BROWN J MARTIN (US)
GRANGE EDWARD W (US)
MARTINEZ ABELARDO P (US)
| US2489359A | 1949-11-29 | |||
| US2489352A | 1949-11-29 | |||
| FR2322140A1 | 1977-03-25 | |||
| EP0001090A2 | 1979-03-21 | |||
| US3482024A | 1969-12-02 | |||
| GB1234845A | 1971-06-09 | |||
| DE2404375A1 | 1974-08-08 |
| 1. | Method of selectively killing hypoxic tumor cellε compriεing administering directly to said cells a compound of the formula wherein X is NH , NHR or NR where each R is independently an alkyl of 14 carbon atoms, an amide, or a morpholino moiety and may further be substituted with hydroxy, alkoxy. amino. or halogeno substituents; n is 1; and Y 1 and Y2 are i.ndependently either H, halogeno. hydrocarbyl (114C) including cyclic and unsaturated hydrocarbyl, optionally substituted with l or 2 substituents selected from the group consisting of halogeno, hydroxy, epoxy, alkoxy, alkylthio, amino (including morpholino). acyloxy, acylamido and their thio analogs, carboxy. alkoxycarbonyl. carbamyl or alkylcarbamyl. and wherein the hydrocarbyl can optionally be interrupted by a single ether (0) 1 2 linkage, or wherein Y and Y are independently either NHR', 0(CO)R', NH(CO)R', 0(SO)R', or 0(POR')R in which R' is a hydrocarbyl optionally substituted as defined above. |
| 2. | Method of radiosensitizing hypoxic tumor cells, comprising administering a compound of the formul : wherein X is OH. OR. NH . NHR or NR where each R is independently an alkyl of 14 carbon atoms or a morpholino moiety and may further be substituted with hydroxy, alkoxy. amino. or halogeno substituents; wherein n is 0 or 1; and Y and Y are independently either halogeno. hydrocarbyl (114C) including cyclic and unsaturated hydrocarbyl. optionally substituted with 1 or 2 substituents selected from the group consisting of halogeno. hydroxy, epoxy. alkoxy, alkylthio, amino (including morpholino). acyloxy. acylamido and their thio analogs, carboxy. alkoxycarbonyl, carbamyl or alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (0) 1 2 linkage, or wherein Y and Y are independently either NHR', 0(CO)R'. NH(CO)R«, 0(SO)R'. or 0(POR')R' in which R' is a hydrocarbyl optionally substituted as defined above. Compoundε given by the following formula wherein when X iε OH. OR, or NR and n is 0 or 1. or when X is NH or NHR and n is 0, where each R is independently an alkyl of 14 carbon atoms or a morpholino moiety and may further be substituted with hydroxy, alkoxy. amino or halogeno substituents, Y 2 and Y are independently either H. halogeno. hydrocarbyl (114C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogeno. hydroxy. epoxy. alkoxy. alkylthio. amino (including morpholino). acyloxy. acylamido and their thio analogs, carboxy. alkoxycarbonyl, carbamyl or alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a single ether (0) linkage, or wherein Y 1 and Y2 are independently either NHR'. 0(CO)R', NH(CO)R'. 0(SO)R'. or 0(POR')R* in which R' is a hydrocarbyl optionally substituted as defined above; and wherein when X is NH. and n is 1. Y and Y 2 are independently either H. saturated or unsaturated hydrocarbyl of between about 7 and 14 carbon atoms, unsaturated hydrocarbyl of between about 1 and 6 carbon atoms, either hydrocarbyl substituent being unsubstituted or substituted with halogen, hydroxy. epoxy. alkoxy, alkylthio. amino. morpholino. acyloxy, acylamido and their thio analogs, and where the hydrocarbyl can be optionally interrupted by a single ether li.kage, or wherein Y 1 and Y2 are independently either NHR« , 0(CO)R' . NH(CO)R' . 0(SO)R' or 0(POR)R', in which R' is a hydrocarbyl optionally substituted as defined above. A method of making a compound of the formula o by reacting the corresponding 3amino 1oxide derivative with hydrogen peroxide in the presence of Na2WO.»2H20 at a temperature of at least about 50°C. |
Reference to Government Grant or Contract
The invention described herein was made in the course of work under grant or contract from the Department of Health and Human Services. The Government has certain rights in this invention.
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 βensitizers.
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.
The effects of radiation are enhanced by the presence of oxygen, and it is established that as the dose of radiation is increased, the effectiveness of the radiation in destroying target cells is enhanced most dramatically when oxygen is present. Therefore, selectivity for tumor cells toward radiation is difficult to achieve — normal cells, in view of their oxygen supply, are generally more susceptible to radiation than the target tumor cells. It is therefore desirable to provide a means of sensitizing tumor cells, but not the surrounding tissue, to radiation treatment. One solution would be to increase the supply of oxygen to these tumor cells. This, however, has proved difficult to do.
Various heterocyclic compounds and in particular those with oxidized nitrogen moieties, have been used to radioεensitize hypoxic tumor cells. Indeed, it has been postulated that the oxidized nitrogen functionality is responsible for this activity. Nitroi idazoles, particularly misonidazole (MIS) and metronidazole have been studied extensively, and MIS is commonly used as a standard in iji vitro and in vivo tests for radioεensitizing 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) £2:171-190; and U.S. patent 4.371.540. The radiosensitizing activities of certain 1-subεtituted 3(5)-nitro-s_-triazoles and of various quinoxaline-1,4-dioxide derivatives have also been disclosed.
In addition, US Serial Nos. 730,761. filed 3 May 1985. and 788.762. filed 18 October 1985 assigned to the same assignee and incorporated by reference disclose a group of radiosensitizets that do not contain oxidized
nitrogen — the substituted benzamides and nicotinamideε and their thio analogs. These compounds, nevertheless, are radiosensitizerε. It is important to distinguish the ability to sensitize hypoxic cells selectively, for instance, by enhancing their oxygen supply, from another mechanism commonly encountered for "sensitizing" cells: inhibition of the enzyme poly(ADP-ribose)polymerase, which is believed to be essential in the repair of irradiated cells after radiation. This repair mechanism is operative in both hypoxic tumor cells and in normal cells. Hence, administration of "radiosensitizerε" which operate according to this latter mechanism does not accompliεh the desired purpoεe of selectively sensitizing the target tumor cells.
A grou ξ of compounds which has not previously been suggested for use in either selectively killing hypoxic cells or in radiosensitizing such cells is 3-amino-l,2,4-benzotriazine 1,4-di-N-oxide and related compounds. Related US patents 3.980,779; 3,868,371; and 4,001,410 disclose the preparation of a group of these compounds and their use as anti-microbial agents, particularly by addition of these materials to livestock fodder. US patents 3.991.189 and 3.957.799 disclose derivatives of these compounds bearing substituents on the nitrogen of the 3-amino group. These compounds also have anti-microbial activity.
The present invention provides additional compounds which specifically radiosensitize hypoxic cells in vitro and which, furthermore, are directly cytotoxic to hypoxic cells both in vitro and jln vivo. Therefore, administration of these compounds prior to or following radiation treatment of tumors selectively kills the hypoxic (tumor) cells which survive the radiation dose. Both the ability of these compounds to
radiosenεitize hypoxic cellε n vitro and eεpecially their ability to selectively kill hypoxic cells directly are unexpected properties of theεe compounds.
Disclosure of the Invention
The invention provides a valuable addition to the group of compounds currently available as selective radroεenεitizers and selective cytotoxic agents for hypoxic tumor cellε. Some of the compounds useful in this regard are known compounds, others are novel. One aspect of the invention, therefore, is a method of radiosensitizing or selectively killing hypoxic tumor cells with a compound of the formula: o
Λ
o
wherein X is OH. OR. NH . NHR or NR 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 O or 1; and
Y 1 and Y2 are i.ndependently either H, halogeno, hyd.rocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl. optionally substituted with 1 or 2 substituents selected from the group consisting of halogeno. hydroxy. epoxy, alkoxy. alkylthio, amino (including morpholino), acyloxy. acylamido and their thio analogs, alkylsulfonyl, alkylphosphonyl. carboxy. alkoxycarbonyl. carbamyl or alkylcarbamyl, and wherein
the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage, or wherein Y 1 and Y2 are independently either NHR'. 0(CO)R'. NH(CO)R'. 0(SO)R', or 0(POR)R' in which R' is a hydrocarbyl optionally substituted as defined above.
The compoundε of the invention, therefore, are the mono- or dioxides of optionally substituted 1.2..-benzotriazine which contain a hydroxyl or amino group, either substituted or unsubεtituted, in the 3 poεition. While all of the compoundε defined by Formula 1 are generally effective as radiosensitizers. only compounds having a 3-amino substituent (i.e., X=NH_, NHR or NR with R as defined above) and which are di-N-oxides (n=l) are effective cytotoxic agents.
Certain of the compounds encompassed by Formula 1 are already known in the art as being * useful for other purposes; other compounds are novel. The novel compounds encompassed 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 NR_ with R as defined above, n is 0 or 1. and Y 1 and Y2 are as defined above; II. X is NH_ or NHR with R as defined above, n is 0, and Y 1 and Y2 are as defined above; III. 3 is NH_. n is 1. and Y and Y are as defined above but not halogeno. saturated alkyl
(1-6C) unsubstitυted or halogen-substituted, alkoxy
(1-6C). carbamyl. carboxy or carboalkoxy (1-6C).
Brief Description of the Drawings
Figures IA. IB and 1C show the selective cytotoxicity of 3-amino-1.2.4-benzotriazine 1,4-dioxide for hypoxic cells derived from hamster, mouse and human tissues.
Figure 2 shows the in vivo efficacy of
3-amino-1.2;4 ' -benzotriazine 1.4-dioxide in enhancing the killing of tumor cells when combined with radiation.
Figure 3 shows the killing of tumor cells in vivo by 3-amino-l-2.4-benzotriazine 1.4-dioxide when the tumor has been made hypoxic by the intraperitoneal administration of the antihypertenεive drug hydralazine.
Modeε of Carrying Out the Invention
A. The Compoundε Useful in the Invention
The compounds useful in radioεensitizing hypoxic tumor cells as deεcribed herein are derivatives of 1.2,4-benzotriazine oxide.
The hydrocarbyl group represented by Y or Y may contain 1-1 'carbon atoms, may be saturated or unsaturated. cyclic or acyclic, and may optionally be interrupted by a single ether linkage. Thus, the unsubεtituted form of Y or Y can 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, tetrahydrofurfuryl, furfuryl. cyclohexenyl. 3-(n-decyloxy)-n-propyl. 4-methyloctyl. 4.7-dimethyloctyl. and the like.
The hydrocarbyl may be subεtituted with one or two substituents as follows: The halogeno substituents are fluoro, chloro. bromo. or iodo. The alkoxy substituents represented by OR' may contain 1 to 4 carbon atoms, and include, for example, methoxy, n-propoxy, and t-butoxy. The amino substituent may be NH . NHR or NR . where each R is independently an alkyl of 1-4 carbons or a morpholino moiety. R may optionally be substituted with 1-2 hydroxy. alkoxy. amino, or halogeno subεtituentε.
The acyloxy and acylamido groups are represented by R'COO- and R'CONH-. respectively, where R' contains 1-4 carbons, and their thio analogs are represented by R'CSO- and R'CSNH-. Alkyl sulfonyl and alkyl phoεphonyl are. respectively. R'SO and R'P(OR')0- wherein each R' is independently as above defined. Carboxy is the group -C(0)OH; alkoxycarbonyl is -C(0)OR' ; carbamyl is -C(0)NH ; and alkylcarbamyl is -C(0)NHR' .
Where X is OH. of course, the compoundε may also be prepared and used as the pharmaceutically acceptable salts formed from inorganic bases, such as sodium, potassium, or calcium hydroxide, or from organic baseε. such as caffeine, ethylamine, and lyεine.
When X is NH2. pharmaceutically 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, succinic acid, mandelic acid, p-toluene sulfonic acid, and so forth. (Amino substituents on the hydrocarbyl εide 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 oxidized, or both the 1- and 4-nitrogens may be oxidized.
Specific particularly preferred compounds which are useful in the radiosensitization and cytotoxic procedures of the invention include 3-hydroxy-1.2.4-benzotriazine 1-oxide; 3-hydroxy-1.2.4-benzotriazine 1.4-dioxide; 3-amino-1.2.4-benzotriazine 1-oxide; 3-amino-l,2,4-benzotriazine 1.4- i-oxide; 6(7)-aethoxy-3-hydroxy-l.2.4-benzotriazine l-oxide;
(7)-βethoxy-3-hydroxy-1.2.4-benzotriazine 1.4-dioxide; (7)-methoxy-3-amino-l,2.4-benzotriazine 1-oxide; (7)-methoxy-3-amino-1.2.4-benzotriazine 1.4-dioxide; (7)-ethoxy-3-hydroxy-1.2.4-benzotriazine 1-oxide; (7)-ethoxy-3-hydroxy-1.2,4-benzotriazine 1.4-dioxide; (7)-ethoxy-3-amino-1.2.4-benzotriazine 1-oxide; (7)-ethoxy-3-amino-1.2.4-benzotriazine 1.4-dio ide; 6(7X-[4-acetamido-n-butanoxy3-3-hydroxy-1.2.4-benzo- triazine 1-oxide; 6(7)-[4-acetamido-n-butanoxy]-3-hydroxy-1.2,4-benzo- triazine 1.4-dioxide; 6{7)-[4-acetamido-n-butanoxy]-3-amino-l,2.4-benzo- triazine 1-oxide; 6(7)-[4-acetamido-n-butanoxy]-3-amino-1.2.4-benzo- triazine 1.4-dioxide; δ(7)-[l-(2.3-dihydroxy)propoxy]-3-hydroxy-lϊ2.4-benzo- triazine 1-oxide; 6(7)-[l-(2.3-dihydroxyJpropoxy]-3-hydroxy-1.2,4-benzo- triazine 1,4-dioxide; 6(7)-[l-(2.3-dihydroxy)propoxy]-3-amino-l,2.4-benzo- triazine 1-oxide; δ(7)-[l-(2.3-dihydroxy)propoxy]-3-amino-1.2.4-benzo- triazine 1.4-dioxide; 6(7)-[(2-furyl)methylamino]-3-hydroxy-1.2. -benzo- triazine 1-oxide; 6(7)-L(2-furyl)methylamino]-3-hydroxy-1.2.4-benzo- triazine 1.4-dioxide; 6(7)-[(2-furyl)methylamino3-3-amino-l,2,4-benzo- triazine 1-oxide; 6(7)-[(2-furyl)methylamino]-3-amino-1.2.4-benzo- triazine 1.4-dioxide; 6(7)-(2-methoxyethylamino)-3-hydroxy-l,2,4-benzotriazine
1-oxide;
6(7)-(2-methoxyethylamino)-3-hydroxy-l.2.4-benzotriazine
1,4-dioxide; 6(7)-(2-methoxyethylamino)-3-amino-1.2.4-benzotriazine l- oxide; 6(7)-(2-methoxyethylamino)-3-amino-1.2.4-benzotriazine
1.4-dioxide; 6(7)-carbethoxymethoxy-3-hydroxy-1.2.4-benzotriazine 1- oxide; 6(7)-ca bethoxymethoxy-3-hydroxy-l,2.4-benzot iazine 1.4- dioxide; 6(7)-ca bethoxymethoxy-3-amino-l,2,4-benzotriazine 1- oxide; 6(7)-carbethoxymethoxy-3-amino-l.2.4-benzotriazine 1.4- dioxide; 6(7)-[(2-methoxyethyl)ca bamylmethoxy3-3-hydroxy-l.2,4- benzotriazine 1-oxide; 6(7)-[ (2-methoxyethyl)carbamylmethoxyJ-3-hyd oxy-l,2.4- benzotriazine 1.4-dioxide; 6(7)-[ (2-methoxyethyl)carbamylme hoxy]-3-araino-l.2.4- benzotriazine 1-oxide; 6(7)-[ (2-methoxyethyl)carbamylraethoxy]-3-amino-l,2.4- benzotriazine 1,4-dioxide; 6(7)-[(2-hydroxyethyl)ca bamylmethoxy -3-hydroxy-l.2.4- benzotriazine 1-oxid ' e; 6(7)-I (2-hydroxyethyl)carbamylmethoxy3-3-hydroxy-1.2,4- benzotriazine 1,4-dioxide; 6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-araino-1.2.4- benzotriazine 1-oxide; 6(7)-[ (2-hydroxyethyl)carbamylmethoxy]-3-amino-1.2,4- benzotriazine 1.4-dioxide; 6(7)-[l-(2-hydroxy-3-mor holino)propoxy]-3-hydroxy-
1.2.4-benzotriazine 1-oxide; δ(7)-[l-(2-hydroxy-3-morpholino)propoxy3-3-hydroxy-
1.2. -benzot iazine 1.4-dioxide;
6(7)-fl-(2-hydroxy-3-βorpholino)propoxy]-3-amino-1.2.4- benzotriazine 1-oxide; 6(7)-[l-(2-hydroxy-3-morpholino)propoxy3-3-amino-1.2.4- benzotriazine 1.4-dioxide; 6(7)-[3-a»ino-n-propoxy3-3-hydroxy-1.2.4- benzotriazine 1-oxide; 6(7)-[3-amino-n-propoxy -3-hydroxy-1.2.4- benzotriazine 1.4-dioxide; δ(7)-[3-amino-n-propoxy3-3-amino-l,2.4- benzotriazine 1-oxide; 6(7)-[3-amino-n-propoxy3-3-amino-1.2.4- benzotriazine 1.4-dioxide; 6(7)-[2.3-epoxypropoxy3-3-hydroxy-1.2.4- benzotriazine 1-oxide; 6(7)-[2.3-epoxypropoxy3-3-hydroxy-1.2,4- benzotriazine 1.4-dioxide; 6(7)-[2.3-epoxypropoxy3-3-amino-l,2,4-benzotriazine
1-oxide; 6(7)-[2.3-epoxypropoxy3-3-amino-1.2.4-benzotriazine
1,4-dioxide; δ(7)-[3-methoxy-2-hydroxy-n-propoxy3-3-hydroxy-l,2.4- benzotriazine 1-oxide; 6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-hydroxy-1.2.4- benzotriazine 1.4-dioxide; 6(7)-[3-methoxy-2-hydroxy-n-propoxy3-3-amino-1.2.4- benzotriazine 1-oxide; 6(7)-[3-methoxy-2-hydroxy-n-propoxy -3-amino-l.2,4- benzotriazine 1,4-dioxide; 6(7)-[4-ethoxy-3-hydroxy-n-butoxy3-3-hydroxy-l,2.4- benzotriazine 1-oxide; 6(7)-[4-ethoxy-3-hydroxy-n-butoxy3-3-hydroxy-l,2.4- benzotriazine 1,4-dioxide; 6(7)-[4-ethoxy-3-hydroxy-n-butoxy3-3-amino-1.2.4- benzotriazine 1-oxide;
(7)-[4-ethoxy-3-hydroxy-n-butoxy -3-amino-1.2,4- benzotriazine 1.4-dioxide; (7)-[3.4-dihydroxy-n-butoxy3-3-hydroxy-l.2,4- benzotriazine 1-oxide; (7)-[3.4-dihydroxy-n-butoxy3-3-hydroxy-1.2.4- benzotriazine 1,4-dioxide; (7)-[3.4-dihydroxy-n-butoxy]-3-amino-l,2.4- benzotriazine 1-oxide; (7)-[3.4-dihydroxy-n-butoxy3-3-amino-l,2.4- benzotriazine 1.4-dioxide; (7)-methyl-3-hydroxy-1.2 4-benzotriazine 1- oxide; (7)-methyl-3-hydroxy-1.2.4-benzotriazine 1.4- dioxide; (7)-methyl-3-amino-l.2.4-benzotriazine 1- oxide; (7)-methyl-3-amino-1.2.4-benzόtriazine 1,4- dioxide; (7)-ethyl-3-hydroxy-1,2.4-benzot iazine 1- oxide; (7)-e hyl-3-hydroxy-1.2.4-benzot iazine 1.4- dioxide; (7)-ethy1-3-amino-1.2.4-benzotriazine 1- oxide; (7)-ethy1-3-amino-l.2.4-benzotriazine 1.4- dioxide; (7)-chloroacetamido-3-hydroxy-1.2.4-benzotriazine 1- oxide; (7)-chloroacetamido-3-hydroxy-1,2.4-benzotriazine 1.4- dioxide; (7)-chloroacetamido-3-amino-1.2.4-benzotriazine 1- oxide; (7)-chloroacetamido-3-amino-l.2,4-benzotriazine 1.4- dioxide;
6(7)-[(2-hyd oxyethyloxy)acetamido3-3-hydroxy-1.2, - benzotriazine 1-oxide; 6(7)-f(2-hydroxyethyloxy)acetamido3-3-hydroxy-1.2,4- benzotriazine 1.4-dioxide; δ(7)-[(2-hydroxyethyloxy)acetamido -3-amino-1.2.4- benzotriazine 1-oxide; 6(7)-[(2-hydroxyethyloxy)acetamido3-3-amino-l.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-benzotriazine 1.4- dioxide; 6.7-dimethoxy-3-amino-1.2.4-benzotriazine 1-oxide; 6.7-dimethoxy-3-amino-1.2.4-benzotriazine 1.4- dioxide; 6.7-diethoxy-3-hydroxy-l,2.4-benzotriazine 1-oxide; 6.7-diethoxy-3-hydroxy-l.2.4-benzotriazine 1,4- dioxide; 6,7-diethoxy-3-amino-1.2.4-benzotriazine 1-oxide; 6.7-diethoxy-3-amino-l,2. -benzotriazine 1,4- dioxide; 6(7)-propionyl-3-hydroxy-l,2,4-benzotriazine 1-oxide; 6(7)-propionyl-3-hydroxy-l,2.4-benzotriazine 1,4- dioxide; 6(7)-propionyl-3-amino-1.2.4-benzot iazine 1-oxide; 6(7)-propionyl-3-amino-1.2.4-benzotriazine 1.4- dioxide; δ(7)-(2-acetoxyethoxy)-3-hydroxy-l,2.4-benzotriazine 1- oxide; 6(7)-(2-acetoxyethoxy)-3-hydroxy-1.2.4-benzotriazine
1.4-dioxide; 6(7)-(2-acetoxyethoxy)-3-amino-1.2.4-benzotriazine 1- oxide; δ(7)-(2-acetoxyethoxy)-3-amino-1.2,4-benzotriazine 1.4- dioxide;
(7 -n-hexyloxy-3-hydroxy-l.2. -benzotriazine 1-oxide; ( 7 -n-hexyloxy-3-hydroxy-l.2,4-benzot iazine 1.4- dioxide; ( 7 -n-hexyloxy-3-amino-l,2,4-benzotriazine 1-oxide; ( 7 -n-hexyloxy-3-amino-l.2.4-benzotriazine 1.4- dioxide; ( 7 -ethylamino-3-hydroxy-l.2.4-benzotriazine 1-oxide; ( 7 -ethylamino-3-hydroxy-l.2.4-benzotriazine 1,4- dioxide; ( 7 -ethylamino-3-amino-l.2.4-benzotriazine 1-oxide; (7 -ethylamino-3-amino-l.2,4-benzotriazine 1.4- dioxide; ( 7 -(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine l- oxide; ( 7 -(2-methoxyethoxy)-3-hydroxy-l.2,4-benzotriazine
1, -dioxide; ( 7 . -(2-methoxyethoxy)-3-amino-l,2.4-benzotriazine 1- oxide; ( 7 -(2-methoxyethoxy)-3-amino-l.2,4-benzotriazine 1.4- dioxide; ( 7 -(aminoacetamido)-3-hydroxy-l.2, -benzotriazine 1- oxide; ( 7 -(aminoacetamido)-3-hydroxy-i,2,4-benzo riazine
1,4-dioxide; ( 7 -(aainoacetamido)-3-araino-l,2, -benzotriazine l- oxide; (7 -(aminoacetamido)-3-amino-l.2. -benzotriazine 1.4- dioxide; ( 7 -(ca ba ylmethoxy)-3-hydroxy-l.2.4-benzotriazine 1- oxide: ( 7 -(ca bamylmethoxy)-3-hydroxy-1,2. -benzotriazine
1.4-dioxide; ( 7 -(carbam lmethoxy)-3-amino-1,2,4-benzotriazine 1- oxide;
6(7)-(carbamyl»ethoxy)-3-amino-1.2. -benzot iazine 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-l.2.4-benzotriazine 1- oxide; 6(7)-carboxymethoxy)-3-amino-1.2.4-benzotriazine 1.4- dioxide; and their pharmaceutically acceptable saltε and the thioamide analogs of the foregoing list of compounds. It should be noted that the M Y 1 or Y2" substituents set forth in most of the above compounds as present in either the " 6 or 7 positions (designated H 6(7)") or in both the 6 and 7 positions (designated "6,7") may also be present at the 5 and/or 8 ring positions.
Of the above compounds useful in the method of the present invention as selective cytotoxic agents or radiosenεitizers. the following compounds are novel: compounds given by the formula above wherein I. X is
OH. OR. or NR . 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. n is 0 or 1. and Y and Y are independently either H, halogeno, hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl. optionally substituted with 1 or 2 substituents selected from the group consisting of halogeno. hydroxy. epoxy, alkoxy, alkylthio. amino
(including morpholino). acyloxy. acylamido and their thio analogs, alkylεul onyl. alkylphosphonyl. carboxy. alkoxycarbonyl. carbamyl or alkylcarbamyl, and wherein the hydrocarbyl can optionally be interrupted by a
single ether (-0-) linkage, or wherein Y1 and Y2 are independently either NHR'. 0(CO)R' . NH(CO)R'. 0(SO)R' or 0(POR)R' in which R' is a hydrocarbyl optionally substituted as defined above; II. X is NH_ or NHR with R as defined above, n is 0. and Y 1 and Y2 are as defined in I; III. X is NH_. n is 1. and Y and
Y are independently either H. hydrocarbyl (7-14C; saturated or unsaturated) ,- unsaturated 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 optionally be interrupted by a single ether (-0-) linkage, or wherein Y 1 and Y2. are independently either NHR*. 0(CO)R'. NH(CO)R'. 0(SO)R'_. or 0(POR)R' in which R' is a hydrocarbyl optionall 'substituted as defined above.
B. Preparation of the Compounds of the Invention
General methods for preparing 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 benzofuroxan of " the formula:
by reaction with a salt of cyanamide. followed by acidi ication of the reaction mixture. The benzofuroxan starting material is not symmetric with respect to its own 5 and 6 positions (which are the 6 and 7 positions
of the resulting 3-amino benzotriazine oxide). Therefore, a mixture of the 6- and 7-εubstituted materials may result. If desired, this mixture can be separated using conventional means into individual components having a substituent in either the 6 or 7 poεition.
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 uεing H NCN; (2) oxidation of the parent compound given by the εtructure
or by controlled reduction of the corresponding dioxide (see Mason, supra, and Wolf et al. J Am Chem Soc 76:355 (1954)).
3-amino-1.2.4-benzotriazines 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-l.2. -benzot iazine oxides may be prepared using peroxide and and tungsten oxide (Scheme II). a novel synthetic procedure for making the 3-hydroxy-l,4-dioxide compound, or concentrated sulfuric acid and sodium nitrate (Scheme III).
Scheme I
Scheme II
Scheme III
C. Formulation and Adminiεtration
As demonstrated below, the oxidized benzotriazines of the invention may be used to radiosenεitize 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 uεe of antihypertensive drugs such as
hydraIazine. or agents which affect the amount of oxygen carried by the blood. While theεe 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 primates, farm animals such as cattle, and sports animals and pets εuch aε horses, dogs, and cats.
Hypoxia is believed to be aεεociated with all typeε of solid malignant neoplasms. The compounds of the invention may. therefore, be used to radioεenεitize . or to kill neoplastic epithelial cells, endothelial cellε. connective tissue cells, bone cells, muscle cells, nerve cells, and brain cells. Examples of carcinomas and sarcomas include carcinomas εuch as epithelial cell, acidic cell, alveolar cell, basal cell, basal squamous cell, cervical, renal, liver. Hurthle, Lucke. mucinous and Walker, and εarco as such as Abernathy's, alveolar εoft part, angiolithic. botyroid. encephaloid. endoraetria εtro a. Swing's fascicular, giant cell, lymphatic. Jensen's, juxtacortical osteogenic. Kaposi'ε. medullary, and synovial. Specific examples of tumors that have been sensitized with other radiosenεitizerε are reported in Adamε. G.E.. Cancer: A Comprehensive Treatise (F. Becker. Ed) vol 6. pp 181-223. Plenum. New York. 1977.
The compoundε may be administered to patients orally or parenterally (intravenously, subcutaneously. intramuscularly, intraspinally, intraperitoneally. and the like). When adminiεtered parenterally the compounds will normally be formulated in a unit dosage injectable form (solution, suspension, emulsion) with a pharmaceutically acceptable vehicle. Such vehicles are typically nontoxic and nontherapeutic. Examples of εuch vehicleε are water, aqueous vehicles εuch as saline.
Ringer's solution, dextrose solution, and Hank's solution and nonaqueouε vehicles such as fixed oils (e.g.. corn, cottonseed, peanut, and sesame), ethyl oleate. and isopropyi myriεtate. Sterile saline is a preferred vehicle and the compounds are sufficiently water soluble to provide a solution for all foreseeable needs. The vehicle may contain minor amounts of additives such as substances that enhance solubility, isotonicity, and chemical stability, e.g.. antioxidants, buffers, and preservatives. When administered orally (or rectally) the compounds will usually be formulated into a unit dosage form such as a tablet, capsule, suppository or cachet. Such formulations typically include a solid, semisolid or liquid carrier or diluent. Exemplary diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobro a, aginates. tragacanth, gelatin, syrup, ethylcellulose, polyoxyethylene εorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, and magnesium stearate.
The amount of compound adminiεtered to the subject is sufficient to radiosensitize or to produce cytotoxicity in the malignant 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 intended and the weight or body surface of the subject. The radiation may be administered to humans in a variety of different 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 benzotriazine will be given before or after each radiation treatment and is likely to be in the range of 0.01 to 20 mmol/kg and usually in the range of 0.1 to 2 mmol/kg.
For use as selective cytotoxic agents, the compounds of the invention can be administered alone, with radiation or other cancer cytotoxic agents, with vaεoactive drugs (e.g., hydralazine) . or,with procedures which reduce the amount of 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 encompasεed by Formula 1 are generally uεeful as radioεensitizerε herein, only those compounds which are (substituted or unsubstituted) 3-amino-1.2,4-benzotriazine.1.4-dioxides (i.e.. X=NH . NHR or NR with R as defined above and n is 1) are useful as selective cytotoxic agents.
Examples
The following examples further illustrate the compounds of the invention and methods for synthesizing using them, and are not intended to limit the invention in any manner.
Example 1: Preparation of 3-Hydroxy-l.2,4-Benzotriazine
1.4-Dioxide o
A stirred mixture of l.SOg (9.25 mole) of 3-amino-1.2.4-benzotriazine 1-oxide (1.), 100.0 ml acetic acid, and 30.0 ml of 30\ hydrogen peroxide was treated with 3.05 g (9.25 mmole) of Na W0 4 *2H O. The mixture was stirred in an oil bath at 60°C for 4 days. The yellowish orange mixture was cooled to about 30° and filtered to remove a light yellow non-UV absorbing solid that was presumably tungstic acid. The orange solution of hydrogen peroxide in acetic acid was evaporated to semi-dryneεε carefully with εeveral additions of water and acetic acid to remove most of the peroxide. The concentrated solution was allowed to stand at room temperature to afford four crops of an orange solid, 0.87g (42 yield of the sodium salt of 2). UV ∑U X (20%
CH OH/H O): 262.2 (r 39.460); 477 (c 7.030).
IR (neat): 3530μ, 3150μ. 2650μ, 2180μ and
1635μ. Anal. (calculated for the sodium salt):
C7H4,N„30.3,Na 1.2SH2O, 223.64: C.37.6; H.2.93; N.
18.79. Found: C. 37.8; H.2.75; N.18.65.
Example 2: Preparation of
3-Amino-7-Trifluoromethyl-1.2.4-Benzotriazine 1-Oxide:
A solution of Na (1.13g. 49.2 mmole) in ethanol (50 ml) was added to a solution of guanidine hydrochloride (4.93g. 51.6 mmole) in ethanol (50 ml). After lh. the mixture was filtered and the filtrate was combined with a εolution of
4-chloro-3-nitro-benzotrifluoride (Aldrich, 5.5g. 24.4 mmole) in ethanol (25 ml). The mixture was stirred and refluxed for 5 h. cooled to 0-5°C. and the precipitated solid collected. The solid was washed with water and ethanol and air-dried to give 0.48g (9%) of 3 . as a light yellow solid. mp>300 β C. TLC: R f 0.60 (9:1 methylene chloride: ethanol on silica gel plates). Mass. Spec: M + «230 (q = 100).
Example 3: Preparation of 3-A__ino-7-Decyl-1.2. -Benzotriazine 1-Oxide
Preparation of 4-(l-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 hexanes (2.41). After stirring for lh. the mixture was cooled and treated over 30 min. at 5-10 β C with 70% nitric acid (34 ml). Stirring was continued at 5-10 β C for lh and at 25 β C for 16h. The mixture was diluted with H O (11). stirred for 5h, poured into an open dish and allowed to stand for 16h. After further dilution with H O (1.51). the solid was collected and recrystallized from an 85% ethanol solution (in water) to give 92g (84%) of the intermediate as an orange solid, m.p. 64°C.
A solution (100 ml) Of 85% KOH (19g. 0.288 mole) in H O was combined with a suspension of 4-(l-decyl)-2-nitroaniline (89g. 0.28 mole), prepared above, in ethanol (900 ml). The mixture was stirred for 6h. neutralized to pH 7-8 with concentrated HCl, and evaporated in vacuo to near dryness. After dilution with H O (400 ml), the solid was collected and air-dried to give 77g (100%) of the intermediate as an orange solid, mp 59°C. l.Og (8.7 mmole) of chloroamidine hydrochloride (previously prepared for use by treating an ether solution of cyanamide with HCl gas and collecting the precipitated solid) was added portionwise over 10 in to a preheated melt (190*C) of 4-(l-decyl)-2-nitroaniline prepared in the preceding step (500 mg. 1.8 mmole). The reaction mixture was heated at 190*C for 5 min. cooled to 25 » C. treated with 6N KOH (10 ml), and heated at 90-95 # C for lh. After cooling to 25°C, the solid was collected, washed with H O and ethanol and air-dried to give 0.25g (46%) of compound 4. as a light yellow
βolid. a.p. 177-C (dec). Mass. spec. M + «285 (q=100) 302 (q»13).
Example 4: Preparation of 3-Amino-7-Carbamyl-l.2.4-Benzotriazine 1-Oxide
Preparation of 4-chloro-3-nitrobenzamide: 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 4tT to give a clear red 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 (-10°C) concentrated ammonium hydroxide (100 ml). After 3h at -10 β C and 16h at 25 β C the mixture was poured into an open dish and allowed to evaporate to dryness. The residue was slurried in H O and the solid was collected and air-dried to give 19.8g (98%) of the intermediate as a light yellow solid, m.p. 153 β C.
A solution of Na (3.45g. 0.15 mole ) in ethanol (75 ml) was added to a solution of guanidine hydrochloride (15.8g. 0.165 mole) in ethanol (75 ml). After lh the mixture was filtered and the filtrate was combined with a suspension of 4-chloro-3-nitrobenzamide (lOg. 0.05 mole) prepared above, in ethanol (50 ml). The mixture was stirred and refluxed for 16h. cooled to 0-5 # C. and acidified with concentrated HCl (8 ml). The
collected solid was combined with K CO (28g. 0.2 mole) and H O (40 ml) and the mixture was stirred and heated at 100 β C for Bh. After cooling to 25 # C. the solid was collected, washed with H O, 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 (6x100ml). The combined filtrate was evaporated 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.>300 β C. TLC: R._=0.23 (methylene chloride: acetone of 2:1. silica gel plates). Masε. Spec: M + 205 (q= 100).
Example 5: Preparation of
7-Acetyl-3-Amino-l,2,4-Benzotriazine 1-Oxide Oxi e
A combined mixture of 7-acetyl-3-amino-l.2.4benzotriazine 1-oxide (prepared in Example 5; 50 mg. 0.25 mmole). hydroxylamine hydrochloride (200 mg. 2.88 mmole), pyridine (1 ml) . and ethanol (1 ml) waε heated at 90-95 β C for lh and then cooled to 25°C. The mixture was diluted with 95% ethanol (5 ml) and the solid waε collected and air-dried to give 30 mg (56%) of compound £. aε a light yellow
solid. .p. 278 β C (dec). TLC: R «0.60 (9:1 ethylene chloride: methanol). Mass Spec: M + «219 (q * 100).
Example 6: Preparation of 3-Amino-6(7)-Decyl-1.2.4-Benzotriazine 1.4-Dioxide
5-(l—decyl)-benzofuroxan: A combined mixture of 4-(l-decyl)-2-nitroaniline (77g, 0.28 mole), 5.25% NaOCl in H 2 0 (476g, 0.34 mole). 85% KOH (20.3g, 0.31 mole). Bn NHSO (4.7g. 0.014 mole), and CH Cl (2.28 1) was stirred rapidly for 6h and diluted with
H 2 0 (500 ml) and CH 2 C1 2 (1 1). The separated organic phase was washed succesεively with IN HCl (1 1) and brine (2 x 1 1)), dried (Na_,SO„). and
2 4 concentrated in vacuo to yield a red oil. 70 g (92%).
A solution of 5-(l-decyl)-benzofuroxan as prepared above (10 g, 0.036 mole) and benzyltriethylammonium chloride (0.36 g. 0.0016 mole) in
DMSO (180 ml) was treated gradually over several hours with cyanamide (13.0 g. 0.31 mole) and K.CO (36.8 g. 0.27 mole). The mixture was stirred for 48h and filtered. The filtrate waε diluted with H O (6.1) and glacial acetic acid (40 ml) and extracted with
CH 2 C1 2 (4 x 500 ml). The combined organic solution waε washed successively with 5% NaHCO solution (1 x
500 ml) and brine (2 x 500 ml), dried (Na SO ). and
2 4 evaporated in vacuo to dryness. The crude product was
purified by chromatography on silica gel using
CH Cl : aethanol (98:2) to give 1.8g (16%) of compound a red solid, .p. 155°C (dec). Mass. Spec: * «318 (q * 4). 285 (q=100).
Example 7: Preparation of
7-Chloro-3-Hydroχy-l.2,4-Benzotriazine 1,4-Pio ide o
m 2
A mixture of 1.50g (7.63 _π_ole) of , 8 in 100 ml acetic acid waε treated with 2.52 g (7.63 mmole) of Na 2_WO4, 2H2„0 and 30 ml of 30% H2„0„. The mixture was stirred and heated for 6 days at 50°C, then slowly evaporated to dryness to remove H O . The residue was boiled in 250 ml H O and filtered to remove about 25 mg of starting material . The aqueous solutions were then extracted with 2 x 250 ml portions of ethyl acetate. A deep red " crystalline material that was characterized as ___ 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 10. which showed good solubility in a mixture of hot isopropyl alcohol and water. Mass. Spec: M + «212 (q*100) (compound ___) TLC: ,_: 0.34 (acetone, silica gel plates).
The ethyl acetate solutions above, separated from the H O layer after the filtration to remove 10..
vere evaporated to dryness. The residue was then treated with isopropyl alcohol at room temperature to afford a dull orange solid. 0.41g (25% yield) of ___. Mass. Spec: M + -213 (q=70); TLC: R f «0.22 (acetone, silica gel plates). Compound g was characterized as the ammonium salt, C 7_H4.C1N_303, NH . .w. 230.61. as follows. The free acid ___ was dissolved in concentrated NH OH and then chilled in
4 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 P ? 0 at 56 β C/1.0 mm. leaving 0.244 g (87% yield) of il o t
11
Anal. Calcd. for c H C1N O NH (230.61): C.
7 4 3 3 3
36.5 H, 3.06; N. 24.30. Found: C. 36.5; H. 3.07; N.
2 233..9944.. UUVVmmaaxx ((HH2z_,,00)):: 221199 (c 12.580); 265.4 (ε
40,000); 4830486 (c 6.640)
Example 8: In Vivo Assay for Activity in Combination with Radiation
The compounds of the invention were tested in vivo for activity by the assay of Brown. J.M.. Radiation Res (1975) 6.4 633-47. incorporated herein by reference. For this asεay, SCCVII carcino aε in female C3H mice
veighing 20-25 g were used. These mice were bred under specific pathogen-free conditions and were 3-4 months old at the beginning of each experiment. The SCVii tumor was grown intradermally in the flank from an inoculation of 2 x 10 tumor cells taken from the 2nd-8th in vitro passage of the tumor cells after removal from the previous n 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 LD (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 2 hr after to.3 hr before injection of the drug. By using these intervals, the results give an indication of both the optimum irradiation time and the extent of extra cell killing compared to radiation alone. The results of such time-course experiments using 3-amino-1.2,4-benzotriazine 1,4-dioxide are shown in Figure 2. They show enhanced cell killing compared to radiation only, more than would have been expected on the basiε of additivity of the two individual cytotoxicities. The similar increased cytotoxicity 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 SCCVII tumors was done by irradiating nonanaesthetized tumor-bearing mice in a Plexiglas box. Irradiation conditionε were 250 kVp X-rays. 15 A. 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 cultured tumor cellε as follows. The tumor-bearing mice were killed 24 hr after irradiation, and tumors were dissected 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) containing 0.02% DNase, 0.05% promase, and 0.02% collagenase. The suspension was stirred for 30 min at 37 β C. filtered, and centrifuged at 1.600 rmp for 10 min at 4 β C. The cell pellet was resuεpended 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 hemacytomete . Suitable dilutions of this serum plated into 60- or 100-mm polystyrene 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 containing 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: Cytotoxicity Tests
Cytotoxicity testε were carried out using 3-amino-l,2.4-benzotriazine 1,4-dioxide and a variety of aerobic and hypoxic cells in culture (human, mouse, and hamster). The cellε in spinner flaskε were gassed for one hour at 37 β C with either air or nitrogen containing 5% CO prior to adding the specified amounts of the drug. Figures 1A, IB and 1C show the results for cell survival of mouse, hamster and human cells at various concentrations of 3-araino-1.2,4-benzotr.iazine
1.4-dioxide. It was found that only 1 to 2% of the drug concentration under aerobic conditions waε 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 literature.
Example 10: Determination of L
LD_ is determined in BALB/c female mice 50
(weighing 20-25 g) following intraperitoneal (ip) injection, unless the compound tested has low lipophilicity and is very soluble, wherein intravenous (iv) administration is used. LD values at 1. 2. 5. and 60 days are determined by administering graded doses of the drug dissolved in physiological saline immediately prior to injection.
Example 11: Radiosensitivity in Vitro
The results of assays to determine the concentration of drug necessary to produce a sensitizer enhancement ratio of hypoxic cells in culture of 1.6 are as follows:
Compound C 1. o _______
7 -chloro-3-amino-l.2, -benzotriazine 1-oxide 3.3 6(7)-aethoxy-3-amino-l,2.4-benzot iazine 1,
4-dioxide ~l.o
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, pharmaceutical, medical, and related arts are intended to be within the scope of the following claims.
Example 12: Enhanced Tumor Cell Toxicity Using HydraIazine
Hydralazine is an antihypertenεive drug which actε by relaxing the εmooth muscle around blood vessels. This has the effect of preferentially shunting blood flow into normal tissues and away from tumors, which proceεε produceε immediate hypoxia in the tumors. If 3-amino-l,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 10 (i.e.. only 1 cell in every 1000 was left viable). The experimental procedures are the same as described in Example 8, and the " results are shown in Figure 3.