Method of treating a mammal having a solid tumor susceptible to treatment with cisplatin

A method of treating a mammal having a solid tumor susceptible to treatment with cisplatin comprising: administering to the mammal an effective amount of 3-amino-1,2,4-benzotriazine 1,4-dioxide; and administering to the mammal an effective amount of cisplatin about 0.5 to about five hours after administration of the 3-amino-1,2,4-benzotriazine 1,4-dioxide.

FIELD OF THE INVENTION 
The present invention relates to the field of treatments for cancer tumors. 
More particularly the present invention relates to treatment of cancer 
tumors with combinations of chemotherapy agents and 1,2,4-benzotriazine 
oxides. 
BACKGROUND OF THE INVENTION 
The most commonly used anticancer drugs are more ctyotoxic toward normally 
oxygenated tumor cells than toward hypoxic tumor cells. Hypoxic cell 
resistance to irradiation is also widely known. Consequently, tumor 
hypoxia and the resultant resistance to treatment is of concern in cancer 
therapeutics. 
Solid cancer tumors contain both adequately oxygenated cells as well as 
varying proportions of inadequately oxygenated or hypoxic cells. Hypoxia 
usually occurs where the tumor cells are furthest away from blood vessels. 
Such cells also tend to have slower rates of proliferation. Although not 
completely understood, resistance of hypoxic cells to anticancer drugs is 
generally thought to be due to inadequate uptake of the drug by the 
hypoxic cells either because they tend to be slowly growing or because of 
their distance from the blood vessels bringing the drug. Thus, the 
relative proportion of hypoxic cells in the tumor can be of great 
importance to the outcome of the treatment. Resistant hypoxic cells that 
survive irradiation or drug treatment may become reoxygenated, thereby 
restoring tumor sensitivity to further treatment. Nonetheless, instead of 
relying on uncertain events, it is desirable to develop cancer treatments 
wherein cancer tumor cells, including hypoxic tumor cells, are killed or 
rendered inactive more reliably at the time the treatment is administered. 
U.S. Pat. No. 5,175,287 issued Dec. 29, 1992 discloses the use of 
1,2,4-benzotriazine oxides in conjunction with radiation for treatment of 
tumors. The 1,2,4-benzotriazine oxides sensitize the tumor cells to 
radiation and make them more amenable to this treatment modality. 
Holden et al (1992) "Enhancement of Alkylating Agent Activity by SR-4233 in 
the FSaIIC Murine Fibrosarcoma" JNCI 84: 187-193 discloses the use of 
SR-4233, also known as tirapazamine, in combination with an antitumor 
alkylating agent. The four antitumor alkylating agents, cisplatin, 
cyclophosphamide, carmustine and melphalan, were each tested to examine 
the ability of tirapazamine to overcome the resistance of hypoxic tumor 
cells to antitumor alkylating agents. Tirapazamine was tested alone and in 
combination with varying amounts of each of the antitumor alkylating 
agents. When SR 4233 was administered just before single-dose treatment 
with cyclophosphamide, carmustine or melphalan marked dose enhancement 
leading to synergistic cytotoxic effects on tumor cells was observed. When 
SR 4233 was administered just prior to single-dose treatment with 
cisplatin, however, the dose enhancement lead to an additive effect, 
except at the highest dose level of cisplatin. 
Nitroimidazole hypoxic cytotoxic agents have been combined with various 
anticancer drugs and it was found that a therapeutic gain could be 
achieved when these agents were combined with various anti-cancer drugs, 
particularly the alkylating agents, cyclophosphamide and melphalan and the 
nitrosoureas, BCNU and CCNU. However, it was later found that the 
therapeutic gain produced was not the consequence of selective killing of 
hypoxic cells by the nitroimidazoles but appeared to be by a mechanism 
involving the potentiation of alkylating agent-induced DNA cross-links by 
metabolites of the nitroimidazoles (Murray et al. (1983) Br. J. Cancer 47: 
195-203). 
SUMMARY OF THE INVENTION 
The present invention provides methods of treating cancer tumors, 
particularly solid tumors comprising adminstering to a mammal in need of 
such treatment an effective amount of a compound having the formula 
##STR1## 
wherein X is H; hydrocarbyl (1-4C); hydrocarbyl (1-4C) substituted with 
OH, NH.sub.2, NHR or NRR; halogen; OH; alkoxy (1-4C); NH.sub.2 ; NHR or 
NRR; wherein the various R groups are independently selected from lower 
alkyl (1-4C) and lower acyl (1-4C) and the R's may themselves be 
substituted with OH, NH.sub.2, 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 
Y.sup.1 and Y.sup.2 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 
(NH.sub.2), 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 Y.sup.1 and 
Y.sup.2 are independently either morpholino, pyrrolidino, piperidino, 
NH.sub.2, 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, NH.sub.2, alkyl 
(1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino, 
pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents, or 
pharmacologically acceptable salts of said compound; and administering to 
the mammal from about one half hour to about twenty-four hours after 
administering the compound of Formula I, as defined herein, an effective 
amount of a chemotherapy agent to which the tumor is susceptible. 
The present invention also provides methods of increasing the toxicity of 
chemotherapy agents towards solid tumors. In this aspect of the invention 
a cytotoxicity-enhancing amount of a compound of Formula I, as defined 
hereinabove, is administered to a mammal having a solid tumor and in need 
of such treatment, the tumor further being susceptible to treatment with 
the chemotherapy agent, about one half hour to about twenty-four hours 
prior to administering the chemotherapy agent, or about one hour to about 
two hours after administering the chemotherapy agent. 
In another aspect, the present invention provides a method of treating 
mammalian cancer tumors comprising administering a compound of Formula I, 
as defined herein, to the mammal one to two hours after administration of 
a chemotherapy agent. 
In a preferred aspect the present invention provides a method of treating a 
mammal having a solid tumor susceptible to treatment with cisplatin, said 
mammal being in need of such treatment, the method comprising: (a) 
administering to said mammal an effective amount of 
3-amino-1,2,4-benzotriazine 1,4-dioxide; and (b) administering to said 
mammal an effective amount of cisplatin about 0.5 to about five hours 
after administration of said 3-amino-1,2,4-benzotriazine 1,4-dioxide. 
Applicants have discovered that administering a compound of Formula I, as 
defined herein, either before or after the administration of a 
chemotherapy agent surprisingly and unexpectedly killed tumor cells to a 
much greater extent than administration of either agent alone, or 
administration of both agents at the same time. When tirapazamine was 
administered up to twenty-four hours prior to administration of cisplatin, 
Applicants found there was a ten to one thousand fold increase in tumor 
cell killing above the amount of tumor cell killing found when 
tirapazamine and cisplatin were administered at the same time. The 
greatest synergistic effect with this combination of agents was found when 
tirapazamine was administered about two and one half hours prior to 
administration of cisplatin. 
Applicants' claimed method represents an enormous increase in anti-tumor 
efficacy of the chemotherapy agent (i.e. its cytotoxic effects upon tumor 
cells). Additionally, in tests of the systemic toxicity of cisplatin 
(serum BUN and acute toxicity) the combination with the optimum separation 
for tumor efficacy showed little or no enhancement of systemic toxicity 
compared to cisplatin alone. Thus, most, if not all, of the additional 
cell kill of the tumor cells translates into a therapeutic gain for this 
combination. The synergistic interaction between tirapazamine and 
cisplatin is also significant since the great increase in tumor cell 
killing was produced at a relatively low dose of cisplatin.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides methods for wearing mammalian cancer tumors, 
including human cancer tumors, particularly solid tumors. In this aspect 
of the invention, an effective amount of a compound having Formula I, as 
defined herein, is administered to a mammal having a cancer tumor and in 
need of such treatment from about one half hour to about twenty-four hours 
before an effective amount of a chemotherapy agent to which the tumor is 
susceptible is administered to the mammal. 
As used herein, susceptibility of a tumor to a chemotherapy agent refers to 
a chemotherapty agent that is capable of exerting a therapeutic effect on 
a tumor by any mechanism such as by killing tumor cells, reducing cell 
proliferation or reducing the size of the tumor. Also as used herein, 
effective amount of the compound of Formula I, as defined herein, refers 
to amounts capable of killing tumor cells or capable of killing tumor 
cells in conjunction with a chemotherapy agent. An effective amount of a 
chemotherapy agent refers to an amount of the chemotherapy agent capable 
of killing cancer cells or otherwise producing a therapeutic effect such 
as by reducing tumor size or slowing tumor cell growth and proliferation. 
Another aspect of the invention provides a method for increasing the 
cytotoxicity of a chemotherapy agent towards a solid tumor susceptible to 
treatment with the chemotherapy agent comprising, administering a 
cytotoxicity-enhancing amount of a compound of Formula I, as defined 
herein, to a mammal having such a tumor from about one hour to about two 
hours after administering the chemotherapy agent. As used herein, the term 
cytotoxicity-enhancing amount refers to an amount of the compound of 
Formula I, as defined herein, that is capable of of increasing the 
cytotoxic effects of the chemotherapy agent on cells. Preferably the 
cytotoxicity-enhancing amount is sufficient to produce a synergistic 
effect, i.e., greater than the sum of the effects of the chemotherapy 
agent and the compound of Formula I when administered singly. 
Cytotoxicity-enhancing amounts of the of the compound of Formula I can be 
assessed by testing such compounds with a chemotherapy agent(s) in in vivo 
and/or in vitro experimental tumor models, such as the one set forth 
herein, or any other tumor model known in the art. The 
cytotoxicity-enhancing amount determined through in vivo and or in vitro 
experimental tumor models is then used as a guide for determining the 
amounts of the two agents that will be administered to the mammal for 
treatment of the tumor. 
Another further aspect of the invention provides methods for increasing the 
cytotoxicity of a chemotherapy agent towards a solid tumor susceptible to 
treatment with the chemotherapy agent, comprising administering to a 
mammal having such a tumor a cytotoxicity-enhancing amount of a compound 
having Formula I, as defined herein, from about one hour to about two 
hours after administering a chemotherapy agent. 
Without wishing to be bound by any theory or mode of action, at the present 
time it is believed that the combination of a benzotriazine chemotherapy 
agent of Formula I, as defined herein, that is specifically cytotoxic to 
hypoxic cancer cells and a chemotherapy agent having its greatest activity 
on normally oxygenated cancer cells provides enhanced or synergistic 
killing of tumor cells. The benzotriazines oxides of Formula I, as defined 
herein, specifically require lower than normal oxygen concentrations in 
order to exert their effects. This requirement for hypoxia is a major 
advantage, since it provides the basis for tumor-specific interaction 
between the two drugs. In general, normal tissues are at an oxygen 
concentration above 10-15 mm Hg. At these and higher oxygen partial 
pressures, the cytotoxicity produced by tirapazamine is very low. On the 
other hand, many tumors have a significant number of cells at oxygen 
concentrations below 10 mm Hg, at which partial pressures the metabolism 
of tirapazamine and the other benzotriazines of Formula I to cytotoxic 
species is greatly increased. As used herein hypoxic tumor cells refers to 
tumor cells at an oxygen partial pressure less than about 10 mm Hg. 
The methods of the present invention are useful in the treatment of 
mammalian cancer tumors, including human cancer tumors, particularly solid 
tumors having hypoxic regions. Examples of such tumors include, but are 
not limited to, adrenocarcinomas, glioblastomas (and other brain tumors), 
breast, cervical, colorectal, endometrial, gastric, liver, lung (small 
cell and non-small cell), lymphomas (including non-Hodgkin's, Burkitt's, 
diffuse large cell, follicular and diffuse Hodgkin's), melanoma 
(metastatic), neuroblastoma, osteogenic sarcoma, ovarian, retinoblastoma, 
soft tissue sarcomas, testicular and other tumors which respond to 
chemotherapy. Thus, the methods of the present invention can be used to 
treat cancer tumors, including experimentally-induced cancer tumors, in 
any type of mammal including humans, commonly used laboratory animals such 
as rats, mice, rabbits and dogs, primates such as monkeys, and horses, 
cats and other animals. 
The methods of the present invention can be practiced with any type of 
chemotherapy agent. In any particular embodiment of the invention, the 
chemotherapy agent will be selected with reference to factors such as the 
type of cancer tumor and the efficacy of the chemotherapy agent for 
treating the cancer tumor involved. The chemotherapy agent may selected 
from alkylating agents, antimetabolites, natural products, hormones and 
antagonists and other types of compounds. 
Examples of alkylating agents include the nitrogen mustards (i.e. the 
2-chloroethylamines) such as, for example, chloromethine, chlorambucil, 
melphalan, uramustine, mannomustine, extramustine phosphate, 
mechlor-thaminoxide, cyclophosphamide, ifosamide and trifosfamide; 
alkylating agents having a substituted aziridine group such as, for 
example, tretamine, thiotepa, triaziquone and mitomycin; alkylating agents 
of the alkyl sulfonate type, such as, for example, busulfan, and 
piposulfan; alkylating N-alkyl-N-nitrosourea derivatives such as, for 
example, carmustine, lomustine, semustine or streptozotocine; alkylating 
agents of the mitobronitole, dacarbazine and procarbazine type; and 
platinum complexes such as, for example, cisplatin and carboplatin. 
Examples of antimetabolites include folic acid derivatives such as, for 
example, methotrexate, aminopterin and 3'-dichloromethotrexate; pyrimidine 
derivatives such as, for example, 5-fluorouracil, floxuridine, tegafur, 
cytarabine, idoxuridine, and flucytosine; purine derivatives such as, for 
example, mercaptopurine, thioguanine, azathioprine, tiamiprine, 
vidarabine, pentostatin and puromycin. 
Examples of natural products include vinca alkaloids such as for example 
vinblastine and vincristine; epipodophylotoxins such as, for example, 
etoposide, and teniposide; antibiotics such as, for example, adrimycin, 
daunomycin, dactinomycin, daunorubicin, doxorubicin, mithramycin, 
bleomycin and mitomycin; enzymes such as, for example, L-asparaginase; 
biological response modifiers such as, for example, alphainterferon; 
camptothecin; taxol; and retinoids such as retinoic acid. 
Examples of hormones and antagonists include adrenocorticoids, such as, for 
example, prednisone; progestins, such as, for example, hydroxyprogesterone 
acetate, medroxyprogesterone acetate and megestrol acetate; estrogens such 
as, for example, diethylstilbestrol and ethinyl estradiol; antiestrogens 
such as for example, tamoxifen; androgens such as, for example, 
testosterone propionate and fluoxymestrone; antiandrogens such as, for 
example, flutamide; and gonadotropin-releasing hormone analogs such as, 
for example, leuprolide. 
Examples of miscellaneous agents include anthracenediones such as for 
example, mitoxantrone; substituted ureas such as, for example, 
hydroxyureas; and adrenocortical suppressants such as, for example, 
mitotane and aminoglutethimide. 
In addition, the chemotherapy agent can be an immunosuppressive drug, such 
as, for example, cyclosporine, azathioprine, sulfasalazine, methozsalen 
and thalidomide. 
The chemotherapy agents useful in the practice of the present invention are 
commercially available or can be prepared by methods known in the art. The 
chemotherapy agent can be used alone or in combination with one or more 
chemotherapy agents. For example, a combination of three different 
chemotherapy agents and one or more of the compounds of Formula I, as 
defined herein, administered in accordance with the methods of the present 
invention could be used to treat a cancer tumor. 
In the compounds of Formula I, 
##STR2## 
X is hydrogen; unsubstituted branched or straight chain hydrocarbyl (1-4C) 
such as methyl, ethyl, s-butyl and iso-propyl; hydroxy; alkoxy (1-4C) such 
as methoxy, ethoxy, propoxy, and t-butoxy; primary amino (NH.sub.2); 
secondary amino (NHR) where R is an alkyl or acyl of 1 to 4 carbons, such 
as methylamino and ethylamino; 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 groups, they 
can be further substituted with OH, NH.sub.2, lower alkyl (1-4C) secondary 
amino and dialkyl (1-4C) tertiary amino, morpholino, pyrrolidino, 
piperidino, alkoxy (1-4C) or halogen (fluoro, chloro, bromo or iodo) 
substituents. 
The hydrocarbyl X groups can be further substituted with OH, NH.sub.2, 
alkyl secondary amino, dialkyl tertiary amino, alkoxy (1-4C) or halogen 
(fluoro, chloro, bromo or iodo) substituents. 
More preferably X is hydrogen, primary amino (NH.sub.2); unsubstituted 
branched or straight chain hydrocarbyl (1-4C) or substituted branched or 
straight chain hydrocarbyl (1-4C). 
n is 0 or 1, preferably 1. 
Y.sup.1 and Y.sup.2 are independently hydrogen; nitro; halogen (e.g. 
fluoro, chloro, bromo or iodo); or hydrocarbyl (1-14C). When hydrocarbyl, 
Y.sup.1 or Y.sup.2 may be saturated or unsaturated, cyclic or acyclic, and 
may optionally be interrupted by a single ether linkage. Thus, the 
unsubstituted hydrocarbyl forms of Y.sup.1 or Y.sup.2 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, and 
4-methyloctyl, 4,7,-dimethyloctyl. 
The hydrocarbyl Y.sup.1 and Y.sup.2 groups may optionally be substituted 
with 1 or 2 substituents selected from halogen such as fluoro, chloro, 
bromo or iodo; hydroxy; epoxy; alkoxy (1-4C) such as, for example, 
methoxy, n-propoxy and t-butoxy; alkyl thio; (1-4C) primary amino 
(NH.sub.2); 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'COO-- and R'CONH--, respectively, and their thiol analogs represented by 
R'CSO-- and R'CSNH-respectively; carboxy (--C(O)OH); alkoxycarbonyl 
(--C--(O)OR'); carbamyl (--C(O)NH.sub.2); alkylcarbamyl (1-4C) 
(--C(O)NHR'); alkylsulfonyl (1-4C) (R'SO.sub.2 --); and alkyl phosphonyl 
(1-4C) (R'P(OR')O--). 
In addition Y.sup.1 and Y.sup.2 can each independently be --NH.sub.2, 
--NHR', --NR'R', --OCOR',--NH(CO)R',--O(SO)R' or --O(ROR')R" in which the 
various R' groups are lower alkyls (1-4C) which themselves may be 
substituted with OH, NH.sub.2, alkyl secondary and tertiary amino, 
pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents. 
More preferably, Y.sup.1 and Y.sup.2 are independently H, nitro, carboxy, 
alkoxycarbonyl, alkylsulfonyl or --NHR' wherein R' is --CH.sub.2 
--(CH.sub.2).sub.m --CH.sub.2 --NR.sub.1 R.sub.2, R.sub.1 and R.sub.2 are 
independently selected from the group consisting of hydrogen, lower alkyl, 
or the R.sub.1 and R.sub.2 groups may be linked to form a piperidino or 
pyrrolidino ring, and m is an integer from 0 to 4, preferably 1 or 2. 
Particularly preferred compounds of Formula I for use in the present 
invention include 1,2,4-benzotriazine 1,4-dioxide (wherein X is hydrogen, 
Y.sup.1 and Y.sup.2 are each hydrogen and n is 1); 
3-amino-1,2,4-benzotriazine 1,4-dioxide (i.e., tirapazamine, SR 4233, 
wherein X is NH.sub.2, Y.sup.1 and Y.sup.2 are each hydrogen and n is 1); 
3-ethyl-1,2,4-benzotriazine 1,4-dioxide (wherein X is ethyl, Y.sup.1 and 
Y.sup.2 are each hydrogen and n is 1); 3-propyl-1,2,4-benzotriazine 
1,4-dioxide (wherein X is propyl, Y.sup.1 and Y.sup.2 are each hydrogen 
and n is 1 ) and; 3-(1-hydroxyethyl)-1,2,4-benzotriazine 1,4-dioxide 
(wherein X is 1-hydroxyethyl, Y.sup.1 and Y.sup.2 are each hydrogen and n 
is 1); most particularly 3-amino-1,2,4-benzotriazine 1,4-dioxide. 
Pharmaceutically acceptable salts of the compounds of Formula I, as defined 
herein, include salts formed from inorganic acids such as hydrochloric, 
hydrobromic, or phosphoric acids; organic acids such as acetic acid, 
pyruvic acid, succinic acid, mandelic acid, and p-toluene sulfonic acid; 
salts formed from inorganic bases such as sodium, potassium or calcium 
hyrdoxide or from organic bases such as caffeine, ethylamine or lysine. 
The compounds of Formula I, as defined herein, 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 vehicle. 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, methylcellulose, polyoxyethylene, sorbitan monolaurate, methyl 
hydroxybenzoate, propyl hydroxybenzoate, talc and magnesium stearate. 
The chemotherapy agent is administered to the mammal by conventional routes 
appropriate for the particular chemotherapy agent. The chemotherapy agent 
and the compound of Formula I, as defined herein, can be administered by 
the same route, or by different routes, depending on the particular 
combination of compound of Formula I, as defined herein, and chemotherapy 
agent. The compound of Formula I, as defined herein, can be administered 
to the mammal alone or in combination with one or more other compounds of 
Formula I, as defined herein. 
The compounds of Formula I, as defined herein, are administered to the 
mammal in amounts effective to kill or produce cytotoxic effects upon 
hypoxic tumor cells. The amount of the compound administered will depend 
on such factors as the type of cancer tumor, the age and health of the 
mammal, the maximum tolerated and/or lethal dosage of the chemotherapy 
agent and the compound of Formula I, and the interaction of the compound 
of Formula I with the chemotherapy agent. In a presently preferred 
embodiment of the invention, tirapazamine is administered in amounts of 
from about 10 mg/m.sup.2 to about 450 mg/m.sup.2 ; more preferably from 
about 20 mg/m.sup.2 to about 350 mg/m.sup.2 ; most preferably from about 
30 mg/m.sup.2 to about 250 mg/m.sup.2. When the compound of Formula I is 
administered to the mammal in divided doses, the lower dosage range may be 
preferably, depending on the maximum tolerated dosage of the compound and 
the interaction of the compound with the chemotherapy agent. 
The chemotherapy agent is administered to the mammal in amounts effective 
to treat susceptible tumors. Such amounts are well-known in the art and 
can be ascertained by reference to product literature furnished by the 
supplier of the chemotherapy agent or scientific literature. In preferred 
embodiments of the invention, the chemotherapy agent and the compound of 
Formula I have a synergistic interaction upon the tumor and it may be 
possible to administer the chemotherapy agent at doses that are lower than 
doses recognized as effective when the chemotherapy agent is administered 
alone. Such lower dosages may be desirable if the chemotherapy agent 
produces severe side effects in the mammal to which it is administered. If 
the chemotherapy agent is to be administered to the mammal in divided 
doses, sufficient amounts of the compound of Formula I, as defined herein, 
is administered to the mammal so that the synergistic effect of the 
combination of two agents is maintained, whether before the initial dose 
of the chemotherapy agent or prior to each individual dose of the 
chemotherapy agent. The methods of the invention can also be employed in 
conjunction with other types of cancer treatments such as radiation 
therapy and surgical removal of the tumor. 
The compound of Formula I is administered to the mammal from about one half 
hour to about twenty-four hours prior to administration of the 
chemotherapy agent. Alternatively, the compound of Formula I can be 
administered to the mammal from about one to about two hours after the 
administration of the chemotherapy agent. For some combinations of 
chemotherapy agent and compound of Formula I it may be possible to 
administer the compound of Formula I more than twenty-four hours prior to 
administration of the chemotherapy agent and still retain the advantages 
of the methods of the present invention. The time differential providing 
the most advantageous increase in cell toxicity can be determined by 
testing the combination of compound of Formula I and chemotherapy agent in 
in vivo and or in vitro experimental tumor models, such as the one set 
forth herein, or any other tumor model. The time differential determined 
in such models is then used as a guide for treatment of tumors in mammals, 
with adjustments made during treatment if necessary. Applicants have found 
that for the combination of tirapazamine and cisplatin, the greatest 
interaction between the two agents was observed when tirapazamine was 
administered between about one and three hours prior to administration of 
the cisplatin, with the greatest increase in cell death occurring when 
tirapazamine was administered about two and one half hours prior to 
cisplatin. When tirapazamine was administered one to two hours after 
administration of cisplatin, an enhanced cytotoxic effect was observed, 
however, the increase was not as large. In some embodiments of the 
invention, it may be desirable to administer the compound of Formula I at 
the same time as the chemotherapy agent. 
The present invention also provides kits for treatment of mammalian tumors 
comprising at least one chemotherapy agent and at least one compound of 
Formula I, as defined herein. The compound of Formula I as defined herein 
is preferably supplied in the kits in cytotoxicity-enhancing amounts or 
doses. Suitable dosage forms for the compounds of Formula I, as defined 
herein, are disclosed herein. The particular dosage form of the 
chemotherapy agent and the compound of Formula I, as defined herein, will 
be determined by the type of cancer tumor to be treated, the preferred 
route of administration and the type of chemotherapy agent. The 
chemotherapy agent and the compound of Formula I, as defined herein, are 
preferably supplied in separate containers to facilitate adminstration of 
the chemotherapy agent and the compound of Formula I at different times in 
accordance with the methods of the invention. 
The compounds of Formula I useful in the practice of the present invention 
can be prepared according to the methods disclosed in U.S. Pat. No. 
5,175,287 issued Dec. 29, 1992, the disclosures of which are hereby 
incorporated by reference. General methods for preparing some 3-amino 
derivatives can be found, for example, in Ley et al., U.S. Pat. No. 
3,980,779. 
The compounds are prepared from benzofuroxan of the Formula: 
##STR3## 
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 mixture 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 parent monoxide or 
1,2,4-benzotriazine by peracid oxidation (see Robbins 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-nitro-2-aminobenzene compound using H.sub.2 
NCN.2HCl; 
(2) oxidation of the parent compound given by the structure 
##STR4## 
or by controlled reduction of the corresponding dioxide (see Mason, supra, 
and Wolf et al, J Am Chem Soc 76:355(1954)). 
The 1,2,4-benzotriazines may be prepared by cyclization of formazan 
precursors using BF.sub.3 /AcOH (see Scheme I and Atallah and Nazer, 
Tetrahedron 38:1793 (1982)). 
3-Amino-1,2,4-benzotriazines may be prepared either by cyclization of a 
parent compound (see Scheme II and Arndt, Chem. Ber. 3522 (1913)) or by 
reduction of the monoxide or dioxide as above. 
The 3-hydroxy-1,2,4-benzotriazine oxides may be prepared using peroxide and 
sodium tungstate (Scheme III), a novel synthetic procedure for making the 
3-hydroxy-1,4-dioxide compound, or concentrated sulfuric acid and sodium 
nitrate (Scheme IV). 
##STR5## 
1,2,4-Benzotriazine oxides unsubtituted at the 3 position (sometimes 
referred to herein as the "3-desamino" compounds) can be prepared by the 
following method. The method involves treating a 1,2,4-benzotriazine oxide 
of Formula(I), wherein X is NH.sub.2, 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 reductive deaminating conditions involve reaction in a 
compatible solvent, e.g., dimethyl formamide, at a temperature of at least 
about 60.degree. C., typically at a temperature in the range of 
60.degree.-65.degree. C. This reaction is illustrated generally at Scheme 
V. 
##STR6## 
EXAMPLES 
The methods of the present invention are exemplified by the following 
non-limiting examples. Examples 1-18 relate to synthesis of compounds of 
Formula I, as defined herein. Example 19 relates to in vitro and in vivo 
tests of tirapazamine and cisplatin. 
EXAMPLE 1 
Preparation of 3-Hydroxy-1,2,4-Benzotriazine 1,4-Dioxide 
##STR7## 
A stirred mixture of 1.50 g (9.25 mmole) of 3-amino-1,2,4-benzotriazine 
1-oxide (1), 100.0 ml acid, and 30.0 ml of 30% hydrogen peroxide was 
treated with 3.05 g (9.25 mmole) of Na.sub.2 WO.sub.4 .cndot.2H.sub.2 O. 
The mixture was stirred in an oil bath at 60.degree. C. for 4 days. The 
yellowish orange mixture was cooled to about 30.degree. and filtered to 
remove a light yellow non-UV absorbing solid. The orange solution of 
hydrogen peroxide in acetic acid was evaporated to semi-dryness carefully 
with several additions of water and acetic acid to remove most of the 
peroxide. The concentrated solution was allowed to stand at room 
temperature to afford four crops of an orange solid, 0.87 g (42% yield of 
the sodium salt of 2). UV: .lambda..sub.max (20% CH.sub.3 OH/H.sub.2 O): 
262.2 (.epsilon.39,460); 477 (.epsilon.7,030). IR (neat): 3530 m, 3150 m, 
2650 m, 2180 m and 1635 m. Anal. (calculated for the sodium salt): C.sub.7 
H.sub.4 N.sub.3 O.sub.3 Na 1.25H.sub.2 O, 223.64: C,37.6; H,2.93; N, 
18.79. Found: C,37.8; H,2.75; N, 18.65. 
##STR8## 
EXAMPLE 2 
Preparation of 3-Amino-7-Trifluoromethyl-1,2,4-Benzotriazine 1-Oxide: 
##STR9## 
A mixture of 4-chloro-3-nitrobenzotrifluoride (Aldrich, 2.70 g, 12.9 mmole) 
and cyanamide dihydrochloride (2.75 g, 24 mmole) (previously prepared by 
treating an ether solution of cyanamide with HCl gas and collecting the 
precipitated solid) was heated at 140.degree. C. for 1 hour. The residue 
was treated with 2N NaOH (45 ml), heated for a further 5 min., and then 
allowed to cool. The precipitate was collected, washed with H.sub.2 O, 
dried, and triturated with acetone-toluene to yield 1.32 g (45%) of 3 as a 
light yellow solid M.P. 301.degree.-302.degree.. TLC R.sub.f 0.60 (9:1 
methylene chloride: methanol on silica gel plates). MS: m/z (relative 
intensity) 230 (100, M.sup.+). 
EXAMPLE 3 
Preparation of 3-Amino-7-Decyl-1,2,4-Benzotriazine 
##STR10## 
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, 80 g, 0.34 mole) in hexanes (2.41). After stirring for 1 h, the 
mixture was cooled and treated over 30 min. at 5.degree.-10.degree. C. 
with 70% nitric acid (34 ml). Stirring was continued at 
5.degree.-10.degree. C. for 1 h and at 25.degree. C. for 16 h. The mixture 
was diluted with H.sub.2 O (11 ), stirred for 5 h, poured into an open 
dish and allowed to stand for 16 h. After further dilution with H.sub.2 O 
(1.51), the solid was collected and recrystallized from an 85% ethanol 
solution (in water) to give 92 g (84%) of the intermediate as an orange 
solid, m.p. 64.degree. C. 
A solution (100 ml) of 85% KOH (19 g, 0.288 mole) in H.sub.2 O was combined 
with a suspension of 4-(1-decyl)-2-nitroaniline (89 g, 0.28 mole), 
prepared above, in methanol (900 ml). The mixture was stirred for 6 h, 
neutralized to pH 7-8 with concentrated HCl, and evaporated in vacuo to 
near dryness. After dilution with H.sub.2 O (400 ml), the solid was 
collected and air-dried to give 77 g (100%) of the intermediate as an 
orange solid, m.p. 59.degree. C. 
1.0 g (8.7 mmole) of cyanamide dihydrochloride (previously prepared for use 
by treating an ether solution of cyanamide with HCl gas and collecting the 
precipitated solid) was added portionwise over 10 min to a preheated melt 
(190.degree. C.) of 4-(1-decyl)-2-nitroaniline prepared in the preceding 
step (500 mg, 1.8 mmole). The reaction mixture was heated at 190.degree. 
C. for 5 min, cooled to 25.degree. C., treated with 6N KOH (10 ml), and 
heated at 90.degree.-95.degree. C. for 1 h. After cooling to 25.degree. 
C., the solid was collected, washed with H.sub.2 O and ethanol and 
air-dried to give 0.25 g (46%) of compound 4 as a light yellow solid, m.p. 
177.degree. C. (dec). MS: m/z (relative intensity) 285 (100, M.sup.+), 302 
(13). 
EXAMPLE 4 
Preparation of 3-Amino-7-Carbamyl-1,2,4-Benzotriazine 1-Oxide 
##STR11## 
Preparation of 4-chloro-3-nitrobenzamide: 20.2 g (0.1 mole) of 
4-chloro-3-nitrobenzoic acid (Aldrich) and thionyl chloride (20 ml) were 
combined, allowed to stand for 16 h, and refluxed for 4 h 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.degree. C.) concentrated ammonium hydroxide (100 ml). 
After 3 h at -10.degree. C. and 16 h at 25.degree. C. the mixture was 
poured into an open dish and allowed to evaporate to dryness. The residue 
was slurried in H.sub.2 O and the solid was collected and air-dried to 
give 19.8 g (98%) of the intermediate as a light yellow solid, m.p. 
153.degree. C. 
A solution of Na(3.45 g, 0.15 mole) in ethanol (75 ml) was added to a 
solution of guanidine hydrochloride (15.8 g, 0.165 mole) in ethanol (75 
ml). After 1 h the mixture was filtered and the filtrate was combined with 
a suspension of 4-chloro-3-nitrobenzamide (10 g, 0.05 mole) prepared 
above, in ethanol (50 ml). The mixture was stirred and refluxed for 16 h, 
cooled to 0.degree.-5.degree. C., and acidified with concentrated HCl (8 
ml). The collected solid was combined with K.sub.2 CO.sub.3 (28 g, 0.2 
mole) and H.sub.2 O (40 ml) and the mixture was stirred and heated at 
100.degree. C. for 8 h. After cooling to 25.degree. C., the solid was 
collected, washed with H.sub.2 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 
(6.times.100 ml). The combined filtrate was evaporated in vacuo to a 
solid. The solid was suspended in 95% ethanol, collected and air-dried to 
give 0.44 g (4.3%) of compound 5 as a light yellow solid, m.p. 300.degree. 
C. TLC: R.sub.f =0.23 (methylene chloride: acetone of 2:1, silica gel 
plates). MS: m/z (relative intensity) 205 (100, M.sup.+). 
EXAMPLE 5 
Preparation of 7-Acetyl-3-Amino-1,2,4-Benotriazine 1-Oxide Oxime 
##STR12## 
A combined mixture of 7-acetyl-3-amino-1,2,4-benzotriazine 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) was heated at 
90.degree.-95.degree. C. for 1 h and then cooled to 25.degree. C. The 
mixture was diluted with 95% ethanol (5 ml) and the solid was collected 
and air-dried to give 30 mg (56%) of compound 6 as a light yellow solid, 
m.p. 278.degree. C. (dec). TLC: R.sub.f =0.60 (9:1 methylene 
chloride:methanol). MS: m/z (relative intensity) 219 (100, M.sup.+). 
EXAMPLE 6 
Preparation of 3-Amino-6(7)-Decyl-1,2,4-Benzotriazine 1,4-Dioxide 
##STR13## 
5-(1-decyl)-benzofuroxan: A combined mixture of 4-(1-decyl)-2-nitroaniline 
(77 g, 0.28 mole), 5.25% NaOCl in H.sub.2 O (476 g, 0.34 mole), 85% KOH 
(20.3 g, 0.31 mole), nBu.sub.4 NHSO.sub.4 (4.7 g, 0.014 mole), and 
CH.sub.2 Cl.sub.2 (2.281) was stirred rapidly for 6 h and diluted with 
H.sub.2 O (500) and CH.sub.2 Cl.sub.2 (11). The separated organic phase 
was washed successively with 1N HCl (11) and brine (2.times.11)), dried 
(Na.sub.2 SO.sub.4), 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, 0.31 
mole) and K.sub.2 CO.sub.3 (36.8 g, 0.27 mole). The mixture was stirred 
for 48 h and filtered. The filtrate was diluted with H.sub.2 O (61) and 
glacial acetic acid (40 ml) and extracted with CH.sub.2 Cl.sub.2 
(4.times.500 ml). The combined organic solution was washed successively 
with 5% NaHCO.sub.3 solution (1.times.500 ml) and brine (2.times.500 ml), 
dried (Na.sub.2 SO.sub.4), and evaporated in vacuo to dryness. The crude 
product was purified by chromatography on silica gel using CH.sub.2 
Cl.sub.2 : methanol (98.2) to give 1.8 g (16%) of compound 7 as a red 
solid, m.p. 155.degree. C. (dec). MS: m/z (relative intensity) 318 (4, 
M.sup.+), 285 (100). 
EXAMPLE 7 
Preparation of 1,2,4-Benzotriazine Dioxide 
##STR14## 
A mixture of 1.80 g (13.73 mmole of 90% H.sub.2 O.sub.2 (9 ml), 
trifluoroacetic anhydride (13.5 ml) and Na.sub.2 WO.sub.4.2-H.sub.2 O 
(12.50 g, 38 mmole) in CHCl.sub.3 (170 ml) was stirred at room temperature 
for 5 days. The reaction mixture was diluted with H.sub.2 O (100 ml) and 
extracted with CHCL.sub.3 (100 ml). The organic layer was washed with 
H.sub.2 O (50 ml), dried (Na.sub.2 SO.sub.4), and the solvent removed in 
vacuo. The residue was chromatographed on silica gel using EtOAcCH.sub.2 
Cl.sub.2 (1:1) to give 0.30 g (13.4%) of compound 9 as a yellow solid, 
m.p. 204.degree.-205.degree. C. Anal. Calc'd. for C.sub.7 H.sub.5 N.sub.3 
O.sub.2 (163.13): C, 51.5; H, 3.09; N, 25.76. Found: C, 51.6; H, 3.36; N, 
26.01. MS: m/z (relative intensity) 163 (100, M.sup.+), 147 (50). TLC: 
R.sub.f =0.27 (EtOAc-CH.sub.2 Cl.sub.2, 1:1, silica gel plates). IR 
(nujol): 1600.mu., 1460.mu., 1300.mu., UV: .lambda..sub.max (H.sub.2 O ): 
227 (e22,900) 252 (e12,950): 392 (e4,080). 
EXAMPLE 8 
Preparation of 7-Chloro-3-Hydroxy-1,2,4-Benzotriazine 1,4-Dioxide 
##STR15## 
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 Na.sub.2 WO4.2H.sub.2 O and 30 ml of 30% 
H.sub.2 O.sub.2. The mixture was stirred and heated for 6 days at 
50.degree. C., then slowly evaporated to dryness to remove H.sub.2 
O.sub.2. The residue was boiled in 250 ml H.sub.2 O and filtered to remove 
about 25 mg of starting material 10. The aqueous solutions were then 
extracted with 2.times.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 12, which showed good solubility in a mixture 
of hot isopropyl alcohol and water. Mass. Spec.: M.sup.+ =212 
(q=100)(compound 10); TLC: R.sub.f =0.34 (acetone, silica gel plates). 
The ethyl acetate solutions above, separated from the H.sub.2 O layer after 
the filtration to remove 12, were evaporated to dryness. The residue was 
then treated with isopropyl alcohol at room temperature to afford a dull 
orange solid, 0.41 g (25% yield) of 11. Mass. Spec.: M.sup.+ =213 (q=70); 
TLC: R.sub.f =0.22 (acetone, silica gel plates). Compound 11 was 
characterized as the ammonium salt, C.sub.7 H.sub.4 ClN.sub.3 O.sub.3 
NH.sub.3, m.w. 230.61, as follows. The free acid 11 was dissolved in 
concentrated NH.sub.4 OH and then chilled in ice and filtrated 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 
P.sub.2 O.sub.5 at 56.degree. C.1/1.0 mm, leaving 0.244 g (87% yield) of 
13 
##STR16## 
Anal. Calc'd. for C.sub.7 H.sub.4 ClN.sub.3 O.sub.3 NH.sub.3 (230.61): C, 
36.5; H, 306; N 24.30 Found: C 36.5; H3.07; N 23.94 UV: .lambda..sub.max 
(H.sub.20): 219 (.epsilon.12,580); 265.4 (.epsilon.40,000); 4830486 
(.epsilon.6,640). 
EXAMPLE 9 
Preparation of 7-Nitro-3-Amino-1,2,4-Benzotriazine 1,4-Dioxide 
##STR17## 
7-Nitro-3-trifluoroacetamido-1,2,4-benzotriazine 1-oxide (15): A solution 
of 7-nitro-3-amino-1,2,4-benzotriazine 1-oxide (14)(4.00 g, 19.3 mmol; 
Parish Chemical Co.), CHCl.sub.3 (125 ml) and trifluoroacetic anhydride 
(12.0 ml, 85.0 mmol) was stirred at room temperature for 44 hr. The 
resultant light yellow solid was filtered, washed with CHCl.sub.3 (50 ml) 
and dried to give 5.35 g (91% (yield) of the product as a yellow solid. 
Anal. Calc'd. for C.sub.9 H.sub.4 F.sub.3 N.sub.5 O.sub.4 : C, 35.7; H, 
1.33; 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-nitro-3-trifluoroacetamido-1,2,4-benzotriazine 1-oxide prepared above 
(15)(2.50 g, 8.25 mmol) in CHCl.sub.3 (200 ml) was added Na.sub.2 
WO.sub.4.2 H.sub.2 O (90 mg, 0.273 mmol) followed by 70% H.sub.2 O.sub.2 
(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 recrystallized in 
acetone to give 1.20 g (65% yield) of the product (16) as an orange solid, 
mp 286.degree.-288.degree. C. (dec.). UV: .lambda..sub.max 259, 300, 345, 
387,472. Anal. Calc'd. for C.sub.7 H.sub.5 N.sub.5 O.sub.4 C, 37.70; H, 
2.26; N, 31.39. Found: C, 7.70; H, 2.13; N, 30.94. 
EXAMPLE 10 
Preparation of 3-(3-N,N-Diethylaminopropylamino)-1,2,4-Benzotriazine 
1,4-Dioxide 
##STR18## 
3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1-oxide (18): A 
solution of 3-chloro-1,2,4-benzotriazine 1-oxide (17)(3.0 g, 16.5 mmol) 
(produced by the method of Sasse et al., U.S. Pat. No. 4,289,771) in 
CH.sub.2 Cl.sub.2 (100 ml) was treated with N,N-diethyl-propylenediamine 
(9.5 ml, 88.3 mmol). After 20 hr at room temperature the mixture was 
diluted with 1,2-dichloroethane (50 ml) and washed successively with 
Na.sub.2 CO.sub.3 and H.sub.2 O. The yellow solution was dried (Na.sub.2 
SO.sub.4), filtered and evaporated in vacuo to give 3.93 g (87% yield) of 
the product as a yellow Solid. Recrystallization (ether/petroleum ether) 
yielded pure material, m.p. 47.degree.-48.degree. C. Anal. Calc'd. for 
C.sub.14 H.sub.21 N.sub.5 O (18): C, 61.10; H, 7.69; N, 25.44. Found: C, 
61.30; H, 7.80; N, 25.61. 
3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1,4-oxide (18a): To a 
stirred solution of 3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 
1-oxide 18 prepared as above ((1.60 g, 6.10 mmol) in CHCl.sub.3 (50 ml) 
was added trifluoroacetic anhdride (22.0 ml). After 15 min the mixture was 
cooled to -10.degree. C., 70% H.sub.2 O.sub.2 (10 ml) added and then 
stirred at room temperature for 20 days. The reaction mixture was dried 
(Na.sub.2 SO.sub.4), filtered and evaporated to dryness. The residue was 
dissolved in saturated NaHCO.sub.3 solution (50 ml) and extracted with 
CH.sub.2 Cl.sub.2 (3.times.150 ml). The organic layer was dried (Na.sub.2 
SO.sub.4), filtered and evaporated to give the product 18a, 0.51 g (29% 
yield) as a red solid. m.p. 92.degree.-94.degree. C. NMR: .delta. (400 
MHz, CDCl.sub.3) 1.11 (6H, t, J=7.1 Hz, CH.sub.3), 1.84-1.90 (2H, m, 
H-2'), 2.48-2.64 (4H, m, NCH.sub.2 CH.sub.3, 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), 
UV: .lambda..sub.max 220, 270, 476. Anal. Calc'd. for C.sub.14 H.sub.21 
N.sub.5 O.sub.2. (1/3 H.sub.2 O): 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-Diethylaminoethylamino)-1,2,4-Benzotriazine 
1,4 Dioxide 
##STR19## 
7-Nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1-oxide 
hydrochloride (20): A solution of 7-nitro-3-chloro-1,2,4-benzotriazine 
1-oxide 919)(1.60 g, 7.06 mmol) (prepared as generally shown in Sasse et 
al, U.S. Pat. No. 4,289,771, with (a) NaNO.sub.2 and H.sub.2 SO.sub.4 at 
40.degree. C., followed by (b) chorination with POCl.sub.3 at 106.degree. 
C.) in CH.sub.2 Cl.sub.2 (50 ml) was treated with 
N,N-diethylethylenediamine (6.0 ml, 42.7 mmol). After 6 hr at room 
temperature the mixture was evaporated to dryness under high vacuum at 
60.degree. 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.degree. C./1.0 mmHg) to give 1.80 g (74% yield) of the product 20 as 
yellow needle crystals. NMR .delta. (90 MHz, d.sub.6 -DMSO/d.sub.4 -MeOH) 
1.25 (6H, t, J=6.0 Hz, CH.sub.3), 3.25 (6H, m, NCH.sub.2), 3.82 (2H, m, 
H-1'), 7.74 (1H, d, J=7.0 H-5), 8.52 (1H, dd, J=7.0 and 2.0 Hz, H-6), 8.91 
(1H, d, J=2.0 Hz, H-8). 
7-Nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1,4-dioxide 
hydrochloride (21): To a stirred solution of 
7-nitro-3-(2-N,N-diethylaminoethylamino) 1,2,4-benzotriazine 1-oxide (20; 
prepared as described above) (0.50 g, 1.46 mmol) in CHCl.sub.3 (50 ml) at 
0.degree. C. was added trifluoroacetic anhydride (9.0 ml). After 30 min 
70% H.sub.2 O.sub.2 (4.0 ml) was added and the mixture stirred at room 
temperature for 3 days, then dried (Na.sub.2 SO.sub.4), filtered, and 
evaporated in vacuo to dryness to give the trifluoraoacetate salt 0.67 g 
(45% yield). This product was dissolved in saturated NaH-CO.sub.3 solution 
(30 ml) and extracted with CH.sub.2 Cl.sub.2 (3.times.30 ml). The 
dichloromethane was washed with H.sub.2 O, dried (Na.sub.2 SO.sub.4), 
filtered, saturated with gaseous HCl and evaporated to dryness to give 
0.35 g (63% yield, 28% overall) of the product as a red solid, m.p. 
194.degree.-195.degree. C. UV: .lambda..sub.max 260, 306, 388, 479. Anal. 
Calc'd. for C.sub.13 H.sub.18 N.sub.6 O.sub.4 HCl: 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 deamination 
reactions for preparing compounds of Formula (I) which are unsubstituted 
at the 3-position, i.e., wherein the substituent "X" is hydrogen. 
EXAMPLE 12 
Preparation of 1,2,4-Benzotriazine 1,4-Dioxide by Reductive Deamination of 
3-Amino-1,2,4-Benzotriazine 1,4-Dioxide 
##STR20## 
To a rapidly stirred solution of t-butyl nitrite (867 mg, 1.0 ml, 8.41 
mmol) in DMF (20 ml) at 60.degree.-65.degree. 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., Anqew. Chem. Internat. Edit. 11 
(1972)) in small portions over 5 min. Following the addition, and 
subsidence of the concomitant effervescence (approx. 5 min), the solution 
was collected and reduced under high vacuum to a dark waxy solid. Flash 
chromatography (30% EtOAc/CH.sub.2 Cl.sub.2) gave a yellow solid, m.p. 
188.degree.-189.5.degree. C. (dec.), which was recrystallized from ethanol 
to give 195 mg (43% yield) of the product 9 as bright yellow platelets, 
m.p. 192.degree.-194.degree. C. (dec.). NMR: (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 Hz), 8.43 (1 H, dd, J=8.5, 1.5 Hz) 9.05 (1 H, s, H-3). UV: 
.lambda..sub.max 405, 300, 225. MS: m/z (relative intensity), 147(13), 
136(19), intensity) 164(9), 163(100, M+), 147(13), 136(19), 90(7), 78(27), 
76(26), 75(8) 64(9), 63(10), 52(12), 51(48), 50(28), 38(8), 37(5), 30(18), 
28(6), 27(7). Anal. Calc'd. for C.sub.7 H.sub.5 N.sub.3 O.sub.2 : 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 
##STR21## 
7-Allyloxy-1,2,4-benzotriazine 1,4-dixoide (24): To a stirred solution of 
t-butyl nitrite (271 mg, 0.312 ml, 2.63 mmol) in DMF (15 ml) at 
60.degree.-65.degree. 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 mmol) was added, and shortly thereafter the deep red 
solution effervesced and lightened appreciably 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/CH.sub.2 CL.sub.2) and crystallized (CH.sub.2 
Cl.sub.2 /petroleum ether) to give 72 mg (38% yield) of the product 24 as 
light orange crystals, m.p 147.degree.-148.degree. C. NMR: .delta. (400 
MHz, d.sub.6 -acetone) 4.89 (2H, ddd, H-1', J.sub.1 '.sub.2 '=5.5, J.sub.1 
'..sub.3 '.sub.cis =J.sub.1 '..sub.3 '.sub.trans =1.5 Hz), 5.36(1 H, ddd, 
H-3', J.sub.3 '..sub.2 '.sub.cis =10.5, J.sub.3 '..sub.3 '=1.5 Hz), 5.52 
(1 H, ddd, H-3', J.sub.3 '..sub.2 '.sub.trans =17.5, J.sub.3 '..sub.3 '=3, 
J.sub.3 '.sub.1 '=1.5 Hz), 6.14 (1 H, ddd, H-2', J.sub.2 ',.sub.3 
'.sub.cis =10.5, J.sub.2 '..sub.1 '-5.5 Hz), 7.70 (1 H, d, H-8, J.sub.8.6 
=2.5 Hz), 7.74 (1 H, dd, H-6, J.sub.6.5 =9.5, J.sub.6.8 -2.5 Hz), 8.33 (1 
H, d, H-5, J.sub.5.6 =9.5 Hz), 8.93 (1 H,s, H-3). UV: .lambda..sub.max 
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 C.sub.10 H.sub.9 N.sub.3 O.sub.3 : C, 54.79; H, 4.14; N, 
19.17. Found: C, 54.73; H, 4.16; N, 9.15. 
EXAMPLE 14 
Preparation of 7-(3-N-Ethylacetamido-2-acetoxypropoxy)-1,2,4-Benzotriazine 
1,4-Dioxide Reductive Amination 
##STR22## 
To a stirred solution of t-butyl nitrite 9185 mg, 1.79 mmol) in DMF (5 ml) 
at 60.degree. 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 through silica gel with 5% MeOH/CH.sub.2 Cl.sub.2 to give CH.sub.2 
Cl.sub.2 /ligroin gave 90 mg yellow solid (75% yield), m.p. 
179.degree.-180.5.degree. C. NMR: .delta. (400 MHZ, d.sub.4 -methanol, 
mixture of rotamers, ratio approx. 2:1) 1.12, 1.22 (t's, 1:2,3 H total, 
J=7 Hz), 2.0-6, 2.07 (s's, 2:1, 3 H total), 2.11, 4.34-4.48 (m, 2 H), 
5.48-5.58 (m, 1 H), 7.76-7.86 (m, 2H), 8.36-8.42 (m, 1 H), 9.04, 9.06 
(s's, 2:1, 1 H total). UV: .lambda..sub.max 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(66), 102(6), 100(21), 
84(30), 63(6), 58(100), 56(8), 43(65), 42(9), 41(9), 41(5), 30(14), 29(5), 
28(8). 
EXAMPLE 15 
Preparation of 7-Nitro-1,2,4-Benzotriazine 1,4-Dioxide via Reductive 
Deamination 
##STR23## 
To a stirred solution of t-butyl nitrite 988 mg, 0.85 mmol) in DMF (5 ml) 
at 60.degree. C. was added 7-nitro-3-amino-1,2,4-benzotriazine 1,4-dioxide 
(14) (38 mg, 0.17 mmol). After 30 min the addition of further t-butyl 
nitrite (175 mg, 1.70 mmol) to the dark red slurry was immediately 
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/CH.sub.2 Cl.sub.2 to give 3 mg of the product 
27 as a yellow solid (10% yield). NMR .delta. (90 MHz, d.sub.6 -dimethyl 
sulfoxide) 7.68 (d, 1 H, J=9.2 Hz), 7.92 (dd, 1 H, J=9.2, 2.2 Hz), 8.10 
(d, 1 H, J=2.2 Hz), 8.65 (s, 1 H). UV: .lambda..sub.max 420, 310, 240, 
205. MS: m/z (relative intensity) 209(9), 208(100, M+), 192(54), 181(14), 
162(16), 105(9), 77(28), 75(52), 74(27), 63(21), 62(16), 30(77), 18(26). 
EXAMPLE 16 
3-ethyl-1,2,4-benzenetriazine-1,4-dioxide (31) 
##STR24## 
The hydrazone (28), formed from the condensation of propionaldehyde and 
phenyl hydrazine, was reacted with benezenediazonium chloride in a mixture 
of acetic acid, sodium nitrite and HCl to give the formazan (29). 
Cyclization with BF.sub.3 -AcOH (boron trifluoride-acetic acid) at 
90.degree.-95.degree. C. gave 3-ethyl-1,2,4-benzenetriazine (30) as an 
oil, which was purified by distillation. Oxidation with 70% hydrogen 
peroxide and trifluoroacetic acid anhydride (TFAA) in CH.sub.2 Cl.sub.2 
gave 3-ethyl-1,2,4-benzenetriazine-1,4-dioxide (31). The title compound 31 
was purified using normal-phase column chormatography and 
recrystallization from aqueous ethanol to give material of 99.8% purity. 
The melting point of 31 was found to be 141.degree.-142.degree. C. 
EXAMPLE 17 
3-propyl-1,2,4-benzotriazine-1,4-dioxide (32) 
3-propyl-1,2,4-benzotriazine-1,4-dioxide (32) was prepared and purified 
according to the method of Example 16 (preparation of 
3-ethyl-1,2,4-benzenetriazine-1,4-dioxide) except that the hydrazone 
formed from the condensation of butyraldehyde and phenyl hydrazine was 
used in the reaction with benzenediazonium instead of the hydrazone formed 
from the condensation of propionaldehyde and phenyl hydrazine. The melting 
point of 32 was found to be 114.degree.-116.degree. C. 
EXAMPLE 18 
3-(1-hydroxyethyl)-1,2,4-benzotriazine 1-oxide 
##STR25## 
3-chloro-1,2,4-benzotriazine 1-oxide was treated with a slight excess of 
tri-N-butylvinyltin in a mixture of acetonitrile, tritoluyl phosgene and 
triethylamine, using palladium II catalysis (Pd II acetate) in a sealed 
tube at 100.degree. C. for 48 hours. Removal of solvent and purification 
by column chromatography gave 3-vinyl-1,2,4-benzotriazine 1-oxide (33). 
Reduction with 9-borabicyclo [3.3.1]nonane (9-BBN) followed by oxidation 
with sodium hydroxide and hydrogen peroxide gave the title compound 
3-(1-hydroxyethyl)-1,2,4-benzotriazine 1-oxide (34). 
EXAMPLE 19 
Tirapazamine and cisplatin were tested in an in vivo RIF-1 tumor model. 
Tirapazamine and cisplatin were also tested in an in vitro assay using 
RIF-1 cells under hypoxic and aerobic conditions. 
Animals and tumors: The RIF-1 fibrosarcoma (developed and maintained in the 
laboratory of Dr. Martin Brown, Department of Radiation Oncology, Stanford 
University, Stanford, Calif.; Twentyman et al. J. Nat'l Cancer Inst. 64: 
595-604) in C3H/Km mice (bred and maintained by the Radiation Biology 
Division at Stanford University Medical School) housed under defined flora 
conditions, was maintained alternately in vivo and in vitro, according to 
a previously published protocol (Twentyman, supra). Tumor cell monolayers, 
growing in Waymouth's medium supplemented with 15% fetal calf serum, were 
harvested with 0.05% trypsin. From this suspension, 2.times.10.sup.5 cells 
in 0.05 ml medium were inoculated intradermally in the back of each mouse 
at a site approximately 2 cm above the tail. Experiments were begun two 
weeks later when the mean tumor volume was approximately 200 mm.sup.3. 
Drugs: Tirapazamine (SR 4233) was supplied by Sterling Drug Inc (New York, 
N.Y.). For animal studies, the drug was dissolved in normal saline at a 
concentration of 1 mg/ml and injected intraperitoneally (i.p.) on a 
mmol/kg basis. Cisplatin (c-DDP) from Bristol Laboratories (Princeton, 
N.J.) was dissolved in sterile water and injected i.p. in 0.01 ml/gm body 
weight. 
Cell Survival: For animal studies, RIF-1 cell survival was evaluated 
according to an in vivo/in vitro excision assay. Toward this end, mice 
were killed 24 hours after cisplatin treatment; tumors were excised, 
minced, and dissociated with an enzyme cocktail (Twentyman supra) and 
cells were plated for clonogenic assay. Resultant tumor cell colonies were 
stained with crystal violet and counted after two weeks incubation at 37C. 
in a 5% CO.sub.2 humidified atmosphere. Relative clonogenic cells per 
tumor was calculated as the product of plating efficiency and tumor cell 
yield for treated tumors relative to that for control untreated tumors 
assayed in parallel. 
For the studies on cells in vitro, RIF-1 cells were seeded into 60 mm glass 
petri dishes in Waymouth's medium supplemented with 15% fetal bovine serum 
at a concentration of 2.times.10.sup.4 cells per dish. The experiments 
were performed 4 to 5 days later when there were approximately 10.sup.6 
cell per dish at the time of treatment. The growth medium was then 
replaced with 2 ml of medium without serum containing tirapazamine at a 
concentration of either 2 or 4 .mu.g/ml. In each experiment, groups were 
included in which treatment with tirapazamine and cisplatin were performed 
both simultaneously and with an interval between the two treatments. In 
those groups in which there was an interval immediately after the exposure 
to tirapazamine, the cells were rinsed twice and the medium replaced with 
full growth medium until the time for the second treatment (with 
cisplatin), which was also performed in medium without serum. Both the 
exposure to tirapazamine and to cisplatin were for one hour under hypoxic 
conditions. To achieve hypoxia, the dishes were loaded into specially 
fabricated, prewarmed aluminum gassing chambers which were placed in a 
shaking table and connected to a gassing manifold comprising a vacuum 
outlet line and inlet lines for air or nitrogen (+5% CO.sup.2). Hypoxia 
was achieved in the aluminum chambers through a series of 5 alternate 
evacuations in 2 to 3 minutes to 0.1 atmosphere followed by gassing with 
nitrogen (+5% CO.sup.2). After gassing, the chambers were sealed and 
incubated for one hour at 37C. Measurement of the oxygen level in the 
medium using a Clarke electrode showed that hypoxia was achieved rapidly 
(in approximately 10 minutes with an average pO.sub.2 level during the one 
hour exposure of less than 200 parts/million oxygen). Immediately after 
the treatment with cisplatin, the cells were trypsinized, counted and 
replated in plastic petri dishes in Waymouth's medium supplemented with 
15% fetal bovine serum and incubated for 14 days at 37C. in a 5% CO2 
humidified atmosphere, after which the colonies were stained with crystal 
violet and counted. 
Normal Tissue: The response of normal tissue to tirapazamine and cisplatin 
was evaluated in the kidney and bone marrow through blood urea nitrogen 
(BUN) assays and peripheral white cell counts. Blood samples were taken 
from tail veins or by cardiac puncture. No anticoagulants were used. 
Peripheral white cell counts for individual mice were determined from 20 
.mu.l whole blood diluted in 0.280 ml 3% acetic acid. For serum BUN 
assays, blood from two mice was pooled, coagulated, vortexed, and 
centrifuged at 830 g for 15 minutes. After the serum was aspirated, BUN 
values were determined by a commercial clinical veterinary laboratory. 
Survival to 30 days was also recorded in another experiment. 
Results: 
(a) Results on tumors in vivo: FIG. 1 shows the pooled results from two 
experiments in which 0.35 mmol/kg tirapazamine (63 mg/kg) was delivered to 
the tumor-bearing mice at various times over an interval from 3 hours 
prior to 2 hours after delivery of 8 mg/kg cisplatin, and clonogenic 
survival was assessed at 24 hours. The x-axis shows the relative number of 
clonogenic cells per tumor, The y-axis shows the time of administration of 
tirapazamine relative to administration of cisplatin (-2 hours represents 
data obtained form mice injected with tirapazamine two hours before the 
cispatin injection). Open circles represent tirapazamine alone; open 
squares represent cisplatin alone; closed squares represent tirapazamine 
and cisplatin. As shown in FIG. 1, when tirapazamine is administered at 
intervals between three hours prior to or one to two hours after 
cisplatin, the relative numbers of clonogenic cells per tumor decreases 
from about 10-4 to about 10-7. These figures represent a 10-fold to a 
1,000-fold decrease in the number of clonogenic cells per tumor in 
comparison with the number of clonogenic cells per tumor when tirapazamine 
is administered at the same time as cisplatin. The synergistic effect of 
tirapazamine and cisplatin was most pronounced when tirapazamine is 
administered from about three hours to one hour prior to cisplatin, with 
the greatest interactive effect being seen at two and one half hours prior 
to administration of cisplatin. 
Because of the large amount of cell killing observed at the nadir in FIG. 1 
(which was on the border of the limits of the clonogenic assay), the dose 
of tirapazamine in the experiment was reduced from 0.35 mmol to 0.27 
mmol/kg (48.6 mg/kg) and the experiments were repeated. In this 
experiment, the time gap between administration of cisplatin and 
tirapazamine was extended to twenty-four hours. The results of this 
experiment are shown in FIG. 2. The x- and y-axes are the same as for FIG. 
1. Open circles represent tirapazamine alone; open squares represent 
cisplatin alone; closed squares represent cisplatin and tirapazamine. As 
shown in FIG. 2, the enhanced interaction between tirapazamine and 
cisplatin was present when tirapazamine was administered up to twenty-four 
hours prior to cisplatin. 
Despite the reduction in amount of tirapazamine administered in the second 
set of experiments, the data from the three experiments show the same 
results: essentially additive toxicity when the drugs are given together, 
and a major cytotoxic interaction when the drugs are separated in time 
with the maximal reduction in the number of clonogenic cells per tumor 
when tirapazamine treatment preceded cisplatin by approximately 2.5 hours. 
Additional experiments were performed with various doses of tirapazamine 
given 2.5 hours before either 4 or 8 mg/kg cisplatin. There was an 
approximately exponential reduction in tumor cell survival at both doses 
of cisplatin with increasing dose of tirapazamine. 
(b) Results on Normal Tissue: Preliminary studies using C3H mice indicated 
that white cell counts reached a nadir on the third day after treatment 
with tirapazamine and cisplatin and then rose again to near control levels 
on day five. A dose response study was therefore done on day 3 of 
cisplatin alone and tirapazamine plus cisplatin with the tirapazamine dose 
(0.35 mmol/kg) given 2.5 hours before cisplatin. Cisplatin was 
administered at three different dose levels--10, 14, and 18 mg/kg. Both 
tirapazamine and cisplatin produced a mild leukopenia and the combination 
produced an affect equal to that predicted from adding the responses to 
the individual drugs. 
Assays of serum BUN were performed on the sixth day after injection of 
tirapazamine and cisplatin, based on a preliminary investigation of the 
time for maximum increase in BUN following high doses of cisplatin. BUN in 
C3H mice six days after a single injection of tirapazamine (0.27 mmol/kg), 
cisplatin (10, 14, or 18 mg/kg) or the two drugs given together (with 
tirapazamine injected two and one half hours before the cisplatin 
injection). BUN levels for doses of 10 and 14 mg/kg cisplatin alone were 
similar to BUN levels for untreated mice (approximately 30 mg/dl). 
However, 18 mg/kg cisplatin alone had a BUN level of about 80 mg/dl. By 
contrast, at each dose of the combination of drugs, the BUN level of the 
treated mice was less than the BUN levels of untreated mice. These results 
show that tirapazamine in combination with cisplatin does not add to 
cisplatin kidney toxicity and may even protect at the highest dose tested. 
As a further test of whether tirapazamine enhanced the systemic toxicity of 
cisplatin, an LD50 experiment was performed with cisplatin alone and 
cisplatin preceded 2.5 hours before injection by tirapazamine. The LD50 
for mice treated with 0.35 mmol/kg tirapazamine plus cisplatin was 17.7 
mg/kg (95% confidence limit: 16.8-18.7 mg/kg), as contrasted with that for 
cisplatin alone which was 17.8 (17.1-18.5 mg/kg). 
(c) Results of in vitro experiments 
Cells were exposed to a one hour period to tirapazamine (2 or 4 .mu.g/ml) 
under hypoxic conditions and also exposed to cisplatin (2 .mu.g/ml) for 
one hour as a function of time later. The concentration of each agent was 
chosen to produce a similar level of cell killing of hypoxic cells as that 
with the RIF-1 tumors in vivo: for tirapazamine (0.3 and 0.009 and at 2 
and 4 .mu.g/ml, respectively) and for cisplatin (3.5.times.10.sup.-3). 
Each experiment contained groups in which there was no separation between 
the exposure of the two agents (i.e., tirapazamine and cisplatin were 
administered simultaneously for one hour under hypoxic conditions), as 
well as a group in which the two exposures were separated from one to four 
hours. The results obtained for the drugs given simultaneously were not 
significantly different from the product of the survivals of the two 
agents given separately (i.e., compatible with additivity); whereas when 
the drugs were separated, there was more cell killing by a factor of up to 
10.sup.2. There is a similar kinetics of enhancement of cell killing as 
observed in the in vivo results, though the absolute magnitude of the 
effect of splitting the two doses is less than that observed in vivo. To 
check that the interaction between the two agents depended on the presence 
of hypoxia, the experiments were repeated with three hours between 
exposure of the cells to tirapazamine under aerobic conditions and 
exposure of the cells to cisplatin under hypoxic conditions. In these 
experiments, there was no cytotoxicity due to the tirapazamine, and there 
was no potentiation of the cell killing compared to that produced by 
cisplatin alone in the same experiments.