CA2121900A1 - Adenosine diphosphoribose polymerase binding nitroso aromatic compounds useful as anti-tumor and anti-retroviral agents - Google Patents
Adenosine diphosphoribose polymerase binding nitroso aromatic compounds useful as anti-tumor and anti-retroviral agentsInfo
- Publication number
- CA2121900A1 CA2121900A1 CA002121900A CA2121900A CA2121900A1 CA 2121900 A1 CA2121900 A1 CA 2121900A1 CA 002121900 A CA002121900 A CA 002121900A CA 2121900 A CA2121900 A CA 2121900A CA 2121900 A1 CA2121900 A1 CA 2121900A1
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- Prior art keywords
- nitroso
- group
- compound
- compounds
- pharmaceutically acceptable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
- A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
- C07C233/65—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
- C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
- C07D217/24—Oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/18—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted otherwise than in position 3 or 7
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Virology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Pyrane Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Other In-Based Heterocyclic Compounds (AREA)
Abstract
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.
Description
W 0 93/07$~8 21 2 1 9 ~ O PCT/US91/0890~
ADENOISE DIPHOSPHOKIBOSE POLYMERASE BINDING NITROSO AROMATIC `~
COMPOUNDS USEFUL AS ANTI-TUMOR AND ANTI-RETROVIRAL AGENTS
Fi~ld of ~he I~io.n The present invention relates generally ts the ~:~
field of retroviral therapeutic agents and their :~
use in treating viral infections and can~ers.
More specifically it xelates to those therapeutic agents which inhibit A~P-ribose transferas~, and in particular variou~ nitroso-benzopyron~s, nitroso-isoquinolinones and ni~roso-benzamides. ;~
'.
BackqrQund of ~he Invention The enzyme ADP-ribose transfera~e (ADPRT) (E.C.4.2.30) is a chromatin-bou~d enzyme located `~
in the nucleus of mo~t eukaryotic cells. ~he enzyme catalyæes the polymerization of ~he ~DP~
ribose moiety o ni~otinamide adenine di~ucleotide ~(NAD~) to form poly lADP-ribo~e~
The polymes is co~alently attached to variou~
nuclear protein~, includi.ng the polymerase i-tself.
~.
; : The many varied roles that ADP-ribosylation plays in cellular metabolism ha~e made ~DPRT :a `:~
target for drugs essentia11y use~ful~ for ~' combating-~ neoplasla ~and viral lnfections.
Numerous physiological ~activi~îes have been detected for compounds that : inhibit the 30 ~ polymerase activity of ADP ~ . ~Such a~:iviti~S
include a~cell cyc1e depend~nt prevention of , carcinogen-induced ma1ignant t~ansformation of : human :fibroblasts (Kun, E., ~irsten, E.) Milo, .E. Kurian:~, P. and Kumari, H. L. (1983)~Qç~ .
N~l. Ac~;. S~i. U~ ~Q:7219-7223), con~erring also carcinogen;resistance (Milo, G.E., Kurian, ~P., Kirsten, E. and Kun, E. (198~3 E~ L~-332-336), inhibition of malignant trans~ormation in hamster embryo and mou~e : : '' W~93/07~68 PCT/US91/~902 212~0 C3HlOTl/2 cell cultures tBorek, C., Morgan, W.F., Ong, A. and Cleaver, J.E. ( 1984) ~QQ, N~tl . ~ad, SC~ ;A 81: 243-247 ~, deletion C: f transfected oncogenes from NIH 3T3 cell~
(Nakayashu, M., Shima, H., Aonuma, S., Nakagama H., Naga~. M. and Sugi.mara, T. (1988) Na~ a~ ~sci. U~ ~5:9066-9070), suppression of the mitog~nic stlmulation of tumor promoter~
(Romano, P., Menapace, L. and Armato, V. (1983) 10Çarci.nQ~nesls 9:~147-2154), inhibition of illegitimate DNA recombinations (Waldman, B.C.
and Waldman, ~. (1990~ ~L~ L8 - 18:59~
5g88) and int~gration (Farzaneh, F., Panayotou, G.N., Bowler, L.D., Harda~, B.D., Broom, ~., Walther, C. and Shall, S. (1988~ ~çl~
Res. 1~: 11319-11326), :induction of sister ohromati~ ~xchange ~ ~:Ikushima, T. ~1990) Çh~nmnzcr~ ~:360-364~ and the loss of certain : ampli~fi~d oncogenes (Grosso, L.E. and Pitot, ~0~ H:.C:. (1984)~ Bio~he~. .Bi~ hys. Res. ommun-473-480; Shima, H.,~Nakayasu, M., Aonums, S ., Sugimura ,~ T . and Nagao,; M. :~198~) ~LQg~
N~tl ! ~Acad. Sci U$~ 6:7~42-7445).
25 :~ : Compounds~nown to:inhibit AD~PRT polymerase :: a~t:iv~ty ~in~lude~benzamide (Kun~ ~E., ~lrs~en, ; E., Milo~ G~.E.~ Kuri~an, P. and;Eumari~, H~.~ L.
1983) ~ 80:7~19-7~:23 substituted benzamid~s (Borek, C~., Morgan,~W.F.,:
3b~ Ong, A. and ~Cl~aver, ~.E. i(l984~ ~Y5L_l~a~
~~L:243-247i Romano~, F., Menapace, :~ ~ L.~:and Armato, V. (1983)~Carcino~enesiQ 9; 2147-: ~ 2154; Farzaneh,~ F., Panayotou,;~:~G.N.~Bowler, L.D.~, Nardas, B.D.,~Bro~om, T., Walther, C.~and . 35~ ; Shall, ~ c1988) ~D~o ~si~hL ~ :11319 ~ 11326.; Grosso,~ L.E. and Pitot, H.C. ~1984) : ~ ; :iQQb~m. BiQ4hy~ Res.~ Com~n. 11~:473-480, ..
~ ~ ' :: :::: ;::~
W~93J~7868 PCT/~S91/0~9~2 2l2l~a :
Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989~ Pxo~.. Natl. ~cad Sci.
~&~ 86:7442-7445), 3-aminonaphthylhydrazide ~:
(Waldman, B.C. and Waldman, A. (1990) ~
Acid~ _~e~ 5981-5988), isoquinoline, ~uercetin, and coumarin (1,2-benzopyrone~ (Milo, G~E., Kurian, P., Kirsten, E. and Kun, B. (1985) EE~Lhç~. 17~: 332-336). The anti-trans~iorming and anti-n~oplastic effect of 1,2 benzopyrone were d~monstxated i~ Yi~Q and La Vi~Q (T6ang, et al., tl987) Other known ADPRT polymera~e activity inhibitors include 5-iodo-6-amino-1,2- :
benzopyrone as d~scribecl in U.S. Patent applioation Serial No. 600,S93, filed October ~
19, 1990 entitl2d "Novel 5-Iodo-6-Amino-1,2- :::
senæopyrone~:and thelr 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 ~i~
: or anti-viral ag~n:t. ::
~,.
The 6-nitroso benzopyrones have ~Q~ b~en -~:
hitherto known or descri~ed. The only ~emotely related compounds found in the litexature are 6-nitro-1,2-~enzopyrone and 6 amino-1~2-benz~pyrone (6-ABP3 (~ ~L__f~ _ Jao ~ 615 (1923)) for which, only scarce medicinal ~;
evaluation~ has been reported. In particular, tes~ing was done for sedative and hypnotic effects (J. Pharm. _Soc. ~apan, 13:351 (1953);
~ 271 (19S4)), hypothermal action (:Yak~q~ Za~shi, 78:491 (1958)), and antipyretic, hypnotic, hypoten~ive and ::
W0~3/07~6~ P~T/US9l/08902
ADENOISE DIPHOSPHOKIBOSE POLYMERASE BINDING NITROSO AROMATIC `~
COMPOUNDS USEFUL AS ANTI-TUMOR AND ANTI-RETROVIRAL AGENTS
Fi~ld of ~he I~io.n The present invention relates generally ts the ~:~
field of retroviral therapeutic agents and their :~
use in treating viral infections and can~ers.
More specifically it xelates to those therapeutic agents which inhibit A~P-ribose transferas~, and in particular variou~ nitroso-benzopyron~s, nitroso-isoquinolinones and ni~roso-benzamides. ;~
'.
BackqrQund of ~he Invention The enzyme ADP-ribose transfera~e (ADPRT) (E.C.4.2.30) is a chromatin-bou~d enzyme located `~
in the nucleus of mo~t eukaryotic cells. ~he enzyme catalyæes the polymerization of ~he ~DP~
ribose moiety o ni~otinamide adenine di~ucleotide ~(NAD~) to form poly lADP-ribo~e~
The polymes is co~alently attached to variou~
nuclear protein~, includi.ng the polymerase i-tself.
~.
; : The many varied roles that ADP-ribosylation plays in cellular metabolism ha~e made ~DPRT :a `:~
target for drugs essentia11y use~ful~ for ~' combating-~ neoplasla ~and viral lnfections.
Numerous physiological ~activi~îes have been detected for compounds that : inhibit the 30 ~ polymerase activity of ADP ~ . ~Such a~:iviti~S
include a~cell cyc1e depend~nt prevention of , carcinogen-induced ma1ignant t~ansformation of : human :fibroblasts (Kun, E., ~irsten, E.) Milo, .E. Kurian:~, P. and Kumari, H. L. (1983)~Qç~ .
N~l. Ac~;. S~i. U~ ~Q:7219-7223), con~erring also carcinogen;resistance (Milo, G.E., Kurian, ~P., Kirsten, E. and Kun, E. (198~3 E~ L~-332-336), inhibition of malignant trans~ormation in hamster embryo and mou~e : : '' W~93/07~68 PCT/US91/~902 212~0 C3HlOTl/2 cell cultures tBorek, C., Morgan, W.F., Ong, A. and Cleaver, J.E. ( 1984) ~QQ, N~tl . ~ad, SC~ ;A 81: 243-247 ~, deletion C: f transfected oncogenes from NIH 3T3 cell~
(Nakayashu, M., Shima, H., Aonuma, S., Nakagama H., Naga~. M. and Sugi.mara, T. (1988) Na~ a~ ~sci. U~ ~5:9066-9070), suppression of the mitog~nic stlmulation of tumor promoter~
(Romano, P., Menapace, L. and Armato, V. (1983) 10Çarci.nQ~nesls 9:~147-2154), inhibition of illegitimate DNA recombinations (Waldman, B.C.
and Waldman, ~. (1990~ ~L~ L8 - 18:59~
5g88) and int~gration (Farzaneh, F., Panayotou, G.N., Bowler, L.D., Harda~, B.D., Broom, ~., Walther, C. and Shall, S. (1988~ ~çl~
Res. 1~: 11319-11326), :induction of sister ohromati~ ~xchange ~ ~:Ikushima, T. ~1990) Çh~nmnzcr~ ~:360-364~ and the loss of certain : ampli~fi~d oncogenes (Grosso, L.E. and Pitot, ~0~ H:.C:. (1984)~ Bio~he~. .Bi~ hys. Res. ommun-473-480; Shima, H.,~Nakayasu, M., Aonums, S ., Sugimura ,~ T . and Nagao,; M. :~198~) ~LQg~
N~tl ! ~Acad. Sci U$~ 6:7~42-7445).
25 :~ : Compounds~nown to:inhibit AD~PRT polymerase :: a~t:iv~ty ~in~lude~benzamide (Kun~ ~E., ~lrs~en, ; E., Milo~ G~.E.~ Kuri~an, P. and;Eumari~, H~.~ L.
1983) ~ 80:7~19-7~:23 substituted benzamid~s (Borek, C~., Morgan,~W.F.,:
3b~ Ong, A. and ~Cl~aver, ~.E. i(l984~ ~Y5L_l~a~
~~L:243-247i Romano~, F., Menapace, :~ ~ L.~:and Armato, V. (1983)~Carcino~enesiQ 9; 2147-: ~ 2154; Farzaneh,~ F., Panayotou,;~:~G.N.~Bowler, L.D.~, Nardas, B.D.,~Bro~om, T., Walther, C.~and . 35~ ; Shall, ~ c1988) ~D~o ~si~hL ~ :11319 ~ 11326.; Grosso,~ L.E. and Pitot, H.C. ~1984) : ~ ; :iQQb~m. BiQ4hy~ Res.~ Com~n. 11~:473-480, ..
~ ~ ' :: :::: ;::~
W~93J~7868 PCT/~S91/0~9~2 2l2l~a :
Shima, H., Nakayasu, M., Aonums, S., Sugimura, T. and Nagao, M. (1989~ Pxo~.. Natl. ~cad Sci.
~&~ 86:7442-7445), 3-aminonaphthylhydrazide ~:
(Waldman, B.C. and Waldman, A. (1990) ~
Acid~ _~e~ 5981-5988), isoquinoline, ~uercetin, and coumarin (1,2-benzopyrone~ (Milo, G~E., Kurian, P., Kirsten, E. and Kun, B. (1985) EE~Lhç~. 17~: 332-336). The anti-trans~iorming and anti-n~oplastic effect of 1,2 benzopyrone were d~monstxated i~ Yi~Q and La Vi~Q (T6ang, et al., tl987) Other known ADPRT polymera~e activity inhibitors include 5-iodo-6-amino-1,2- :
benzopyrone as d~scribecl in U.S. Patent applioation Serial No. 600,S93, filed October ~
19, 1990 entitl2d "Novel 5-Iodo-6-Amino-1,2- :::
senæopyrone~:and thelr 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 ~i~
: or anti-viral ag~n:t. ::
~,.
The 6-nitroso benzopyrones have ~Q~ b~en -~:
hitherto known or descri~ed. The only ~emotely related compounds found in the litexature are 6-nitro-1,2-~enzopyrone and 6 amino-1~2-benz~pyrone (6-ABP3 (~ ~L__f~ _ Jao ~ 615 (1923)) for which, only scarce medicinal ~;
evaluation~ has been reported. In particular, tes~ing was done for sedative and hypnotic effects (J. Pharm. _Soc. ~apan, 13:351 (1953);
~ 271 (19S4)), hypothermal action (:Yak~q~ Za~shi, 78:491 (1958)), and antipyretic, hypnotic, hypoten~ive and ::
W0~3/07~6~ P~T/US9l/08902
2 1 2 1 ~ O O
adrenolytic action (Ibid) 83:1124 (1963)). No significant application for any of these compounds has been de~cribed except for 6 ABP.
2-nitrosobenzamide (Irne-Ra~a, K.M. and Koubek, E. (1963) ~ _5~L_I~b~m. 28:3240-32~1), and 4-nitrosobenzamide (Wubbels, G.G., Kalhorn, T.F., Johnson, D.E. and Campbell, D. (lg82~ .J.
Q~ _ÇhQm. 47:4664-4670), have been reported in the chemical literature, but no commercial use of these i omer~ i^e known. Neither of these articles ~u~g~st the use of ~itrosobenzamid~s as ADPRT inhibitors.
The anti-viral ana ant~-tumorig~nic actions of substituted and unsub~tituted 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone is the ~ubject of copending U.S. patent applications Serial No. 5B5,231 ;filed on 20~ September ~ 21, 1990 entitle~ "6-Amlno-1~2-~enzopyrones U~eful for Treatmen~ of Viral Diseases" :and Serial No. 600,593 filed on October l9,:1g90:entitled i'Novel 5-Iodo-6-Amino-1,2~-Benz~opyrones and Thelr M~tabolites~U~eful~as : : ~ 25 ~ Cytostatic ~and AntiviraI Agents"~ which are : ~ ~ incorporated hereln by refer~nce. ;
: : : ThP. precursoL moleculs, 1,2-benzop~rone : :~coumarinj, was ~hown to he an:inhibitory ligand ~0 of l adenosinediphosphor~bosyl t~rans~era~le (ADPRT), a DNA-binding ~uclear protein present :
: in all mammalian:cells (Ts~ng,:~ al~.,;(lg37) PrQ~. Nat.~A~ad _S~i USA, 84:1107-llll).: ~`
; ; ~; ~ ~ :',
adrenolytic action (Ibid) 83:1124 (1963)). No significant application for any of these compounds has been de~cribed except for 6 ABP.
2-nitrosobenzamide (Irne-Ra~a, K.M. and Koubek, E. (1963) ~ _5~L_I~b~m. 28:3240-32~1), and 4-nitrosobenzamide (Wubbels, G.G., Kalhorn, T.F., Johnson, D.E. and Campbell, D. (lg82~ .J.
Q~ _ÇhQm. 47:4664-4670), have been reported in the chemical literature, but no commercial use of these i omer~ i^e known. Neither of these articles ~u~g~st the use of ~itrosobenzamid~s as ADPRT inhibitors.
The anti-viral ana ant~-tumorig~nic actions of substituted and unsub~tituted 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone is the ~ubject of copending U.S. patent applications Serial No. 5B5,231 ;filed on 20~ September ~ 21, 1990 entitle~ "6-Amlno-1~2-~enzopyrones U~eful for Treatmen~ of Viral Diseases" :and Serial No. 600,593 filed on October l9,:1g90:entitled i'Novel 5-Iodo-6-Amino-1,2~-Benz~opyrones and Thelr M~tabolites~U~eful~as : : ~ 25 ~ Cytostatic ~and AntiviraI Agents"~ which are : ~ ~ incorporated hereln by refer~nce. ;
: : : ThP. precursoL moleculs, 1,2-benzop~rone : :~coumarinj, was ~hown to he an:inhibitory ligand ~0 of l adenosinediphosphor~bosyl t~rans~era~le (ADPRT), a DNA-binding ~uclear protein present :
: in all mammalian:cells (Ts~ng,:~ al~.,;(lg37) PrQ~. Nat.~A~ad _S~i USA, 84:1107-llll).: ~`
; ; ~; ~ ~ :',
3$ ~aka~, et ~ .~ ~EBS~ ., 21~:7~ (1987) has :
:sh~own that 6-amino~ benzopyrone (6-ABP) ~i.nd~ :
speciii~ally to ADRPT at the site that also . .
W093/~7868 PCT/~S91/08~02 ;
!
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 5~l-, (1991) ~ _ Çell. R~ 4~-Subsequently, these ligands were found to i~hibit viral replication and are the subject of the copending U.S. patent application entitle~
"6-Amino-1-2-Benzopyrones useful for Tr~atment of Viral Disea~es," Serial No. 585,~31, filsd on September 21, 1990 which îs hereby incorporatea by refere~ce.
Thus it is of int~re~t to provide ADPRT
polymerase activity inhibitors or use~as anti-viral a~d anti-tumor agents. The-: sub~eet invention provide~ for novel nitroso-1,2-benzopyrone, nitr~oso-benz;~mide and nitroso-;; isoquinolinone::~ compounds, and Yarious20 ~: structural~ly related o~her nitroso compounds for e 8 anti-viral and anti-tumor therapeutic ~agents. The compounds taught for use:~in the subject ~ nvention ~are believed to~ be gnifi~can~ly: l:ess toxic ~and far more (500 to 25~ ; 1000 :fold)~potent: than structurally analogous amino compounds. ~ `
Sum~ t~ IDV en Th~ sub~ect invention:proYides fsr ~ovel anti~
3~0 ~ tumo~ and anti-viral compounds. These compounds :in~lude ~ 6-ni.tro~o-1~ : 2-~enzopyro~é, 3-itr~osobenza~mide, 5-ni~troso-~1:(2H)~
isoqulnol~inone,~7-nitroso-1~(2H)-isoquinolinone, 8-nit~o~o-1(2H)-isoquinolinone.~: Ths invention~ :
;~35 ~ :a1~o provides for compo~itions containing one or : ~~ more:o~ t~e compounds, and for methods o~
.
: :: treating viral in~ections and cancer with these ::::~ ::
.:~
W0~3~07868 PCT/US91/08902 2121~00 ~
-6- -~
compounds and compos.itions.
Also provided for ar~ methods of treating cancer and viral infections with 2- --nitro~obenæamide and 4-~itrosobenzamide.
Composition containing one or more of the~e compounds is al~o provid~d for.
D~ riptio.n cf Figur~6 ~.
Figure 1 i~ a graph comparing the degr~e of AD~PT polymeras~ activity ~ADPRP) inactiv~tlon exhibited by different concentrations of 6-~itroso 1~2-benzopyrone, 3-nitroso be~zam~de, and nitro o-1[2H)-isoquinQlinones (~OQ) (a mixture of the 5 and 7 nitroso i~omers).
: , .
Fig~re 2 is a compositei of graphs di~playi~g :~:
the inhlbi~ory effeots of the ADRPT ligands on ) 855 2 ells la cell ~line~of human~B-cell ~, 20~ linèage acute lymphobl~stic leu~emla~, (B) N9 cells~(a c:ell line of human T-oell lineage acute : ly~pho~lastic;leukemia~, (C) HL-60 cells (a oell .~; 1ine~of ~uman ac~t~ nonlympho~lastic~leu~emia~
and~(D)~K562~oells~(a oell;~line~.~of human~chronio 25~ ;:: myelogenou;6 le:ukemia3~ These~ ~c~lls~;~werc :
: culture~ whil:e:un~er ~he influen~e of th~ ~rowth Eaotors~in~ 0%~fetal;~ovlne ~rum (~FCS~), whereas:~
: : :~ ;:~in (E) and~(F) thc 855-2 oe:11s were:cultured~ln~
the presence of autocrine growth actor actiYit i 30~ (A~F) or low molecular weight B-cell growth~
faotor ~(BCGF~, a T-oell derived lymphokinc) resp2CtiVQly ~ s.7 ~ ~ ~ : Figure 3 ~i~s ~a graph showing the inhlbition of : ;35~ creasing lcvels~of :leukemio cell growth (ln:
~ :response to~inoreaslng concentrations of FCS) of .
:~ .
i W~93/07868 PCT/US91/089~2 2121~9ûD
I
855-2 cells by 6-nitroso~ -benzopyrone (NOBP~ -and 3-~itrosobenzamide ~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 c lls in a semi-solid medium.
Figure 5 shows graph of the r01ative inhibitory effects of anti-leukemic dosss of ADRPT ligands on the ability of (A3 normal ~:
rhesus bone marrow stem cells or (B~ human peripheral blood stem cells to form coloni~s ln soft agar. Note that the NOBP and NOBA had lS minimal effect on normal cells. :~
Figure 6 shows graph~; displaying the inhibi~ory ef~ects of NOBP, NOBA and NOQ on f~ur human brain ~umor cell line~
20:: Figure 7 is a graph comparing t~e e~fe~tiveness:of NOBP with vincri6~ine. :~
'~.;`
Figure ~ is a g:raph displaying t~e effects of NO~P, NOBA and MOQ on human breast tumor cel~
: 2s li~e M~A 468. ~ -.
: Figure 9 is a graph displayinq the ef~eots of NOBP, NOBA and NOQ on murine leukema ~ell line L 1210. ` :
~ De6~ri~ipn_cf SpQcifi~ ~mbQdimQn~
, .
The ~ubject in~ention pro~ides for ~everal : :: nitroso aom~ounds that are ADPRT pol~mer~6e :: activity inhibitors. These a~pounds find use a6~anti-tumor and antl-viral compounds.
~:
~;
'..
WO93/07B68 P~T/US91/08902 ~8- :~
Compound (I) has the following formulaO -~
~4--y~
R3 }~
S wherein ~, ~, ~, R~, ~, and ~ a~ selected from th~ ~roup con~isting of hydrogen and nitroso, and only;one of ~ , R4, ~, a~d is a nitroso group.
~ ':`
A preferred embodiment of compound I i5 where R4 is the nitroso gxoup, i.e., the molecule 6 nitro~o-1,2-be~zopyrone.
Compound II~has~the formula~
15;~: :(}I~
0;~ ~ ~wherein~ 2~:and ~are ~selected~rom the group~consisting of~ hy~rogen~:and:nitroso,:~and~
only on~of~ , and~RJ:~Is:a~D~troso~gro~p.
2~5~Compound~ has the~formula~
:.
~;
WC~93/07B68 PCT/US91/08gO2 2121~00 ! -:
_g_ :
wherein ~, ~, R3, R~, and ~ are selected from the group consisting of hydrogen and nitro60 t and only one of R1, ~, ~, R4, and R5 is a nitroso group.
Preferred embodim~nt~ of compound III are where either R~ or ~ is the ni~roso group, i.~
5-nitroso-1(2H)-isoquinolinone and 7-nitroso-1(2H~-isoquin41inone, re~pectively.
The disclosed synthPsis for 5-nitroso-1(2H~
isoquinolinone may produc~ 2 closely related structural isomers, 7-nitroso-1(2H~
isoquinolinone and 8-nitroso-1(2H~-isoqui~olinon~. Although experiments testing the biological activity of 5-nitro~o-1(2H)-iso~uinolino~ may ha~e contained significant quantities of 8-nitroso-1(2H)-isoquinolinone or 20: ~ 7-nitro~o-1(2H)-isoquinolinone, all three isomers are b~lieved to:possess similar anti~
tumor and anti-viral activity on the basis of their cIose ~tructural :similarity. This : : : hypQ~hesi~ may b~ ~ conveni~ntly ~ted by ;~~25: ~ s parating~ the isomers by thin layer chromatography or similar methods, and comparing the anti-tumor and~anti-~iral:activities of the : : separated compounds.
3~ Detail~zd synthesis of 6-nitrofio-1~2 benzopyrone:, 3-nitroso-benzamide, 5-nitr~so~
1(2H~- isoqui-nolinQne ~ 7-nitroso-~1(2H):-i~oquinol1nonez, and 8-nitroso-1(2H)-iso~uinolinone~ are providea in the ex~mple : 35 section bslow.
,.
W~93/0786~ PCT/US91/O~gO~ .
2121~00 In general, the nitroso compounds of the subje~t of invention may be synthesized by oxidizing a corre~pondlng amino compound to~a compound of the ~ubject invPntion by oxidation S with 3-chloroperoxy~enzoic acid (or other pexoxyacids) in ethyl acetate or a halocarbon solvent. Syntheses of thes~ precursor amino compounds are described in the chemi~ l literature and some of the compounds are commercially available. Some precur~or amino compound~ for oxidation to nitroso compounds of the subject inven~ion are as follows: 3-amino-1,2-b~æopyrone tSpectrum Ch~mic~l Mfg. Corp. t Gardena~ CA 902483; 4-amino-1,2-b~nzopyrone (Aldrich, Rare Che~i¢al Catalog3; 5-ami~o-1,2-benzopyro~e (by reduction of 5-nitro-1,2-benzopyrone, Çh~=L~ E~ ~7 16536d (1952)3; 7-amino-1,2-benzopyrone ~Gottlieb, ~ al., ~hem. Soc.~ ~P~rkin~_~r~ns. I 435 (~1979)); 8-amino-1,2-benzopyrone lby reduction of 8-amino--benzopyrone, ~bdel~Megid, e~ al-, ~gy~~
~hQm~ 20:453-462~(1977)), and. 4-amlno-1(2H)-i~quinolinone, by redu~tion of:~ the : : correspond~i~ng~4:~nitro analog (Horning~
~1971) Ca~ J. .~hem. 49::2785-2796).
In ~ddltion to compounds (l) to (III), the~
subject in~ention contemplates various structurally related compounds that have similar ` ~ : 30 carcino~tatic and/or antl-viral activities These ~tru~turally related compounds could~be~
c~nv~nien~ly scre~n~d ~on the ~basis of: their : highly pot~ent~lnhibitory~ ef~ect on ~ADPRT
~ polyme*ase activity. Structurally related compounds o~ ;int~rest include derivatives : : substituted by: additional nitroso groups and small, e.g., ~C1-C3 alkyl groups. A1BO of :
W~93/0786g PCTJUS91/08~02 ~21~)0 interest are various nitroso substituted stxucturally related heterocyclic ring~ such as 3 , 4 - d i h y d r o - 1 ( 2 H ~ - i 5 o q u i n o 1 i n o n e s , nicotinamides, pthalhydrazide~, and 1,~-benzoxazine-2,4-diones.
Another aspect of the compound~ of th~ ~ubject invention are the ea~e with which they permeate cell membranes and their relative absence of lQ non spe~ific binding to protelns and nucl~ic acid.
In practiae, the ADPRT polymeras~ i~hibitors of this inventio~, namely compounds (I) to (III), and any of their pharmaceuti~ally acceptable salts, may b~ adminis~tered in amount~, either~alone or in combination with each other, and in the ph rmaceutical form which will be suf f ic ient and effective to 1nhibit ~ ~ 20 neoplastl~ gro~ h or v:iral replication or prevent ~he development of the cancerous growth or vlr~1 inection in the mammalian ~o~t.
:.
Admi~istration of the active compounds~and 6alts described herein can be Yl~ any of the : accepted modes;of admlnistra~lon for therapeutic agent~. These methods include s:ys:temic or looal : administration: such a~ oral, parenter~
tran~dermal, subcutaneous, or topi~al administration modes. The preferr~d method of administxation o these drugs is intravenous,~
: except in~ tho~ cases :where th~ subjec~ has .
topiaa~ ~:umor~ or lesions, ~where the topi~al ~ ~ admin~tration~ may : be : proper. In other ~ a~oes, it may be ~ecessary to adminl~ter the oompo3ition in other parenteral or~even oral ~`
forms.
' '~
W093/07~68 PCTJVS~1/08902 2121900 ~ :
Depending on the intended mode, the compositions may be in the solid, se~i-solid ox liquid dosage form, such as, for example, injectables, tablets, suppositorles, pills, time-release capsules~ powders, liquid~, suspensions, or the like, preferably in unit do~a~es. ~he compositions will include an effecti~e ~mount of at least one of compound~
(I) to (III), or pharmaceutically acceptabl~
~alts t~ereof, and in addition it may i~clude any conventional pharmaceutical excipientæ and other medicinal or pharmaceutical drugs or agents, carr~ers, adjuvants, diluents, etc., as cuætomary in the pharmacautical sciences.
'".
~or solid compositionæ, in addition to the oompouna~ tI) to (III), such exGipientæ as~ for : exampl~, pharmaceutical gradss of mannitol, :~ 20: laatose, s:tarch, magnesium stearate, sodium : saccharin, talcum, cellulose, glucose, ~ucrose, , ~
magnssium:carb~na~e, and the like may~be used.
~he compounds of:th~e subject invention may be als~ formuIate~ ~as:; suppositories using, for ~25 example,~ polyalkylene glycols~ for example, propylene glycol, as the carrier. ~ ~ ~s :: Liquid, partioularly in~ectable compo~itions can,:for example, be prepared by dissolving, ~30 dis`persing, etc., at least one of act~è
compounas (~ o ~III) ~in a pharmaceutical ~;
solution such~ as, for example,~water, saline, aqueous dextrose, glycerol, ethanol,~DMS0 and the like, ~to thereby form the in~ctable ` ~-~
3~ : solution or ~uspension.
~'~
~:
:
W093/07~8 PCT/U~9l/08902 2121~
If desiredO the pharmaceutical composition to be administered may also contain ~inor amou~ts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buff~ring agent~, and other substances such as, for example, ~odium acetate, triethanolamine oleate, etc.
.
Parenteral injectable administration is generally used for subcutaneou~, intramu~cular or intravenous injections and infu ions.
Injec~ables can be prepar~d in convsntional ~orms, either as liquid solutions or suspensions or solid forms suitable for dissol~ing in liquid prior to injection.
A more recently devi.sed approach for parenteral administration employs the implan~atlon of a slow-release or sustained-r~lease systems, which assure~ that a constant level of d~sage ls maintained, accoxding to U.S.
~Patent No. 3,710j795, which is incorporated herein by reference.
:
Any of the above pharmaceutical composition~
: may contain 0.1-99%, preferably 1-70% o~ the : ~ 25~ active ingredient.:
: Actual method~ of preparing such dosage forms are known, ox will be apparent to those skill~d ' in this art, and ~are ~described in detail in~
R~mina~Q 'Q ~h~rmacçu~i~al~ ien~, : Mack Publishing~Company, Easton, Pennsylvania, 17th Edition, 19~5. The ~omposition or ~ormulation to be adm1nistered will, in any event, contain such quantity of the active compound(s) that will ~ssure: ~hat a therapeutically effective amou~t will be delive~red to a patient.
W093/07868 PCT/US91/08902 ~
21 21!~0~
therapeutically effective amount mean~ an amount effective to prevent development of or to :-~
alleviate the existing symptoms of the subject being txeated. ~;
The amount of active compound administered will, of course, be dependent on the subject ~-being trea~ed, on the subj~ct's weight, the ~everity of the affliction, the mann~r of administration and the judgm~t of the -~
~0 prescribing physician. HoweverO an effective --.
dos~ge may b~ in the range of 1 to 12 mg/kgJday, preferably 1 to 5 mg/kg/day, given o~ly for 1- -2 days at one treatm~nt cycle. Generally, the upper limit for the drug dos~e determination~
it~ ~fficacy ~ balanced with its pos~ible toxicity.
The i~vsntion having been descri~bed, the ollo~in~ examples are off~red to illustra~e the ~20~ subJect invention by way of illustration, not ~y ~.-way o limita~ion.
: EX~YPL S;:
~ ~ lU5~ J
An example of a~method fsr the preparatlon:of 6 nitroso-1,2-benzopyr~n~s ~is provided as~
: ~ollows~
30~ To a stirred s:olution~ of :6-amino~ 2-~b~enzopyrone~hydrochloride (~.00 g, 20 mmol) in water~(40~ml) ~at ~2'C was: added a æo1ution o~
: ~odium tung~ate (5.93 ~, 20 mm~1) in water ~(20 : ~ : m1~):f~110wed by 30% a~u~ous hydrogen peroxide (5 ;~35~: ml)~and s~irring~was continued for 1.5 hours.
: The oxida~ion product was extracted rom the ~
~ :' ~93/~7868 PC~/V~91/OX902 2121L900 '.:
green-colored mixture with two lOO ml volumes of .-~
ethyl acetate, the combined extracts washed with O.l N HCl (50 ml) and than water (lOO ml). ~he ethyl acetate was removed by rot~ry evaporation and the residue recrystallized rom warm ethanol (250 ml~
Analysi~ of Rea~iQn P~oduc~
The green crystals obtained from the ~`
recrystallization ~t~p (l.48 g, 42~ yield) displayecl light a~sorption at 750 nm characteri~tic of monomeric arylnitroso compounds. Mass: ~pectrum: m/z (~elative .:
intensity): 175 (M+, ~loo?, 1~1 ( 16.88), 145 (33.77), 133 110.38), 117 (S6.09), 89 (7~.71), , 63 (57.13). High re~olution data for the M+
peak: calculated for C~H5NO~: l7500268; ~ound:
l75.0271 (de~iation - i.l ppm). 1H-NMR (CDCl3, : ~: 300:MHz3 ~ (pp~) from TMS:~doublet (6.S72 and -. 6.604) H-4 split:by H~3; ~doub1~t ~7.~472 aDd :~ : 7.5013 H-8 split by H-7; doublet of doublet~ . `
(7.860/7.866 and ~7.8B9/7.798) H-7 split by H-8~ j`.
and ~inely~ ~pl1t ~by~H:-5; double~ (7.91~0 and ; 7.9~423 H-3 split by~H-:4; doublet (8~.308 and ~
25~ 8~.315)~H~5~fine1y ~plit by H-7. ~V/VIS spectrum ~ ~ d : in e~hanol, ~:max (~)~: 75~ nm (46)j 316 nm 1~.96 x:103), 27~ nm (2.24~x lO4):. Melting Poin~:~ The :compound polymerizes a~ove I60-;C, blacken~ and~
melts in the range of 325-340 C.
~ 30: This nitroso-compound:may also be prepared by~
react1ng 6-~amino-1,2-benzopyrone (as the fxee :
:ba~e) wi~h 3-chloroperoxybenzoic acid in ethyl aceta~e or halocarbo~ solvents.
: ~ .
~.
~ 3/0786~ PCI~/~Sgl/0~902.
2 1 2 1 9 0 0 ! ~
II. Synthe~is of 3-nitrosobenzamide To a stirred solution of 3-aminobenzamide (Aldrich Chemical Co.) (0.476 g, 3.50 mmol) in ethyl a~etate (50 mL) at ambient temperature wa~
added 1.208 g of 3-chloroperoxybenzoic aaid (commercial grade, 50-60% purity, Aldrich~, whereupon the solution turn~d green. After 10 minutes the mixture was extracted with 0.14M
aqueous sodium bicarbonate (58 mL), washed with three succ~ sive 40-mL portions of water, dri~d over sodium sulfate, then reduced in voluma to 20 mL by rotary evaporation and placed in the freezer t-20~C), whereupon the product slowly deposiked a~ a iight y~llow solid during a period of 72 hours ~0.180 g, 34% yield~
Th~ 2-nitro~obenzamide and 4-nitrosobenzamide i~omers may be similarly prepared by p~rforming the abo~e:oxidat~on on 2-aminobenzamid0 and 4-20~ aminobenzamide, respectiYely.
Melting poi:nt: The substance darken~ above~
13~5 ~, softens~and apparently polymeri~es in:the :: 25~ range::lS0-1~60 ~, and melts at 240-250^C (with decompo~ition3. In solution~th~ compound ~ 1B
:: gre~n-blue. ~ Ma~s: spectrum: m/z ~relative : in~ensity): 150 (M+,lOOj, ~36 (10.9), 12Q
. ~ I
(77.2), I03 (31.6)j 92 (46.5), 85 ~:2~.83, 71 :~ 3b (33~3). Hi~h resolution data~or the M~peak: :
:~ : calcula~ed~fr~C7H6~22: 150.042928; found:
~1$0.042gOO;~deviation = 0.2 ppm~. NMR~spectrum~
,.
1H-NMR t~MSO-d6,~300 MHæ) ~ (ppm:) from TMS: broad :
: ~ sin~l~t~(7.737) N-H; t (7.824, 7.850, 7.~75) H- :
~ ~5 sp~it ~y H-4 and~H-6; d (8.059 and 8.086~
6 split:by H-5;~d~(B.357 and 8.383) H-4 split by :H-S:;: s (8.472) H-2. The single~ at 7.737 : :
:
.
~0~3~(~7X6X PCT/US91~90~
2 1 2 ~
corresponds to 1 proton; th0 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 re~olved by addition of D20 to ths DMS0 solution. W-VIS
absorption spectrum in absolute ethanol, ~max ~ 750nm ~37.6), 304nm ~5.35 x 103) and 218nm (1.50 x 104~. An absorptlon maximum at 750nm i5 charac~eristic of monomeric aryl~itroso compound~
III.
l(~H)-I~oquinolinone (isocarbostyril) (Aldrich) was nitrated using a general m~thod for i~oquinoline compounds (C.G. ~e~evre and R.J.W. LeFevre, ~. Chçm. ~Q~. 1470 (1935)). The nitration product ~a mixture of the 5-nitro and 7-nitro isomers, as assig~ed by Y. ~awazoe and Y. Yoshioka, Çh~m__~hg~DL_~yll. (Tokyo3 16:715-720 (196B), althou~h one:of the isomers could be the 8-nitro isom rj was then reduced to the : : corre~ponding amino-1(2H)-isoqui~olinones using a combination of potassium borohydrid~ and ~ palladium-on-carbon catalyst in aqueous : m~thanol.~ To ~ the resultant amino-1(2H)~
~ isoquinolinones (as free ba~ses) (0.560 g, 3.50 : mmol) in ethyl acetate :(175 mL) a~ 30 C was .~
3.0 1 added 1.208 g of 3-chloroperoxybenzoic:a~id ~Aldrich). The mixture became cloudy and after ~ -20 ~inute~ it was ~iltere~, extracted with 0.14M
sodium bicarbonate (5~8 ~L), washed with two 50-mL portio~ of water, and dried o~er sodium ;.
: 35 sulfate. The volume of the solution was reduced to 50 mL by rot~ry evaporation and then pl~ced ~.
in the freezer (-20 C), w~ereupon an orange ~;
solid product was deposited (0.102 g).
.
:~;
W093/~7X6X PCT~VS91/08902.
2l2lsno 1 ,-., -18- ~
Analysis of_R~action Product ~;
Meltiny point: substance darkens above 175-C, -~
softens, blackens and ~pparently polymerizes above lg5'C, and finally melts in the range 310-335 C. NMR analysis: lH-NMR (DMSO-d6/D20, 300 MH2) ~ ~ppm~ from TMS: m (6.723, 6.741, 6.752); ~
m (7.511, 7.518, 7.533, 7.539, 7.~47, 7.559, , 7.577, 7.585); m 17.663, 7.674, 7.686. 7.698, 7.707); d (7.818, 7.846). In the absenc~ of D20, the compound also di~pl~ys a b~oad 8ingl~t at 11.90 ppm. The i~omeric components were analytically resolved by thin-lay~r chromatog~aphy (silica gel plates, ethyl acetat~
solvent), givi~g two bands, ~ 0.82 and ~ 0~72.
Mass spectrum for ~ ~ 0.82: m/~ (relative intensity): 174 tM~, 100), 160 ~2~.8), 144 : ~93.0), l17 ~9;0;.8~, 97 l(2~.91~ 89 (96~1)J 71 ~24.1). Hi~h resolution data for the M+ peak: ~`
calculated fo~ C~H6N2O2- 174004292B; ~ound:
174.043200 :(deviation =~-0.3 ppm). For the :;~
: component ~having ~ 0.72, M+, calculated fox : CgH6N2O2 174.04~928;~Found: 174.043200 (devia~ion = -1.6 ppm).~ ~These data confirm that the compounds ar~ mono-nitroso isomers.
I~. ADPRT I~active S~udi~
The compound~ of the subject inv~ntion were : tested for their ability to inactivate the ~ 30 polymerae~ activity of adenosinediphosphori~o~yl;~
:~: tran~ferage ~AD-PRT~). Assays were: performed according~ ~to the~:method of Buki an~d Xun, ÇÇhÇm.~27:5990-5995 (19883, using cal~ thymus ADPRT. The assay result~ as ~i~en in ~able I
::~ :: 35~ provide the I~ (the co~centration of the : compound ~hat inhibits enzyme activity 50%j ~.
: values for ADPRT of the nitroso precursox (6-: : ~: :
~-93/07B68 2 1 219 O ~ PCT/US91/08902 -19- ' ~' amino-1,2-benzopyrone) and the mor~ potent 5-iodo-derivative (Table I, compounds 1 and 2, respectively). The nitxoso compounds (3,4,5 in Table I) are all hi~hly active as anti-tumor and anti-HIV molecules (as ~hown in later s~ctions) and are effective ~ven after expo~ure of cells for a period as ~hort as 30 minutes. 5-I-6~
nitro~o-1,2-benæopyrons (compound 6) in these studies has been shown to b~ a relatively poor inhibitor of ADRPT (It is believed that ~he iodo subst~ tution deactivat~s th~ NO group as an electrophile) and it~ biological action i~ 10 :~:
times wea}c~r than that o~ 6-MO-1,2-benzopyrone.
For these reasons, the compositions of the present invention are believed to be superior to ::
5-I-6-nitroso-1,2 benzopyxone, which has be~n shown to be a p~or permeant molecule.
~ ' I50 data for aromatic inhibitors of ADPRT ~:
No . IIlhi~i~r 6-NH2- 1, 2-benzopyrone* 370 2 5-I-6:-NH2-1,2~benzopyrone* 41 ::
3 3-NO-benzamide 15
:sh~own that 6-amino~ benzopyrone (6-ABP) ~i.nd~ :
speciii~ally to ADRPT at the site that also . .
W093/~7868 PCT/~S91/08~02 ;
!
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 5~l-, (1991) ~ _ Çell. R~ 4~-Subsequently, these ligands were found to i~hibit viral replication and are the subject of the copending U.S. patent application entitle~
"6-Amino-1-2-Benzopyrones useful for Tr~atment of Viral Disea~es," Serial No. 585,~31, filsd on September 21, 1990 which îs hereby incorporatea by refere~ce.
Thus it is of int~re~t to provide ADPRT
polymerase activity inhibitors or use~as anti-viral a~d anti-tumor agents. The-: sub~eet invention provide~ for novel nitroso-1,2-benzopyrone, nitr~oso-benz;~mide and nitroso-;; isoquinolinone::~ compounds, and Yarious20 ~: structural~ly related o~her nitroso compounds for e 8 anti-viral and anti-tumor therapeutic ~agents. The compounds taught for use:~in the subject ~ nvention ~are believed to~ be gnifi~can~ly: l:ess toxic ~and far more (500 to 25~ ; 1000 :fold)~potent: than structurally analogous amino compounds. ~ `
Sum~ t~ IDV en Th~ sub~ect invention:proYides fsr ~ovel anti~
3~0 ~ tumo~ and anti-viral compounds. These compounds :in~lude ~ 6-ni.tro~o-1~ : 2-~enzopyro~é, 3-itr~osobenza~mide, 5-ni~troso-~1:(2H)~
isoqulnol~inone,~7-nitroso-1~(2H)-isoquinolinone, 8-nit~o~o-1(2H)-isoquinolinone.~: Ths invention~ :
;~35 ~ :a1~o provides for compo~itions containing one or : ~~ more:o~ t~e compounds, and for methods o~
.
: :: treating viral in~ections and cancer with these ::::~ ::
.:~
W0~3~07868 PCT/US91/08902 2121~00 ~
-6- -~
compounds and compos.itions.
Also provided for ar~ methods of treating cancer and viral infections with 2- --nitro~obenæamide and 4-~itrosobenzamide.
Composition containing one or more of the~e compounds is al~o provid~d for.
D~ riptio.n cf Figur~6 ~.
Figure 1 i~ a graph comparing the degr~e of AD~PT polymeras~ activity ~ADPRP) inactiv~tlon exhibited by different concentrations of 6-~itroso 1~2-benzopyrone, 3-nitroso be~zam~de, and nitro o-1[2H)-isoquinQlinones (~OQ) (a mixture of the 5 and 7 nitroso i~omers).
: , .
Fig~re 2 is a compositei of graphs di~playi~g :~:
the inhlbi~ory effeots of the ADRPT ligands on ) 855 2 ells la cell ~line~of human~B-cell ~, 20~ linèage acute lymphobl~stic leu~emla~, (B) N9 cells~(a c:ell line of human T-oell lineage acute : ly~pho~lastic;leukemia~, (C) HL-60 cells (a oell .~; 1ine~of ~uman ac~t~ nonlympho~lastic~leu~emia~
and~(D)~K562~oells~(a oell;~line~.~of human~chronio 25~ ;:: myelogenou;6 le:ukemia3~ These~ ~c~lls~;~werc :
: culture~ whil:e:un~er ~he influen~e of th~ ~rowth Eaotors~in~ 0%~fetal;~ovlne ~rum (~FCS~), whereas:~
: : :~ ;:~in (E) and~(F) thc 855-2 oe:11s were:cultured~ln~
the presence of autocrine growth actor actiYit i 30~ (A~F) or low molecular weight B-cell growth~
faotor ~(BCGF~, a T-oell derived lymphokinc) resp2CtiVQly ~ s.7 ~ ~ ~ : Figure 3 ~i~s ~a graph showing the inhlbition of : ;35~ creasing lcvels~of :leukemio cell growth (ln:
~ :response to~inoreaslng concentrations of FCS) of .
:~ .
i W~93/07868 PCT/US91/089~2 2121~9ûD
I
855-2 cells by 6-nitroso~ -benzopyrone (NOBP~ -and 3-~itrosobenzamide ~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 c lls in a semi-solid medium.
Figure 5 shows graph of the r01ative inhibitory effects of anti-leukemic dosss of ADRPT ligands on the ability of (A3 normal ~:
rhesus bone marrow stem cells or (B~ human peripheral blood stem cells to form coloni~s ln soft agar. Note that the NOBP and NOBA had lS minimal effect on normal cells. :~
Figure 6 shows graph~; displaying the inhibi~ory ef~ects of NOBP, NOBA and NOQ on f~ur human brain ~umor cell line~
20:: Figure 7 is a graph comparing t~e e~fe~tiveness:of NOBP with vincri6~ine. :~
'~.;`
Figure ~ is a g:raph displaying t~e effects of NO~P, NOBA and MOQ on human breast tumor cel~
: 2s li~e M~A 468. ~ -.
: Figure 9 is a graph displayinq the ef~eots of NOBP, NOBA and NOQ on murine leukema ~ell line L 1210. ` :
~ De6~ri~ipn_cf SpQcifi~ ~mbQdimQn~
, .
The ~ubject in~ention pro~ides for ~everal : :: nitroso aom~ounds that are ADPRT pol~mer~6e :: activity inhibitors. These a~pounds find use a6~anti-tumor and antl-viral compounds.
~:
~;
'..
WO93/07B68 P~T/US91/08902 ~8- :~
Compound (I) has the following formulaO -~
~4--y~
R3 }~
S wherein ~, ~, ~, R~, ~, and ~ a~ selected from th~ ~roup con~isting of hydrogen and nitroso, and only;one of ~ , R4, ~, a~d is a nitroso group.
~ ':`
A preferred embodiment of compound I i5 where R4 is the nitroso gxoup, i.e., the molecule 6 nitro~o-1,2-be~zopyrone.
Compound II~has~the formula~
15;~: :(}I~
0;~ ~ ~wherein~ 2~:and ~are ~selected~rom the group~consisting of~ hy~rogen~:and:nitroso,:~and~
only on~of~ , and~RJ:~Is:a~D~troso~gro~p.
2~5~Compound~ has the~formula~
:.
~;
WC~93/07B68 PCT/US91/08gO2 2121~00 ! -:
_g_ :
wherein ~, ~, R3, R~, and ~ are selected from the group consisting of hydrogen and nitro60 t and only one of R1, ~, ~, R4, and R5 is a nitroso group.
Preferred embodim~nt~ of compound III are where either R~ or ~ is the ni~roso group, i.~
5-nitroso-1(2H)-isoquinolinone and 7-nitroso-1(2H~-isoquin41inone, re~pectively.
The disclosed synthPsis for 5-nitroso-1(2H~
isoquinolinone may produc~ 2 closely related structural isomers, 7-nitroso-1(2H~
isoquinolinone and 8-nitroso-1(2H~-isoqui~olinon~. Although experiments testing the biological activity of 5-nitro~o-1(2H)-iso~uinolino~ may ha~e contained significant quantities of 8-nitroso-1(2H)-isoquinolinone or 20: ~ 7-nitro~o-1(2H)-isoquinolinone, all three isomers are b~lieved to:possess similar anti~
tumor and anti-viral activity on the basis of their cIose ~tructural :similarity. This : : : hypQ~hesi~ may b~ ~ conveni~ntly ~ted by ;~~25: ~ s parating~ the isomers by thin layer chromatography or similar methods, and comparing the anti-tumor and~anti-~iral:activities of the : : separated compounds.
3~ Detail~zd synthesis of 6-nitrofio-1~2 benzopyrone:, 3-nitroso-benzamide, 5-nitr~so~
1(2H~- isoqui-nolinQne ~ 7-nitroso-~1(2H):-i~oquinol1nonez, and 8-nitroso-1(2H)-iso~uinolinone~ are providea in the ex~mple : 35 section bslow.
,.
W~93/0786~ PCT/US91/O~gO~ .
2121~00 In general, the nitroso compounds of the subje~t of invention may be synthesized by oxidizing a corre~pondlng amino compound to~a compound of the ~ubject invPntion by oxidation S with 3-chloroperoxy~enzoic acid (or other pexoxyacids) in ethyl acetate or a halocarbon solvent. Syntheses of thes~ precursor amino compounds are described in the chemi~ l literature and some of the compounds are commercially available. Some precur~or amino compound~ for oxidation to nitroso compounds of the subject inven~ion are as follows: 3-amino-1,2-b~æopyrone tSpectrum Ch~mic~l Mfg. Corp. t Gardena~ CA 902483; 4-amino-1,2-b~nzopyrone (Aldrich, Rare Che~i¢al Catalog3; 5-ami~o-1,2-benzopyro~e (by reduction of 5-nitro-1,2-benzopyrone, Çh~=L~ E~ ~7 16536d (1952)3; 7-amino-1,2-benzopyrone ~Gottlieb, ~ al., ~hem. Soc.~ ~P~rkin~_~r~ns. I 435 (~1979)); 8-amino-1,2-benzopyrone lby reduction of 8-amino--benzopyrone, ~bdel~Megid, e~ al-, ~gy~~
~hQm~ 20:453-462~(1977)), and. 4-amlno-1(2H)-i~quinolinone, by redu~tion of:~ the : : correspond~i~ng~4:~nitro analog (Horning~
~1971) Ca~ J. .~hem. 49::2785-2796).
In ~ddltion to compounds (l) to (III), the~
subject in~ention contemplates various structurally related compounds that have similar ` ~ : 30 carcino~tatic and/or antl-viral activities These ~tru~turally related compounds could~be~
c~nv~nien~ly scre~n~d ~on the ~basis of: their : highly pot~ent~lnhibitory~ ef~ect on ~ADPRT
~ polyme*ase activity. Structurally related compounds o~ ;int~rest include derivatives : : substituted by: additional nitroso groups and small, e.g., ~C1-C3 alkyl groups. A1BO of :
W~93/0786g PCTJUS91/08~02 ~21~)0 interest are various nitroso substituted stxucturally related heterocyclic ring~ such as 3 , 4 - d i h y d r o - 1 ( 2 H ~ - i 5 o q u i n o 1 i n o n e s , nicotinamides, pthalhydrazide~, and 1,~-benzoxazine-2,4-diones.
Another aspect of the compound~ of th~ ~ubject invention are the ea~e with which they permeate cell membranes and their relative absence of lQ non spe~ific binding to protelns and nucl~ic acid.
In practiae, the ADPRT polymeras~ i~hibitors of this inventio~, namely compounds (I) to (III), and any of their pharmaceuti~ally acceptable salts, may b~ adminis~tered in amount~, either~alone or in combination with each other, and in the ph rmaceutical form which will be suf f ic ient and effective to 1nhibit ~ ~ 20 neoplastl~ gro~ h or v:iral replication or prevent ~he development of the cancerous growth or vlr~1 inection in the mammalian ~o~t.
:.
Admi~istration of the active compounds~and 6alts described herein can be Yl~ any of the : accepted modes;of admlnistra~lon for therapeutic agent~. These methods include s:ys:temic or looal : administration: such a~ oral, parenter~
tran~dermal, subcutaneous, or topi~al administration modes. The preferr~d method of administxation o these drugs is intravenous,~
: except in~ tho~ cases :where th~ subjec~ has .
topiaa~ ~:umor~ or lesions, ~where the topi~al ~ ~ admin~tration~ may : be : proper. In other ~ a~oes, it may be ~ecessary to adminl~ter the oompo3ition in other parenteral or~even oral ~`
forms.
' '~
W093/07~68 PCTJVS~1/08902 2121900 ~ :
Depending on the intended mode, the compositions may be in the solid, se~i-solid ox liquid dosage form, such as, for example, injectables, tablets, suppositorles, pills, time-release capsules~ powders, liquid~, suspensions, or the like, preferably in unit do~a~es. ~he compositions will include an effecti~e ~mount of at least one of compound~
(I) to (III), or pharmaceutically acceptabl~
~alts t~ereof, and in addition it may i~clude any conventional pharmaceutical excipientæ and other medicinal or pharmaceutical drugs or agents, carr~ers, adjuvants, diluents, etc., as cuætomary in the pharmacautical sciences.
'".
~or solid compositionæ, in addition to the oompouna~ tI) to (III), such exGipientæ as~ for : exampl~, pharmaceutical gradss of mannitol, :~ 20: laatose, s:tarch, magnesium stearate, sodium : saccharin, talcum, cellulose, glucose, ~ucrose, , ~
magnssium:carb~na~e, and the like may~be used.
~he compounds of:th~e subject invention may be als~ formuIate~ ~as:; suppositories using, for ~25 example,~ polyalkylene glycols~ for example, propylene glycol, as the carrier. ~ ~ ~s :: Liquid, partioularly in~ectable compo~itions can,:for example, be prepared by dissolving, ~30 dis`persing, etc., at least one of act~è
compounas (~ o ~III) ~in a pharmaceutical ~;
solution such~ as, for example,~water, saline, aqueous dextrose, glycerol, ethanol,~DMS0 and the like, ~to thereby form the in~ctable ` ~-~
3~ : solution or ~uspension.
~'~
~:
:
W093/07~8 PCT/U~9l/08902 2121~
If desiredO the pharmaceutical composition to be administered may also contain ~inor amou~ts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buff~ring agent~, and other substances such as, for example, ~odium acetate, triethanolamine oleate, etc.
.
Parenteral injectable administration is generally used for subcutaneou~, intramu~cular or intravenous injections and infu ions.
Injec~ables can be prepar~d in convsntional ~orms, either as liquid solutions or suspensions or solid forms suitable for dissol~ing in liquid prior to injection.
A more recently devi.sed approach for parenteral administration employs the implan~atlon of a slow-release or sustained-r~lease systems, which assure~ that a constant level of d~sage ls maintained, accoxding to U.S.
~Patent No. 3,710j795, which is incorporated herein by reference.
:
Any of the above pharmaceutical composition~
: may contain 0.1-99%, preferably 1-70% o~ the : ~ 25~ active ingredient.:
: Actual method~ of preparing such dosage forms are known, ox will be apparent to those skill~d ' in this art, and ~are ~described in detail in~
R~mina~Q 'Q ~h~rmacçu~i~al~ ien~, : Mack Publishing~Company, Easton, Pennsylvania, 17th Edition, 19~5. The ~omposition or ~ormulation to be adm1nistered will, in any event, contain such quantity of the active compound(s) that will ~ssure: ~hat a therapeutically effective amou~t will be delive~red to a patient.
W093/07868 PCT/US91/08902 ~
21 21!~0~
therapeutically effective amount mean~ an amount effective to prevent development of or to :-~
alleviate the existing symptoms of the subject being txeated. ~;
The amount of active compound administered will, of course, be dependent on the subject ~-being trea~ed, on the subj~ct's weight, the ~everity of the affliction, the mann~r of administration and the judgm~t of the -~
~0 prescribing physician. HoweverO an effective --.
dos~ge may b~ in the range of 1 to 12 mg/kgJday, preferably 1 to 5 mg/kg/day, given o~ly for 1- -2 days at one treatm~nt cycle. Generally, the upper limit for the drug dos~e determination~
it~ ~fficacy ~ balanced with its pos~ible toxicity.
The i~vsntion having been descri~bed, the ollo~in~ examples are off~red to illustra~e the ~20~ subJect invention by way of illustration, not ~y ~.-way o limita~ion.
: EX~YPL S;:
~ ~ lU5~ J
An example of a~method fsr the preparatlon:of 6 nitroso-1,2-benzopyr~n~s ~is provided as~
: ~ollows~
30~ To a stirred s:olution~ of :6-amino~ 2-~b~enzopyrone~hydrochloride (~.00 g, 20 mmol) in water~(40~ml) ~at ~2'C was: added a æo1ution o~
: ~odium tung~ate (5.93 ~, 20 mm~1) in water ~(20 : ~ : m1~):f~110wed by 30% a~u~ous hydrogen peroxide (5 ;~35~: ml)~and s~irring~was continued for 1.5 hours.
: The oxida~ion product was extracted rom the ~
~ :' ~93/~7868 PC~/V~91/OX902 2121L900 '.:
green-colored mixture with two lOO ml volumes of .-~
ethyl acetate, the combined extracts washed with O.l N HCl (50 ml) and than water (lOO ml). ~he ethyl acetate was removed by rot~ry evaporation and the residue recrystallized rom warm ethanol (250 ml~
Analysi~ of Rea~iQn P~oduc~
The green crystals obtained from the ~`
recrystallization ~t~p (l.48 g, 42~ yield) displayecl light a~sorption at 750 nm characteri~tic of monomeric arylnitroso compounds. Mass: ~pectrum: m/z (~elative .:
intensity): 175 (M+, ~loo?, 1~1 ( 16.88), 145 (33.77), 133 110.38), 117 (S6.09), 89 (7~.71), , 63 (57.13). High re~olution data for the M+
peak: calculated for C~H5NO~: l7500268; ~ound:
l75.0271 (de~iation - i.l ppm). 1H-NMR (CDCl3, : ~: 300:MHz3 ~ (pp~) from TMS:~doublet (6.S72 and -. 6.604) H-4 split:by H~3; ~doub1~t ~7.~472 aDd :~ : 7.5013 H-8 split by H-7; doublet of doublet~ . `
(7.860/7.866 and ~7.8B9/7.798) H-7 split by H-8~ j`.
and ~inely~ ~pl1t ~by~H:-5; double~ (7.91~0 and ; 7.9~423 H-3 split by~H-:4; doublet (8~.308 and ~
25~ 8~.315)~H~5~fine1y ~plit by H-7. ~V/VIS spectrum ~ ~ d : in e~hanol, ~:max (~)~: 75~ nm (46)j 316 nm 1~.96 x:103), 27~ nm (2.24~x lO4):. Melting Poin~:~ The :compound polymerizes a~ove I60-;C, blacken~ and~
melts in the range of 325-340 C.
~ 30: This nitroso-compound:may also be prepared by~
react1ng 6-~amino-1,2-benzopyrone (as the fxee :
:ba~e) wi~h 3-chloroperoxybenzoic acid in ethyl aceta~e or halocarbo~ solvents.
: ~ .
~.
~ 3/0786~ PCI~/~Sgl/0~902.
2 1 2 1 9 0 0 ! ~
II. Synthe~is of 3-nitrosobenzamide To a stirred solution of 3-aminobenzamide (Aldrich Chemical Co.) (0.476 g, 3.50 mmol) in ethyl a~etate (50 mL) at ambient temperature wa~
added 1.208 g of 3-chloroperoxybenzoic aaid (commercial grade, 50-60% purity, Aldrich~, whereupon the solution turn~d green. After 10 minutes the mixture was extracted with 0.14M
aqueous sodium bicarbonate (58 mL), washed with three succ~ sive 40-mL portions of water, dri~d over sodium sulfate, then reduced in voluma to 20 mL by rotary evaporation and placed in the freezer t-20~C), whereupon the product slowly deposiked a~ a iight y~llow solid during a period of 72 hours ~0.180 g, 34% yield~
Th~ 2-nitro~obenzamide and 4-nitrosobenzamide i~omers may be similarly prepared by p~rforming the abo~e:oxidat~on on 2-aminobenzamid0 and 4-20~ aminobenzamide, respectiYely.
Melting poi:nt: The substance darken~ above~
13~5 ~, softens~and apparently polymeri~es in:the :: 25~ range::lS0-1~60 ~, and melts at 240-250^C (with decompo~ition3. In solution~th~ compound ~ 1B
:: gre~n-blue. ~ Ma~s: spectrum: m/z ~relative : in~ensity): 150 (M+,lOOj, ~36 (10.9), 12Q
. ~ I
(77.2), I03 (31.6)j 92 (46.5), 85 ~:2~.83, 71 :~ 3b (33~3). Hi~h resolution data~or the M~peak: :
:~ : calcula~ed~fr~C7H6~22: 150.042928; found:
~1$0.042gOO;~deviation = 0.2 ppm~. NMR~spectrum~
,.
1H-NMR t~MSO-d6,~300 MHæ) ~ (ppm:) from TMS: broad :
: ~ sin~l~t~(7.737) N-H; t (7.824, 7.850, 7.~75) H- :
~ ~5 sp~it ~y H-4 and~H-6; d (8.059 and 8.086~
6 split:by H-5;~d~(B.357 and 8.383) H-4 split by :H-S:;: s (8.472) H-2. The single~ at 7.737 : :
:
.
~0~3~(~7X6X PCT/US91~90~
2 1 2 ~
corresponds to 1 proton; th0 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 re~olved by addition of D20 to ths DMS0 solution. W-VIS
absorption spectrum in absolute ethanol, ~max ~ 750nm ~37.6), 304nm ~5.35 x 103) and 218nm (1.50 x 104~. An absorptlon maximum at 750nm i5 charac~eristic of monomeric aryl~itroso compound~
III.
l(~H)-I~oquinolinone (isocarbostyril) (Aldrich) was nitrated using a general m~thod for i~oquinoline compounds (C.G. ~e~evre and R.J.W. LeFevre, ~. Chçm. ~Q~. 1470 (1935)). The nitration product ~a mixture of the 5-nitro and 7-nitro isomers, as assig~ed by Y. ~awazoe and Y. Yoshioka, Çh~m__~hg~DL_~yll. (Tokyo3 16:715-720 (196B), althou~h one:of the isomers could be the 8-nitro isom rj was then reduced to the : : corre~ponding amino-1(2H)-isoqui~olinones using a combination of potassium borohydrid~ and ~ palladium-on-carbon catalyst in aqueous : m~thanol.~ To ~ the resultant amino-1(2H)~
~ isoquinolinones (as free ba~ses) (0.560 g, 3.50 : mmol) in ethyl acetate :(175 mL) a~ 30 C was .~
3.0 1 added 1.208 g of 3-chloroperoxybenzoic:a~id ~Aldrich). The mixture became cloudy and after ~ -20 ~inute~ it was ~iltere~, extracted with 0.14M
sodium bicarbonate (5~8 ~L), washed with two 50-mL portio~ of water, and dried o~er sodium ;.
: 35 sulfate. The volume of the solution was reduced to 50 mL by rot~ry evaporation and then pl~ced ~.
in the freezer (-20 C), w~ereupon an orange ~;
solid product was deposited (0.102 g).
.
:~;
W093/~7X6X PCT~VS91/08902.
2l2lsno 1 ,-., -18- ~
Analysis of_R~action Product ~;
Meltiny point: substance darkens above 175-C, -~
softens, blackens and ~pparently polymerizes above lg5'C, and finally melts in the range 310-335 C. NMR analysis: lH-NMR (DMSO-d6/D20, 300 MH2) ~ ~ppm~ from TMS: m (6.723, 6.741, 6.752); ~
m (7.511, 7.518, 7.533, 7.539, 7.~47, 7.559, , 7.577, 7.585); m 17.663, 7.674, 7.686. 7.698, 7.707); d (7.818, 7.846). In the absenc~ of D20, the compound also di~pl~ys a b~oad 8ingl~t at 11.90 ppm. The i~omeric components were analytically resolved by thin-lay~r chromatog~aphy (silica gel plates, ethyl acetat~
solvent), givi~g two bands, ~ 0.82 and ~ 0~72.
Mass spectrum for ~ ~ 0.82: m/~ (relative intensity): 174 tM~, 100), 160 ~2~.8), 144 : ~93.0), l17 ~9;0;.8~, 97 l(2~.91~ 89 (96~1)J 71 ~24.1). Hi~h resolution data for the M+ peak: ~`
calculated fo~ C~H6N2O2- 174004292B; ~ound:
174.043200 :(deviation =~-0.3 ppm). For the :;~
: component ~having ~ 0.72, M+, calculated fox : CgH6N2O2 174.04~928;~Found: 174.043200 (devia~ion = -1.6 ppm).~ ~These data confirm that the compounds ar~ mono-nitroso isomers.
I~. ADPRT I~active S~udi~
The compound~ of the subject inv~ntion were : tested for their ability to inactivate the ~ 30 polymerae~ activity of adenosinediphosphori~o~yl;~
:~: tran~ferage ~AD-PRT~). Assays were: performed according~ ~to the~:method of Buki an~d Xun, ÇÇhÇm.~27:5990-5995 (19883, using cal~ thymus ADPRT. The assay result~ as ~i~en in ~able I
::~ :: 35~ provide the I~ (the co~centration of the : compound ~hat inhibits enzyme activity 50%j ~.
: values for ADPRT of the nitroso precursox (6-: : ~: :
~-93/07B68 2 1 219 O ~ PCT/US91/08902 -19- ' ~' amino-1,2-benzopyrone) and the mor~ potent 5-iodo-derivative (Table I, compounds 1 and 2, respectively). The nitxoso compounds (3,4,5 in Table I) are all hi~hly active as anti-tumor and anti-HIV molecules (as ~hown in later s~ctions) and are effective ~ven after expo~ure of cells for a period as ~hort as 30 minutes. 5-I-6~
nitro~o-1,2-benæopyrons (compound 6) in these studies has been shown to b~ a relatively poor inhibitor of ADRPT (It is believed that ~he iodo subst~ tution deactivat~s th~ NO group as an electrophile) and it~ biological action i~ 10 :~:
times wea}c~r than that o~ 6-MO-1,2-benzopyrone.
For these reasons, the compositions of the present invention are believed to be superior to ::
5-I-6-nitroso-1,2 benzopyxone, which has be~n shown to be a p~or permeant molecule.
~ ' I50 data for aromatic inhibitors of ADPRT ~:
No . IIlhi~i~r 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 6 NO-1,2-benzopyron~ 40 `, ~ !
6 5-I~6-NO-1,2-benzopyron~ ~ 400 *biochemic~l precursor of nitroso compounds 5 and 6 **a mixture of the 5- and 7-nitroso compounds Assay condition~: ADPRT~ 0.4 ~g; coDNA~ 4 ~g; ~' inhibitor diluted betw~en 0.8 and 600 ~Mj in 50 :-~l of 50 mM Tri~-HC-l/ gO mM KÇl, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.1 mM NAD (~32~
P)-labelled~, pH 7.5. Polymerization at 25 C
for 4 minutes.
;
. ., WV93/0786X PCT/U~91/08902 Figure 1 illustrates the % inactivati~n of ADPRT polymerase activity observed after 2 hour~ :
of incubation with the nitroso-compound inhibitors at s~veral concentrations. ~:~
Additional experiments in~olving the equilibration between 65Zllt2 and ADPRT-bound ~n~2 suggest that the ADPRT inhibition activi~y of the hitroso compounds appears to act by ~;~
destabili~ing the protein through the ejecting of Zn~2. (Buki K.G,, Bauer PoT~ I Mendeleyev, F.;
Hakam, H. and Kun E. (1991) FEB$ L~. 290~
185). The above m~chani~m of action for ADPRT
inhibitor~ is speculative and does n~t constitute any limitatio~ on claimed subject matter.
~ ~`
V. ~i515~Uh~LL An~i-C~ncer Ac~ivi~i~ of ~:
Ni~osQb~nzoeyr~es, Ni~;rQs~2k~amisl~
and NitrosQ~ uinolin~-ne~
: :
:.. ~:
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 (~IABPj, 6-nitro~ -benzopyrone : ~ ~ 25 (NO2BP), 6~nitroso-1,2-benzopyrone :(NOBP)~ 3-nitrosobPnzamide (NOB~? or 5~7)-nitroso-1(~H)~
isoquinolinone (NOQ) (a mixture of the 5-nitroso and 7 nitroso ieomer~ nd the level of 3H], thymidine~ uptake ~wasi determin~d as a measure of oellular proliferation. ~B shown in : ~igure 2, ~or ea:ch of the cell lines tssted (855-2 ~ells, Fig.~2A; H9 cells, Fig.~2B; H~-- .;
60 c lls, Fig. 2C; K562 cells, Fig. 2D~ the ~`;
nitroso-containi~g ligands ~NOBP, NOBA, NOQ) were ~able to inhibit 3H-thymidine uptake in lower molar concentrati~ns than the other compounds. NOBP, NOBA and NOQ powerfully ~V~93/07868 PCT/U~91/O~gO2 2121~0~ ~
inhibited 3H-thymidine uptake ~t a concentration of 1~ ~M, a concentration at which the other compounds exhibited compara tively slight ~:.
inhibitory ef :f ectE~ .
Experiments with H9 cells grown in 10% fetal bovine ~erum (FCS) (Fig. 2B~ found NOQ to be the most pot~nt inhibitor, demonstrating al~o~t complete inhi~ition a~ 10 ~M levels. NOBP
demon~trated about a 30% decrease in thy~idi~e uptake at 10 ~M, and an almost complste inhibition of uptake at 100 ~M. NOBA
demonstrated ab~ut 75% level of inhibition at 10 pM, about 85% inhibition at 100 ~M, and almoBt :.
complete inhibition at 250 ~M. The remaini~g amino and nitro compounds were ~ignificantly le~ potent and did not display complst~
inhibition until concentration~ of 100:0 ~M were reached. : : ~
',, ;20 Experiments with ~562 cel:Ls grown in;1~% f~tal ~ bo~ine serum (Fig. 2~) fou~d NO~ and NOBP to be :: the most pot~nt inhibitors of cell gr~wth. Both ~ ~ ~OQ and:NOBP~resulted in the almost~complcte : ~ : inhibition~at ~on~entratlons of 10 ~M.~ NOBP was:
; ~5 almost: as potent~as NO~ and prsduced~about 90#
: : inhibition at a concentration~ of;~10 :pM, a~d almos~ complete inhib~ition at a concentration of 100 ~M. ~Th~ other 3 compounds test~d were signifid~ntly less potent.
.
: ~, Exp~riments with 855-2 cells grown in 10%
fetal bovine~serum:(Fig. 2A~ found that ~OQ,~and NOBP producea almost ~omplete lnhibl~t~ion at a ~concentratlon of lO ~M. At a concentration of 1 ~ ~M, ~ produced some~hat more inhibition than : 35 NOBP, and NOBP produced ~omewhat more inhibition .
.. . . .. ..... ............ . . . . .... ... . .... .. . . ..... . . . . . . . . ... . .. . .... .
WO ~3/07~68 P~/US91/089~2 .
~12~!30Q
than NOBA. Experiments using HL-60 cells ~Fig.
2C) provided similar corlclusions. The other 3 compounds tested wsre significantly less potent.
The effect of differ~nt growth factor~ OI~ the growth inhibitory effect~ of NOBP was te~ted. -~.
855-2 cells that were grown in media with ( 1 10% fetal bovine serum, (2~ autocrine growth ~:~
factor (At;F) and (3) low mol~cular weigh1~-BCGF
(a T cell derived lymphokir~e) wl~re expo8~d to increa~ing conc~ntrations of the ADRPT ligands.
The results are provided in Figure 2 (~, E, F) .
Cells grown in each o the growth faotox~ we~
all potently inhibited by the nitroso containing compounds, with concentrations of 5 to 10 ~M
resultin~ in 100% inhibit:ion, Thus, NOBP, NOBA
and NOQ ex~rt potent inhibitory effect~
regardless of the sourt~e of growth factor ~-activity. ;:
~: ~ 20 ~ In order to ex~lude the possibility that NOBP :~:
and NOBA manifest their growth inhibitor~ ;
: effects through inactivation of gxowth factors, the effects of lO ~M NOBP or NOBA (constant : ~ concentration)~on 855-2 cell~s in the presenae of ~25 : increasing concentrations~:of:~et:aI bovine ~erum FCS) were tes~d (FCS) contains growth factors or 855 2 cells). The data are pro~ided ~in : Figure 3. Growth arrest occurs irrespective of~
the csncentration of FCS. Thus, the mode of ~30 action of :NOBP, does not ~appear to be~ by antagoni~m -~f ~rowth factors but at ADPRT si~es : ~ related to DNA-repllcation. ~;
umor cell inhibitory concentratio~s of NOBP
~ and NOBA were shown not to a~~ct adver~ely the viability ~f normal cells. Experiments w2re ~
~, :`
~ 3/078~s PCr/US91/~8902 2 ~ 2 ~L ~
performed in which the functions of various cancer cells ( 855-2 and HL-60 leukemia cells, D32, D37 and CRL 7712 glioblastoma c ll lin~s, 185 medulla tumor cell line, L1210 murine leukemia cell line, MDA-468 human breast tumor cell line) and normal cells (neutrophil leukocytes and bon~ marro~ or peripheal blood stream cells) wexe asseæ~ed in the absence or presence of the compounds. The results are ~hown in Figures 4-9. Together, the data indicate that a concentration of 10 ~M o~ the n$troso-containing ligands effectively suppressed cancer cell grow~h but demonstrated only modest effects the functions on normal cells.
VI. ~9~iLJ ~ Y~
The cytotoxicity of Oj 2 ~M, 4 ~M, 8 ~M and 10 ~M NOBP wa~ maasured by examining the effect of : 20 the compound on the colony f~rmation (CFU-GM) of.
normal human stem:~ells (PBSC~. The results of : ~ the experiment~ are pro~ided i~ figure 5B.
Toxicity was not detected,~e~en though levels of : NOBP suf~ficient to ~ : block fl55-2~ cell ~: 25 :~ proliferatlon completely were tested.
: ' ~ ::
~similar ~FU :stem cell toxicity assay ~was : pe~formed in which~comparisons were made betw~en (A~P) 6-amino-1,2-benzopyrone l mM; (~ABP)~ 5 I~6~amino-1,2-benzop~xone 250 ~M, (NO2BP) 6 :
nitro-1,2-benzopyrone ~weakly active) 250 ~M, NOBP 10 ~M, and NOBA lO:~M. The results of:the experiments axe provided in figure ~A. ~Whereas the 6-amino~ enzopyrone, 5-I~6-amino-1,2-benzopyrone and the 6-~itro deri~ative were ~oxic at the tested given doses, the almost inefeative (again~t t~mor cells) 6-nitro ::
21219UO "
I
derivative and the highly effective (against tumor cells) NOBP and NOBA were non-toxic.
The effects of lO~m Nosp and NO~A on superoxide generation by normal human peripheral blood neutrophil leukocytes was te~ted. The results are provided in tabla X I . Only minor reductions in sup~roxide generation were observed.
1 0 ~gkL~
Effects of 10 ~M NOBP and NOBA on the Generation of Superoxide by Human Neutrophils nmol 02/hr/105 cells I~ LLi 105 PMN ~ PMA: 5S.9 ~ 7.7 +10 ~M NOBP 34.1 ~ 14.1 +10 ~M NOBA 44.4 ~ 10.0 ~20 VII. ' Vincristine, a highly toxic chemetherapeutic compound, is the currently:used in the treiatment o~ leukemia and other malignancies. Studie:s ~5 were :performed i~ order to dete;rmine the : ~ concentration of vincristine that produ¢es the same level of growth inhibition a~ lO ~N NOBP,:~ :
when a~sayed on: 855-~ leukemia:cells grown i~
Vi~rQ. Vincristine was~tested~in doses of ~.l, :30 : ~1, 10 and lOO~M. ~s shown in Fig~e~:7. 100 ~M
of vincristine (a highly toxi~ co~cent~ation) :~ was re~uired~ to produce the~:~ame level of : inhibition a~ 10 ~M o NOBP, thus NO~P is about : 10 times more potent than an e~ual ~ioncentration o~ vincristine, and is ~t toxic to normal cells. :;
; Thus certain aromatic nitroso molecules tha~
are also inhibitors of ADPRT polymerase activity ~0~3/07~68 PCT/US9l/OB902 2 :1219 ~ U
-25- ;~
may be useful chemotherapeutic cytostatic agents ~:~ because of their effactiveness combined wi~h low toxicity.
,'",,~
VIII. Anti-HIV_action of_NOBP/ NOBA and NOO ~n The ability of NOBP (6-nitroso-1,2-benzopyrone3 and NOBA (3-nitrosobenæamide~ to inhibit HIV infections wer~ tested using the methods described in the ~9~ L___ Immu~l~iç~l M~hod~ 76:171-183 (1985).
Exposure to the two drugs was only for 30 minutes at the commencement of viral inf~ction, and dxugs were never re-added. The results given in ~able III pro~ide t:he ID~ of HIV ti~er 10 days after infection of cell cultures with HIV. The data in Table III demonstrate that 10 ~M of the ~itroso-containinS ligands cause~
a three log decrease in ~the HIV-1 infectivity ~: ti~er.
: :
~ ~ ABLE 111 ~ç~=~am~ Virus Titer lloq ID ~L~ Q~ ~
: Virus ~lone~ 5~25 :~.
500 ~M ABP ~ : 4.50 ~ ~
250 ~M:IABP ~ 4.66 ::.
~ ~250 ~M NO~BP 4.93 +10 ~M NO~P : 2.01 :: : +10 ~M NO~ 1.05 ; ~ +10 ~M~NOQ : ~ 1.73 : 35 IX. ~ : : ~ , :~ Exp rimetns were performed to determine i~the ~:
:inhibition~of prollferation o~ 855-2 ce;lls seen 4;0 ~: in cu1ture~nd in soft agar is due to the ~ : cytostatic or cytocidal effect of the nitroso : ~ : compounds NOBP, :NOBA, and NOQ. Cells at ~.
:
W093/0786~ PCT/~S91/08902.
2 1 ~ 1 ~3 0 û
- 2 6 - ~
lxlO5/ml (concentration used in bone marrow assay) were treated with NOBP, NOBA and NOQ at 1, 2.5, 5 and lO~m fox 2 hours then ~timulated :
with 10% fetal calf serum a~d 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. ~he absorbance of the pellete~ cell was then measured at 550nm after adding DMSO to solubilze the cells Results: With lO~M NOBP, NOBA and NOQ, complete killing was observed in 85S-2 cell~ at lOO,OOO/ml.
..;
All publications, patents, and patent applications cited abo~e are herein incorporated by reference.
The foregoing writt~n specification i5 ~.
~20 co:nsidered to be su~ficient to enable one ;: skilled in~ the art to pract~ice the invention.
ndeed, various modifi:~ations of the above-de~cribed modes~for carrying:out the invention which are obvlou6 to tho~e killed :in the ~ield :: 25 o~ pharmaceutical ~ormul~ation or rel~ated fields : ~.
are intended to be within the scope of the ~ ~ .
following claims. ~ ~
: ~ : :
.:
~:, .':
6 5-I~6-NO-1,2-benzopyron~ ~ 400 *biochemic~l precursor of nitroso compounds 5 and 6 **a mixture of the 5- and 7-nitroso compounds Assay condition~: ADPRT~ 0.4 ~g; coDNA~ 4 ~g; ~' inhibitor diluted betw~en 0.8 and 600 ~Mj in 50 :-~l of 50 mM Tri~-HC-l/ gO mM KÇl, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.1 mM NAD (~32~
P)-labelled~, pH 7.5. Polymerization at 25 C
for 4 minutes.
;
. ., WV93/0786X PCT/U~91/08902 Figure 1 illustrates the % inactivati~n of ADPRT polymerase activity observed after 2 hour~ :
of incubation with the nitroso-compound inhibitors at s~veral concentrations. ~:~
Additional experiments in~olving the equilibration between 65Zllt2 and ADPRT-bound ~n~2 suggest that the ADPRT inhibition activi~y of the hitroso compounds appears to act by ~;~
destabili~ing the protein through the ejecting of Zn~2. (Buki K.G,, Bauer PoT~ I Mendeleyev, F.;
Hakam, H. and Kun E. (1991) FEB$ L~. 290~
185). The above m~chani~m of action for ADPRT
inhibitor~ is speculative and does n~t constitute any limitatio~ on claimed subject matter.
~ ~`
V. ~i515~Uh~LL An~i-C~ncer Ac~ivi~i~ of ~:
Ni~osQb~nzoeyr~es, Ni~;rQs~2k~amisl~
and NitrosQ~ uinolin~-ne~
: :
:.. ~:
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 (~IABPj, 6-nitro~ -benzopyrone : ~ ~ 25 (NO2BP), 6~nitroso-1,2-benzopyrone :(NOBP)~ 3-nitrosobPnzamide (NOB~? or 5~7)-nitroso-1(~H)~
isoquinolinone (NOQ) (a mixture of the 5-nitroso and 7 nitroso ieomer~ nd the level of 3H], thymidine~ uptake ~wasi determin~d as a measure of oellular proliferation. ~B shown in : ~igure 2, ~or ea:ch of the cell lines tssted (855-2 ~ells, Fig.~2A; H9 cells, Fig.~2B; H~-- .;
60 c lls, Fig. 2C; K562 cells, Fig. 2D~ the ~`;
nitroso-containi~g ligands ~NOBP, NOBA, NOQ) were ~able to inhibit 3H-thymidine uptake in lower molar concentrati~ns than the other compounds. NOBP, NOBA and NOQ powerfully ~V~93/07868 PCT/U~91/O~gO2 2121~0~ ~
inhibited 3H-thymidine uptake ~t a concentration of 1~ ~M, a concentration at which the other compounds exhibited compara tively slight ~:.
inhibitory ef :f ectE~ .
Experiments with H9 cells grown in 10% fetal bovine ~erum (FCS) (Fig. 2B~ found NOQ to be the most pot~nt inhibitor, demonstrating al~o~t complete inhi~ition a~ 10 ~M levels. NOBP
demon~trated about a 30% decrease in thy~idi~e uptake at 10 ~M, and an almost complste inhibition of uptake at 100 ~M. NOBA
demonstrated ab~ut 75% level of inhibition at 10 pM, about 85% inhibition at 100 ~M, and almoBt :.
complete inhibition at 250 ~M. The remaini~g amino and nitro compounds were ~ignificantly le~ potent and did not display complst~
inhibition until concentration~ of 100:0 ~M were reached. : : ~
',, ;20 Experiments with ~562 cel:Ls grown in;1~% f~tal ~ bo~ine serum (Fig. 2~) fou~d NO~ and NOBP to be :: the most pot~nt inhibitors of cell gr~wth. Both ~ ~ ~OQ and:NOBP~resulted in the almost~complcte : ~ : inhibition~at ~on~entratlons of 10 ~M.~ NOBP was:
; ~5 almost: as potent~as NO~ and prsduced~about 90#
: : inhibition at a concentration~ of;~10 :pM, a~d almos~ complete inhib~ition at a concentration of 100 ~M. ~Th~ other 3 compounds test~d were signifid~ntly less potent.
.
: ~, Exp~riments with 855-2 cells grown in 10%
fetal bovine~serum:(Fig. 2A~ found that ~OQ,~and NOBP producea almost ~omplete lnhibl~t~ion at a ~concentratlon of lO ~M. At a concentration of 1 ~ ~M, ~ produced some~hat more inhibition than : 35 NOBP, and NOBP produced ~omewhat more inhibition .
.. . . .. ..... ............ . . . . .... ... . .... .. . . ..... . . . . . . . . ... . .. . .... .
WO ~3/07~68 P~/US91/089~2 .
~12~!30Q
than NOBA. Experiments using HL-60 cells ~Fig.
2C) provided similar corlclusions. The other 3 compounds tested wsre significantly less potent.
The effect of differ~nt growth factor~ OI~ the growth inhibitory effect~ of NOBP was te~ted. -~.
855-2 cells that were grown in media with ( 1 10% fetal bovine serum, (2~ autocrine growth ~:~
factor (At;F) and (3) low mol~cular weigh1~-BCGF
(a T cell derived lymphokir~e) wl~re expo8~d to increa~ing conc~ntrations of the ADRPT ligands.
The results are provided in Figure 2 (~, E, F) .
Cells grown in each o the growth faotox~ we~
all potently inhibited by the nitroso containing compounds, with concentrations of 5 to 10 ~M
resultin~ in 100% inhibit:ion, Thus, NOBP, NOBA
and NOQ ex~rt potent inhibitory effect~
regardless of the sourt~e of growth factor ~-activity. ;:
~: ~ 20 ~ In order to ex~lude the possibility that NOBP :~:
and NOBA manifest their growth inhibitor~ ;
: effects through inactivation of gxowth factors, the effects of lO ~M NOBP or NOBA (constant : ~ concentration)~on 855-2 cell~s in the presenae of ~25 : increasing concentrations~:of:~et:aI bovine ~erum FCS) were tes~d (FCS) contains growth factors or 855 2 cells). The data are pro~ided ~in : Figure 3. Growth arrest occurs irrespective of~
the csncentration of FCS. Thus, the mode of ~30 action of :NOBP, does not ~appear to be~ by antagoni~m -~f ~rowth factors but at ADPRT si~es : ~ related to DNA-repllcation. ~;
umor cell inhibitory concentratio~s of NOBP
~ and NOBA were shown not to a~~ct adver~ely the viability ~f normal cells. Experiments w2re ~
~, :`
~ 3/078~s PCr/US91/~8902 2 ~ 2 ~L ~
performed in which the functions of various cancer cells ( 855-2 and HL-60 leukemia cells, D32, D37 and CRL 7712 glioblastoma c ll lin~s, 185 medulla tumor cell line, L1210 murine leukemia cell line, MDA-468 human breast tumor cell line) and normal cells (neutrophil leukocytes and bon~ marro~ or peripheal blood stream cells) wexe asseæ~ed in the absence or presence of the compounds. The results are ~hown in Figures 4-9. Together, the data indicate that a concentration of 10 ~M o~ the n$troso-containing ligands effectively suppressed cancer cell grow~h but demonstrated only modest effects the functions on normal cells.
VI. ~9~iLJ ~ Y~
The cytotoxicity of Oj 2 ~M, 4 ~M, 8 ~M and 10 ~M NOBP wa~ maasured by examining the effect of : 20 the compound on the colony f~rmation (CFU-GM) of.
normal human stem:~ells (PBSC~. The results of : ~ the experiment~ are pro~ided i~ figure 5B.
Toxicity was not detected,~e~en though levels of : NOBP suf~ficient to ~ : block fl55-2~ cell ~: 25 :~ proliferatlon completely were tested.
: ' ~ ::
~similar ~FU :stem cell toxicity assay ~was : pe~formed in which~comparisons were made betw~en (A~P) 6-amino-1,2-benzopyrone l mM; (~ABP)~ 5 I~6~amino-1,2-benzop~xone 250 ~M, (NO2BP) 6 :
nitro-1,2-benzopyrone ~weakly active) 250 ~M, NOBP 10 ~M, and NOBA lO:~M. The results of:the experiments axe provided in figure ~A. ~Whereas the 6-amino~ enzopyrone, 5-I~6-amino-1,2-benzopyrone and the 6-~itro deri~ative were ~oxic at the tested given doses, the almost inefeative (again~t t~mor cells) 6-nitro ::
21219UO "
I
derivative and the highly effective (against tumor cells) NOBP and NOBA were non-toxic.
The effects of lO~m Nosp and NO~A on superoxide generation by normal human peripheral blood neutrophil leukocytes was te~ted. The results are provided in tabla X I . Only minor reductions in sup~roxide generation were observed.
1 0 ~gkL~
Effects of 10 ~M NOBP and NOBA on the Generation of Superoxide by Human Neutrophils nmol 02/hr/105 cells I~ LLi 105 PMN ~ PMA: 5S.9 ~ 7.7 +10 ~M NOBP 34.1 ~ 14.1 +10 ~M NOBA 44.4 ~ 10.0 ~20 VII. ' Vincristine, a highly toxic chemetherapeutic compound, is the currently:used in the treiatment o~ leukemia and other malignancies. Studie:s ~5 were :performed i~ order to dete;rmine the : ~ concentration of vincristine that produ¢es the same level of growth inhibition a~ lO ~N NOBP,:~ :
when a~sayed on: 855-~ leukemia:cells grown i~
Vi~rQ. Vincristine was~tested~in doses of ~.l, :30 : ~1, 10 and lOO~M. ~s shown in Fig~e~:7. 100 ~M
of vincristine (a highly toxi~ co~cent~ation) :~ was re~uired~ to produce the~:~ame level of : inhibition a~ 10 ~M o NOBP, thus NO~P is about : 10 times more potent than an e~ual ~ioncentration o~ vincristine, and is ~t toxic to normal cells. :;
; Thus certain aromatic nitroso molecules tha~
are also inhibitors of ADPRT polymerase activity ~0~3/07~68 PCT/US9l/OB902 2 :1219 ~ U
-25- ;~
may be useful chemotherapeutic cytostatic agents ~:~ because of their effactiveness combined wi~h low toxicity.
,'",,~
VIII. Anti-HIV_action of_NOBP/ NOBA and NOO ~n The ability of NOBP (6-nitroso-1,2-benzopyrone3 and NOBA (3-nitrosobenæamide~ to inhibit HIV infections wer~ tested using the methods described in the ~9~ L___ Immu~l~iç~l M~hod~ 76:171-183 (1985).
Exposure to the two drugs was only for 30 minutes at the commencement of viral inf~ction, and dxugs were never re-added. The results given in ~able III pro~ide t:he ID~ of HIV ti~er 10 days after infection of cell cultures with HIV. The data in Table III demonstrate that 10 ~M of the ~itroso-containinS ligands cause~
a three log decrease in ~the HIV-1 infectivity ~: ti~er.
: :
~ ~ ABLE 111 ~ç~=~am~ Virus Titer lloq ID ~L~ Q~ ~
: Virus ~lone~ 5~25 :~.
500 ~M ABP ~ : 4.50 ~ ~
250 ~M:IABP ~ 4.66 ::.
~ ~250 ~M NO~BP 4.93 +10 ~M NO~P : 2.01 :: : +10 ~M NO~ 1.05 ; ~ +10 ~M~NOQ : ~ 1.73 : 35 IX. ~ : : ~ , :~ Exp rimetns were performed to determine i~the ~:
:inhibition~of prollferation o~ 855-2 ce;lls seen 4;0 ~: in cu1ture~nd in soft agar is due to the ~ : cytostatic or cytocidal effect of the nitroso : ~ : compounds NOBP, :NOBA, and NOQ. Cells at ~.
:
W093/0786~ PCT/~S91/08902.
2 1 ~ 1 ~3 0 û
- 2 6 - ~
lxlO5/ml (concentration used in bone marrow assay) were treated with NOBP, NOBA and NOQ at 1, 2.5, 5 and lO~m fox 2 hours then ~timulated :
with 10% fetal calf serum a~d 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. ~he absorbance of the pellete~ cell was then measured at 550nm after adding DMSO to solubilze the cells Results: With lO~M NOBP, NOBA and NOQ, complete killing was observed in 85S-2 cell~ at lOO,OOO/ml.
..;
All publications, patents, and patent applications cited abo~e are herein incorporated by reference.
The foregoing writt~n specification i5 ~.
~20 co:nsidered to be su~ficient to enable one ;: skilled in~ the art to pract~ice the invention.
ndeed, various modifi:~ations of the above-de~cribed modes~for carrying:out the invention which are obvlou6 to tho~e killed :in the ~ield :: 25 o~ pharmaceutical ~ormul~ation or rel~ated fields : ~.
are intended to be within the scope of the ~ ~ .
following claims. ~ ~
: ~ : :
.:
~:, .':
Claims (20)
1. A compound having the formula:
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
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
PCT/US91/08920
3. A compound according to claim 2, wherein R2 or R4 is a nitroso group.
4. 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.
, 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.
5. 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.
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.
6. 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.
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.
7. 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.
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.
8. 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.
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.
9. 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.
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.
10. The method of claim 9 wherein R4 a nitroso group.
11. 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.
, 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.
12. The method of claim 11 wherein R2 is a nitroso group.
13. 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.
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.
14. The method of claim 13 wherein R2 is a nitroso group.
15. The method of claim 13 wherein the virus is human immunodeficiency virus.
16. 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 nitroso group.
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 nitroso group.
17. The method of claim 16 wherein R2 or R4 is a nitroso group.
18. 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.
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.
19. The method of claim 18 wherein R2 or R4 a nitroso group.
20. The method of claim 18 wherein the virus is human immunodeficiency virus.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US78080991A | 1991-10-22 | 1991-10-22 | |
| US780,809 | 1991-10-22 |
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| Publication Number | Publication Date |
|---|---|
| CA2121900A1 true CA2121900A1 (en) | 1993-04-29 |
Family
ID=25120767
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002121900A Abandoned CA2121900A1 (en) | 1991-10-22 | 1991-11-26 | Adenosine diphosphoribose polymerase binding nitroso aromatic compounds useful as anti-tumor and anti-retroviral agents |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0609211A4 (en) |
| JP (1) | JP3070767B2 (en) |
| AU (1) | AU676992B2 (en) |
| CA (1) | CA2121900A1 (en) |
| WO (1) | WO1993007868A1 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5464871A (en) | 1993-05-12 | 1995-11-07 | Octamer, Inc. | Aromatic nitro and nitroso compounds and their metabolites useful as anti-viral and anti-tumor agents |
| US5753674A (en) * | 1991-10-22 | 1998-05-19 | Octamer, Inc. | Adenosine diphosphoribose polymerase binding nitroso aromatic compounds useful as retroviral inactivating agents, anti-retroviral agents, anti-retroviral agents and anti-tumor agents |
| US5877185A (en) * | 1991-10-22 | 1999-03-02 | Octamer, Inc. | Synergistic compositions useful as anti-tumor agents |
| JPH10504517A (en) * | 1992-11-02 | 1998-05-06 | オクタマー，インコーポレイティド | Adenosine diphosphoribose polymerase binding nitrosoaromatic compounds useful as antiretroviral and antitumor agents |
| GB9404485D0 (en) * | 1994-03-09 | 1994-04-20 | Cancer Res Campaign Tech | Benzamide analogues |
| IL129871A (en) * | 1994-05-06 | 2003-11-23 | Pharmacia & Upjohn Inc | Process for preparing 4-phenyl-substituted octanoyl-oxazolidin-2-one intermediates that are useful for preparing pyran-2-ones useful for treating retroviral infections |
| EP1904468A4 (en) * | 2005-06-10 | 2009-04-22 | Bipar Sciences Inc | Parp modulators and treatment of cancer |
| WO2008030887A2 (en) * | 2006-09-05 | 2008-03-13 | Bipar Sciences, Inc. | Methods for designing parp inhibitors and uses thereof |
| US11433074B2 (en) | 2017-06-22 | 2022-09-06 | Triact Therapeutics, Inc. | Methods of treating glioblastoma |
| EP3687501A4 (en) | 2017-09-29 | 2021-06-23 | Triact Therapeutics, Inc. | Iniparib formulations and uses thereof |
-
1991
- 1991-11-26 CA CA002121900A patent/CA2121900A1/en not_active Abandoned
- 1991-11-26 EP EP92904892A patent/EP0609211A4/en not_active Ceased
- 1991-11-26 AU AU12609/92A patent/AU676992B2/en not_active Ceased
- 1991-11-26 JP JP4505220A patent/JP3070767B2/en not_active Expired - Lifetime
- 1991-11-26 WO PCT/US1991/008902 patent/WO1993007868A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07502975A (en) | 1995-03-30 |
| AU676992B2 (en) | 1997-04-10 |
| EP0609211A4 (en) | 1995-02-01 |
| JP3070767B2 (en) | 2000-07-31 |
| EP0609211A1 (en) | 1994-08-10 |
| WO1993007868A1 (en) | 1993-04-29 |
| AU1260992A (en) | 1993-05-21 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |