CA1338445C - 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents - Google Patents
1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agentsInfo
- Publication number
- CA1338445C CA1338445C CA000540932A CA540932A CA1338445C CA 1338445 C CA1338445 C CA 1338445C CA 000540932 A CA000540932 A CA 000540932A CA 540932 A CA540932 A CA 540932A CA 1338445 C CA1338445 C CA 1338445C
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- Prior art keywords
- amino
- hydrocarbyl
- hydroxy
- independently
- halogen
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D253/00—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00
- C07D253/08—Heterocyclic compounds containing six-membered rings having three nitrogen atoms as the only ring hetero atoms, not provided for by group C07D251/00 condensed with carbocyclic rings or ring systems
- C07D253/10—Condensed 1,2,4-triazines; Hydrogenated condensed 1,2,4-triazines
-
- 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
- A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
-
- 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
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
A method of using 1,2,4-benzotriazine oxides of the formula wherein X is NH2, NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms or an amide, or wherein in the case of NRR the two R groups may be linked together to form a morpholino ring, and wherein R may be further substituted with OH, alkoxy, amino or halogen substituents;
n is 1; and Y1 and Y2 are independently either H;
halogen; hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy, alkylthio, amino, morpholino, acyloxy, acylamido and thio analogs thereof, carboxy, alkoxycarbonyl, carbamyl, alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-O-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above or a pharmaceutically acceptable salt of said compound, as radiosensitizers and selective cytotoxic agents is disclosed. The compounds are shown to specifically radiosensitive hypoxic tumor cells and are additionally disclosed to be useful as specific cytotoxic agents for these cells.
n is 1; and Y1 and Y2 are independently either H;
halogen; hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy, alkylthio, amino, morpholino, acyloxy, acylamido and thio analogs thereof, carboxy, alkoxycarbonyl, carbamyl, alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-O-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above or a pharmaceutically acceptable salt of said compound, as radiosensitizers and selective cytotoxic agents is disclosed. The compounds are shown to specifically radiosensitive hypoxic tumor cells and are additionally disclosed to be useful as specific cytotoxic agents for these cells.
Description
1,2,4-BENZOTRIAZINE OXIDES AS 13 3 8 4 4 S
RADIOSENSITIZERS AND SELECTIVE CYTOTOXIC AGENTS
Technical Field The invention relates to cytotoxic agents and radiotherapy effective against hypoxic cells. Specifically, the invention relates to selectively killing tumor cells and to sensitizing tumor cells to radiation using 1,2,4-benzotriazine oxides.
Background Art Hypoxic cell radiosensitizers are compounds that selectively increase the sensitivity of hypoxic cells to destructive radiation. Cytotoxins which have enhanced activity under hypoxic conditions also provide a means for selective destruction of cells under low oxygen pressure. This specificity for hypoxic cells is important because it is tumors that are typically characterized by such cells. Virtually all tumors which are present as solid masses contain these cells, while normal cells generally have an adequate supply of oxygen. Accordingly, anti-tumor agents can be made selective for tumors by virtue of high activity under hypoxic conditions, and radiation can be employed more effectively in the presence of these sensitizers.
Of course, the use of radiation treatment to destroy tumor cells is only practical if damage to the surrounding normal tissue can be minimized or avoided.
.f~, /, ''IS~, `~. 1338445 The effects of radiation are enhanced by the presence of oxygen, and it is established that as the dose of radiation is increased, the ~rreclivelless of the radiation in destroying target cells is enhanced most dramatically when oxygen is present. Therefore, selectivity for tumor cells toward radiation is difficult toachieve -- normal cells, in view of their oxygen supply, are generally more ~uscel~tible to radiation than the target tumor cells. It is therefore desirable to provide a means of sen~iti7ing tumor cells, but not the surrounding tissue, to radiation treatment One solution would be to incrcase the supply of oxygen to these tumors cells. This, however, has proved difficult to do.
Various heterocyclic compounds and in particular those with oxidized nitrogen moieties, have been used to radiosensitize hypoxic tumor cells. Indeed,it has been postulated that the oxidized nitrogen functionality is responsible for this activity. Nitroimifl~oles, particularly misonidazole (MIS) and metronidazole have been studied extensively, and MIS is commonly used as a standard in in vitro andin vito tests for radiosensitizing activity. (See, e.g., Asquith, et al. Radiation Res (1974) 60:108-118; Hall, et al. Brit J. Cancer (1978) 37: 567-569; Brown, et al.Radiation Res (1980) 82:171-190; and U.S. patent 4,371,540. The radiosensitizingactivities of certain l-substituted 3(5)-nitro-_-triazoles and of various quinoxaline-1,4-dioxide derivatives have also been disclosed.
In addition, C~n~ n Patent Application No. 519,514 assigned to the same assignee discloses a group of radiosensitizers that do not contain oxidized nitrogen -- the substituted be~ litles and nicotinamides and their thio analogs. These compounds, nevertheless, are radiosen~ ,s. It is important to distinguish the ability to s~ ;L;~ hypoxic cells selectively, for instance, by enhancing their oxygen supply, from another merh~ni~m commonly encountered for "sellsili~ g"
cells: inhibition of the enzyme poly(ADP-ribose)polymerase, which is believed tobe essenti~l in the repair of irradiated cells after radiation. This repair mech~ni~m is operated in both hypoxic tumor cells and in normal cells. Hence, a~lmini.~tration of "radiosen~iti7ers" which operate according to this latter mechanism does not accomplish the desired purpose of selectively se~ ;llg the target tumor cells.
A group of compounds which has not previously been suggested for use in either selectively killing hypoxic cells or in radiosensitizing such cells is 3-amino-1,2,4-ben7otria_ine 1,4-di-N-oxide and related compounds. Related US patents 3,980,779; 3,868,371, and 4,001,410 disclose the prepal~lion of a group of thesecompounds and their use as anti-microbial agents, particularly by addition of these m~tçri~l~ to livestock fodder. US patents 3,991,189 and 3,957,799 disclose derivatives of these compounds bearing substitu.ont~ on the nitrogen of the 3-amino group. These compounds also have anti-microbial activity.
The present invention provides additional compounds which specifically radiosçn~iti7~ hypoxic cells in vitro and which, furthermore, are directed cytotoxic to hypoxic cells both in vitro and in vito. Therefore, ~flmini.ctration of thesecompounds prior to or following radiation tre~tn ent of tumors selectively kills the hypoxic (tumor) cells which survive the radiation dose. Both the ability of these compounds to radiosçn.~iti7~ hypoxic cells in vitro and especially their ability to selectively kill hypoxic cells directly are unexpected pr~ellies of these compounds.
Disclosure of the Invention The invention provides a valuable addition to the group of compounds c~ lly available as selective radiose~ and selective cytotoxic agents for hypoxic tumor cells. Some of the compounds useful in this regard are known compounds, others are novel. One aspect of the invention, therefore, is a methodof radiosen~ g or selectively killing hypoxic tumor cells with a compound of the formula: o lr ~N j--X
J~
n wherein X is OH, OR, NH2, NHR or NR2 where each R is independently an alkyl of 1-4 carbon atoms, an amide, or a morpholino moiety and may further be substituted with hydroxy, alkoxy, amino, or halogeno substituents;
wherein n is 0 or 1; and .. _.
yl and y2 are independently either H, halogeno, hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2substit~l~nt.e selected from the group coneieting of halogeno, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino), acyloxy, acylamido and their thio analogs, alkylsulfonyl, alkylphosphonyl, carboxy, alkoxycarbonyl, c~l,~llyl or alkylc~l,~llyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage, or wherein Y1 and y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR)R' in which R' is a hydrocarbyl optionally substituted as defined above.
The compounds of the invention, therefore, are the mono- or dioxides of optionally substituted 1,2,4-benzotriazine which contain a hydroxyl or amino group, either substituted or unsubstituted, in the 3 position. While all of the compounds defined by Formula 1 are generally effective as radiosensitizers, onlycompounds having a 3-amino substituent (i.e., X=NH2, NHR or NR2 with R as defined above) and which are di-N-oxides (n=1) are effective cytotoxic agents.
Certain of the compounds enco~ eed by Formula 1 are already known in the art as being useful for other purposes; other compounds are novel. The novel compounds encomp~se(l by the present invention and which may be prepared by methods disclosed herein include compounds represented by the formula above, in the following three classes: I, X is OH, OR, or NR2 with R as defined above, n is 0 or 1, and yl and y2 are as defined above, II. X is NH2 or NHR with R as defined above, n is o, and yl and y2 are as defined above: III. X
is NH2, n is 1, and yl and y2 are as defined above but not halogen, sdluldled alkyl (1-6C) unsubstituted or halogen-sub~liluled, alkoxy (1-6C), c~balllyl, carboxy or carboaLkoxy (1-6C).
Brief Description of the Drawin~s Figure lA, lB and lC show the selective cytotoxicity of 3-amino-1,2,4-benzotriazine 1,4-dioxide for hypoxic cells derived from h~m~ter, mouse and human tissues.
Figure 2 shows the in vito efficacy of 3-amino-1,2,4-benzotriazine 1,4-dioxide in enh~ncin~ the killing of tumor cells when combined with radiation.
J, `` 1338445 _.
RADIOSENSITIZERS AND SELECTIVE CYTOTOXIC AGENTS
Technical Field The invention relates to cytotoxic agents and radiotherapy effective against hypoxic cells. Specifically, the invention relates to selectively killing tumor cells and to sensitizing tumor cells to radiation using 1,2,4-benzotriazine oxides.
Background Art Hypoxic cell radiosensitizers are compounds that selectively increase the sensitivity of hypoxic cells to destructive radiation. Cytotoxins which have enhanced activity under hypoxic conditions also provide a means for selective destruction of cells under low oxygen pressure. This specificity for hypoxic cells is important because it is tumors that are typically characterized by such cells. Virtually all tumors which are present as solid masses contain these cells, while normal cells generally have an adequate supply of oxygen. Accordingly, anti-tumor agents can be made selective for tumors by virtue of high activity under hypoxic conditions, and radiation can be employed more effectively in the presence of these sensitizers.
Of course, the use of radiation treatment to destroy tumor cells is only practical if damage to the surrounding normal tissue can be minimized or avoided.
.f~, /, ''IS~, `~. 1338445 The effects of radiation are enhanced by the presence of oxygen, and it is established that as the dose of radiation is increased, the ~rreclivelless of the radiation in destroying target cells is enhanced most dramatically when oxygen is present. Therefore, selectivity for tumor cells toward radiation is difficult toachieve -- normal cells, in view of their oxygen supply, are generally more ~uscel~tible to radiation than the target tumor cells. It is therefore desirable to provide a means of sen~iti7ing tumor cells, but not the surrounding tissue, to radiation treatment One solution would be to incrcase the supply of oxygen to these tumors cells. This, however, has proved difficult to do.
Various heterocyclic compounds and in particular those with oxidized nitrogen moieties, have been used to radiosensitize hypoxic tumor cells. Indeed,it has been postulated that the oxidized nitrogen functionality is responsible for this activity. Nitroimifl~oles, particularly misonidazole (MIS) and metronidazole have been studied extensively, and MIS is commonly used as a standard in in vitro andin vito tests for radiosensitizing activity. (See, e.g., Asquith, et al. Radiation Res (1974) 60:108-118; Hall, et al. Brit J. Cancer (1978) 37: 567-569; Brown, et al.Radiation Res (1980) 82:171-190; and U.S. patent 4,371,540. The radiosensitizingactivities of certain l-substituted 3(5)-nitro-_-triazoles and of various quinoxaline-1,4-dioxide derivatives have also been disclosed.
In addition, C~n~ n Patent Application No. 519,514 assigned to the same assignee discloses a group of radiosensitizers that do not contain oxidized nitrogen -- the substituted be~ litles and nicotinamides and their thio analogs. These compounds, nevertheless, are radiosen~ ,s. It is important to distinguish the ability to s~ ;L;~ hypoxic cells selectively, for instance, by enhancing their oxygen supply, from another merh~ni~m commonly encountered for "sellsili~ g"
cells: inhibition of the enzyme poly(ADP-ribose)polymerase, which is believed tobe essenti~l in the repair of irradiated cells after radiation. This repair mech~ni~m is operated in both hypoxic tumor cells and in normal cells. Hence, a~lmini.~tration of "radiosen~iti7ers" which operate according to this latter mechanism does not accomplish the desired purpose of selectively se~ ;llg the target tumor cells.
A group of compounds which has not previously been suggested for use in either selectively killing hypoxic cells or in radiosensitizing such cells is 3-amino-1,2,4-ben7otria_ine 1,4-di-N-oxide and related compounds. Related US patents 3,980,779; 3,868,371, and 4,001,410 disclose the prepal~lion of a group of thesecompounds and their use as anti-microbial agents, particularly by addition of these m~tçri~l~ to livestock fodder. US patents 3,991,189 and 3,957,799 disclose derivatives of these compounds bearing substitu.ont~ on the nitrogen of the 3-amino group. These compounds also have anti-microbial activity.
The present invention provides additional compounds which specifically radiosçn~iti7~ hypoxic cells in vitro and which, furthermore, are directed cytotoxic to hypoxic cells both in vitro and in vito. Therefore, ~flmini.ctration of thesecompounds prior to or following radiation tre~tn ent of tumors selectively kills the hypoxic (tumor) cells which survive the radiation dose. Both the ability of these compounds to radiosçn.~iti7~ hypoxic cells in vitro and especially their ability to selectively kill hypoxic cells directly are unexpected pr~ellies of these compounds.
Disclosure of the Invention The invention provides a valuable addition to the group of compounds c~ lly available as selective radiose~ and selective cytotoxic agents for hypoxic tumor cells. Some of the compounds useful in this regard are known compounds, others are novel. One aspect of the invention, therefore, is a methodof radiosen~ g or selectively killing hypoxic tumor cells with a compound of the formula: o lr ~N j--X
J~
n wherein X is OH, OR, NH2, NHR or NR2 where each R is independently an alkyl of 1-4 carbon atoms, an amide, or a morpholino moiety and may further be substituted with hydroxy, alkoxy, amino, or halogeno substituents;
wherein n is 0 or 1; and .. _.
yl and y2 are independently either H, halogeno, hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2substit~l~nt.e selected from the group coneieting of halogeno, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino), acyloxy, acylamido and their thio analogs, alkylsulfonyl, alkylphosphonyl, carboxy, alkoxycarbonyl, c~l,~llyl or alkylc~l,~llyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage, or wherein Y1 and y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR)R' in which R' is a hydrocarbyl optionally substituted as defined above.
The compounds of the invention, therefore, are the mono- or dioxides of optionally substituted 1,2,4-benzotriazine which contain a hydroxyl or amino group, either substituted or unsubstituted, in the 3 position. While all of the compounds defined by Formula 1 are generally effective as radiosensitizers, onlycompounds having a 3-amino substituent (i.e., X=NH2, NHR or NR2 with R as defined above) and which are di-N-oxides (n=1) are effective cytotoxic agents.
Certain of the compounds enco~ eed by Formula 1 are already known in the art as being useful for other purposes; other compounds are novel. The novel compounds encomp~se(l by the present invention and which may be prepared by methods disclosed herein include compounds represented by the formula above, in the following three classes: I, X is OH, OR, or NR2 with R as defined above, n is 0 or 1, and yl and y2 are as defined above, II. X is NH2 or NHR with R as defined above, n is o, and yl and y2 are as defined above: III. X
is NH2, n is 1, and yl and y2 are as defined above but not halogen, sdluldled alkyl (1-6C) unsubstituted or halogen-sub~liluled, alkoxy (1-6C), c~balllyl, carboxy or carboaLkoxy (1-6C).
Brief Description of the Drawin~s Figure lA, lB and lC show the selective cytotoxicity of 3-amino-1,2,4-benzotriazine 1,4-dioxide for hypoxic cells derived from h~m~ter, mouse and human tissues.
Figure 2 shows the in vito efficacy of 3-amino-1,2,4-benzotriazine 1,4-dioxide in enh~ncin~ the killing of tumor cells when combined with radiation.
J, `` 1338445 _.
Figure 3 shows the killing of tumor cells i vito by 3-amino-1,2,4-benzotriazine 1,4-dioxide when the tumor has been made hypoxic by the hlll~eliLoneal ~flmini~tration of the antihypertensive drug hydralazine.
Modes of Carryin~ Out the Invention A. The Compounds Useful in the Invention The compounds useful in radiosensitizing hypoxic tumor cells as described herein are derivatives of 1,2,4-bel~ulliazine oxide.
The hydrocarbyl group re~lesenled by yl or y2 may contain 1-14 carbon atoms, may be saturated or unsdluldl~d, cyclic or acyclic, and may optionally beinterrupted by a single ether linkage. Thus, the unsubstituted form of yl and y2can be, for example, methyl, ethyl, n-propyl, s-butyl, n-hexyl, 2-methyl-n-pentyl, 2-ethoxyethyl, 3-(n-propoxy)-n-propyl, 4-methoxybutyl, cyclohexyl, tetrahydrorulrul~l, rulrul~l, cyclohexenyl, 3-(n-decyloxy)-n-propyl, 4-methyloctyl, 4,7-dimethyloctyl, and the like.
The hydrocarbyl may be substituted with one or two substituents as follows:
The halogeno substituents are fluoro, chloro, bromo, or iodo. The alkoxy substituents leplcsell~d by OR' may contain 1 to 4 carbon atoms, and include, for example, methoxy, n-propoxy, and t-butoxy. The amino substituent may be NH2, NHR or NR2, where each R in independently and alkyl of 1-4 carbons or a morpholino moiety. R may optionally be substituted with 1-2 hydroxy, alkoxy, amino, or halogeno substi~ent.~.
The acyloxy and acylamido groups are represented by R'COO- and R'CONH-, re~e~ ely, where R' contains 1-4 carbons, and their thio analogs are represented by R'CSO- and R'CSNH-. Alkyl sulfonyl and alkyl phosphonyl are, respectively, R'SO2 and R'P(OP')O- wherein each R' is independently as above defined. Carboxy is the group -C(O)OH; alkoxycarbonyl is -C(O)OR'; ca,b~llyl is -C(O)NH2; and alkylc~b~llyl is -C(O)NHR'.
Where X is OH, of course, the compounds may also be ~lepaled and used as the ph~rm~çeutically acceptable salts formed from inorganic bases, such as sodium, potassium, or calcium hydroxide, or from organic bases, such as caffeine, ethylamine, and lysine.
When X is NH2, ph~rm~celltically acceptable acid addition salts may be used. These salts are those with inorganic acids such as hydrochloric, hydrobromic or phosphoric acids or organic acids such as acetic acid, pyruvic acid, succinicacid, mandelic acid, p-toluene sulfonic acid, and so forth. (Amino substituents on the hydrocarbyl side chain can also, of course, be converted to salts).
The 1,2,4-benzotriazine may be used as the mono- or dioxide. Either the 1-nitrogen of the triazino ring may be oxidiæd, or both the 1- and 4-nitrogens may be oxidized.
Specific particularly p-~,r~ d compounds which are useful in the radiosensitization and cytotoxic procedures of the invention include 3-hydroxy-1,2,4-benzoll;azine 1-oxide;
3-hydroxy-1,2,4-bel.~.otl;azine 1,4-dioxide;
3-amino-1,2,4-bel~oll;azine 1-oxide;
3 -amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-methoxy-3-hydroxy- 1 ,2,4-be.lz~ ;azine 1 -oxide;
6(7)-methoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-methoxy-3-amino-1,2,4-bel,~.oll;azine 1-oxide;
6(7)-methoxy-3-amino-1,2,4-bellzotl;azine 1,4-dioxide;
6(7)-ethoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-ethoxy-3-hydroxy-1,2,4-belwtl;azine 1,4-dioxide;
6(7)-ethoxy-3-amino-1,2,4-belwll;~zine 1-oxide;
6(7)-ethoxy-3-amino-1,2,4-bellzull;azine 1,4-dioxide;
6(7)-[4-~cet~mido-n-butanoxy]-3-hydroxy-1,2,4-belwll;azine 1,-oxide;
6(7)- [4-~cet~mido-n-butanoxy] -3 -hydroxy- 1 ,2,4-benzull ;a~ e 1 ,4-dioxide;
6(7)-[4-~r.et~mido-n-butanoxy]-3-amino- 1 ,2,4-bellGull;azine 1 ,-oxide;
6(7)-[4-acetamido-n-butanoxy]-3-amino-1,2,4-bel~o~liazine 1,4-dioxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-hyd~oxy-1,2,4-benzotriazine 1-oxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-hydroxy-1,2,4-bel.~olliazine 1,4-dioxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-amino-1,2,4-belwlliazine 1-oxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-amino-1,2,4-bel~olliazine 1,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[(2-furyl)methylamino]-3-hydroxy- 1 ,2,4-bel,~olliazine 1 ,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-amino-1,2,4-bel~oll;azine l-oxide;
6(7)-[(2-furyl)methylamino]-3-amino-1,2,4-bell~olliazine 1,4-dioxide;
6(7)-(2-methoxyethylamino)-3-hydroxy- 1 ,2,4-bel~ulliazine 1 -oxide;
6(7)-(2-methoxyethylamino)-3-hydroxy- 1 ,2,4-bel,70lliazine 1 ,4-dioxide;
6(7)-(2-methoxyethylamino)-3-amino- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-carbethoxymethoxy-3-hydroxy-1,2,4-bel,zolliazine l-oxide;
6(7)-carbethoxymethoxy-3-hydlo~y-1,2,4-bell~olliazine 1,4-dioxide;
6(7)-carbethoxymethoxy-3-amino- 1 ,2,4-benzolliazine 1 -oxide;
6(7)-carbethoxymethoxy-3-amino- 1 ,2,4-bel~ll iazine 1 ,4-dioxide;
6(7)-[(2-methoxyethyl)c~b~llyllllethoxy]-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-[(2-methoxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[(2-methoxyethyl)c~balllyllllethoxy]-3-amino-1,2,4-bell~olliazine l-oxide;
6(7)-[(2-methoxyethyl)c~b~llyllllethoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)c~b~llyllllethoxy]-3-hydroxy-1,2,4-benzotriazine l-oxide;
6(7)- [(2-hydroxyethyl)carbamylmethoxy] -3 -hydroxy- l ,2 ,4-benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)c~balllyllllethoxy]-3-amino-1,2,4-bell~olliazine l-oxide;
6(7)-[(2-hydroxyethyl)c~l,~llyLllethoxy]-3-amino-1,2,4-bell~olliazine 1,4-dioxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-hydroxy-1,2,4-bell~o~liazine l-oxide;6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-hydroxy-1,2,4-benGolliazine 1,4-dioxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-amino-1,2,4-bell~olliazine l-oxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)- [3 -amino-n-propoxy] -3 -hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-[3-amino-n-propoxy]-3-hydroxy-1,2,4-bel,~olliazine 1,4-dioxide;
6(7)-[3-amino-n-propoxy]-3-amino-1,2,4-bel~olliazine l-oxide;
6(7)-[3-amino-n-propoxy]-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-[2,3-epo~y~iopoxy]-3-hydroxy-1,2,4-benzotriazine l-oxide;
~ 13384~
6(7)-[2,3-epoxypropoxy]-3-hydroxy-1,2,4-be~ iazine 1,4-dioxide;
6(7)- [2,3 -epoxypropoxy] -3 -amino- 1 ,2,4-bel, ~oll i~ine 1 -oxide;
6(7)-[2,3-epoxypropoxy]-3-amino-1,2,4-bell~ulli~zine 1,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-hydroxy-1,2,4-bel~oll;æine 1-oxide;
6(7)- [3 -methoxy-2-hydroxy-n-propoxy]-3-hydroxy- 1 ,2,4-be,~ol, iazine 1 ,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-amino- 1 ,2~4-bell~oll;azine 1 -oxide;
6(7)- [3 -methoxy-2-hydroxy-n-propoxy]-3 -amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-bell~olliazine 1-oxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-amino-1,2,4-bell~olliazine 1-oxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-amino-1,2,4-benzol~;azine 1,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-hydroxy- 1 ,2,4-bel~ol,;azine 1 -oxide;
6(7)-[3 ,4-dihydroxy-n-butoxy]-3-hydroxy- 1 ,2,4-bel, ~oll;azine 1 ,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-bellzolliazine 1-oxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-bel~lliazine 1,4-dioxide;
6(7)-methyl-3-hydroxy-1,2,4-bell~oll;azine 1-oxide;
6(7)-methyl-3-hydroxy-1,2,4-benzol,;azine 1,4-dioxide;
6(7)-methyl-3-arnino-1,2,4-bel~zol,;azine 1-oxide;
6(7)-methyl-3-arnino-1,2,4-bel~utl;azine 1,4-dioxide;
6(7)-ethyl-3-hydroxy-1,2,4-bel,70ll;azine 1-oxide;
6(7)-ethyl-3-hydroxy- 1 ,2,4-be~ ~ulli~7ine 1 ,4-dioxide;
6(7)-ethyl-3-amino- 1 ,2,4-bell~oll;azine 1 -oxide;
6(7)-ethyl-3-amino- 1 ,2,4-bel~ol, ;azine 1 ,4-dioxide;
6(7)-chloro~cet~mido-3-hydroxy-1,2,4-be~ iazine 1-oxide;
6(7)-chloro~cet~mido-3-hyd~ y-1,2,4-be~oLI;azine 1,4-dioxide;
6(7)-chloroacetamido-3-amino-1,2,4-bel~oll;azine 1-oxide;
6(7)-chloro~cet~mido-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-[(2-hydroxyethyloxy)acet~mido]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[(2-hydroxyethyloxy)~cet~mido]-3-hydroxy-1,2,4-belL~oll;azine 1,4-dioxide;
6(7)-[(2-hy-ilo~y~lhyloxy)~cet~mido]-3-amino-1,2,4-bel.~oll;azine 1-oxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6,7-dimethoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6,7-dimethoxy-3-hydroxy-1,2,4-bel.~oll;azine 1,4-dioxide;
6,7-dimethoxy-3-amino-1 ,2,4-be,, ~oLl;æine 1-oxide;
6,7-dimethoxy-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6,7-diethoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6,7-diethoxy-3-hydroxy- 1 ,2,4-bel~ull;azine 1 ,4-dioxide;
6,7-diethoxy-3-amino- 1 ,2,4-benzotriazine 1 -oxide;
6,7-diethoxy-3-amino-1,2,4-bel,~ull;azine 1,4-dioxide;
6(7)-propionyl-3 -hydroxy- 1 ,2,4-benzotriæine 1 -oxide;
6(7)-propionyl-3-hydroxy- 1 ,2,4-benzotriæine 1 ,4-dioxide;
6(7)-propionyl-3-amino- 1 ,2,4-benzotriæine 1 -oxide;
6(7)-propionyl-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-bell70ll;azine 1-oxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-bell~oll;azine 1-oxide;
6(7)-(2-acetoxyethoxy)-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-bel~oll;azine 1-oxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-bel,~oll;azine 1,4-dioxide;
6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-n-hexyloxy-3 -amino- 1 ,2,4-bel~oll ;æine 1 ,4-dioxide;
6(7)-ethylamino-3-hydroxy-1,2,4-bell~oll;azine 1-oxide;
6(7)-ethylamino-3 -hydroxy- 1 ,2,4-bel~oll ;azine 1 ,4-dioxide;
6(7)-ethylamino-3-amino-1,2,4-bel,~oll;æine 1-oxide;
6(7)-ethylamino-3-amino-1,2,4-bel~oll;æine 1,4-dioxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-bell~oll;æine 1-oxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotl;azine 1,4-dioxide;
6(7)-(2-methoxyethoxy)-3-amino-1,2,4-bel.~oll;azine 1-oxide;
6(7)-(2-methoxyethoxy)-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(amino~cet~mido)-3-hydroxy-1,2,4-bel~oll;æine 1-oxide;
6(7)-(amino~cet~mido)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(amino~cet~mido)-3-amino-1,2,4-bell~.oll;æine 1-oxide;
6(7)-(amino~cet~mido)-3-amino-1,2,4-bell~olliæine 1,4-dioxide;
6(7)-(c~l,alllylmethoxy)-3-hydroxy-1,2,4-bell~otl;æine 1-oxide;
13384~5 g 6(7)-(c~l,alllyhllethoxy)-3-hydroxy- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(call,alllyLllethoxy)-3-amino-1,2,4-bell~ulliazine 1-oxide;
6(7)-(c~l,~llyhllethoxy)-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-carboxymethoxy)-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-carboxymethoxy)-3-hydroxy-1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-carboxymethoxy)-3-amino-1,2,4-bel~oll;azine 1-oxide;
6(7)-carboxymethoxy)-3-amino-1,2,4-bellzoll;azine 1,4-dioxide;
and their ph~ ce~ltically acceptable salts and the thioamide analogs of the foregoing list of compounds. It should be noted that the IlYl or y2ll substituents set forth in most of the above compounds as present in either the 6 or 7 positions (-lesign~te.1 "6(7)") or in both the 6 and 7 positions (design~ted "6,7") may also be present at the S and\or 8 ring positions.
Of the above compounds useful in the method of the present invention as selective cytotoxic agents or radiosensitizers, the following compounds are novel:
compounds given by the formula above wherein I, X is OH, OR, or NR2, where each R is independently an alkyl of 1-4 carbon atoms, an amide, or a morpholino moiety and may further be substituted with hydroxy, alkoxy, amino, or halogen substitll~nt~, n is 0 or 1, and Y~ and y2 are independently either H, halogeno, hydrocarbyl (1-14C) including cyclic and uns~ d hydrocarbyl, optionally substituted with 1 or 2 substitlleMt~ selected from the group con~ ting of halogeno, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino), acyloxy, acylamido and their thio analogs, alkylsulfonyl, alkylphosphonyl, carboxy, alkoxycarbonyl, c~balllyl or alkylc~b~llyl, and wherein the hydrocarbyl can optionally be illlellu~ted by a single ether (-O-) linkage, or wherein Y~ and y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R' or O(POR)R' in which R' is a hydrocarbyl optionally sllbstit~lted as defined above; II. X is NH2, or NHR with R as defined above, n is O, and yl and y2 are as defined in I; III.
X is NH2, n is 1. and yl and y2 are independently either H, hydrocarbyl (7-14C;
~alu~ d or unsaturated), ul~s~ al~d hydrocarbyl (1-6C), either hydrocarbyl substituent being either unsubstituted or substituted with halogen, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino), acyloxy, acylamido and their thio analogs, alkylsulfonyl or alkylphosphonyl, and wherein the hydrocarbyl can `-- 133844~
optionally be i,lle~ t~d by a single ether (-O-) linkage, or wherein yl and y2 are independently either NHR', O(CO)R', O(SO)R', or O(POR)R' in which R' is a hydrocarbyl optionally substituted as defined above.
B. Pr~a~ion of the Compounds of the Invention General methods for prep~ing some 3-amino derivatives are found in the above reference patents to Ley et al.. for example US 3,980,779. The compounds are prepared from bel.~or~uoxan of the formula:
N
~J~N
by reaction with a salt of cyanamide, followed by acidification of the reaction mixture. The benzofuroxan starting material is not symmetric with respect to itsown 5 and 6 positions (which are the 6 and 7 positions of the resulting 3-amino bel.~olliazine oxide). Therefore, a ~llixLule of the 6- and 7-substituted m~tPri~l~
may result. If desired, this mixlule can be sepa~dled using conventional means into individual components having a substituent in either the 6 or 7 position.
The dioxide may also be prepared from the parent monoxide by peracid oxidation (see Robbins et al. J Chem Soc 3186 (1975) and Mason et al. J Chem Soc B 911 (1970)).
In addition, the monoxide may be prepared by: (1) cyclization of a l-nitro-2-aminobenzene compound using H2NCN, (2) oxidation of the parent compound given by the structure.
or by controlled reduction of the corresponding dioxide (see Mason, ~E~, and Wolf et al. J Am Chem Soc 76:355 (1954)).
X
-11- 1338~
3-amino-1,2,4-bel~o~ 7ines may be prepared either by cyclization of a parent compound (see Scheme I and Arndt. Chem. Ber. 3522 (1913)) or by reduction of the monoxide or dioxide as above.
The 3-hydroxy-1,2,4-benzotriazine oxides may be prepared using peroxide and tllng~t~n oxide (Scheme II), a novel synthetic procedure for m~king the 3-hydroxy-1,4-dioxide compound, or concentrated sulfuric acid and sodium nitrate (Scheme III).
NH2 I;Me ~ > ~O~
~N-C-N-N~NH N 2 ~;ct~e~e I
o o Y--~H N~2~ 2H2 ~--OH
~50C O
Sche~e I I
o y2~,G, ~, 4 > y2~ N j~ OH
Scheme III
1~38445 C. Formulation and Administration As demonstrated below, the oxidized bcllzo~ 7inee of the invention may be used to radiosensitize or selectively kill hypoxic tumor cells in warm-blooded animal hosts. A way in which they may be used is in conjunction with agents known to selectively create hypoxia in tumors. Such methods include the use of antihypertensive drugs such as hydral~ine, or agents which affect the amount of oxygen carried by the blood. While these compounds will typically be used in cancer therapy of human patients, they may be used to kill hypoxic tumor cells in other warm blooded animal species such as other prim~tes, farm ~nim~le such as cattle, and sports ~nim~le and pets such as horses, dogs, and cats.
Hypoxia is believed to be associated with all types of solid malignant neoplasms. The compounds of the invention may, therefore, be used to radiosen.eiti7e or to kill neoplastic epithelial cells, endothelial cells, connective tissue cells, bone cells, muscle cells, nerve cells, and brain cells. Examples of carcinomas and sarcomas include carcinomas such as epithelial cell, acidic cell,alveolar cell, basal cell, basal squamous cell, cervical, renal, liver, Hurthle, Lucke, mucinous and Walker, and sarcomas such as Ab~ lhy~s~ alveolar soft part, angiolithic, botyroid, encephaloid, endometria stroma, Ewing's fascicular, giantcell, lymphatic, Jensen's, juxtacortical osteogenic, Kaposi's medullary, and synovial. Specific examples of tumors that have been sensitized with other radiosensitizers are reported in Adams, G.E., Cancer: A Comprehensive Treatise (F. Becker, Ed) vol. 6, pp. 181-223, Plenum, New York, 1977.
The compounds may be ~llminict~red to patients orally or palclll~lally (hllldvellously, subcutaneously, inlld~llus~iularly~ hlll~illally, hl~apel;loneally, and the like). When ~lmini~t~red p~clll~ldlly the compounds will normally be form~ tecl in a unit dosage injectable form (solution, suspension, emulsion) with a ph~rm~ceutically acceptable vehicle. Such vehicles are typically nontoxic and nontherapeutic. Examples of such vehicles are water, aqueous vehicles such as saline. Ringer's solution, dextrose solution, and Hank's solution and nonaqueousvehicles such as fixed oils (e.g., corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate. Sterile saline is a plcrelled vehicle and the compounds are sufficiently water soluble to provide a solution for all foreseeable -13- 13384~5 needs. The vehicle may contain minor amounts of additives such as substances that enhance solubility, isotonicity, and chemical stability, e.g., antioxi(l~nt~, buffers, and presel~lives. When ~(1miniqt~red orally (or rectally) the compoundswill usually be formulated into a unit dosage form such as a tablet, capsule, suppository or cachet. Such formulations typically include a solid, semisolid orliquid carrier or diluent. Exemplary diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, ~gin~tes, tr~g~nth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxyben70~te, propyl hydroxyben_oate, talc, and m~gneqium stearate.
The amount of compound ~-lmini~t~red to the subject is sufficient to radiosen.qiti7e or to produce cytotoxicity in the m~lign~nt neoplasm to be treated but below that which may elicit toxic effects. This amount will depend upon the type of tumor, the species of the subject being treated, the indication dosage inten(led and the weight or body surface of the subject. The radiation may be ~lminiqtered to hllm~nq in a variety of dirr~ fractionation regimes, i.e., the total radiation dose is given in portions over a period of several days to several weeks.
These are most likely to vary from daily (i.e., five times per week) doses for up to six weeks, to once weekly doses for four to six weeks. An individual dose of the ben_otria_ine will be given before or after each radiation treatment and is likely to be in the range of 0.01 to 20 mmoVkg and usually in the range of 0.1 to 2 mmoVkg.
For use as selective cytotoxic agents, the compounds of the invention can be ~rlminiqtered alone, with radiation or other cancer cytotoxic agents, with vasoactive drugs (e.g. hydralazine), or, with procedures which reduce the amountof available oxygen carried by the blood such as anemia or drugs which increase the binding of oxygen to hemoglobin, all of which can enhance selectively the degree of hypoxia in the tumor. As noted above, while all of the compounds encompassed by Formula 1 are generally useful as radiosensitizers herein, only those compounds which are (sul~slilul~d or unsubstituted) 3-amino-1,2,4-benzotriazine 1,4-dioxides (i.e., X=NH2, NHR or NR2 with R as defined above and n is 1) are useful as selective cytotoxic agents.
~ `
-14- 13381~5 Examples The following examples further illustrate the compounds of the invention and methods for synthesi~ing using them, and are not intended to limit the invention in any manner.
Example 1: Pl~aLdlion of 3-Hydroxy-1,24-Benzotriazine 1,4-Dioxide o t ~N ~ N
~l A stirred ~ixlule of 1.50 g (9.25 mmole) of 3-amino-1,2,4-benzotriazine 1-oxide (V, 100.0 ml acetic acid and 30.0 ml of 30% hydrogen peroxide was treated with 3.05 g (9.25 mmole) of Na2WO4 2H2O. The mixlule was stirred in an oil bath at 60C for 4 days. The yellowish orange lllixlule was cooled to about 30 and filtered to remove a light yellow non-W absorbing solid that was presumably tungstic acid. The orange solution of hydrogen peroxide in acetic acid was evaporated to semi-dryness carefully with several additions of water and acetic acid to remove most of the peroxide. The concentrated solution was allowed to stand at room lelll~eldlul~, to afford four crops of an orange solid, 0.87 g (42%
yield of the sodium salt of ;~). W,na,~ (20% CH30H/H20): 262.2 ( 39,460): 477 ( 7,030). IR (neat): 353011, 3150~1, 2650~, 2180,u and 163511. Anal. (calculated for the sodium salt): C7H4N303 Na 1.25H2O, 223.64: C.37.6; H,2.93; N,18.79.
Found: C,37.8; H,2.75; N,18.65.
`~ -15- 133~445 Example 2: Pr~aldlion of 3-Amino-7-Trifluoromethyl-1,2~4-Benzotriazine 1-Oxide:
~3c ~Q~2 A solution of Na (1.13g, 49.2 mmole) in ethanol (50 ml) was added to a solution of gu~ni(line hydrochloride (4.93g, 51.6 mmole) in ethanol (50 ml). After 1 h, the ~ luie was filtered and the filtrate was combined with a solution of 4-chloro-3-nitro-bellzoll;fluoride (Aldrich, 5.5g, 24.4 mmole) in ethanol (25 ml).The llli~lul~ was stirred and refluxed for 5 h, cooled at 0-5C, and the plecipildled solid collected. The solid was washed with water and ethanol and air-dried to give 0.48 g (9%) of 3 as a light yellow solid, mp>300C. TLC: Rf 0.60 (9:1 methylene chloride: methanol on silica gel plates). Mass. Spec.: M+=230 (q = 100).
Example 3: P~ep~dlion of 3-Amino-7-Decyl-1~2.4-Benzotriazine l-Oxide ClOH21 ~[~0~ H2 P~epaLalion of a 4-(1-decyl)-2-nitroaniline: Acetic anhydride (400 ml) was added over a 30-minute period to a stirred solution of 4-decylaniline (Aldrich, 80g, 0.34 mole) in he~n~s (2.41). After stirring for lh, the mixture was cooled and treated over 30 min. at 5-10C with 70% nitric acid (34 ml.). Stirring was continue~l at 5-10C for lh and at 25C for 16h. The llli~lule was diluted with H2O (11), stirred for 5h, poured into an open dish and allowed to stand for 16h.After further dilution with H20 (1.51), the solid was collected and recryst~lli7e~1 from an 85% ethanol solution (in water) to give 92g (84%) of the intermediate as Q~
an orange solid, m.p. 64C.
A solution (100 ml) of 85% KOH (19 g, 0.288 mole) in H2O was combined with a suspension of 4-(1-decyl)-2-nitroaniline (89g, 0.28 mole), plepa~ed above, in methanol (900 ml). The llliXLUlC was stirred for 6h, neutralized to pH 7-8 with concentrated HCl, and evaporated in vacuo to near dryness. After dilution with H2O (400 ml), the solid was collected and air-dried to give 77g (100%) of the intermediate as an orange solid, mp 59C.
1.0g (8.7 mmole) of chloroamidine hydrochloride (previously prepaled for use by treating an ether solution of cyanamide with HCl gas and collecting the precipitated solid) was added portionwise over 10 min to a preheated melt (190C) of 4-(1-decyl)-2-nitroaniline prepared in the preceding step (500 mg, 1.8 mmole).
The reaction lllixLu~e was heated at 190C for S min, cooled to 25C, treated with 6N KOH (10 ml), and heated at 90-95C for lh. After cooling to 25C, the solid was collected, washed with H2O and ethanol and air-dried to give 0.25g (46%) of compound _ as a light yellow solid, m.p. 177C (dec). Mass. spec. M+=285 (q=100), 302 (q=13).
Example 4: Plepalalion of 3-Amino-7-Ca~ llyl-1~2~4-Benzotriazine 1-Oxide o ~
Plep~Lion of 4-chloro-3-nitrobe~ide:
20.2g (0.1 mole) of 4-chloro-3-nitrobenzoic acid (Aldrich) and thionyl chloride (20 ml) were combined, allowed to stand for 16h, and refluxed for 4h to give a clearred solution. The solution was evaporated in vacuo and azeotroped with benzene.
The residue was dissolved in acetonitrile (20 ml) and added over 30 min to cold (-10C) concellLldled ammonium hydroxide (100 ml). After 3h at -10C and 16h at 25C the lllixlule was poured into an open dish and allowed to evaporate to dryness. The residue was slurried in H20 and the solid was collected and air-dried to give 19.8g (98%) of the intermediate as a light yellow solid, m.p. 153C.
A solution of BNa (3.45g, 0.15 mole) in ethanol (75 ml) was added to a solution of quanidine hydrochloride (15.8g, 0.165 mole) in ethanol (75 ml). After lh the llliXlUle was filtered and the filtrate was combined with a suspension of 4-chloro-3-nitrobenzamide (lOg, 0.05 mole) pl~ared above, in ethanol (50 ml). The ixlule was stirred and refluxed for 16h, cooled to 0-5C, and acidified with concentrated HCl (8 ml). The collected solid was combined with K2CO3 (28g, 0.2 mole) and H20 (40 ml) and the llliX~ , was stirred and heated at 100C for 8h.
After cooling to 25C, the solid was collected, washed with H20, and air-dried.
The solid was suspended in boiling ethyl acetate, collected and washed with hot ethyl acetate. The solid was repeatedly suspended in boiling dioxane and collected (6xlOOml). The combined filtrate was e~oldl~d in vacuo to a solid. The solid was suspended in 95% ethanol, collected and air-dried to give 0.44g (4.3%) of compound 5 as a light yellow solid, m.p.>300C. TLC: Rf=0.23 (methylene chloride: acetone of 2:1, silica gel plates). Mass. Spec.: M+ 205 (q= 100).
Example 5: Pl~aldlion of 7-acetyl-3-Amino-1,2,4-Benzotriazine l-Oxide Oxime NOH
A combined mixlule of 7-acetyl-3-amino-1,2,4benzotriazine l-oxide (~r~aled in Example 5; 50 mg, 0.25 mmole), hydroxylamine hydrochloride (200 mg, 2.88 mole), pyridine (1 ml), and ethanol (1 ml) was heated at 90-95C for lhand then cooled to 25C. The lllixlule was diluted with 95% ethanol (S ml) and the solid was collected and air-dried to give 30 mg (56%) of compound 6 as a light yellow solid, m.p. 278C (dec). TLC: Rf=0.60 (9:1 methylene chloride:
methanol). Mass. Spec.: M+=219 (q=100).
q~
~_ 1338445 Example 6: pl~al~lion of 3-Amino-6(7)-Decyl-1~2,4-Benzotriazine 1~4-Dioxide t ~ !O~i -5-(1-decyl)-benzofuroxan: A combined lllixlule Ol ~-(1-decyl)-2-nitroaniline (77g, 0.28 mole), 5.25% NaOCl in H2O (476g, 0.34 mole), 85% KOH
(20.3g, 0.31 mole), Bn4NHSO4 (4.7g, 0.014 mole), and CH2Cl2 (2.28 1) was stirredrapidly for 6h and diluted with H2O (500 ml) and Ch2Cl2 (1 1). The separated organic phase was washed successively with lN HC1 (1 1) and brine (2 x 1 1)), dried (Na2SO4), and concenlldled in vacuo to yield a red oil, 70 g (92%).
A solution of 5-(1-Decyl)-belL~oruloxall as prepared above (10 g, 0.036 mole) and benzyltriethylammonium chloride (0.36 g, 0.0016 mole) in DMSO (1180 ml) was treated gradually over several hours with cyanamide (13.0 g, 0.31 mole) and K2CO3 (36.8 g, 0.27 mole). The lllixlure was stirred for 48h and filtered. The filtrate was diluted with H2O (6 1) and glacial acetic acid (40 ml) and extracted with CH2Cl2 (4 x 500 ml). The combined organic solution was washed s~lcces~ively with 5% NaHCO3 solution (1 x 500 ml) and brine (2 x 500 ml), dried(Na2SO4), and evaporated in vacuo to dryness. The crude product was purified by chromatography on silica gel using CH2Cl2: methanol (98:2) to give 1.8g (16%) of compound 7 as a red solid, m.p. 155C (dec.) Mass. Spec.: N+=318 (q=4), 285 (q=100).
Example 7: Pl~al~lion of 7-Chloro-3-HydroxY- 1 ~2,4-Benzotriazine 1.4-Dioxide o Cl ~N~ 8202 ;N t 8 ~N~ff ~NlNH
9 0 1o 0 ~ 133844~
A nlixlule of l.SOg (7.63 mole) of 8 in 100 ml acetic acid was treated with 2.52 g (7.63 mmole) of Na2WO4 2H2O and 30 ml of 30% H2O2. The nlixlure was stirred and heated for 6 days at 50C, then slowly evaporated to dryness to remove H2O2. The residue was boiled in 250 ml H2O and filtered to remove about 25 mg of starting material 8. The aqueous solutions were then extracted with 2 x 250 ml portions of ethyl acetate. A deep red crystalline m~t~ri~l that was characterized as 10 by TLC and Mass. Spec. analysis formed in the partitioning mixture above and was collected by filtration to afford 60.0 mg of a yellowish orange solid (3.7%
yield), characterized as follows as ~Q which showed good solubility in a mixluleof hot isopropyl alcohol and water. Mass. Spec.: M+=212 (q=lOO)(compound ~):
TLC: Rf=0.34 (acetone, silica gel plates).
The ethyl acetate solutions above, separated from the H2O layer after the filtration to remove ~, were evaporated to dryness. The residue was then treatedwith isopropyl alcohol at room telll~.dlure to afford a dull orange solid, 0.41g(25% yield) of 2. Mass. Spec.: M+=213 (q=70); TLC: Rf=0.22 (acetone, silica gel plates). Compound 9 was char~ct~ri7~d as the ammonium salt, C7H4ClN303 NH3, m.w. 230.61, as follows. The free acid_ was dissolved in concentrated NH40H
and then chilled in ice and filtered to remove a trace of insoluble 10. The red filtrate and washings were evaporated to dryness, leaving a reddish-orange solid.
The solid was treated with 50 ml of boiling 1,2-dimethoxyethane, collected on a filter and washed with an additional 25 ml of hot 1,2-dimethyl ether. The solid was dried over P2O5 at 56C/1.0 mm, leaving 0.244 g (87% yield) of 11 Anal.
t Cl ~N~
~O N
i Calcd. for C7H4ClN303 NH3 (230.61): C, 35.6: H, 3.06: N, 24.30. Found: C, 1338~4~
36.5; H, 3.07; N, 23.94. W"""~ (H20): 219 (~ 12,580); 265.4 (~ 40,000); 4830486 6,640) Example 8: In Vivo Assay for Activity in Combination with Radiation The compound of the invention were tested Ln vivo for activity by the assay of Brown, J.M., Radiation Res (1975) 64:633-47. For this assay, SCCVII
carcinomas in female C3H mice weighing 20-25 g were used. These mice were bred under specific pathogen-free conditions and were 3-4 months old at the be~inning of each eXperim~nt The SCVII tumor was grown intr~derm~lly in the flank from an inoculation of 2 x 105 tumor cells taken from the 2nd-8th i vito passage of the tumor cells after removal from the previous i vivo tumor. Two tumors per mouse were implanted, and were used as subject tumors when they reached a volume of approximately 100 ml. At this point the tumors contained approximately 20% hypoxic cells.
The test compound was tested at a fixed injected dose of either 5 mmol/kg or 2/3 of the LD50 (whichever is lower). Suitable controls of test compound-injected but nonirradiated and saline-injected and irradiated mice were also included. A fixed radiation dose of 20 Gy was applied at variable intervals of 2hr after to 3 hr before injection of the drug. By using these intervals, the results give an indication of both the o~ ll irradiation time and the extent of extra cell killing compared to radiation alone. The result of such time-course ~x~elilllents using 3-amino-1,2,4-benzotriazine 1,4-dioxide are shown in Figure 2. They show enhanced cell killing colllpared to radiation only, more than would have been expected on the basis of additivity of the two individual cytotoxicities. The similar increased cyl()l()~icity when the drug is given before or after radiation indicates selective toxicity to the hypoxic cells rather than a radiosensitizing effect of the benzotriazine dioxide.
Irradiation of the SCVII tumors was done by irrarli~ting nonanaesthetized tumor-bearing mice in a Plexiglas box. Irradiation conditions were 250 kVp X-rays, 15 mA, FSC 33 cm, added filtration of 0.35 mm Cu, half value layer 1.3 mm Cu, and a dose rate of 317 rad/min.
The amount of cell killing was judged by survival rate of dissected and ~ 1338445 cultured tumor cells as follows. The tumor-bearing mice were killed 24 hr after irradiation, and tumors were ~ .sected from the skin, cut into several pieces, and made into a fine brei by high speed chopping with a razor blade attached to a jigsaw. The brei was added to 30 ml of Hank's buffered salt solution (HBSS) co~ il-g 0.02% DNase, 0.05% promase, and 0.02% collagenase. The suspension was stirred for 30 min at 37C, filtered, and centrifuged at 1,600 rmp for 10 min at 4C. The cell pellet was resuspended in complete Waymouth's medium plus 15% fetal calf serum (FCS) and an aliquot mixed with trypan blue and counted with the use of a hemacytometer. Suitable dilutions of this serum plated into 60-or 100-mm poly~ly~ene petri dishes (Lux Scientific Corp) in 5 or 15 ml of medium. After incubation for 13 days, the colonies were fixed and stained, and those co~ 50 cells or more were counted. The dilution yielding an average count of 25-100 colonies in a 60 mm dish was used in calculation of results.
Example 9: C,vtotoxicit,v Tests Cytotoxicity tests were carried out using 3-amino-1,2-bel~olliazine 1,4-dioxide and a variety of aerobic and hypoxic cells in culture (human, mouse, andh~m~ter). The cells in spinner flasks were gassed for one hour at 3rC with either air or nitrogen co,,~ g 5% CO2 prior to adding the specified amounts of the drug. Figures lA, lB and lC show the results for cell survival of mouse, hamsterand human cells at various concentrations of 3-amino-1,2,4-bel,~oll;azine 1,4-dioxide. It was found that only 1 to 2% of the drug concentration under aerobic conditions was required to get equal cell killing under hypoxia. This ratio of selective hypoxic toxicity (50-100) is higher than that for any compound so far reported in the lil~alule.
Example 10: Delr. "~i"~tion of LD,o LD50 is determined in BALB/c female mice (weighing 20-25 g) following hllld~l;loneal (ip) injection, unless the compound tested has low lipophilicity and is very soluble, wherein h~l~dvellous (iv) ~minictration is used. LD50 values at 1, 2, 5, and 60 days are ~let~rmined by ~-lmini~tering graded doses of the drug dissolved in physiological saline immediately prior to injection.
Example 11: Radiosensitivity inVitro The results of assays to clet.ormine the concentration of drug necessary to produce a sensili~l enhancement ratio of hypoxic cells in culture of 1.6 are as follows:
Compound C, 6 (mM) 7-chloro-3-amino-1,2,4-benzotriazine 1-oxide 3.3 6(7)-methoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide ~1.0 3-hydroxy-1,2,4-benzotriazine 1,4-dioxide ~2.0 Modifications of the above described modes for carrying out the invention that are apparent to those of skill in the chemical, ph~ eutical, medical, and related arts are intended to be within the scope of the following claims.
Example 12: Enhanced Tumor Cell Toxicity Usin~ Hydralazine Hydralazine is an antihypertensive drug which acts by relaxing the smooth muscle around blood vessels. This has the effect of preferentially ~hlmting blood flow into normal tissues and away from tumors, which process produces immediate hypoxia in the tumors. If 3-amino-1,2,4-benzotriazine 1,4-dioxide is given in conjunction with this agent, there is a massive increase in tumor cell killing. In this experiment, neither hydralazine nor the aforementioned benzotriazine compound produced any significant cell killing in the SCCVII tumor, whereas the combination of the two reduced survival by a factor of 103 (i.e., only 1 cell inevery 1000 was left viable). The e~,;mental procedures are the same as described in Example 8, and the results are shown in Figure 3.
Modes of Carryin~ Out the Invention A. The Compounds Useful in the Invention The compounds useful in radiosensitizing hypoxic tumor cells as described herein are derivatives of 1,2,4-bel~ulliazine oxide.
The hydrocarbyl group re~lesenled by yl or y2 may contain 1-14 carbon atoms, may be saturated or unsdluldl~d, cyclic or acyclic, and may optionally beinterrupted by a single ether linkage. Thus, the unsubstituted form of yl and y2can be, for example, methyl, ethyl, n-propyl, s-butyl, n-hexyl, 2-methyl-n-pentyl, 2-ethoxyethyl, 3-(n-propoxy)-n-propyl, 4-methoxybutyl, cyclohexyl, tetrahydrorulrul~l, rulrul~l, cyclohexenyl, 3-(n-decyloxy)-n-propyl, 4-methyloctyl, 4,7-dimethyloctyl, and the like.
The hydrocarbyl may be substituted with one or two substituents as follows:
The halogeno substituents are fluoro, chloro, bromo, or iodo. The alkoxy substituents leplcsell~d by OR' may contain 1 to 4 carbon atoms, and include, for example, methoxy, n-propoxy, and t-butoxy. The amino substituent may be NH2, NHR or NR2, where each R in independently and alkyl of 1-4 carbons or a morpholino moiety. R may optionally be substituted with 1-2 hydroxy, alkoxy, amino, or halogeno substi~ent.~.
The acyloxy and acylamido groups are represented by R'COO- and R'CONH-, re~e~ ely, where R' contains 1-4 carbons, and their thio analogs are represented by R'CSO- and R'CSNH-. Alkyl sulfonyl and alkyl phosphonyl are, respectively, R'SO2 and R'P(OP')O- wherein each R' is independently as above defined. Carboxy is the group -C(O)OH; alkoxycarbonyl is -C(O)OR'; ca,b~llyl is -C(O)NH2; and alkylc~b~llyl is -C(O)NHR'.
Where X is OH, of course, the compounds may also be ~lepaled and used as the ph~rm~çeutically acceptable salts formed from inorganic bases, such as sodium, potassium, or calcium hydroxide, or from organic bases, such as caffeine, ethylamine, and lysine.
When X is NH2, ph~rm~celltically acceptable acid addition salts may be used. These salts are those with inorganic acids such as hydrochloric, hydrobromic or phosphoric acids or organic acids such as acetic acid, pyruvic acid, succinicacid, mandelic acid, p-toluene sulfonic acid, and so forth. (Amino substituents on the hydrocarbyl side chain can also, of course, be converted to salts).
The 1,2,4-benzotriazine may be used as the mono- or dioxide. Either the 1-nitrogen of the triazino ring may be oxidiæd, or both the 1- and 4-nitrogens may be oxidized.
Specific particularly p-~,r~ d compounds which are useful in the radiosensitization and cytotoxic procedures of the invention include 3-hydroxy-1,2,4-benzoll;azine 1-oxide;
3-hydroxy-1,2,4-bel.~.otl;azine 1,4-dioxide;
3-amino-1,2,4-bel~oll;azine 1-oxide;
3 -amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-methoxy-3-hydroxy- 1 ,2,4-be.lz~ ;azine 1 -oxide;
6(7)-methoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-methoxy-3-amino-1,2,4-bel,~.oll;azine 1-oxide;
6(7)-methoxy-3-amino-1,2,4-bellzotl;azine 1,4-dioxide;
6(7)-ethoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-ethoxy-3-hydroxy-1,2,4-belwtl;azine 1,4-dioxide;
6(7)-ethoxy-3-amino-1,2,4-belwll;~zine 1-oxide;
6(7)-ethoxy-3-amino-1,2,4-bellzull;azine 1,4-dioxide;
6(7)-[4-~cet~mido-n-butanoxy]-3-hydroxy-1,2,4-belwll;azine 1,-oxide;
6(7)- [4-~cet~mido-n-butanoxy] -3 -hydroxy- 1 ,2,4-benzull ;a~ e 1 ,4-dioxide;
6(7)-[4-~r.et~mido-n-butanoxy]-3-amino- 1 ,2,4-bellGull;azine 1 ,-oxide;
6(7)-[4-acetamido-n-butanoxy]-3-amino-1,2,4-bel~o~liazine 1,4-dioxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-hyd~oxy-1,2,4-benzotriazine 1-oxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-hydroxy-1,2,4-bel.~olliazine 1,4-dioxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-amino-1,2,4-belwlliazine 1-oxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-amino-1,2,4-bel~olliazine 1,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[(2-furyl)methylamino]-3-hydroxy- 1 ,2,4-bel,~olliazine 1 ,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-amino-1,2,4-bel~oll;azine l-oxide;
6(7)-[(2-furyl)methylamino]-3-amino-1,2,4-bell~olliazine 1,4-dioxide;
6(7)-(2-methoxyethylamino)-3-hydroxy- 1 ,2,4-bel~ulliazine 1 -oxide;
6(7)-(2-methoxyethylamino)-3-hydroxy- 1 ,2,4-bel,70lliazine 1 ,4-dioxide;
6(7)-(2-methoxyethylamino)-3-amino- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-carbethoxymethoxy-3-hydroxy-1,2,4-bel,zolliazine l-oxide;
6(7)-carbethoxymethoxy-3-hydlo~y-1,2,4-bell~olliazine 1,4-dioxide;
6(7)-carbethoxymethoxy-3-amino- 1 ,2,4-benzolliazine 1 -oxide;
6(7)-carbethoxymethoxy-3-amino- 1 ,2,4-bel~ll iazine 1 ,4-dioxide;
6(7)-[(2-methoxyethyl)c~b~llyllllethoxy]-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-[(2-methoxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[(2-methoxyethyl)c~balllyllllethoxy]-3-amino-1,2,4-bell~olliazine l-oxide;
6(7)-[(2-methoxyethyl)c~b~llyllllethoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)c~b~llyllllethoxy]-3-hydroxy-1,2,4-benzotriazine l-oxide;
6(7)- [(2-hydroxyethyl)carbamylmethoxy] -3 -hydroxy- l ,2 ,4-benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)c~balllyllllethoxy]-3-amino-1,2,4-bell~olliazine l-oxide;
6(7)-[(2-hydroxyethyl)c~l,~llyLllethoxy]-3-amino-1,2,4-bell~olliazine 1,4-dioxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-hydroxy-1,2,4-bell~o~liazine l-oxide;6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-hydroxy-1,2,4-benGolliazine 1,4-dioxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-amino-1,2,4-bell~olliazine l-oxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)- [3 -amino-n-propoxy] -3 -hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-[3-amino-n-propoxy]-3-hydroxy-1,2,4-bel,~olliazine 1,4-dioxide;
6(7)-[3-amino-n-propoxy]-3-amino-1,2,4-bel~olliazine l-oxide;
6(7)-[3-amino-n-propoxy]-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-[2,3-epo~y~iopoxy]-3-hydroxy-1,2,4-benzotriazine l-oxide;
~ 13384~
6(7)-[2,3-epoxypropoxy]-3-hydroxy-1,2,4-be~ iazine 1,4-dioxide;
6(7)- [2,3 -epoxypropoxy] -3 -amino- 1 ,2,4-bel, ~oll i~ine 1 -oxide;
6(7)-[2,3-epoxypropoxy]-3-amino-1,2,4-bell~ulli~zine 1,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-hydroxy-1,2,4-bel~oll;æine 1-oxide;
6(7)- [3 -methoxy-2-hydroxy-n-propoxy]-3-hydroxy- 1 ,2,4-be,~ol, iazine 1 ,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-amino- 1 ,2~4-bell~oll;azine 1 -oxide;
6(7)- [3 -methoxy-2-hydroxy-n-propoxy]-3 -amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-bell~olliazine 1-oxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-amino-1,2,4-bell~olliazine 1-oxide;
6(7)-[4-ethoxy-3-hydroxy-n-butoxy]-3-amino-1,2,4-benzol~;azine 1,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-hydroxy- 1 ,2,4-bel~ol,;azine 1 -oxide;
6(7)-[3 ,4-dihydroxy-n-butoxy]-3-hydroxy- 1 ,2,4-bel, ~oll;azine 1 ,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-bellzolliazine 1-oxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-bel~lliazine 1,4-dioxide;
6(7)-methyl-3-hydroxy-1,2,4-bell~oll;azine 1-oxide;
6(7)-methyl-3-hydroxy-1,2,4-benzol,;azine 1,4-dioxide;
6(7)-methyl-3-arnino-1,2,4-bel~zol,;azine 1-oxide;
6(7)-methyl-3-arnino-1,2,4-bel~utl;azine 1,4-dioxide;
6(7)-ethyl-3-hydroxy-1,2,4-bel,70ll;azine 1-oxide;
6(7)-ethyl-3-hydroxy- 1 ,2,4-be~ ~ulli~7ine 1 ,4-dioxide;
6(7)-ethyl-3-amino- 1 ,2,4-bell~oll;azine 1 -oxide;
6(7)-ethyl-3-amino- 1 ,2,4-bel~ol, ;azine 1 ,4-dioxide;
6(7)-chloro~cet~mido-3-hydroxy-1,2,4-be~ iazine 1-oxide;
6(7)-chloro~cet~mido-3-hyd~ y-1,2,4-be~oLI;azine 1,4-dioxide;
6(7)-chloroacetamido-3-amino-1,2,4-bel~oll;azine 1-oxide;
6(7)-chloro~cet~mido-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-[(2-hydroxyethyloxy)acet~mido]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[(2-hydroxyethyloxy)~cet~mido]-3-hydroxy-1,2,4-belL~oll;azine 1,4-dioxide;
6(7)-[(2-hy-ilo~y~lhyloxy)~cet~mido]-3-amino-1,2,4-bel.~oll;azine 1-oxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6,7-dimethoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6,7-dimethoxy-3-hydroxy-1,2,4-bel.~oll;azine 1,4-dioxide;
6,7-dimethoxy-3-amino-1 ,2,4-be,, ~oLl;æine 1-oxide;
6,7-dimethoxy-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6,7-diethoxy-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6,7-diethoxy-3-hydroxy- 1 ,2,4-bel~ull;azine 1 ,4-dioxide;
6,7-diethoxy-3-amino- 1 ,2,4-benzotriazine 1 -oxide;
6,7-diethoxy-3-amino-1,2,4-bel,~ull;azine 1,4-dioxide;
6(7)-propionyl-3 -hydroxy- 1 ,2,4-benzotriæine 1 -oxide;
6(7)-propionyl-3-hydroxy- 1 ,2,4-benzotriæine 1 ,4-dioxide;
6(7)-propionyl-3-amino- 1 ,2,4-benzotriæine 1 -oxide;
6(7)-propionyl-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-bell70ll;azine 1-oxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-bell~oll;azine 1-oxide;
6(7)-(2-acetoxyethoxy)-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-bel~oll;azine 1-oxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-bel,~oll;azine 1,4-dioxide;
6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-n-hexyloxy-3 -amino- 1 ,2,4-bel~oll ;æine 1 ,4-dioxide;
6(7)-ethylamino-3-hydroxy-1,2,4-bell~oll;azine 1-oxide;
6(7)-ethylamino-3 -hydroxy- 1 ,2,4-bel~oll ;azine 1 ,4-dioxide;
6(7)-ethylamino-3-amino-1,2,4-bel,~oll;æine 1-oxide;
6(7)-ethylamino-3-amino-1,2,4-bel~oll;æine 1,4-dioxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-bell~oll;æine 1-oxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotl;azine 1,4-dioxide;
6(7)-(2-methoxyethoxy)-3-amino-1,2,4-bel.~oll;azine 1-oxide;
6(7)-(2-methoxyethoxy)-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(amino~cet~mido)-3-hydroxy-1,2,4-bel~oll;æine 1-oxide;
6(7)-(amino~cet~mido)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(amino~cet~mido)-3-amino-1,2,4-bell~.oll;æine 1-oxide;
6(7)-(amino~cet~mido)-3-amino-1,2,4-bell~olliæine 1,4-dioxide;
6(7)-(c~l,alllylmethoxy)-3-hydroxy-1,2,4-bell~otl;æine 1-oxide;
13384~5 g 6(7)-(c~l,alllyhllethoxy)-3-hydroxy- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-(call,alllyLllethoxy)-3-amino-1,2,4-bell~ulliazine 1-oxide;
6(7)-(c~l,~llyhllethoxy)-3-amino- 1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-carboxymethoxy)-3-hydroxy- 1 ,2,4-benzotriazine 1 -oxide;
6(7)-carboxymethoxy)-3-hydroxy-1 ,2,4-benzotriazine 1 ,4-dioxide;
6(7)-carboxymethoxy)-3-amino-1,2,4-bel~oll;azine 1-oxide;
6(7)-carboxymethoxy)-3-amino-1,2,4-bellzoll;azine 1,4-dioxide;
and their ph~ ce~ltically acceptable salts and the thioamide analogs of the foregoing list of compounds. It should be noted that the IlYl or y2ll substituents set forth in most of the above compounds as present in either the 6 or 7 positions (-lesign~te.1 "6(7)") or in both the 6 and 7 positions (design~ted "6,7") may also be present at the S and\or 8 ring positions.
Of the above compounds useful in the method of the present invention as selective cytotoxic agents or radiosensitizers, the following compounds are novel:
compounds given by the formula above wherein I, X is OH, OR, or NR2, where each R is independently an alkyl of 1-4 carbon atoms, an amide, or a morpholino moiety and may further be substituted with hydroxy, alkoxy, amino, or halogen substitll~nt~, n is 0 or 1, and Y~ and y2 are independently either H, halogeno, hydrocarbyl (1-14C) including cyclic and uns~ d hydrocarbyl, optionally substituted with 1 or 2 substitlleMt~ selected from the group con~ ting of halogeno, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino), acyloxy, acylamido and their thio analogs, alkylsulfonyl, alkylphosphonyl, carboxy, alkoxycarbonyl, c~balllyl or alkylc~b~llyl, and wherein the hydrocarbyl can optionally be illlellu~ted by a single ether (-O-) linkage, or wherein Y~ and y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R' or O(POR)R' in which R' is a hydrocarbyl optionally sllbstit~lted as defined above; II. X is NH2, or NHR with R as defined above, n is O, and yl and y2 are as defined in I; III.
X is NH2, n is 1. and yl and y2 are independently either H, hydrocarbyl (7-14C;
~alu~ d or unsaturated), ul~s~ al~d hydrocarbyl (1-6C), either hydrocarbyl substituent being either unsubstituted or substituted with halogen, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino), acyloxy, acylamido and their thio analogs, alkylsulfonyl or alkylphosphonyl, and wherein the hydrocarbyl can `-- 133844~
optionally be i,lle~ t~d by a single ether (-O-) linkage, or wherein yl and y2 are independently either NHR', O(CO)R', O(SO)R', or O(POR)R' in which R' is a hydrocarbyl optionally substituted as defined above.
B. Pr~a~ion of the Compounds of the Invention General methods for prep~ing some 3-amino derivatives are found in the above reference patents to Ley et al.. for example US 3,980,779. The compounds are prepared from bel.~or~uoxan of the formula:
N
~J~N
by reaction with a salt of cyanamide, followed by acidification of the reaction mixture. The benzofuroxan starting material is not symmetric with respect to itsown 5 and 6 positions (which are the 6 and 7 positions of the resulting 3-amino bel.~olliazine oxide). Therefore, a ~llixLule of the 6- and 7-substituted m~tPri~l~
may result. If desired, this mixlule can be sepa~dled using conventional means into individual components having a substituent in either the 6 or 7 position.
The dioxide may also be prepared from the parent monoxide by peracid oxidation (see Robbins et al. J Chem Soc 3186 (1975) and Mason et al. J Chem Soc B 911 (1970)).
In addition, the monoxide may be prepared by: (1) cyclization of a l-nitro-2-aminobenzene compound using H2NCN, (2) oxidation of the parent compound given by the structure.
or by controlled reduction of the corresponding dioxide (see Mason, ~E~, and Wolf et al. J Am Chem Soc 76:355 (1954)).
X
-11- 1338~
3-amino-1,2,4-bel~o~ 7ines may be prepared either by cyclization of a parent compound (see Scheme I and Arndt. Chem. Ber. 3522 (1913)) or by reduction of the monoxide or dioxide as above.
The 3-hydroxy-1,2,4-benzotriazine oxides may be prepared using peroxide and tllng~t~n oxide (Scheme II), a novel synthetic procedure for m~king the 3-hydroxy-1,4-dioxide compound, or concentrated sulfuric acid and sodium nitrate (Scheme III).
NH2 I;Me ~ > ~O~
~N-C-N-N~NH N 2 ~;ct~e~e I
o o Y--~H N~2~ 2H2 ~--OH
~50C O
Sche~e I I
o y2~,G, ~, 4 > y2~ N j~ OH
Scheme III
1~38445 C. Formulation and Administration As demonstrated below, the oxidized bcllzo~ 7inee of the invention may be used to radiosensitize or selectively kill hypoxic tumor cells in warm-blooded animal hosts. A way in which they may be used is in conjunction with agents known to selectively create hypoxia in tumors. Such methods include the use of antihypertensive drugs such as hydral~ine, or agents which affect the amount of oxygen carried by the blood. While these compounds will typically be used in cancer therapy of human patients, they may be used to kill hypoxic tumor cells in other warm blooded animal species such as other prim~tes, farm ~nim~le such as cattle, and sports ~nim~le and pets such as horses, dogs, and cats.
Hypoxia is believed to be associated with all types of solid malignant neoplasms. The compounds of the invention may, therefore, be used to radiosen.eiti7e or to kill neoplastic epithelial cells, endothelial cells, connective tissue cells, bone cells, muscle cells, nerve cells, and brain cells. Examples of carcinomas and sarcomas include carcinomas such as epithelial cell, acidic cell,alveolar cell, basal cell, basal squamous cell, cervical, renal, liver, Hurthle, Lucke, mucinous and Walker, and sarcomas such as Ab~ lhy~s~ alveolar soft part, angiolithic, botyroid, encephaloid, endometria stroma, Ewing's fascicular, giantcell, lymphatic, Jensen's, juxtacortical osteogenic, Kaposi's medullary, and synovial. Specific examples of tumors that have been sensitized with other radiosensitizers are reported in Adams, G.E., Cancer: A Comprehensive Treatise (F. Becker, Ed) vol. 6, pp. 181-223, Plenum, New York, 1977.
The compounds may be ~llminict~red to patients orally or palclll~lally (hllldvellously, subcutaneously, inlld~llus~iularly~ hlll~illally, hl~apel;loneally, and the like). When ~lmini~t~red p~clll~ldlly the compounds will normally be form~ tecl in a unit dosage injectable form (solution, suspension, emulsion) with a ph~rm~ceutically acceptable vehicle. Such vehicles are typically nontoxic and nontherapeutic. Examples of such vehicles are water, aqueous vehicles such as saline. Ringer's solution, dextrose solution, and Hank's solution and nonaqueousvehicles such as fixed oils (e.g., corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate. Sterile saline is a plcrelled vehicle and the compounds are sufficiently water soluble to provide a solution for all foreseeable -13- 13384~5 needs. The vehicle may contain minor amounts of additives such as substances that enhance solubility, isotonicity, and chemical stability, e.g., antioxi(l~nt~, buffers, and presel~lives. When ~(1miniqt~red orally (or rectally) the compoundswill usually be formulated into a unit dosage form such as a tablet, capsule, suppository or cachet. Such formulations typically include a solid, semisolid orliquid carrier or diluent. Exemplary diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, ~gin~tes, tr~g~nth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxyben70~te, propyl hydroxyben_oate, talc, and m~gneqium stearate.
The amount of compound ~-lmini~t~red to the subject is sufficient to radiosen.qiti7e or to produce cytotoxicity in the m~lign~nt neoplasm to be treated but below that which may elicit toxic effects. This amount will depend upon the type of tumor, the species of the subject being treated, the indication dosage inten(led and the weight or body surface of the subject. The radiation may be ~lminiqtered to hllm~nq in a variety of dirr~ fractionation regimes, i.e., the total radiation dose is given in portions over a period of several days to several weeks.
These are most likely to vary from daily (i.e., five times per week) doses for up to six weeks, to once weekly doses for four to six weeks. An individual dose of the ben_otria_ine will be given before or after each radiation treatment and is likely to be in the range of 0.01 to 20 mmoVkg and usually in the range of 0.1 to 2 mmoVkg.
For use as selective cytotoxic agents, the compounds of the invention can be ~rlminiqtered alone, with radiation or other cancer cytotoxic agents, with vasoactive drugs (e.g. hydralazine), or, with procedures which reduce the amountof available oxygen carried by the blood such as anemia or drugs which increase the binding of oxygen to hemoglobin, all of which can enhance selectively the degree of hypoxia in the tumor. As noted above, while all of the compounds encompassed by Formula 1 are generally useful as radiosensitizers herein, only those compounds which are (sul~slilul~d or unsubstituted) 3-amino-1,2,4-benzotriazine 1,4-dioxides (i.e., X=NH2, NHR or NR2 with R as defined above and n is 1) are useful as selective cytotoxic agents.
~ `
-14- 13381~5 Examples The following examples further illustrate the compounds of the invention and methods for synthesi~ing using them, and are not intended to limit the invention in any manner.
Example 1: Pl~aLdlion of 3-Hydroxy-1,24-Benzotriazine 1,4-Dioxide o t ~N ~ N
~l A stirred ~ixlule of 1.50 g (9.25 mmole) of 3-amino-1,2,4-benzotriazine 1-oxide (V, 100.0 ml acetic acid and 30.0 ml of 30% hydrogen peroxide was treated with 3.05 g (9.25 mmole) of Na2WO4 2H2O. The mixlule was stirred in an oil bath at 60C for 4 days. The yellowish orange lllixlule was cooled to about 30 and filtered to remove a light yellow non-W absorbing solid that was presumably tungstic acid. The orange solution of hydrogen peroxide in acetic acid was evaporated to semi-dryness carefully with several additions of water and acetic acid to remove most of the peroxide. The concentrated solution was allowed to stand at room lelll~eldlul~, to afford four crops of an orange solid, 0.87 g (42%
yield of the sodium salt of ;~). W,na,~ (20% CH30H/H20): 262.2 ( 39,460): 477 ( 7,030). IR (neat): 353011, 3150~1, 2650~, 2180,u and 163511. Anal. (calculated for the sodium salt): C7H4N303 Na 1.25H2O, 223.64: C.37.6; H,2.93; N,18.79.
Found: C,37.8; H,2.75; N,18.65.
`~ -15- 133~445 Example 2: Pr~aldlion of 3-Amino-7-Trifluoromethyl-1,2~4-Benzotriazine 1-Oxide:
~3c ~Q~2 A solution of Na (1.13g, 49.2 mmole) in ethanol (50 ml) was added to a solution of gu~ni(line hydrochloride (4.93g, 51.6 mmole) in ethanol (50 ml). After 1 h, the ~ luie was filtered and the filtrate was combined with a solution of 4-chloro-3-nitro-bellzoll;fluoride (Aldrich, 5.5g, 24.4 mmole) in ethanol (25 ml).The llli~lul~ was stirred and refluxed for 5 h, cooled at 0-5C, and the plecipildled solid collected. The solid was washed with water and ethanol and air-dried to give 0.48 g (9%) of 3 as a light yellow solid, mp>300C. TLC: Rf 0.60 (9:1 methylene chloride: methanol on silica gel plates). Mass. Spec.: M+=230 (q = 100).
Example 3: P~ep~dlion of 3-Amino-7-Decyl-1~2.4-Benzotriazine l-Oxide ClOH21 ~[~0~ H2 P~epaLalion of a 4-(1-decyl)-2-nitroaniline: Acetic anhydride (400 ml) was added over a 30-minute period to a stirred solution of 4-decylaniline (Aldrich, 80g, 0.34 mole) in he~n~s (2.41). After stirring for lh, the mixture was cooled and treated over 30 min. at 5-10C with 70% nitric acid (34 ml.). Stirring was continue~l at 5-10C for lh and at 25C for 16h. The llli~lule was diluted with H2O (11), stirred for 5h, poured into an open dish and allowed to stand for 16h.After further dilution with H20 (1.51), the solid was collected and recryst~lli7e~1 from an 85% ethanol solution (in water) to give 92g (84%) of the intermediate as Q~
an orange solid, m.p. 64C.
A solution (100 ml) of 85% KOH (19 g, 0.288 mole) in H2O was combined with a suspension of 4-(1-decyl)-2-nitroaniline (89g, 0.28 mole), plepa~ed above, in methanol (900 ml). The llliXLUlC was stirred for 6h, neutralized to pH 7-8 with concentrated HCl, and evaporated in vacuo to near dryness. After dilution with H2O (400 ml), the solid was collected and air-dried to give 77g (100%) of the intermediate as an orange solid, mp 59C.
1.0g (8.7 mmole) of chloroamidine hydrochloride (previously prepaled for use by treating an ether solution of cyanamide with HCl gas and collecting the precipitated solid) was added portionwise over 10 min to a preheated melt (190C) of 4-(1-decyl)-2-nitroaniline prepared in the preceding step (500 mg, 1.8 mmole).
The reaction lllixLu~e was heated at 190C for S min, cooled to 25C, treated with 6N KOH (10 ml), and heated at 90-95C for lh. After cooling to 25C, the solid was collected, washed with H2O and ethanol and air-dried to give 0.25g (46%) of compound _ as a light yellow solid, m.p. 177C (dec). Mass. spec. M+=285 (q=100), 302 (q=13).
Example 4: Plepalalion of 3-Amino-7-Ca~ llyl-1~2~4-Benzotriazine 1-Oxide o ~
Plep~Lion of 4-chloro-3-nitrobe~ide:
20.2g (0.1 mole) of 4-chloro-3-nitrobenzoic acid (Aldrich) and thionyl chloride (20 ml) were combined, allowed to stand for 16h, and refluxed for 4h to give a clearred solution. The solution was evaporated in vacuo and azeotroped with benzene.
The residue was dissolved in acetonitrile (20 ml) and added over 30 min to cold (-10C) concellLldled ammonium hydroxide (100 ml). After 3h at -10C and 16h at 25C the lllixlule was poured into an open dish and allowed to evaporate to dryness. The residue was slurried in H20 and the solid was collected and air-dried to give 19.8g (98%) of the intermediate as a light yellow solid, m.p. 153C.
A solution of BNa (3.45g, 0.15 mole) in ethanol (75 ml) was added to a solution of quanidine hydrochloride (15.8g, 0.165 mole) in ethanol (75 ml). After lh the llliXlUle was filtered and the filtrate was combined with a suspension of 4-chloro-3-nitrobenzamide (lOg, 0.05 mole) pl~ared above, in ethanol (50 ml). The ixlule was stirred and refluxed for 16h, cooled to 0-5C, and acidified with concentrated HCl (8 ml). The collected solid was combined with K2CO3 (28g, 0.2 mole) and H20 (40 ml) and the llliX~ , was stirred and heated at 100C for 8h.
After cooling to 25C, the solid was collected, washed with H20, and air-dried.
The solid was suspended in boiling ethyl acetate, collected and washed with hot ethyl acetate. The solid was repeatedly suspended in boiling dioxane and collected (6xlOOml). The combined filtrate was e~oldl~d in vacuo to a solid. The solid was suspended in 95% ethanol, collected and air-dried to give 0.44g (4.3%) of compound 5 as a light yellow solid, m.p.>300C. TLC: Rf=0.23 (methylene chloride: acetone of 2:1, silica gel plates). Mass. Spec.: M+ 205 (q= 100).
Example 5: Pl~aldlion of 7-acetyl-3-Amino-1,2,4-Benzotriazine l-Oxide Oxime NOH
A combined mixlule of 7-acetyl-3-amino-1,2,4benzotriazine l-oxide (~r~aled in Example 5; 50 mg, 0.25 mmole), hydroxylamine hydrochloride (200 mg, 2.88 mole), pyridine (1 ml), and ethanol (1 ml) was heated at 90-95C for lhand then cooled to 25C. The lllixlule was diluted with 95% ethanol (S ml) and the solid was collected and air-dried to give 30 mg (56%) of compound 6 as a light yellow solid, m.p. 278C (dec). TLC: Rf=0.60 (9:1 methylene chloride:
methanol). Mass. Spec.: M+=219 (q=100).
q~
~_ 1338445 Example 6: pl~al~lion of 3-Amino-6(7)-Decyl-1~2,4-Benzotriazine 1~4-Dioxide t ~ !O~i -5-(1-decyl)-benzofuroxan: A combined lllixlule Ol ~-(1-decyl)-2-nitroaniline (77g, 0.28 mole), 5.25% NaOCl in H2O (476g, 0.34 mole), 85% KOH
(20.3g, 0.31 mole), Bn4NHSO4 (4.7g, 0.014 mole), and CH2Cl2 (2.28 1) was stirredrapidly for 6h and diluted with H2O (500 ml) and Ch2Cl2 (1 1). The separated organic phase was washed successively with lN HC1 (1 1) and brine (2 x 1 1)), dried (Na2SO4), and concenlldled in vacuo to yield a red oil, 70 g (92%).
A solution of 5-(1-Decyl)-belL~oruloxall as prepared above (10 g, 0.036 mole) and benzyltriethylammonium chloride (0.36 g, 0.0016 mole) in DMSO (1180 ml) was treated gradually over several hours with cyanamide (13.0 g, 0.31 mole) and K2CO3 (36.8 g, 0.27 mole). The lllixlure was stirred for 48h and filtered. The filtrate was diluted with H2O (6 1) and glacial acetic acid (40 ml) and extracted with CH2Cl2 (4 x 500 ml). The combined organic solution was washed s~lcces~ively with 5% NaHCO3 solution (1 x 500 ml) and brine (2 x 500 ml), dried(Na2SO4), and evaporated in vacuo to dryness. The crude product was purified by chromatography on silica gel using CH2Cl2: methanol (98:2) to give 1.8g (16%) of compound 7 as a red solid, m.p. 155C (dec.) Mass. Spec.: N+=318 (q=4), 285 (q=100).
Example 7: Pl~al~lion of 7-Chloro-3-HydroxY- 1 ~2,4-Benzotriazine 1.4-Dioxide o Cl ~N~ 8202 ;N t 8 ~N~ff ~NlNH
9 0 1o 0 ~ 133844~
A nlixlule of l.SOg (7.63 mole) of 8 in 100 ml acetic acid was treated with 2.52 g (7.63 mmole) of Na2WO4 2H2O and 30 ml of 30% H2O2. The nlixlure was stirred and heated for 6 days at 50C, then slowly evaporated to dryness to remove H2O2. The residue was boiled in 250 ml H2O and filtered to remove about 25 mg of starting material 8. The aqueous solutions were then extracted with 2 x 250 ml portions of ethyl acetate. A deep red crystalline m~t~ri~l that was characterized as 10 by TLC and Mass. Spec. analysis formed in the partitioning mixture above and was collected by filtration to afford 60.0 mg of a yellowish orange solid (3.7%
yield), characterized as follows as ~Q which showed good solubility in a mixluleof hot isopropyl alcohol and water. Mass. Spec.: M+=212 (q=lOO)(compound ~):
TLC: Rf=0.34 (acetone, silica gel plates).
The ethyl acetate solutions above, separated from the H2O layer after the filtration to remove ~, were evaporated to dryness. The residue was then treatedwith isopropyl alcohol at room telll~.dlure to afford a dull orange solid, 0.41g(25% yield) of 2. Mass. Spec.: M+=213 (q=70); TLC: Rf=0.22 (acetone, silica gel plates). Compound 9 was char~ct~ri7~d as the ammonium salt, C7H4ClN303 NH3, m.w. 230.61, as follows. The free acid_ was dissolved in concentrated NH40H
and then chilled in ice and filtered to remove a trace of insoluble 10. The red filtrate and washings were evaporated to dryness, leaving a reddish-orange solid.
The solid was treated with 50 ml of boiling 1,2-dimethoxyethane, collected on a filter and washed with an additional 25 ml of hot 1,2-dimethyl ether. The solid was dried over P2O5 at 56C/1.0 mm, leaving 0.244 g (87% yield) of 11 Anal.
t Cl ~N~
~O N
i Calcd. for C7H4ClN303 NH3 (230.61): C, 35.6: H, 3.06: N, 24.30. Found: C, 1338~4~
36.5; H, 3.07; N, 23.94. W"""~ (H20): 219 (~ 12,580); 265.4 (~ 40,000); 4830486 6,640) Example 8: In Vivo Assay for Activity in Combination with Radiation The compound of the invention were tested Ln vivo for activity by the assay of Brown, J.M., Radiation Res (1975) 64:633-47. For this assay, SCCVII
carcinomas in female C3H mice weighing 20-25 g were used. These mice were bred under specific pathogen-free conditions and were 3-4 months old at the be~inning of each eXperim~nt The SCVII tumor was grown intr~derm~lly in the flank from an inoculation of 2 x 105 tumor cells taken from the 2nd-8th i vito passage of the tumor cells after removal from the previous i vivo tumor. Two tumors per mouse were implanted, and were used as subject tumors when they reached a volume of approximately 100 ml. At this point the tumors contained approximately 20% hypoxic cells.
The test compound was tested at a fixed injected dose of either 5 mmol/kg or 2/3 of the LD50 (whichever is lower). Suitable controls of test compound-injected but nonirradiated and saline-injected and irradiated mice were also included. A fixed radiation dose of 20 Gy was applied at variable intervals of 2hr after to 3 hr before injection of the drug. By using these intervals, the results give an indication of both the o~ ll irradiation time and the extent of extra cell killing compared to radiation alone. The result of such time-course ~x~elilllents using 3-amino-1,2,4-benzotriazine 1,4-dioxide are shown in Figure 2. They show enhanced cell killing colllpared to radiation only, more than would have been expected on the basis of additivity of the two individual cytotoxicities. The similar increased cyl()l()~icity when the drug is given before or after radiation indicates selective toxicity to the hypoxic cells rather than a radiosensitizing effect of the benzotriazine dioxide.
Irradiation of the SCVII tumors was done by irrarli~ting nonanaesthetized tumor-bearing mice in a Plexiglas box. Irradiation conditions were 250 kVp X-rays, 15 mA, FSC 33 cm, added filtration of 0.35 mm Cu, half value layer 1.3 mm Cu, and a dose rate of 317 rad/min.
The amount of cell killing was judged by survival rate of dissected and ~ 1338445 cultured tumor cells as follows. The tumor-bearing mice were killed 24 hr after irradiation, and tumors were ~ .sected from the skin, cut into several pieces, and made into a fine brei by high speed chopping with a razor blade attached to a jigsaw. The brei was added to 30 ml of Hank's buffered salt solution (HBSS) co~ il-g 0.02% DNase, 0.05% promase, and 0.02% collagenase. The suspension was stirred for 30 min at 37C, filtered, and centrifuged at 1,600 rmp for 10 min at 4C. The cell pellet was resuspended in complete Waymouth's medium plus 15% fetal calf serum (FCS) and an aliquot mixed with trypan blue and counted with the use of a hemacytometer. Suitable dilutions of this serum plated into 60-or 100-mm poly~ly~ene petri dishes (Lux Scientific Corp) in 5 or 15 ml of medium. After incubation for 13 days, the colonies were fixed and stained, and those co~ 50 cells or more were counted. The dilution yielding an average count of 25-100 colonies in a 60 mm dish was used in calculation of results.
Example 9: C,vtotoxicit,v Tests Cytotoxicity tests were carried out using 3-amino-1,2-bel~olliazine 1,4-dioxide and a variety of aerobic and hypoxic cells in culture (human, mouse, andh~m~ter). The cells in spinner flasks were gassed for one hour at 3rC with either air or nitrogen co,,~ g 5% CO2 prior to adding the specified amounts of the drug. Figures lA, lB and lC show the results for cell survival of mouse, hamsterand human cells at various concentrations of 3-amino-1,2,4-bel,~oll;azine 1,4-dioxide. It was found that only 1 to 2% of the drug concentration under aerobic conditions was required to get equal cell killing under hypoxia. This ratio of selective hypoxic toxicity (50-100) is higher than that for any compound so far reported in the lil~alule.
Example 10: Delr. "~i"~tion of LD,o LD50 is determined in BALB/c female mice (weighing 20-25 g) following hllld~l;loneal (ip) injection, unless the compound tested has low lipophilicity and is very soluble, wherein h~l~dvellous (iv) ~minictration is used. LD50 values at 1, 2, 5, and 60 days are ~let~rmined by ~-lmini~tering graded doses of the drug dissolved in physiological saline immediately prior to injection.
Example 11: Radiosensitivity inVitro The results of assays to clet.ormine the concentration of drug necessary to produce a sensili~l enhancement ratio of hypoxic cells in culture of 1.6 are as follows:
Compound C, 6 (mM) 7-chloro-3-amino-1,2,4-benzotriazine 1-oxide 3.3 6(7)-methoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide ~1.0 3-hydroxy-1,2,4-benzotriazine 1,4-dioxide ~2.0 Modifications of the above described modes for carrying out the invention that are apparent to those of skill in the chemical, ph~ eutical, medical, and related arts are intended to be within the scope of the following claims.
Example 12: Enhanced Tumor Cell Toxicity Usin~ Hydralazine Hydralazine is an antihypertensive drug which acts by relaxing the smooth muscle around blood vessels. This has the effect of preferentially ~hlmting blood flow into normal tissues and away from tumors, which process produces immediate hypoxia in the tumors. If 3-amino-1,2,4-benzotriazine 1,4-dioxide is given in conjunction with this agent, there is a massive increase in tumor cell killing. In this experiment, neither hydralazine nor the aforementioned benzotriazine compound produced any significant cell killing in the SCCVII tumor, whereas the combination of the two reduced survival by a factor of 103 (i.e., only 1 cell inevery 1000 was left viable). The e~,;mental procedures are the same as described in Example 8, and the results are shown in Figure 3.
Claims (15)
1. A use of a pharmaceutical composition comprising a compound of the formula where X is NH2, NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms or an amide, or wherein in the case of NRR the two R groups may be linked together to form a morpholino ring, and wherein R may be further substituted with OH, alkoxy, amino or halogen substituents;
n is 1; and Y1 and Y2 are independently either H; halogen; hydrocarbyl (1-14C) comprising cyclic and unsaturated hydrocarbyl, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy, alkylthio, amino morpholino, acyloxy, acylamino and thio analogs thereof, carboxy, alkoxycarbonyl, carbamyl, alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above or a pharmaceutically acceptable salt of said compound;
for selectively killing hypoxic tumor cells.
n is 1; and Y1 and Y2 are independently either H; halogen; hydrocarbyl (1-14C) comprising cyclic and unsaturated hydrocarbyl, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy, alkylthio, amino morpholino, acyloxy, acylamino and thio analogs thereof, carboxy, alkoxycarbonyl, carbamyl, alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above or a pharmaceutically acceptable salt of said compound;
for selectively killing hypoxic tumor cells.
2. The use of claim 1, wherein X is NH2.
3. The use of claims 1 or 2, wherein Y1 and Y2 are both H.
4. A use of a pharmaceutical composition comprising a compound of the formula:
wherein X is halogen, OH, alkoxy (1-4C), NH2, NHR or NRR, wherein each R is independently an alkyl of 1-4 carbon atoms or an amide, or wherein in the case of NRR, the two R groups may be linked together to form a morpholino ring, and wherein R may be further substituted with OH, alkoxy, amino or halogen substituents;
n is 0 or 1; and Y1 and Y2 are independently either H; halogen; hydrocarbyl (1-14C) comprising cyclic and unsaturated hydrocarbyl, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy, alkylthio, amino, morpholino, acyloxy, acylamino and thio analogs thereof, carboxy, alkoxycarbonyl, carbamyl, alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above;
or a pharmacologically acceptable salt of said compound;
for radiosensitizing hypoxic tumor cells.
wherein X is halogen, OH, alkoxy (1-4C), NH2, NHR or NRR, wherein each R is independently an alkyl of 1-4 carbon atoms or an amide, or wherein in the case of NRR, the two R groups may be linked together to form a morpholino ring, and wherein R may be further substituted with OH, alkoxy, amino or halogen substituents;
n is 0 or 1; and Y1 and Y2 are independently either H; halogen; hydrocarbyl (1-14C) comprising cyclic and unsaturated hydrocarbyl, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy, alkylthio, amino, morpholino, acyloxy, acylamino and thio analogs thereof, carboxy, alkoxycarbonyl, carbamyl, alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above;
or a pharmacologically acceptable salt of said compound;
for radiosensitizing hypoxic tumor cells.
5. The use of claim 4, wherein X is OH or OR.
6. The use of claim 4, wherein X is NH2, NHR or NRR.
7. The use of claim 6, wherein X is NH2.
8. The use of claims 4, 5 or 6, wherein Y1 and Y2 are H.
9. A compound having the structural formula:
wherein X is OH, alkoxy (1-4C), or NRR where each R is independently an alkyl of 1-4 carbon atoms, or where the two R
groups are linked through an oxygen to form a morpholino ring, and wherein R may be further substituted with OH, alkoxy (1-4C), amino or halogen substituents;
n is 1; and Y1 and Y2 are independently either H; halogen; hydrocarbyl (1-14C) comprising cyclic and unsaturated hydrocarbyl, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), amino, morpholino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above;
or a pharmacologically acceptable salt thereof.
wherein X is OH, alkoxy (1-4C), or NRR where each R is independently an alkyl of 1-4 carbon atoms, or where the two R
groups are linked through an oxygen to form a morpholino ring, and wherein R may be further substituted with OH, alkoxy (1-4C), amino or halogen substituents;
n is 1; and Y1 and Y2 are independently either H; halogen; hydrocarbyl (1-14C) comprising cyclic and unsaturated hydrocarbyl, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), amino, morpholino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above;
or a pharmacologically acceptable salt thereof.
10. A compound according to claim 9, wherein X is OH or alkoxy.
11. A compound according to claim 9, wherein X is NRR.
12. A compound according to claims 9, 10 or 11, wherein Y1 and Y2 are both H.
13. A compound having the structural formula:
X is NH2;
n is 1; and Y1 and Y2 are independently hydrogen, saturated or unsaturated hydrocarbyl of 7-14C, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), amino, morpholino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage with the proviso that only one of Y1 and Y2 can be hydrogen; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above;
or a pharmacologically acceptable salt thereof.
X is NH2;
n is 1; and Y1 and Y2 are independently hydrogen, saturated or unsaturated hydrocarbyl of 7-14C, unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), amino, morpholino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), and wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage with the proviso that only one of Y1 and Y2 can be hydrogen; or wherein Y1 and Y2 are independently either NHR', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-14C) which may be substituted as defined above;
or a pharmacologically acceptable salt thereof.
14. A compound according to claim 13, wherein Y1 is H and Y2 is saturated or unsaturated hydrocarbyl of 7-14C.
15. A compound according to claim 13, wherein Y1 is H and Y2 is unsaturated hydrocarbyl of 2-6C.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US91190686A | 1986-09-25 | 1986-09-25 | |
| US911,906 | 1986-09-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1338445C true CA1338445C (en) | 1996-07-09 |
Family
ID=25431084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000540932A Expired - Fee Related CA1338445C (en) | 1986-09-25 | 1987-06-30 | 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP2668232B2 (en) |
| CA (1) | CA1338445C (en) |
| DE (2) | DE3790581B4 (en) |
| GB (1) | GB2203151B (en) |
| WO (1) | WO1988002366A1 (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5175287A (en) * | 1986-09-25 | 1992-12-29 | S R I International | Process for preparing 1,2,4-benzotriazine oxides |
| US5624925A (en) | 1986-09-25 | 1997-04-29 | Sri International | 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents |
| IE62911B1 (en) * | 1988-03-18 | 1995-03-08 | Stanford Res Inst Int | 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents |
| GR1000941B (en) * | 1989-03-17 | 1993-03-16 | Stanford Res Inst Int | Method for preparing 1,2,4-benzotriazine oxides |
| AU646794B2 (en) | 1989-09-18 | 1994-03-10 | Sri International | 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents |
| US5672702A (en) * | 1995-12-04 | 1997-09-30 | Sanofi | Process for preparing 3 amino 1, 2, 4-benzotriazine dioxide |
| WO2013086467A1 (en) * | 2011-12-07 | 2013-06-13 | Sri International | Benzotriazine oxides as drugs targeting mycobacterium tuberculosis |
| WO2015098963A1 (en) | 2013-12-26 | 2015-07-02 | 東亞合成株式会社 | Method for promoting expression of calreticulin, and synthetic peptide for use in method for promoting expression of calreticulin |
| JP6872713B2 (en) | 2015-05-29 | 2021-05-19 | 東亞合成株式会社 | Synthetic peptides that increase the radiosensitivity of tumor cells and their use |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2489352A (en) * | 1946-04-10 | 1949-11-29 | Merck & Co Inc | Halogen compounds of 3 aminobenzotriazine 1, 2, 4 oxide-1 |
| US2489359A (en) * | 1947-01-10 | 1949-11-29 | Merck & Co Inc | Benzotriazines |
| CH487171A (en) * | 1963-10-22 | 1970-03-15 | Siegfried Ag | Process for the preparation of therapeutically active 1,2-dihydro-1,2,4-benzotriazines |
| DE1809390A1 (en) * | 1968-11-16 | 1970-06-11 | Bayer Ag | Phosphorus, phosphonic or thionophosphorus (- phosphonic) acid esters and process for their production |
| IE38778B1 (en) * | 1973-02-02 | 1978-05-24 | Ciba Geigy | New 1,2,4-benzotriazine derivatives |
| DE2538179C2 (en) * | 1975-08-27 | 1986-05-28 | Bayer Ag, 5090 Leverkusen | 3-alkoxy-benzo-1,2,4-triazines, process for their preparation and their use as fungicides |
| DE2740887A1 (en) * | 1977-09-10 | 1979-03-22 | Bayer Ag | AGENTS FOR CONTROLLING PLANT BACTERIOS |
-
1987
- 1987-06-16 DE DE3790581A patent/DE3790581B4/en not_active Expired - Lifetime
- 1987-06-16 WO PCT/US1987/001412 patent/WO1988002366A1/en active Application Filing
- 1987-06-16 DE DE19873790581 patent/DE3790581T1/de active Pending
- 1987-06-16 GB GB8810634A patent/GB2203151B/en not_active Expired - Lifetime
- 1987-06-16 JP JP62504083A patent/JP2668232B2/en not_active Expired - Lifetime
- 1987-06-30 CA CA000540932A patent/CA1338445C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE3790581T1 (en) | 1988-08-25 |
| DE3790581B4 (en) | 2004-12-23 |
| GB2203151B (en) | 1991-05-29 |
| WO1988002366A1 (en) | 1988-04-07 |
| JP2668232B2 (en) | 1997-10-27 |
| JPH01500826A (en) | 1989-03-23 |
| GB8810634D0 (en) | 1988-07-06 |
| GB2203151A (en) | 1988-10-12 |
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