CA2001903C - 1,2,4-benzotriazine oxides as radiosensitizers and selective cytotoxic agents - Google Patents

Abstract

A method of using 1,2,4-benzotriazine oxides, some of which are novel compounds, as radiosensitizers and selective cytotoxic agents is disclosed. These compounds are shown to specifically radiosensitize hypoxic tumor cells. Some are additionally disclosed to be useful as specific cytotoxic agents for these cells. They also show an unexpected ability to radiosensitize aerobic cells following or preceding a hypoxic incubation of the cells with the drug. This provides a basis for selective radiosensitization of tumors compared to normal cells.
Novel compounds of a novel method for preparing the 1,2,4-benzotriazine oxides is also disclosed. Novel compounds of the formula wherein X is OH, alkoxy (1-4C), NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms, or aryl of 1-4 carbon atoms, or where the two R groups are alkyls linked together to form a pyrrolidino or piperidino ring or linked through an oxygen to form a morpholino ring, and the R
groups may be further substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents;
n is 1; and Y1 is H and Y2 is nitro;
or a pharmacologically acceptable salt thereof are also disclosed. A novel method for preparing the 1,2,4-benzotriazine oxides is also disclosed.

Description

t v 1,2,4-BENZOTRIAZINE OXIDES AS
RADIOSENSITIZERS AND SELECTIVE CYTOTOXIC AGENTS
Technical Field The invention relates to cytotoxic agents and radiotherapy effective against hypoxic cells. More specifically, the invention relates to certain novel 1,2,4-benzotriazine oxides, to methods of selectively killing tumor' cells and/or sensitizing tumor cells to radiation using selected 1,2,4-benzotriazine oxides, and to novel synthetic methods.

~i~f~~.~3 _2_ 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 fo;c 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.
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 radiosensitize hypoxic tumor cells. Indeed, it has been postulated that the oxidized nitrogen functionality is responsible for this activity. Nitroimidazoles, particularly mison_idazole (MIS) and metronidazole have been studied extensively, and MIS is commonly used as a standard in in vitro and in vivo 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, Radi<~tion Res (1980) 82: 171-190; and U.S.
patent 4,371,540. The radiosensitizing activities of certain 1-substituted 3(5)-nitro-s-triazoles and of various quinoxaline-1,4-dioxide derivatives have also been disclosed.
In addition WO 86/06628 published November 20, 1986 assigned to the same assignee discloses a group of radiosensitizers that do not contain oxidized nitrogen--the substituted benzam:ides and nicotinamides and their thio analogs. These compounds, nevertheless, are radiosensitizers. 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 "radiosensitizers" which operate according to this latter mechanism does '':
t:

~Q~~.~~~ 4 not accomplish the desired purpose of selectively sensitizing t:he target tumor cells.
A croup of compounds which has not previously been suggestead for use in either selectively killing hypoxic cells or in radiosensitizing such cells is 3-amino-1,2,96-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. U.S. Patent Nos. 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.
They present invention provides additional compounds which specifically radiosensitize hypoxic cells and which, furthermore, are directly cytotoxic to hypoxic cellsc both in vitro and in 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 abi7.ity of these compounds to radiosensitize hypoxic cells; and especially their ability to selectively hall hypoxic cells directly are unexpected properties of these compounds.
they invention also provides novel 1,2,4-benzotriazine oxides useful as radiosensitizers and/or selective cytotoxic agents; methods of synthesizing the compounds; and methods of administering the compound's to achieve radiosensitization and/or selective ce7.1 killing.

. _g-Disclosure oi: the Invention Then invention provides a valuable addition to the group of compounds currently available as selective radiosensiti:.ers and selective cytotoxic agents for hypoxic tumor cells. Some of the compounds now newly shown to be useful in this regard are known compounds.
Others are themselves novel.
Accordingly, one aspect of the invention is a method of ra<iiosensitizing hypoxic tumor cells by administering to these cells a compound of the formula:
O
T
y1 N \
N
y2 (I) N X
O
n wherein X is H; hydrocarbyl (1-4C);
hYdrocarbyl (1-4C) substituted with OH, NH2, NHR or NRR;
halogen; OH; alkoxy (1-4C); NH2; NHR or NRR; wherein the various iEt groups are independently selected from lower alkyl (1-4C) and lower acyl (1-4C) and the R's may themselves bye substituted with OH, NH2, alkyl (1-4C) secondary and dialkyl (1-4C) tertiary amino groups, alkoxy (1-4C) or halogen. In the case of NRR, the two R's can be linked together directly or through a bridge oxygen into a morpholino ring, pyrrolidino ring or piperidino ring;
n is 0 or 1; and Y1 and Y2 are independently either H; nitro;
halogen; hydrocarbyl (1-14C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of .

~~~~.~C~~ -s_ halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl (1-4C) tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C:), acylamido (1-4C) and thio analogs thereof, acet:ylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfony7. (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (-O-) linkage; or wherein Yl and Y2 are independently either morpholino, pyrrolidino, piperidino, rtH2, NHR,', NR'R' 0(CO)R', NH(CO)R', 0(SO)R', or 0(POR')R' in which R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino,~piperidino, alkoxy (1-4C), or halogen substitutents, or pharmacologically acceptable salts of said compound.
In another aspect, the invention provides an improved method of fractionated radiotherapy which involves treating the cells requiring radiotherapy with a 1,2,4-benzc>triazine oxide of Formula (I), as just defined, before or after subjecting the treated cells to a plurality of distinct radiation doses over an extended period of time, each of the radiation doses being less than about 5 Gy.
The! compounds useful in conjunction with the presently disclosed radiosensitizing methods, therefore, are the mono-~ or dioxides of optionally substituted 1.2,4-benzotriazine which may contain a hydrocarbyl (1-4C), hydroxyl., alkoxy or amino group, either substituted or unsubstituted, in the 3-position, and their pharmacologically acceptable salts as set forth in Formula I
Then invention also provides a method for selectively killing hypoxic tumor cells using certain of these 1,2,4-benzotriazine oxides. The compounds which are useful as selective cytotoxic agents are a subset of the above-dei:ined compounds useful as radiosensitizers.
That is, while all of the compounds defined by Formula (I) are generally effective as radiosensitizers, only those compounds unsubstituted at the 3-position or having a 3-annino or 3-hydrocarbyl (1-4C) substituent (i.e., X=H, hydrocarbyl (1-4C), NH2, NHR or NRR with each R as dei'ined above) and which are di-N-oxides (n=1) are effective cytotaxic agents. In this aspect, the invention provides a method of selectively killing hypoxic tumor cells by administering one or more of these compounds (or its salts) to the hypoxic tumor cells.
Certain of the compounds encompassed by Formula (I) 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 Formula (I), in which the substitu~ents fall into the following three classes:
I. X is OH, alkoxy (1-4C), NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms, yr acyl of l-4 carbon atoms, or where the two R groups are alkyls linked together to form a pyrrolidino or piperidino ring or linked through an oxygen to form a morpholino ring, and the R groups may be further substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents;
n i s 1; and Yl and Y2 are independently either H; nitro;
halogen; hyd:rocarbyl. (1-14C) including cyclic and unsaturated lzydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hyd:roxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialk:yl (1-4C) tertiary amino, dialkyl tertiary amino where 'the two alkyls are linked together to produce a mo:rpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (l.-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, al'.kylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (-0-) linkage; or wherein Y1 and Y2 are independently either morpholino, ~ayrrolidino, piperidino, NH2, NHR', NR'R' 0(CO)R', NH(CO)R', 0(SO)R', or 0(POR')R' in which R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (:1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents.
Pharmacologically acceptable salts of these compounds are also included in this class of compounds.
II. X is NH2;
n is 1; and Y1 and Y2 are chosen such that one but not both may be hydrogen and one or both may independently be either vitro, saturated or unsaturated hydrocarbyl of ~-14C, or unsaturated hydrocarbyl of 2-6C, optionally ~1~:~.°~~
substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylth.io (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) and alkylphosphonyl (1-4C), wherein the hydrocarbyl. can optionally be interrupted by a single ether (-~0-) linkage; or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2;, NHR', NR'R' 0(CO)R', NH(CO)R', O(SO)R', or 0(POR')R' in which R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, pi.peridino, alkoxy (1-4C), or halogen substitutents. Pharmacologically acceptable salts of these compound's are also included in this class of compounds.
III. X is hydrogen or hydrocarbyl (2-4C) optionally substituted with OH, NH2, alkoxy (1-4C) or halogen substit:uents;
n is 1; and Yl and Y2 are independently either H; vitro;
halogen; hydroc:arbyl (1-14C) including cyclic and unsaturated hyctrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primary amino (NH2), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl tertiary amino where t:he two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoa7.ky1 (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, al~;ylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally beg interrupted by a single ether (-O-) linkage; or urherein Yl and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHR', NR'R' 0(CO)R', NH(C:0)R', 0(SO)R', or 0(POR')R' in which R' is a hydrocarby7. (1-4C) which may be substituted with OH, NH2, alkyl (7.-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents.
Pharmacologically acceptable salts of these compounds are also inc7.uded in this class of compounds.
The' invention also provides a straightforward, one-step synthesis for preparing 1,2,4-benzotriazine oxides unsub:ctituted at the 3-position (i.e., the compounds of Formula (I) wherein X=H) by treating the corresponding 3-amino-1,.2,4-benzotriazine oxide with a lower alkyl nitrite under reductive deaminating conditions.

2~ Brief Descrix~tion of the Drawin4s Figures 1A, 1B 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-1,2,56-benzotriazine 1,4-dioxide in enhancing the killing of tumor cells when combined with radiation.

, , _11-Figure 3 shows the killing of tumor cells in vivo by 3-amino-1,2,4-benzotriazine 1,4-dioxide when the tumor has been made hypoxic by the intraperitoneal administration of the antihypertensive drug hydralazine.
Figure 4 is a graph illustrating aerobic radi.osensitiz:ation of CHO cells by hypoxic pretreatment or post-treatment with 3-amino-1,2,4-benzotriazine 1,4-dioxide, as described in Example 22.
Figure 5 illustrates tumor cell survival of SCCVII tumor irradiated with 8x2.5 Gy in four days, also as described in Example 22.
Figure 6 graphically illustrates the growth delay of SCCVII tumors irradiated with 8x2.5 Gy in four days, also asp described in Example 22.
Figure 7 shows in graph form the average skin reaction of normal mouse skin irradiated with 8 fractions (every 12 hours) of between 3 and 6 Gy per fraction.
Modes of Carryinct Out the Invention A. The Compounds Useful in the Invention They compounds useful as radiosensitizers and selective cyt:otoxic agents as described herein are derivatives of 1,2,4-benzotriazine oxide represented by Formula (I).
These compounds, as shown in the Formula (I), contain a group X in their 3 position. X will vary specifically as set forth above, depending upon the activity desired. Subject to the above-recited selection criteria, X is chosen in general from hydrogen; un~~ubstituted hydrocarbyls (1-4C) such as methyl, ethyl., s-butyl and the like; hydroxy; alkoxy (1-4C) such as methoxy, ethoxy, propoxy, t-butoxy and the like; primary amino (NH2); secondary amino (NHR) where R is are alkyl or acyl of 1 to 4 carbons, such as methylamino, ethylamino and the like; tertiary amino (NRR) where each of the R groups is an alkyl or acyl of 1 to 4 carbons, for example diethylamino and the like, or the two R's join to form a morpholino, pyrrolidino or piperidino ring. In the case of the various alkyl and acyl R group:., they can be further substituted with OH, NH2, lower a:~kyl (i-4C) secondary amino and dialkyl (1-4C) tertiary amino, morpholino pyrrolidino, piperidino, alkoxy (1-4C) or halogen (fluoro, chloro, bromo or iodo) substituents.
In the case of the hydrocarbyl X groups, they can be furthe r substituted with OH, NH2, alkyl secondary amino, dialk~~l tertiary amino, alkoxy (1-4C) or halogen (fluoro, chloro, bromo or iodo) substituents.
The compounds of Formula (I) additionally contain groups Yl and Y2. These groups are selected specifically according to the criteria set forth above, depending upon the utility desired.
Subject to these criteria, Yl and Y2 may be selected from hydrogen; vitro; halogen (e. g. fluoro, chloro, bromo or iodo); or hydrocarbyl (1-14C). When hydrocarbyl, Y1 or Y2 may be saturated or unsaturated, cyclic or-ac:yclic, and may optionally be interrupted by a single ether linkage. Thus, the unsubstituted hydrocarbyl forms of Yl or Y2 can be, for example, methyl, ethyl, n-propyl, s-butyl, n-hexyl, 2-methyl-n-pentyl, 2-et:hoxyethyl, 3-(n-propoxy)-n-propyl, 4-methoxybutyl, cyclohexyl, tetrahydrofurfuryl, furfuryl, cyclohexenyl, 3-(n-decyloxy)-n-propyl, 4-methyloctyl, 4,7-dimethyloctyl, or the like.

. ~ -13-The hydrocarbyl Y1 and Y2 groups may optionally be substituted with 1 or 2 substituents selected from halogen such as fluoro, chloro, bromo or iodo; hydrox;~r; epoxy; alkoxy (1-4C) such as, for example, metlzoxy, n-~propoxy and t-butoxy; alkyl thio;
(1-4C) primary amino (NH2); morpholino; pyrrolidino;
piperidino; secondary amino (NHR') where R' is a 1-4C
alkyl, such as methylamino, propylamino and the like;
tertiary amino (NR'R'); acyloxy and acylamido groups represented by R'C00- and R'CONH-, respectively, and their thiol analogs represented by R'CSO- and R'CSNH-respectively;; carboxy (-C(0)OH); alkoxycarbonyl (-C(0)OR'): carbamyl (-C(0)NH2); alkylcarbamyl (1-4C) (-C(0)NHR'); alkylsulfonyl (1-4C) (R'SOZ-); and alkyl phosphonyl (:l-4C) ( R'P(OR')0-).
In addition Y1 and Y2 can each independently be -NH2, -NHI~', -NR'R', -OCOR', -NH(CO)R', -0(SO)R' or -0(POR')R' in which the various R' groups are lower alkyls (1-4C) which themselves may be substituted with OH, NH2, alkyl secondary and tertiary amino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutenta.
A particularly promising class of compounds for use both as radiosensitizers and selective cytotoxic agents include those represented by the following structural Formula (II):

y1 ~ N ~
~~ N
(II) Y ~ N ~yH ~ (CH ) ~ NR R
2 m 1 2 ~~~~1.~'~
In Formula (II), one of Y1 and Y2 is H, the other an electron-withdrawing group (e. g., vitro, carboxy, alkoxycarbonyl, alkylsulfonyl), R1 and R2 are independently selected from the group consisting of hydrogen and lower alkyl, or the R1 and R2 groups may be linked to form a piperidino or pyrrolidino ring, and m is an integer from a-4 inclusive, preferably 1 or 2.
Where X is OH, of course, the compounds may also be prepared and used as the pharmaceutically acceptable salts foamed from inorganic bases, such as sodium, potassium, or calcium hydroxide, or from organic bases, such as caffeine, ethylamine, and lysine.
When X is NH2, NHR, or NRR, e.g., NH-CH2-(CH2)m-CH2NR1R2 as in Formula (II), pharmaceutically l~ 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 20 substituents on the hydrocarbyl side chain can also, of course, be converted to salts.) The 1,2,4--benzotriazines may be usedin the practice of this invention as the mono- or dioxides;
i.e. either the 1-nitrogen of the triazino ring may be 25 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;
30 3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
3-amino-1,2,4-benzotriazine 1-oxide;
3-amino-1,2,4-benzotriazine 1,4-di-oxide;
6(7)-methoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;

6(7)-methoxy--3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methoxy--3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-methoxy--3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-ethoxy-'.3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethoxy-'.3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-ethoxy-".3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[4-acetamido-n-butanoxy]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[4-acetamido-n-butanoxy]-3-hydroxy-1,2,4- .
benzotriazine 1,4-dioxide;
6(7)-[4-acet<imido-n-butanoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[4-acetamido-n-butanoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-(2,3--dihydroxy)propoxy]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-(2,3-~dihydroxy)propoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[1-(2,3-~dihydroxy)propoxy]-3-amino-1,2,4-benzotriazine 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-ber~zotriazine 1,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-amino°1,2,4-benzotriazine 1-oxide;
6(7)-[(2-furyl)methylamino]-3-amino-1,2,4-benzotriazine 1,9:-dioxide;
6(7)-(2-methoxyethylamino)-3-hydroxy-1,2,4 benzotriazine 1-oxide;

6(7)-(2-methoxyethylamino)-3-hydroxy-1,2,4-benzotriazine 1, 96-dioxide;
6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine 1, 96-dioxide;
6(7)-carbethoxymethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-carbethoxymethoxy-3-hydraxy-1,2,4-benzotriazine 1, 9b-dioxide;
6(7)-carbethoxymethoxy-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-carbethoxymethoxy-3-amino-1,2,4-benzotriazine 1, 96-dioxide;
6(7)-[(2-methoxyethyl)carbamylmethoxy]-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)carbamylmethoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(~)-[(2-methoxyethyl)carbamylmethoxy]-3-amino-1,2,4 benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[(2.-hydroxyethyl)carbamylmethoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(~)-[1-(2-h~rdroxy-3-morpholino)propoxy]-3-hydroxy-1,:?,4-benzotriazine 1-oxide;

6(7)-[1-(2-h;Ydroxy-3-morpholino)propoxy]-3-hydroxy l,;Z,4-benzotriazine 1,4-dioxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-amino-1,2,4 benzotriazine 1-oxide;
6(7)-[1-(2-h;Ydroxy-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-benzotriazine 1,4-dioxide;
6(7)-[3-amino-n-propoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[3-amino-n-propoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[2,3-epoxypropoxy]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[2,3-epoxypropoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[2,3-epoxypropoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[2,3-epoxypropoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-hydroxy-1,2,4-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-propoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6i7)-[3-methoxy-2-hydroxy-n-propoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[4-ethosKy-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[4-ethos~y-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-~~~~~ ~~;~ -la-benzotriazine 1,4-dioxide;
6(7)-[4-ethos:y-3-hydroxy-n-butoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[4-ethos:y-3-hydroxy-n-butoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-(3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-methyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methyl-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-methyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-ethyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethyl-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-ethyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-chloroac.etamido-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-chloroacetamido-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-chloroacetamido-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-chloroacetamido-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-hydroxy-1,2,4-ben.zotriazine 1-oxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide; .

6(7)-[(2-hydroxyethyloxy)acetamido]-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6,7-dimethox;~r-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-1,2,4-benzotriazine 1-oxide;
6,7-diethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6,7-diethoxy~-3-amino-1,2,4-benzotriazine 1-oxide;
6,7-diethoxy-3-amin~-1,2,4-benzotriazine 1,4-dioxide;
6(7)-propionyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-propionyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-propion:Y1-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-propion;Y1-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-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;
6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-benzotriazine 1, ~4-dioxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-benzotriazine 1, ~4-dioxide;
6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethylamino-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-ethylamino-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;

6(7)-ethylamino-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-ethylamino-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine 1, ~4-dioxide;
6(7)-(2-methoxyethoxy)-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-(Z-methoxyethoxy)-3-amino-1,2,4-benzotriazine 1, 4-dioxide;
6(7)-(aminoacetamido)-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-(aminoacetamido)-3-hydroxy-1,2,4-benzotriazine 1, 4-dioxide;
6(7)-(aminoacetamido)-3-amino-1,2,4-benzotriazine -1-ox ide;
6(7)-(aminoacetamido)-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(carbam;Ylmethoxy)-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-(carbamylmethoxy)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(carbamylmethoxy)-3-amino-1,2,4-benzotriazine 1-ox ide;
6(7)-(carbamylmethoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(carboxyrmethoxy)-3-hydroxy-1,2,4-benzotriazine 1-ox ide;
6(7)-(carbox;ymethoxy)-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(carbox~ymethoxy)-3-amino-1,2,4-benzotriazine 1-oxide;

2pp1903 6(7)-(carboxymethoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1,2-dih,ydroxyethyl]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-(3-ethylamino-2-hydroxypropoxy)]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[2-ethylamino-1-hydroxyethyl]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7) -[2-hydroxyethyl]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-hydro~xyethyl]-3-amino-1,2,4-benzotriazine 1,4-dioxide;
3-(2-hydroxye~thylamino)-1,2,4-benzotriazine 1-oxide;
3-(2-hydroxye~thylamino)-1,2,4-benzotriazine 1,4-dioxide;
6(7)-chloro-3-(2-hydroxyethylamino)-1,2,4-benzotriazine 1-oxide;
6(7)-chloro-3-(2-hydroxyethylamino)-1,2,4-benzotriazine 1,4-dioxide;
3-(1-hydroxye~thylamino)-1,2,4-benzotriazine 1-oxide;
3-(1-hydroxye~thylamino)-1,2,4-benzotriazine 1,4-dioxide;
1,2,4-benzotriazine 1-oxide;
1,2,4-benzotriazine 1,4-dioxide;
3-methyl-1,2,4-benzotriazine 1,4-dioxide;
3-ethyl-1,2,4-benzotriazine 1,4-dioxide;
3-propyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ami.no-3-methyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-amino-3-ethyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methoxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methoxy-~3-methyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-(2,3-~dihydroxypropoxy]-1,2,4-benzotriazine 1, 9~-dioxide;' 6(7)-[1,2-dihydroxyethyl]-1,2,4-benzotriazine 1, 9~-dioxide;
6(7)-[1-(3-et:hylamino-2-hydroxypropoxy)]-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[2-ethyl.amino-1-hydroxyethyl]-1,2,4-benzotriazine 1-9r dioxide;
6(7)-chloro-1.,2,4-benzotriazine 1,4-dioxide;
6(7)-[2-hydroxyethyl]=1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-hydroxyethyl]-1,2,4-benzotriazine 1,4-dioxide;
6(7)-vitro-3-amino-1,2,4-benzotriazine 1,4-dioxide;
3-(3-N,N-diet:hylaminopropylamino)-1,2,4-benzotriazine 1, 96-dioxide;
6(7)-vitro-3--(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1,4-dioxide and their pharmaceutically acceptable salts and the thioamide analogs of the foregoing list of compounds. It should be noted that the Yl or Y2 substituents set forth in most of the above compounds as present in either the 6 or 7 positiions (designated ~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.

_23_ B. Preoaratio~n of the Compounds of the Invention General methods for preparing some 3-amino derivatives are found in the above-referenced patents to Ley et al., for example US 3,980,779. The compounds are prepared from benzofuroxan of the formula:

'f N ,~
~ ~ /O
by reaction with a salt of cyanamide, followed by acidification 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 nuixture of the 6- and 7-substituted 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 position.
The dioxide may also be prepared from the parent monoxidle or 1,2,4-benzotriazine by peracid oxidation (seep Bobbins et al, J Chem Soc 3186 (1957) and Mason et al, J' Chem Soc B 911 (1970)).

In addition, the monoxide may be prepared by:
(1) cyclization of a 1-vitro-2-aminobenzene compound using H2NCN~2HC1;
(2) oxidation of the parent compound given by the structure s~ N -~ ~J
or by controlled reduction of the corresponding dioxide (see Mason, aupra, and Wolf et al, J Am Chem Soc 76:355 (1954)).
They 1,2,4-benzotriazines may be prepared by cyclization of formazan precursors using BF3/AcOH (see Scheme I and Atallah and Nazer, Tetrahedron _38:1793 (1982)).
3-amino-1,2,4-benzotriazines may be prepared either by cyc;lization of a parent compound (see Scheme II and Arndt,. Chem. Ber. 3522 (1913)) or by reduction of the monoxide or dioxide as above.
They 3-hydroxy-1,2,4-benzotriazine oxides may be prepared using peroxide and sodium tungstate (Scheme III), a novel. synthetic procedure for making the 3-hydroxy-1,9~-dioxide compound, or concentrated sulfuric acid and sodium nitrate (Scheme IV).

~ll~'~t.~'~~
N'N BF~/AcOH
N ~L,~ X
N~ X
NH

Scheme I
H
NH SMe O N
O . 2 . ---~. 0 0 ~N=c-N=IN-c=NH N

Scheme II

YL_ ~N 1 N
~N
~ N H202 Y
2 (/1 ~ 2 Y s Y N~\ OH
Na2WOQ 2H20 > 50°C p Scheme III

f YL N L _ NWN
WN H ~ Y

2 , Y= Y N/ \ 0 H
N ~ NdNO

goo ~ 90 The invention also encompasses a novel method of preparing 1,2,4-benzotriazine oxides unsubstituted at the 3 position (sometimes referred to herein as the "3-desamino" compounds). The novel synthesis involves reductive deaminatian of the corresponding 3-amino structure. In contrast to prior methods of synthesizing 3-desamino-1,2,4-benzotriazine oxides, the present method enables a simple, straightforward one-step method which gives 'the desired product in a high yield. The method invol~~es treating a 1,2,4-benzotriazine oxide of Formula (I), wherein X is NH2, with a lower alkyl nitrite under reductive deaminating conditions. By "reductive deaminating conditions" is meant reaction conditions which will give rise to at least about 10%, preferably at least about 50%, of the desired 3-unsubstituted reaction product. A preferred lower alkyl nitrite for use in said method is t-butyl nitrite.
Exemplary re<3uctive deaminating conditions involve reaction in a compatible solvent, e.g., dimethyl formamide, at a temperature of at least about 60°C, typically at a temperature in the range of 60-65°C.
This reaction is illustrated generally at Scheme V, and is exemplified in Examples 12-15 herein.
O O
1 N ?
\ \
y I N t-BuN02.. ~ y1 N
y ~ DIdF y ~ N ' "N'H 60-65oC

On Scheme V On _27_ C. Formulation and Administration As demonstrated below, the oxidized benzotriazine~s of the invention may be used to radiosensitiz;e or selectively kill hypoxic tumor cells in warm-bloodied animal hosts. A way in which they may be used is ir,~ conjunction with agents known to selectively create hypoxia in tumors. Such methods include the use of antihypertensive drugs such as hydralazine, or agents which affect the amount of oxygen carried by tree 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 primates, farm animals such as cattle, and sports animals 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 radiosensitize or to kill ne~oplastic 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, ~~1.~~~

Lucke, mucinous and Walker, and sarcomas such as Abernathy's, alveolar soft part, angiolithic, botyroid, encephaloid, endometria stroma, Ewing's fascicular, giant cell, 7.ymphatic, Jensen's, juxtacortical osteogenic, y:aposi's, medullary, and synovial. Specific examples of tumors that have been sensitized with other radiosensiti::ers are reported in Adams, G.E., Cancer: A
ComprehensivEa Treatise (F. Hecker, Ed) vol 6, pp 181-2Z3, Plenum, New York, 1977.
The compounds may be administered to patients orally or parenterally (intravenously, subcutaneously, intramuscularly, intraspinally, intraperitoneally, and the like). When administered 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 such vehicles are water, aqueous vehicles such as saline, Ringer's solution, dextrose solution, and Hank's solution and nonaqueous vehicles such as fixed oils (e. g., corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate. Sterile saline is a preferred velhicle 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 theobroma, alginates, tragacanth, gelatin, methylcellulo;se, polyoxyethylene sorbitan monolaurate, methyl hydrox:ybenzoate, propyl hydroxybenzoate, talc, and magnesium stearate.
The amount of compound administered to the subject is su:Eficient to radiosensitize or to produce cytotoxicity :in the malignant neoplasm to be treated but below that which may elicit toxic effects to normal tissue. This amount will depend upon the type of tumor, the species o1: the subject being treated, the indicated 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 weeha, 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 i,n the range of 0.01 to 20 mmol/kg and usually in they range of 0.1 to 2 mmol/kg. In these treatment regimens, each radiation dose is typically 1-5 Gy, preferably, less than 2.5 Gy and more preferably 2-2.5Gy. Typically, one dose of radiation is administered pier day although 2 or more might be used if tolerated by the patient It has now been found that the compounds disclosed herein as radiosensitizers, particularly 3-amino-1,2,4-benzotriazine 1,4-dioxide, both sensitize o' -30-tumors to radiation without increasing the sensitivity of normal skin, and work in highly fractionated radiation regimens. As demonstrated in Example 22 herein, pre- or post-irradiation treatment of cells with, for example, 3-amino-1,2,4-benzotriazine 1,4-dioxide, under hypoxic conditions, radiosensitizes cells even when the drug is not present during the radiation exposure and the cells are aerobic.
For use as selective cytotoxic agents., the compounds of the invention can be administered alone, ~~rith radiation or other cancer cytotoxic agents, with vasoactive drugs (e. g., hydralazine), or under other conditions which reduce the amount of available oxygen carried by tile 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.
Examples The following examples further illustrate the compounds of the invention and methods for synthesizing and using them, and are not intended to limit the invention in any manner.
Experimental: All reactions were carried out i~ flame-dried glassware and under a blanket of Argon.
t-butyl nitrite (90%) was purchased from the Aldrich CtfEemicai Company. ~imethylformamide was distilled from calcium hydride. 7-Nitro-3-amino-1,2,4-benzotriazine-3-amine 1-oxide was purchased from Parish Chemical Company, trifluoroacetic anhydride, N,N-diethylethylene-diamine, N,N~-diethylpropylenediamine and sodium..
tungstate di,hydrate were purchased from Aldrich Chemical Company and 70% hydrogen peroxide was a gift from ~~~~~~~ -31-Interox America. All reactants were used without further purification. Flash chromatography was carried out on E. Merck 230-400 mesh silica gel under a positive pressure of argon. NMR spectra were obtained on a Varian XL-400 or Jeol FX90Q spectrometer and in d6-acetone, d4-methanol, or d6-dimethyl sulfoxide, as indicated, and are reported relative to the central peak in the appropriate multiplet (2.04, 3.30, and 2.49 ppm, respectively), W spectra were obtained on a Perkin-Elmer 552 spectrophotometer in 95% ethanol, mass spectra were obtained on an LKH 9000 mass spectrometer, and elemental analyses were carried out by Desert Analytics, Tucson, AZ.
Example 1: Preparation of 3-Hydroxv-1,2.4-Benzotriazine 1,4-Dioxide T
I~~ t ~"''~~ N''1' Nl,li A stirred mixture of 1.50g (9.25 mmole) 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 1;9.25 mmole) of Na2W04 2Fi20. 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-W absorbing solid. The orange solution of hydrogen peroxide in ~~Q~~.~~~ -32-acetic acid was evaporated to semi-dryness carefully with several. additions of water and acetic acid to remove most of the peroxide. The eoncentrated 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). Wmax (20% CH30H/H20): 262.2 (e 39,460);
477 (E 7,030). IR (neat): 3530u, 3150u, 2650u, 2180u and 1635u. Anal. (calculated for the sodium salt):
C7H4N303Na 1..25H20, 223.64: C,37.6; H,2.93; N, 18.79.
_fund: C, ?'.7.8; H,2.75; N,18.65.

T
N
(~~,~Nf)~
~ O~
O

Examflle 2: Preparation of 3-Amino-7-Trifluoromethvl-1,2.4-Henzot:riazine 1-Oxide:

r o ~~' A mixture of 4-chloro-3-nitrobenzotrifluoride (Aldrich, 2..70 g, I2.9 mmole) and eyanamide dihydrochloride (2.75 g, 24 mmole) (previously prepared by treating am ether solution of cyanamide with HC1 gas and collecting the precipitated solid) was heated at 140°C for 1 h~,. The residwe was treated with 2N NaOH (45 ml), heated for a further 5 min, and then allowed to cool. The precipitate was collected, washed with H20, dried, and triturated with acetone-toluene to yield I.32 g (45%) of 3 as a light yellow solid M.P. 301-302°, TLC: Rf 0.60 (9:1 methylene chloride: methanol on silica gel plates). Mass. Spec.: M+=230 (q = 100).
Example 3: Pretaaration of 3-amino-7-Decyl-1,2.4-Benzotriazine~ 1-Oxide 1 S ~LOH21 ~ ~~J~

Preparation of 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 hexanes (2.41). After stirring for Ih, the mixture was cooled and treated over 30 min. at 5-10°C with T0% nitric acid (34 ml). Stirring was continued at 5-10°C for 1h and at 25°C for 16h. The mixture was dliluted with H20 (11), stirred for 5h, poured,into am open dish and allowed to stand for 16h.
After further' dilution with H20 (1.51), the solid was collected andl 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 H20 was combined with a suspension of 4-(1-decyl)-2;-nitroaniline (89g, 0.28 mole), prepared above, in methanol (900 ml). The mixture was stirred for 6h, neutralized to pH 7-8 with concentrated HC1, and evaporated iru vacuo to near dryness. After dilution with H20 (400 ml), the solid was collected and air-dried to give i7g (I00%) of the intermediate as an orange solid, m.p. ~~9°C.
l.C~g (8.7 mmole) of cyanamide dihydrochloride (previously F~repared for use by treating an ether solution of c:yanamide with HC1 gas and collecting the precipitated solid) was added portionwise over 10 min to a preheated melt (190°C) of 4-(1-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 1.h. After cooling to 25°C, the solid was collected, waished with H20 and ethanol and air-dried to give 0.258 (9:6%) of compound 4 as a light yellow solid, m.p. 177°C (clec). Mass. spec. M+=285 (q=100), 302 (q=13).
Example 4: F~reparation of 3-Amino-7-Carbamyl-1.2,4-Benzotriazine~ 1-~Yide r ', -35-~~U~1.~~
Preparation of 4-chloro-3-nitrobenzamide:
2~r2g (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 clear red solution. The solution was evaporated in vacuo and aze~otroped 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°C and 16h at 25°C the mixture 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. 153°C.
A solution of Na (3.45g, 0.15 mole ) in ethanol (75 ml) was added to a solution of guanidine I5 hydrochloride' (15.88, 0.165 mole) in ethanol (75 ml).
After 1h the mixture was filtered and the filtrate was combined with a suspension of 4-chloro-3-nitrobenzamide (10g, 0.05 merle) prepared above, in ethanol (50 ml). The mixture was stirred and refluxed for 16h, cooled to 0-5°C, and acidified with concentrated HC1 (8 ml). The collected solid was combined with K2CO3 (28g, 0.2 mole) and H20 (40 ml) and the mixture was stirred and heated at 100°C for 8h. After cooling to 25°C, the solid was collected, washed with H20, and air-dried. The solid was suspendedt in boiling ethyl acetate, collected and washed with hoot ethyl acetate. The solid was repeatedly suspended in boiling dioxane and collected (6x100m1).
TFi~ combined filtrate was evaporated in vacuo to a solid. The solid was suspended in 95% ethanol, collected andl air-dried to give 0.44g (4.3%) of compound 5 as a light yellow solid, m.p. 300°C. TLC: Rf=0.23 ' -36-(methylene chloride: acetone of 2:1, silica gel plates).
Mass. Spec.: M+ 205 (q~ 100).
Example 5: 1?reparation of 7-Acetyl-3-Amino-1.2.4-Benzotriazine 1-Oxide Oxime H c'"~ . H., CD C~~
1O ' N ~2 A combined mixture of 7-acetyl-3-amino-12.4-benzotriazine 1-oxide (prepared in Example 5;
50 mg, 0.25 rnmole), hydroxylamine hydrochloride (200 mg, 2.88 mmole), pyridine (1 ml) , and ethanol (1 ml) was heated at 90--95°C for 1h and then cooled to 25°C. The mixture was <iiluted with 95% ethanol (5 ml) and the Solid was collected and air-dried to give 30 mg (56%) of compound 6 a:a a light yellow solid, m.p. 278°C (dec).
TLC: Rf=0.60 (9:1 methylene chloride: methanol). Mass Spec . : M+=2719 ( q=100 ) .

i ' _37_ Eaauaule 6: ~?reparation of 3-Amino-6(7)-Decvl-1,2,4-Henzotriazine 1,4-Dioxide T
N
~~aH2i ~NHz 5-i;l-decyl)-benzofuroxan: A combined mixture of 4-(1-decy7L)-2-nitroaniline (779, 0.28 mole), 5.25%-Na0C1 in H20 (4769, 0.34 mole), 85% KOH (20.38, 0.31.
mole), nBu4NHS04 (4.79, 0.014 mole), and CH2C12 (2.28 1) was stirred rapidly for 6h and diluted with H20 (500 ml) and CH2C12 (7. 1). The separated organic phase was washed successively- with 1N HC1 (1 1) and brine (2 x 1 1)), dried (Ma2S04), and concentrated in vacuo to yield a red oil, 70 g (92%).
A :solution of 5-(1-decyl)-benzofuroxan as prepared above (10 g, 0.036 mole) and benzyltriethyl ammonium chloride (0.36 g, 0.0016 mole) in DMSO (180 ml) was treated gradually over several hours with cyanamide (13.0 g, 0.37. mole) and K2C03 (36.8 g, 0.27 mole). The mix~ure~-waa atirred for 48h and filtered. The filtrate wa~..diluted with H20 (6 1) and glacial acetic acid (40 ml~ and extracted with CH2C12 (4 x 500 ml). The combined organic solution was washed successively with 5% NaHC03 solution (1 x 500 ml) and brine (2 x 500 ml), dried (Na2S09~), and evaporated in vacuo to dryness. The crude product: was purified by chromatography on silica geZ using CH2C1.2: methanol (98:2) tv give 1.8g (16%) of compound 7 as a~ red solid, m.p. 155°C (dec). Mass.
Spec.: M+=318 (q=4), 285 (q=100).
Example 7: Pre~~aration of 1.2.4-Benzotriazine 1.4-Dioxide of ~ -~ 0 0 A mi~;ture of 1.80 g (13.73 mmole) of 8, 90$
H202 (9 ml), trifluoroacetic anhydride (13.5 ml) and Na2W04.2H20 (li!.50g, 38 mmole) in CHC13 (170 ml) was stirred at room temperature for 5 days. The reaction mixture was diluted with H20 (100 ml) and extracted with CHC13 (100 ml). The organic layer was washed with H20 (50 ml), dried (Na2S04), and the solvent removed in vacuo. The residue was chromatographed on silica gel using EtOAc-CH~!C12 (1:1) to give 0.30 g (I3.4%) of compound 9 as a~ yellow solid, m.p. 204-205°C. Anal.
Calc'd. for C7~t5N302 (163.13): C, 51.5; H, 3.09; N, 25.76. Found: C, 51.6; H, 3.36; N, 26.01. Mass Spec.
M+=I63 (q~100), 147 (q=50). TLC: Rf=0.27 (E~t~-CH2C12, 1:1, silica gel plates). IR (nujol):
160~'p, 1460u, 1.300u, 1230u. wmax (H20): 227 22,900) 252 (E 12,950), 392 (E 4,080).

Eaamflle 8: F~reparation of 7-Chloro-3-Hvdroxv-1.2~4-HenzotriazinEe 1,4-Dioxide T
--: ~
O O~
~2 a 0 ~L ~ T
CL
O ON ~ O O 1N
OH ~NH2 10 ~ 1 A mixture of 1.50 g (7.63 mmole) of 10 in 100 ml acetic acid was treated with 2.52 g (7.63 mmole) of Na2W04~2H20 and 30 ml of 30% H202. The mixture was stirred and heated for 6 days at 50~C, then slowly evaporated to dryness to remove H202. The residue was boiled in 250 ml H2C and filtered to remove about 25 mg of starting nnaterial 12. The aqueous solutions were then extracted with 2 x 250 ml portions of ethyl acetate. A deep red crystalline material that was characterized as 12 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 1~?x which showed good solubility in a mixture of hot isopropyl alcohol and water. Mass. Spec.: M+=212 (q=100)(compound 10); TLC: Rf= 0.34 (acetone, silica gel plates).
The ethyl acetate solutions above, separated from the H20 layer after the filtration to remove 12, wire 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 11.
Mass. Spec.: M+=213 (q=70); TLC: Rf=0.22 (acetone, silica gel plates). Compound 11 was characterized as the ammonium salt, C7H4C1N303~NH3, m.w. 230.61, as follows. The free acid 11 was dissolved in concentrated NH40H and then chilled in ice and filtered to remove a trace of insoluble 12. 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 P205 at 56°C/1.0 mm, leaving 0.244 g (87% yield) of 13 , °
c i .~./w G ON
-l~.o b a Anal. Calc'd. for C7H4C1N303 NH3 (230.61): C, 36.5; H, 3.06; N, 24.30. Found: C, 36.5; H, 3.07; N, 23.94.
Wmax (HBO): 219 (~ 12,580); 265.4 (e 40,000); 4830486 (~ 6,640).

200j90~
Example 9: 1?reparation of 7-Nitro-3-Amino-1,2,4-Benzotriazine 1,4-Dioxide O
T
T
--~ DiN -'~
~'' ' ~.W N ~ ~
~'N~NHCOCF3 n l5 T
Oy N
-'!
'~.~' L NOON
c r6 7-Nitro-3-trifluoroacetamido-1,2,4-benzotriazine 1-oxide (15): A solution of 7-vitro-3-amino-1,2,4-benzotriazine 1-oxide (14) (4.00g, 19.3 mmol; Parish Chemical Co.), CHC13 (125 ml) and trifluoroacet:ic anhydride (12.0 ml, 85.0 mmol) was stirred at room temperature for 44 hr. The resultant light yellow solid was filtered, washed with CHC13 (50 ml) and dried to give 5.35g (91% yield) of the product as a yellow :3olid. Anal. Calc'd, for C9H4F3N504: C, 35.7; H, 1.3:3; N, 23.10. Found: C, 35.7; H, 1.23; N, 23.06.
7-Nitro-3-amino-1,2,4-benzotriazine 1,4-oxide (16): To a stirred solution of 7-vitro-3-trifluoroacet~amido-1,2,4-benzotriazine 1-oxide prepared above (15) (:Z.50g, 8.25 mmol) in CHC13 (200 ml) was S
42 2UU 1 ~Q~
added Na2W04.2H20 (90 mg, 0.273 mmol) followed by 70%
H20~ (10 ml). After 15 min the solution was treated with trifluoroacetic anhydride (8.0 ml, 56.7 mmol) and stirring was continued at room temperature for 64 hr.
The reaction mixture was chromatographed (EtOAc, 20%
MeOH/acetone, and finally 20% DMF/acetone) then recrystallizeld in acetone to give 1.20g (65% yield) of the product (16) as an orange solid, mp 286-28S°C
(dec.). UV: a 259, 300, 345, 387, 472. Anal. Calc'd.
for C7H5N504: C, 37.70; H, 2.26; N, 31.39. Found: C, 37.70; H, 2.1.3; N, 30.94.
Example 10: Preparation of 3-(3-N,N-Diet.hylaminopropvlamino)-1,2,4-Benzotriazine 1,4-Dioxide O
N''"N
.--~, '~ ~ "~ N
O 1D , ek t8 1' ,,.., N .., ~
~'''~'~~ R,~-w N NEty N
V
lea.
3-(,3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine! 1-oxide (18): A solution of 3-chloro-~~~1.:~~ i -43-1,2,4-benzotriazine 1-oxide (17) (3.0g, 16.5 mmol) (produced by the method of Sasse et al., U.S. Patent 4,288,771) in CH2C12 (100 ml) was treated with N,N-diethylpropylenediamine (9.5 rnl, 88.3 mmol). After 20 hr at room temperature the mixture was diluted with 1,2-dichloros~thane (50 ml) and washed successively with Na2C03 and H;~O. The yellow solution was dried (Na2S04), filtered and evaporated in vacuo to give 3.938 (87%
yield) of the product as a yellow solid.
Recrystallizartion (ether/petroleum ether) yielded pure material, mp 47-48°C. Anal Calc'd. for CI4H21N50 (18):
C, 61.10; H, 7.69; N, 25.44. Found: C, 61.30; H, 7.80;
N, 25.61.
3-1;3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1,4-dioxide (18a): To a stirred solution .
of 3-(3-N,N-cliethylaminopropylamino)-I,2,4-benzotriazine 1-oxide 18 prepared as above (1.608, 6.10 mmol) in CHC13 (50 ml) was added trifluoroacetic anhydride (22.0 ml).
After 15 min the mixture was cooled to -10°C, 70% H202 (10 ml) added and then stirred at room temperature for 20 days. The reaction mixture was dried (Na2S04), filtered and evaporated to dryness. The residue was dissolved in saturated NaHC03 solution (50 ml) and extracted wii:h CH2C12 (3x150 ml). The organic layer was dried (Na2S0~~),_filtered and evaporated to give the product LBa, 0.518 (29% yield) as a red solid, mp 92-94°C. NM~t: d ( 400 t~Iz, CDC13 ) 1.11 ( 6H, t, J=7 .1 Hz, CH3), 1.84-1.90 (2H,. m, H-2'), 2.48-2.64 (4H, m, NCH2CH3.and H-3'), 3.68 (2H, br t, J=5.5 Hz, H-1'), 7.46 (1H, ddd, J="7.1, 7.0 and 1.2 Hz, H-6), 7.84, ddd, J=7.0, 6.9 and 1.2 Hz, H-7), 8.31 (2H, m, H-5 and 8), 8.80 (1H, br s, NH). (1V: 7~ 220, 270, 476. Anal. Calc'd. for C14H21N502. (1/3 H20): C, 56.50; H, 7.34; N, 23.55.
Found: C, 56.90; H, 7.15; N, 23.40.
Example 11: Preparation of 7-Nitro-3-(2-N,N-_ Diethvlaminoethvlamino)-1,2.4-Henzotriazine ' ' Dioxide r Oi N v ~_ ~ ' N ~ ~ N'- ~1 '~ .~~~ C~.
~ ~t 19 r U Z Al .v ~ M ~. ,~
< < [~ ( ---"y er-''\N NE:Ei~ HCt is .10 1~ N ,.- N ,' 2 5 s ~ ~ JU ''~-' N ~' Ey a EI C t 1, ~
ai 7-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1-oxide hydrochloride (20): A

solution of 7-vitro-3-chloro-1,2,4-benzotriazine 1-oxide (13~ (1.60g, 7.06 mmol) (prepared as generally shown in Sasse et al., U.S. Patent 4,289,771, with (a) NaN02 and H2S04 at 40°C, follawed by (b) chlorination with POC13 at 106°C) in CH2C12 (50 ml) was treated with N,N-diethylethylenediamine (6.0 ml, 42.7 mmol). After 16 hr at room temperature the mixture was evaporated to dryness under high ~racuum at 60°C. The yellow solid was stirred in 20% iPrOH/ether (150 ml) for 5 hr, filtered, washed with iPrOH then petroleum ether and dried (80°C/1.0 mm:Eig) to give 1.808 (74% yield) of the product 20 as yellow needle crystals. NMR 8 (90 MHz, d6-DMSO/d4-MeOH) 1.25 (6H, t, J=6.0 Hz, CH3), 3.25 (6H, m, NCH2), 3.82 (2H, m, H-1'), 7.74 (1H, d, J=7.0 Hz, 15 H-5), 8.52 (1H, dd, J=7.0 and 2.O Hz, H-6), 8.91 (1H, d,.
J=2.0 Hz, H-8).
7-vitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1,4-dioxide hydrochloride (21). To a stirred solution of 7-vitro-3-(2-N,N-diethylamino-Zp ethylamino)-1,2,4-benzotriazine 1-oxide hydrochloride (20; prepared as described above) (0.50g, 1.46 mmol) in CHC13 (50 ml) at 0°C was added trifluoroacetic anhydride (9.0 ml). After 30 min 70% H202 (4.0 ml) was added and the mixture stirred at room temperature for 3 days, then ~5 dried (Na2S04), filtered, and evaporated in vacuo to dryness to give the trifluoroacetate salt 0.678 (45%
yield?. This product was dissolved in saturated NaHC03 solution (30 ml) and extracted with CH2C12 (3x30 ml).
The dich_loromethane was washed with H20, dried (Na2S04), 3J filtered, saturated with gaseous HC1 and evaporated to dryness to give 0.35g (63% yield, 28% overall) of the product as a red solid, m.p. 194-195°C. UV: a 260, 306, 388, 479. Anal. Calc'd. for C13H18N604~HC1: C, 43.50;
H, 5.34; N, 23.43. Found: C, 43.20; H, 5.37; N, 23.11.
The following Examples 12-15 are directed to reductive de,amination reactions for preparing compounds of Formula (I) which are unsubstituted at the 3-position, i.e.. wherein the substituent "X" is hydrogen.
Example 12: Pretaaration of 1,2,4-8enzotriazine 1,4-Dioxide by Reductive Deamination of 3-Amino-1.2.4-Benzotriazin~e 1,4-Dioxide o ~ o T T
~ N '."' m n~ "'~ N
~~ 1 C3 '~-/ ~ ~ .-~ N N ~ ~ N i-L ~ ~, To a rapidly stirred solution of t-butyl nitrite (867 mg, 1.0 ml, 8.41 mmol) in DMF (20 ml) at 60-65°C was .added 3-amino-1,2,4-benzotriazine 1,4-dioxide ("SR 4233") (500 mg, 2.81 mmol) (prepared by the method of Seng et al., Anctew. Chem. internat. Edit.
11:11 (1972)) in small portions over 5 min. Following the addition, and subsidence of the concomitant effervescence (approx. 5 min), the solution was cooled and reduced 'under high vacuum to a dark waxy solid.
Flash chromatography (30~ EtOAc/CH2C12) gave a yellow solid, mp 188-189.5°C (dec.), which was recrystallized , -47_ v Zp(~ 1 g 4~3 from methanol to give 195 mg (43% yield) of the product 9 as bright ~~ellow platelets, mp 192-194°C (dec.). NMR:
d (400 MHz, d6-acetone) 8.04 (1 H, ddd, J=8.5, 7, 1.5 Hz), 8.15 (1 H, ddd, J=8.5, 7, 1.5 Hz), 8.42 (1 H, dd, J=8.5, 1.5 H:c), 8.43 (1 H, dd, J=8.5, 1.5 Hz) 9.05 (1 H, s, H-3). W.: J~ 405, 300, 225. MS, m/z (relative intensity) lfi4(9), 163(100, M'~), 147(13), 136(19), 90(7), 78(27;1, 76(26), 75(8), 64(9), 63(10), 52(12), 51(48), 50(2E3), 38(8), 37(5), 30(18), 28(6), 27(7).
Anal. Calc'd,. for C7H5N302: C, 51.54; H, 3.09; N, 25.76. Found: C, 51.42; H, 3.02; N, 25.66.
Example 13:- Preparation of 7-Allyloxy-1,2,4-Benzotriazine 1,4-Dioxide Via Reductive Deamination N~
C~ 1 ---~. ~ ~ ~ a t ~N/~N~ ~N/

7-Allyloxy-1,2,4-benzotriazine 1,4-dioxide 24:
To a stirred solution of t-butyl nitrite (271 mg, 0.312 ml, 2.63 mmo'.l) in DMF (15 ml) at 60-65°C was added 7-allyloxy-3--amino-1,2,4-benzotriazine- 1,4-dioxide 23 (205 mg, 0.875 mmol) in small portions over 5 min.
After 30 min additional t-butyl nitrite (271 mg, 0.312 ml, 2.63 mmo:l) was added, and shortly thereafter the deep red solution effervesced and lightened appreciably ~1~~~.~~;~ -48-in color over' a period of a few minutes. After an additional 30 min the resultant orange solution was reduced under' vacuum to a brown solid which was sequentially flash chromatographed (10% EtOAc/CH2C12) and crystallized (CH2C12/petroleum ether) to give 72 mg (38% yield) of the product 24 as light orange crystals, mp 147-148°C. NMR: d (400 MHz, d6-acetone) 4.89 (2 H, ddd, H-1', J1,',2'=5.5, J1~,3'cis=Jl°,3'trans=1.5 Hz), 5.36 (1 H, dctd, H-3', J3~,2'cis=10.5, J3~,3~=3, J3~,1~=1.5 H~:), 5.52 (1 H, ddd, H-3°, J3~,2'trans=17~5, J3',3'=3~ J3'1'=1.5 Hz), 6.14 (1 H, ddt, H-2', J2',3'cis=10.5, J2',1'=5.5 Hz), 7.70 (1 H, d, H-8, Jg,6=2.5 Hz), 7.74 (1 H, dd, H-6, J6,5=9.5, J6,g=2,5 Hz), 8.33 (1 H, d, H-5, J5,6= 9.5 Hz), 8.93 (1 H, s, H-3). W: a, 425, 410, 365, 355, 320, 245, 200. MS
m/z/ (relative intensity) 220(4), 219(34,M+), 103(4), 77(4), 75(4),. 63(13), 62(4), 42(3), 41(100), 39(16).
Anal. Calc'd. for ClpHgN303: C, 54.79; H, 4.14; N, 19.17. Found: C, 54.73; H, 4.16; N. 19.15.
Example 14: Preparation of 7-(3-N-Ethylacetamido-2-acetoxvpropoxv)-1.2.4-Benzotriazine 1.4-Dioxide Via Reductive Amination OAc ( o ~/'~ ~, ~'J' N O ~ ~ N ~ N
l ~ ._..~,. ( ~ 0 a a Et w .J~
V G N la, ~" i I~CN6'E~

i4~~'~.~~~ -49-To a stirred solution of t-butyl nitrite (185 mg, 1.79 mmo:l) in DMF (5 ml) at 60°C was added via syringe a solution of 7-(3-N-ethylacetamido-2-acetoxypropoxy)-3-amino-1,2,4-benzotriazine 1,4-dioxide (25) (125 mg, 0.329 mmol) in DMF (5 ml) over a period of 1 min. After 5 min additional t-butyl nitrite (217 mg, 2.10 mmol) was added and an immediate reaction occurred, as evidenced by the evolution of a gas and a change in color of the solution from red to :light orange. After an additional 10 min the solution was stripped to a yellow/brown solid and eluted throu<~h silica gel with 5% MeOH/CH2C12 to give 119 mg of a yellow oil. Recrystallization from CH2C12/ligroin gave 90 mg yellow solid (75% yield), mp 179-180.5°C. NMR: d (400 MHz, d4-methanol, mixture of rotamers, ratio approx. 2:1) 1.12, 1.22 (t's, 1:2, 3 H
total, J=7 Ha), 2.06, 2.07 (s's, 2:1, 3 H total), 2.11, 2.17 (s's, 2.:1, 3 H total), 3.41-3.92 (m, 4 H), 4.34-4.48 (m" 2 H), 5.48-5.58 (m, 1 H), 7.76-7.86 (m, 2 H), 8.36-8.42 (m, 1 H), 9.04, 9.06 (s's, 2:1, 1 H
total). W: a 420, 405, 365, 350, 315, 240, 200. MS:
m/z (relativE~ intensity) 365(0.5), 364(1.4, M+), 349(0.5), 348(1.1), 347(0.5), 332(1.2), 331(3.6), 187(7), 186(ES6), 102(6), 100(21), 84(30), 63(6), 58(100), 56(8), 43(65), 42(9)~ 41(5), 30(14), 29(5), 28(8).

r~~~~~. ~~~
8:a~~le 15: Preparation of 7-Nitro-1.2.4-Benzotriazine 1t4~-Dioxide 'via Reductive Deamination o C
r T
---~ ~z t'~ ~ d ~~ N~~N11 ~'~-~ /
i ~ N
O

To a stirred solution of t-butyl nitrite (88 mg, 0.85 mmo:1) in DMF (5 ml) at 60°C was added 7_vitro-3-amino-1,2,4-benzotriazine 1,4-dioxide (14) (38 mg, 0.17 mmo:l). After 30 min the addition of further t-butyl nitrate (175 mg, 1.70 mmol) to the dark red slurry was irnmediately followed by a change in coloration and effervescence. After an additional 10 min the orange solution was reduced to a red solid _in vacuo and chromatographed with 1% AcOH/CH2CI2 to give 3 mg of the product 27 as a yellow solid (10% yield). NMR
S (90 MHz, d6-dimethyl sulfoxide) 7.68 (d, 1H, J = 9.2 Hz), 7.92 (dd, 1H, J' = 9.2, 2.2 Hz), 8.10 (d, 1H, J =
2.2 Hzf, 8.65 (s, 1H)). W: a 420, 310, 240, 205.
M~:m~z (relai:ive intensity) 209 (9), 208 (100, M+), 192 f~'4~, I81 (1!E), 162 (16), 105 (9), 77 (28), 75 (52), 74 (2?~, G3~(21;1, 62 (16), 30 (77), 18 (26).

Example 16: In Vivo Assav for Activity in Combination with Radiation Compounds of the invention were tested in 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 beginning of each experiment. The SCCVII tumor was grown intradermally in the flank from an inoculation of 2 x 105 tumor cells taken from the 2nd-8th in vitro passage of t:he tumor cells after removal from the previous in vivo tumor. 7.'wo 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 20o hypoxic cells.
The test compound was tested at a fixed injected does of either 5 m~nol/kg or 2/3 of the LDSO (whichever was lower). Suitabl°e control of the test compound-injected but non-irradiated and saline-injected and irradiated mice were also included. A fixed radiation does 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 basis of additivity of the two individual cytotoxicities. T:he similar increased cytotoxicity when .....
iC:~!~~.~~~~s °52-the4-drug is given before or after radiation indicates serl~ctive:to:Kicity to the hypoxic cells rather than a radiosensiti;sing effect of the benzotriazine dioxide.
Irradiation of the SCCVIi tumors was done by irradiating nonanaesthetized tumor-bearing mice in a Plexiglas bo:K. irradiation conditions were 250 kVp X-rays, 15 mid, 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 cells 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 (HESS) containing 0.02% DNase, 0.05%
promase, and 0.02% collagenase. The suspension was stirred for :30 min at 37°C, filtered, and centrifuged at 1,600 rmp for 10 min at 4°C. 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 polystyrene petri dishes (Lux Scientific Corp) in 5 or 15 ml of med3~um. After incubation for 13 days, the co~.anies were fixed and stained, and those containing 50 cl~~~i or mores were counted. The dilution yielding an avesrage count: of 25-100 colonies in a 60 mm dish was used im calculation of results.

~~~~.~~~ _53_ Example I7: Cvtotoxicitv Tests Cytotoxici.ty tests were carried out using 3-amino-1,2,4-benzotriazine 1,4-dioxide and a variety of aerobic and ;hypoxic cells in culture (human, mouse, and hamster). T'.he cells in spinner flasks were gassed for one hour at 37°C with either air or nitrogen containing 5% C02 prior to adding the specified amounts of the drug. Figures 1A, 18 and 1C show the results for cell survival of mouse, hamster and human cells at various concentrations of 3-amino-1,2,4-benzotriazine 1,4-dioxide. It was found that only 1 to 2% of the drug concentration under aerobic conditions was required to get equal c ell 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 18: Determination of LD50 LD~~p is determined in BALB/c female mice (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. LD50 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 19: Radiosensitivity in Vitro The results of assays to determine the concentration of drug necessary to produce a sensitizer enhancement ratio of 1.6 of hypoxic cells in culture are as follows:

Compound C1.6 ((mM)) 7-chloro-3-amino-1,2;4-benzotriazine 3.3 1-oxide 6(7)-methoxy~-3-amino-1,2,4-benzotriazine 1,4-dio:Kide '1.0 3-hydroxy-:1,2,4-benzotriazine 1,4-dioxide '"2.0 Modifications of the above described modes for carrying out the invenltion 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 20: Enhanced Tumor Cell Toxicitv Using Hvdralazine Hydralazine is an antihypertensive drug which acts by rela:King the smooth muscle around blood vessels.
This has the effect of preferentially shunting blood flow into normal tissues and away from tumors, which process produces immediate hypoxia in the tumors. If 3-amino-1,2,n~-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 in every 1000 was lefit viable). The experimental procedures are the same as described in Example 9, and the results are shown in Figure 3.

~:~~~~.~~~ -55-Example 21: Physicochemical and Biolocrical Properties of Some I,2,4-Benzotriazine 1,4-Dioxides The i:ollowing table sets forth various properties of compounds 22, 14, 18a, and 21 as determined by t:he inventors herein:
Solubility El/2b C.~,d. Mol. _ (mM) Log Pa (mV) RHTo HCRd LD50e Wt.

22 178.2 13.50 -0.32 -332 1 100 0.50 14 223.2 2.00 -0.97 -133 2 140 1.00 18a 291.4 >181 -1.34 -348 3 100 0.25 21 358.9 96.40 -0.20 -140 3 211 0.40 a.) Log of the octanol-water partition coefficient as measured by the method of Fujita et al., J. Amer. Chem. Soc. 86:5175 (1964), using pH 7.4 buffer.
b.) Polarographic half-wave reduction potentials measured in Britton & Robinson pH 7.4 buffer using a dropping mercury electrode.
c.) Relative Hypoxic Cytoxicity: Ratio of equitoxic concentrations of 22: analog for HA-1 cells attached, under, hypoxic conditions. Exponentially growing cells were placed in suspension culture and gassed for 90 nnin in nitrogen or air prior to addition of drugs. Samples were removed periodically for a survival determination, and the ratios determined from a comparison oi: the resulting survival curves.
d.) Hypoxic Cytoxicity Ratio: Ratio of equitoxic concentrations of each analog for HA-1 cells atta~,ned under' hypoxic:aerobic conditions. Treatment conditions as above.
e.) l3alb/c female mice 3-5 months of age were used in the LDSp experiments. LDSp was evaluated as described in Example 18.
As may be readily deduced from the table, novel compounds 14, 1~8a,and 21 exhibit significantly enhanced cytotoxicity against hypoxic HA-1 tumor cells in vitro compared to 3-amino-1,2,4-benzotriazine 1,4-dioxide (22), while retai:~ing the high differential cytotoxicity against hypoxic cells compared to aerobic cells. These results suggest that these drugs will be more tumor-specific and therefore more effective as antitumor agents in vivo.
Example 22: Fractionated Radiotheratay Usina 3-Amino-1.2.4-Benzotriazine 1,4-Dioxide The following experimental work establishes that pre- or post-irradiation treatment of cells _in vitro with compound 22 under hypoxic conditions radiosensitizes the cells even when drug is not present during radiation exposure and the cells are aerobic.
a.) Figure 4 shows the results of experiments in which the survival of Chinese hamster ovary (CHO) cells after graded doses of x-rays was determined either with a hypoxic compound 22 exposure given before or after irradiation. The preirradiation treatments consisted of 20 uM drug for exposure times of 1.0 (D
in Figure 4), 1.5 (~), 2.0 (a) and 2.5 (Q) h duration prior to cells being reaerated and irradiated.
These drug treatments alone reduced cell survival to approximately 32, 19, 8 and 2~ respectively. The ~~~'~~.~~ -57-postirradiation drug treatment consisted of 20 uM for 1.5 h (+), which alone reduced cell survival to 23%.
Compared to the survival curve for cells exposed to pre- or post-irradiation hypoxia only (t ), treatment with compound X22 sensitized cells to aerobic irradiation. The sensitization was predominantly a change in the :slope of the radiation survival curve.
Do decreased from 1.34-Gy (95% confidence limits:
1.09-1.76 Gy) 1:o 0.80 Gy (95% confidence limits:
0.73-0.88 Gy), based on least-squares regression analysis of pooled data for the exponential portions of the survival curves for control and treated cells.
However, sensitization at low radiation doses was also readily apparent. Survival ratios for "no drug:drug-treated" CHO csalls irradiated with doses of 1-3 Gy averaged a factor of 3.6. The amount of the radiosensitizat:ion produced did not vary with the severity of them drug treatment.
b.) The data of section (a.) on radiosensitizat:ion of aerobic cells at low radiation doses by hypoxi.c activation led us to test the feasibility of obtaining preferential radiosensitizat:ion of tumors in vivo. Our protocol was to use eight doses of 2.5 Gy/dose in four days (irradiating 2 x/day). Because of the in vitro data showing that ra~diosensitization could only be achieved by hypoxia activation, either before or after irradiation, and because we did not want to make the tumors hypoxic :before irradiation (as this would make them resistant), we made the tumors hypoxic after ~Q~~.~~~ 58 irradiationusing the vasoactive drug hydralazine (HDZ) at the same time as injecting compound 22. We used two different types of controls. First, compound 22 alone before each dose of radiation, and second, the potent hypoxic cell sensitizer SR 2508 (a 2-nitroimidazole, DuPont, undergoing Phase III clinical trials in Europe) before each dose. The effectiveness, or lack of effectiveness, or SR 2508 indicates whether the radiation response of the tumor is being governed by hypoxic, or .aerobic, cells respectively. We assayed the efficacies of the treatments using clonogenic cell survival and also regrowth delay. Figures 5 and 6 show the results.
As Figure 5 illustrates, there was no effect of SR 2508 (:1000 mg/kg) given before each radiation dose, but compound ~2 (0.08 mmole/kg) given alone or with hydrala;zine (HDZ) produced a large enhancement of the radiation response. Part of this is attributable to an additive response (crosses), but the additional cell killing is the result of radiosensitization of aerobic cells.
In Figure 6, it may be seen that SR 2508 (1000 mg/kg) before each radiation dose had no radiosensiti;zing effect, but compound 22 (0.08 mmole/kg) alone before irradiation or with HDZ after irradiation produced a large increase in effect compared to radiation or drug alone.
The major and unexpected result of these experiments is the radiosensitization of the tumors by compound 22 .given before each radiation dose without the addition of hydralazine. This cannot be accounted l ~ ~~~~~.~~~~ -59-for by a radiosensitization of hypoxic cells, since SR
2508 (a hypoxic radiosensitizer) is ineffective. Thus, it is a radiosensitization of aerobic tumor cells.
This would not be useful if it radiosensitized aerobic normal cells. We tested this by performing the same eight fraction protocol on the response of normal mouse skin using a skin reaction scoring scale previously used by us. Figure 7 shows the result.
There is no radiosensitization of normal skin.
In conclusion, the data show that the aerobic cells of tumors can be radiosensitized in multifraction regimes similar to those used in radiotherapy. The radiosensiti;zation i.s tumor specific (i.e., does not occur in nornnal cel7.s), and appears to be the result of activation b;y hypoxic areas in the tumors.

Claims (43)

1. The use of a pharmaceutical composition comprising a compound of the formula wherein X is NH2, NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms or acyl of 1-4 carbon atoms, or wherein in the case of NRR the two R groups may be linked together to form a morpholino, pyrrolidino or piperidino ring, and wherein R may be further substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents:
n is 1; and Y1 is H and Y2 is nitro;
or a pharmacologically acceptable salt of said compound;
to selectively kill hypoxic tumor cells.

2. The use of claim 1, wherein X is NH2.

3. Use of a pharmaceutical composition comprising a compound of the formula wherein m is an integer in the range of 0-4 inclusive, and R1 and R2 are independently selected from hydrogen or lower alkyl or together form a piperidino or pyrrolidino ring;
Y1 is H and Y2 is nitro;
or a pharmaceutically acceptable salt thereof , for selectively killing hypoxic tumor cells.

4. The use of claim 3, wherein m is 0, 1 or 2.

5. The use of claim 4, wherein the compound is 7-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine-1,4-dioxic

6. The use of claim 2, wherein the compound is 7-nitro-3-amino-1,2,4-benzotriazine-1,4-dioxide.

7. The use of a pharmaceutical composition comprising a compound of the formula wherein X is H or hydrocarbyl (1-4C), and, if hydrocarbyl, may be substituted with OH, NH2, alkoxy (1-4C), or halogen substituents;
n is 1; and Y1 is H and Y2 is nitro;
or a pharmacologically acceptable salt of said compound;
to selectively kill hypoxic tumor cells

8. The use of claim 7, wherein X is H.

9. The use of claim 7, wherein X is hydrocarbyl (1-4C).

10. The use of claim 8, wherein the compound is 7-nitro-1,2,4-benzotriazine-1,4-dioxide.

11. The use of a pharmaceutical composition comprising a compound of the formula:

wherein X is halogen; OH; alkoxy (1-4C); NH2; NHR or NRR, wherein the R groups are independently selected from alkyl (1-4C) and acyl (1-4C) and the R's may themselves be substituted with OH, NHZ, lower alkyl (1-4C) secondary and dialkyl (1-4C) tertiary amino groups, alkoxy (1-4C) or halogen, and in the case of NRR, the two R's can be linked together directly or through a bridge oxygen into a morpholino ring, pyrrolidino ring or piperidino ring;
wherein n is 0 or 1; and Y1 is H and Y2 is vitro;
or a pharmacologically acceptable salt of said compound;
to radiosensitize hypoxic tumor cells.

12. The use of claim 11, wherein X is OH ar OR.

13. The use of claim 11, wherein X is NH2, NHR or NRR.

14. The use of claim 13, wherein X is NHZ.

15. The use of claim 14, wherein Y1 is H, Y2 is vitro, and n is 1.

16. Use of a pharmaceutical composition comprising a compound of the formula wherein m is an integer in the range of 0-4 inclusive, and R1 and R2 are independently selected from hydrogen or lower alkyl or together form a piperidino or pyrrolidino ring;
n is 0 or 1;
Y1 is H and Y2 is nitro;
or a pharmaceutically acceptable salt thereof, for selectively killing hypoxic tumor cells.

17. The use of claim 16, wherein m is 0, 1 or 2.

18. The use of claim 17, wherein the compound is 7-nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine-1,4-dioxide.

19. The use of claim 15, wherein the compound is 7-nitro-3-amino-1,2,4-benzotriazine-1,4-dioxide.

20. The use of a pharmaceutical composition comprising a compound of the formula:
wherein X is H; hydrocarbyl (1-4C); or hydrocarbyl (1-4C) substituted with OH, NH2; NHR or NRR, wherein the R
groups are independently selected from alkyl (1-4C) and acyl (1-4C), optionally substituted with OH, NH2, alkyl (1-4C) secondary and dialkyl (1-4C) tertiary amino groups, alkoxy (1-4C) or halogen, and in the case of NRR, the two R's can be linked together directly or through a bridge oxygen into a morpholino ring, pyrrolidino ring or piperidino ring;
wherein n is 0 or 1; and Y1 is H and Y2 is nitro;
or a pharmacologically acceptable salt of said compound;

to radiosensitize hypoxic tumor cells.

21. The use of claim 20, wherein X is H.

22. The use of claim 20, wherein X is hydrocarbyl (1-4C).

23. The use of claim 21, wherein the compound is 7-nitro-1,2,4-benzotriazine 1,4-dioxide.

24. A compound having the structural formula:
wherein X is OH, alkoxy (1-4C), NHR or NRR where each R is independently an alkyl of 1-4 carbon atoms, or acyl of 1-4 carbon atoms, or where the two R groups are alkyls linked together to form a pyrrolidino or piperidino ring or linked through an oxygen to form a morpholino ring, and the R groups may be further substituted with OH, NH2, alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents;
n is 1; and Y1 is H and Y2 is nitro;
or a pharmacologically acceptable salt thereof.

25. A compound according to claim 24, wherein X is OH or alkoxy.

26. A compound according to claim 24, wherein X is NRR.

27. A compound of the formula wherein m is an integer in the range of 0-4 inclusive, and R1 and R2 are independently selected from hydrogen or lower alkyl or together form a piperidino or pyrrolidino ring;
Y1 is H and Y2 is nitro;
or a pharmaceutically acceptable salt thereof.

28. A compound according of claim 27, wherein m is 0, 1 or 2.

29. 7-Nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine-1,4-dioxide according to claim 28.

30. A compound having the structural formula:
X is NF.2, n is 1; and Y1 hydrogen and Y2 is nitro;
or a pharmacologically acceptable salt thereof.

31. 7-Nitro-3-amino-1,2,4-benzotriazine-1,4-dioxide according to claim 30.

32. A compound having the structural formula:
X is hydrogen or hydrocarbyl (2-4C) optionally substituted with OH, NH2, alkoxy (1-4C) or halogen substituents;
n is 1; and Y1 is H and Y2 is nitro, or a pharmacologically acceptable salt thereof.

33. A compound according to claim 32, wherein X is H.

34. A compound according to claim 32, wherein X is hydrocarbyl (2-4C).

35. 7-Nitro-1,2,4-benzotriazine-1,4-dioxide according to claim 33.

36. A method of synthesizing a 1,2,4-benzotriazine oxide having the structure wherein X is H; n is 1 and Y1 and Y2 are independently either H; nitro; 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 (1-4C), alkylthio (1-4C), primary amino (NH2), lower alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl (1-4C) tertiary amino where the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (-O-) linkage, or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHR', NR'R',O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-4C) which may be substituted with one or more OH, NH2, alkyl-(1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents, or a pharmacologically acceptable salt of said compound, said method comprising:
treating a 3-amino-1,2,4-benzotriazine oxide having the structure - with a lower alkyl nitrite under reductive deaminating conditions.

37. The method of claim 36, wherein said lower alkyl nitrite is t-butyl nitrite.

38. The method of claim 36, wherein said reductive deaminating conditions comprise reaction in a compatible solvent at a temperature of at least about 60°C.

39. The use of a pharmaceutical composition comprising a 1,2,4-benzotriazine oxide having the structure wherein X is H; hydrocarbyl (1-4C); hydrocarbyl (1-4C) substituted with OH, NH2, NHR or NRR; halogen; OH; alkoxy (1-4C); NH2; NHR or NRR, wherein the R groups are independently selected from alkyl (1-4C) and acyl (1-4C), optionally substituted with OH, NH2, alkyl (1-4C) secondary and dialkyl (1-4C) tertiary amino groups, alkoxy (1-4C) or halogen, and in the case of NRR, the two R's can be linked together directly or through a bridge oxygen into a morpholino ring, pyrrolidino ring or piperidino ring;
n is 0 or 1; and Y1 and Y2 are independently either H; vitro, 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 (1-4C), alkylthio (1-4C), primary amino (NH2), lower alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, dialkyl (1-4C) tertiary amino wherein the the two alkyls are linked together to produce a morpholino, pyrrolidino or piperidino, acyloxy (1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbyl can optionally be interrupted by a single ether (-O-) linkage; or wherein Y1 and Y2 are independently either morpholino, pyrrolidino, piperidino, NH2, NHR', NR'R', O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH2, alkyl-(1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), or halogen substitutents to radiosensitize tumor cells in a warm-blooded mammal wherein said tumor cells are subjected to distinct radiation doses; and wherein the mammal receives a plurality of doses of drug and radiation over an extended period of time, wherein each of said radiation doses is less than about 5 Gy.

40. The use of claim 39, wherein the pharmaceutical composition is used prior to radiation.

41. The use of claim 39, wherein the pharmaceutical composition is used after radiation.

42. The use of claim 39, wherein each of said radiation doses is less than about 2.5 Gy, and said extended period of time is at least about 3 days.

43. The use of claim 39, wherein said 1,2,4-benzotriazine oxide is 3-amino-1,2,4-benzotriazine-1,4-dioxide.