kang, Ernest United States of America, California 94941, Mill Valley, Helens Lane 8 (72) Inventor Mendereiev, Jerome United States of America, California 94117, San Francisco, Stannign Street 1292 (72) Inventor Rice, William G. United States, California 30278, Sneelville, Code Loop 2848 Examiner Takeshi Nimi (56) References Special Table 10-504517 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB Name) CA (STN) REGISRY (STN)

Claims (3)

(57) [Claims] 1. The following formula: A compound having the formula: 2. A composition for the treatment of cancer and retroviral diseases, comprising a compound according to claim 1. 3. A method for the treatment of cancer and retroviral diseases comprising administering to a non-human animal a compound according to claim 1.