The title compounds are provided as well as a method for their production, pharmaceutical compositions comprising the compounds, and a method of treatment using the compounds in dosage form. Compounds of the invention have pharmacological properties and are useful antileukemic agents.

DESCRIPTION 
1. Technical Field 
The invention relates to novel 
3-(2-haloethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide 
compounds, to a method for their production, to pharmaceutical 
compositions comprising the compounds, and to methods of treatment using 
the compounds in dosage form. The compounds of the invention have 
pharmacological properties and are useful antileukemic agents. 
2. Background of the Invention 
Azolo[5,1-d][1,2,3,5,]tetrazin-4-ones are known from Tetrahedron Letters 
No. 44, pp. 4253-4256, Pergamon Press Ltd. 1979 and Chemical Abstracts, 
95, 698, 1981. The title compounds are unknown in the prior art. 
SUMMARY OF THE INVENTION 
The invention in one aspect relates to 
3-(2-haloethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide 
compounds having the structural formula: 
##STR1## 
where Hal is a halogen. According to the invention, the halogen may be 
chloro, bromo, iodo or fluoro, preferably chloro. 
PROCESS FOR PREING THE COMPOUNDS 
The invention in another aspect comprises a process for preparing the title 
compounds having the above structural formula, which comprises reacting 
3-diazapyrazole-4-carboxamide and a haloethylisocyanate having the 
structural formula Hal--CH.sub.2 --CH.sub.2 --N.dbd.C.dbd.O under 
cyclizing conditions and isolating the product where Hal has the above 
meaning. The reaction is suitably carried out in a dry inert organic 
solvent such as ethyl acetate, with mixing at room temperature for a short 
period and then at higher temperature, for example, at about 50 to about 
60 degrees celsius. Heating is continued until the reaction is complete at 
which time the mixture becomes a solution. The starting materials either 
are known or can be prepared from known materials by procedures described 
herein starting from ethoxymethylene malononitrile (I) and hydrazine (II). 
The procedure is illustrated as follows: 
##STR2## 
PHYSICAL AND PHARMACOLOGICAL PROPERTIES OF THE COMPOUNDS 
The compounds of the invention typically are light stable, have 
leukemia-inhibiting properties, and are useful as pharmacological agents 
in dosage form for the treatment of leukemia in warm-blooded animals. The 
activity of test compounds is established by the in vivo assay for 
anticancer (antileukemic) activity. This assay is carried out with male 
DC.sub.2 F.sub.1 mice (six per treatment group) that weigh 22-24 grams at 
first treatment. L1210 leukemia cells are harvested from the peritoneal 
ascites fluid of a leukemic male DBA.sub.2 mouse and diluted with sterile 
0.9% saline containing 2.1% W/V bovine serum albumin, 200U/ml penicillin, 
and 0.3 mg/ml streptomycin. The cells are counted with a Coulter.sup.R 
counter. The mice are randomized, inoculated with 10.sup.4 L1210 cells 
(0.5 ml, i.p) and rerandomized to treatment or control groups on day zero. 
The test compound is dissolved in 10% aqueous dimethylsulfoxide. Treatment 
groups are injected i.p. with 0.5 ml of freshly made DMSO solutions of the 
test compound once daily on days 3-7. Control mice are treated with 0.5 ml 
10% dimethylsulfoxide. All mice are weighed on days 3 and 7 and all dying 
mice are autopsied to confirm the presence of advnaced leukemia. A %T/C 
value [T/C computed as (median lifespan of the treated group/median 
lifespan of the control group)] greater than 125 is considered as showing 
significant activity. The results for compounds and compositions of the 
invention exemplified by 
3-(2-chloroethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, 
are presented in Table 1. 
TABLE 1 
______________________________________ 
L1210 Activity* 
Dose D Weight** Median 21-Day 
(mg/kg/inj.) 
g Lifespan % T/C Survivors 
______________________________________ 
Control +1.0 10.4 -- 0/20 
40 -4.3 &gt;21.5 &gt;207 6/6 
20 -1.8 20.7 199 0/6 
10 -0.1 13.2 127 0/6 
5 +0.7 12.2 117 0/6 
2.5 +1.7 11.7 112 0/6 
______________________________________ 
*L1210 10.sup.4 cells, i.p., day zero 
Treatment i.p., days 3-7. 
**Dwt = (mean, day 3) (mean, day 7). 
The invention in its composition aspect relates to a pharmaceutical 
composition for treating leukemia in a dosage form comprising a compound 
of the invention having the above structural formula, preferably 
3-(2-chloroethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, in 
combination with a pharmaceutically acceptable carrier. 
The invention in another method aspect relates to a method for treating 
leukemia in a mammal which comprises administering a dosage form 
containing a leukemia-inhibiting amount of compound of the invention 
having the above structural formula, preferably 
3-(2-chloroethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, in 
combination with a pharmaceutically acceptable carrier, to a mammal in 
need thereof. 
PREATION OF PHARMACEUTICAL COMPOSITIONS 
When being utilized as pharmacological agents, the compounds of the 
invention can be prepared and administered in a wide variety of topical, 
oral, and parenteral dosage forms. It will be clear to those skilled in 
the art that the following dosage forms may comprise as the active 
component, one or more compounds of the above formula. 
For preparing pharmaceutical compositions from the compounds described by 
this invention, inert, pharmaceutically acceptable carriers can be either 
solid or liquid. Solid form preparations include powders, tablets, 
dispersible granules, capsules, cachets, and suppositories. A solid 
carrier can be one or more substances which may also act as diluents, 
flavoring agents, solubilizers, lubricants, suspending agents, binders, or 
tablet disintegrating agents; it can also be an encapsulating material. In 
powders, the carrier is a finely divided solid which is in admixture with 
the finely divided active compound. In the tablet the active compound is 
mixed with carrier having the necessary binding properties in suitable 
proportions and compacted in the shape and size desired. The powders and 
tablets preferably contain from 5 or 10 to about 70 percent of the active 
ingredient. Suitable solid carriers are magnesium carbonate, magnesium 
stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, 
tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low 
melting wax, cocoa butter, and the like. The term "preparation" is 
intended to include the formulation of the active compound with 
encapsulating material as carrier providing a capsule in which the active 
component (with or without other carriers) is surrounded by carrier, which 
is thus in association with it. Similarly, cachets are included. Tablets, 
powders, cachets, and capsules can be used as solid dosage forms suitable 
for oral administration. 
Liquid form preparations include solutions, suspensions, and emulsions. As 
an example may be mentioned water or water-propylene glycol solutions for 
parenteral injection. Liquid preparations can also be formulated in 
solution in aqueous polyethylene glycol solution. Aqueous solutions 
suitable for oral use can be prepared by dissolving the active component 
in water and adding suitable colorants, flavors, stabilizing, and 
thickening agents as desired. Aqueous suspensions suitable for oral use 
can be made by dispersing the finely divided active component in water 
with viscous material, i.e., natural or synthetic gums, resins, methyl 
cellulose, sodium carboxymethyl cellulose, and other well-known suspending 
agents. 
Topical preparations include dusting powders, creams, lotions, gels, and 
sprays. These various topical preparations may be formulated by well-known 
procedures. See for example Remington's Pharmaceutical Sciences, Chapter 
43, 14th Ed., Mack Publishing Co., Easton, Pa. 18042, USA. 
Preferably, the pharmaceutical preparation is in unit dosage form. In such 
form, the preparation is subdivided into unit doses containing appropriate 
quantities of the active component. The unit dosage form can be a packaged 
preparation, the package containing discrete quantities of preparation, 
for example, packeted tablets, capsules, and powders in vials or ampoules. 
The unit dosage form can also be a capsule, cachet, or tablet itself or it 
can be the appropriate number of any of these packaged forms. 
The quantity of active compound in a unit dose of preparation may be varied 
or adjusted from 50 mg to 500 mg according to the particular application 
and the potency of the active ingredient. 
In therapeutic use as pharmacological agents the compounds utilized in the 
pharmaceutical method of this invention are administered at the initial 
dosage of about 0.1 mg to about 100 mg per kilogram. A dose range of about 
4.0 mg to about 40 mg per kilogram is preferred. The dosages, however, may 
be varied depending upon the requirements of the patient, the severity of 
the condition being treated, and the compound being employed. 
Determination of the proper dosage for a particular situation is within 
the skill of the art. Generally, treatment is initiated with smaller 
dosages which are less than the optimum dose of the compound. Thereafter, 
the dosage is increased by small increments until the optimum effect under 
the circumstances is reached. For convenience, the total daily dosage may 
be divided and administered in portions during the day if desired. 
The active compounds may also be administered parenterally or 
intraperitoneally. Solutions of the active compound can be prepared in 
water suitably mixed with a surfactant such as hydroxypropylcellulose. 
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, 
and mixtures thereof and in oils. Under ordinary conditions of storage and 
use, these preparations contain preservative to prevent the growth of 
microorganisms. 
The pharmaceutical forms suitable for injectable use include sterile 
aqueous solutions or dispersions and sterile powders for the 
extemporaneous preparation of sterile injectable solutions or dispersions. 
In all cases the form must be sterile and must be fluid to the extent that 
easy syringability exists. It must be stable under the conditions of 
manufacture and storage and must be preserved against the contaminating 
action of microorganisms such as bacteria and fungi. The carrier can be a 
solvent or dispersion medium containing, for example, water, ethanol, 
polyol (for example, glycerol, propylene glycol, and liquid polyethylene 
glycol, and the like), N,N-dimethylacetamide, suitable mixtures thereof 
and vegetable oils. The proper fluidity can be maintained, for example, by 
the use of a coating such as lecithin, by the maintenance of the required 
particle size in the case of dispersion and by the use of surfactants. The 
prevention of the action of microorganisms can be brought about by various 
antibacterial and antifungal agents, for example, parabens, chlorobutanol, 
phenol, sorbic acid, thimerosal, and the like. In many cases, it will be 
preferable to include isotonic agents, for example, sugars or sodium 
chloride. Prolonged absorption of the injectable compositions can be 
brought about by the use in the compositions of agents delaying 
absorption, for example, aluminum monostearate and gelatin. 
Sterile injectable solutions are prepared by incorporating the active 
compound in the required amount in the appropriate solvent with various of 
the other ingredients enumerated above, as required, followed by 
sterilization accomplished by filtering. Generally, dispersions are 
prepared by incorporating the various sterilized active ingredient into a 
sterile vehicle which contains the basic dispersion medium and the 
required other ingredients from those enumerated above. In the case of the 
sterile powders for the preparation of sterile injectable solutions, the 
preferred methods of preparation are vacuum drying and the freeze-drying 
technique which yield a powder of the active ingredient plus any 
additional desired ingredient from a previously sterile-filtered solution 
thereof. 
As used herein, "pharmaceutically acceptable carrier" includes any and all 
solvents, dispersion media, coatings, antibacterial and antifungal agents, 
isotonic and absorption delaying agents and the like. The use of such 
media and agents for pharmaceutically active substances is well-known in 
the art. Except insofar as any conventional media or agent is incompatible 
with the active ingredient, its use in the therapeutic compositions is 
contemplated. Supplementary active ingredients can also be incorporated 
into the compositions. 
It is especially advantageous to formulate parenteral compositions in unit 
dosage form for ease of administration and uniformity of dosage. Unit 
dosage forms used herein refers to physically discrete units suitable as 
unitary dosages for the mammalian subjects to be treated; each unit 
containing a predetermined quantity of active material calculated to 
produce the desired therapeutic effect in association with the required 
pharmaceutical carrier. The specification for the novel unit dosage forms 
of the invention are dictated by and directly dependent on (a) the unique 
characteristics of the active material and the particular therapeutic 
effect to be achieved, and (b) the limitation inherent in the art of 
compounding such an active material for the treatment of disease in living 
subjects having a diseased condition in which bodily health is impaired as 
herein disclosed in detail. 
The principal active ingredient is compounded for convenient and effective 
administration in effective amounts with a suitable 
pharmaceutically-acceptable carrier in unit dosage form as hereinbefore 
disclosed. A unit dosage form can, for example, contain the principal 
active compound in amounts ranging from about 0.1 to about 500 mg, with 
from about 0.5 to about 250 mg being preferred. Expressed in proportions, 
the active compound is generally present in from about 0.1 to about 500 
mg/ml of carrier. In the case of compositions containing supplementary 
active ingredients, the dosages are determined by reference to the usual 
dose and the manner of administration of the said ingredients. The daily 
parenteral doses for mammalian subjects to be treated ranges from 4.0 
mg/kg to 40 mg/kg.

The invention and the best mode of practicing the same are illustrated by 
the following examples of preferred embodiments of selected compounds and 
their preparation. 
EXAMPLE 1 
3-Amino-4-cyanopyrazole (III) 
This compound was reported by R. K. Robins, J. Am. Chem. Soc., 78: 784 
(1956). Following is a modified procedure. 
To 74 g of 64% hydrazine (II) in water (1.5 mole) was added portionwise 100 
g (0.82 mole) of ethoxymethylenemalononitrile (I) with stirring. An 
exothermic reaction occurred during the addition. After the addition was 
complete, the brown solution was heated in a water bath for 4 hours. The 
resulting solution was placed in a refrigerator overnight. Light brown 
crystals, which formed from the reaction mixture, was collected by 
filtration, washed with cold water and ether, and dried to give 63 g (71% 
yield) of III as light brown crystals, m.p. 169-170 degrees C. It was used 
for the next reaction without further purification. 
EXAMPLE 2 
3-Amino-4-pyrazolecarboxamide Hemisulfate (IV) 
This compound was reported by R. K. Robins, J. Am. Chem. Soc., 78: 784 
(1956). Following is a modified procedure. 
To 180 ml of concentrated sulfuric acid cooled in an ice bath was added 
portionwise, with stirring, 56 g (0.52 mole) of finely powdered III. The 
addition was at such a rate that the temperature of the stirred reaction 
mixture remained at or below 20 degrees C. Total addition time: 2.5 hrs. 
After the addition was complete, the reaction mixture, which still 
contained some solids, was stirred at 10-20 degrees C. for 30 minutes, 
then at room temperature for 4 hrs. The resulting dark brown solution was 
slowly poured, with vigorous stirring, onto 1 kg of crushed ice in a large 
beaker. Light brown solid product separated immediately. The resulting 
reaction mixture was stored in a refrigerator overnight. The solid was 
then collected by filtration, washed successively with water, a small 
amount of ethanol, andether. It was then dried at 50 degrees C. under 
reduced pressure for 18 hrs to give 102 g (100% yield) of IV as light 
brown solids, m.p. 217-218 degrees C. dec. It was used for the next 
reaction without further purification. 
EXAMPLE 3 
3-Diazapyrazole-4-carboxamide(3-Diazonium,pyrazole-4-carboxamide hydroxide 
inner salt) (V) and 4-Hydroxypyrazolo[3,4-d]-v-triazine (VI) 
These compounds were reported by C. C. Cheng, R. K. Robins, K. C. Cheng, 
and D. C. Lin, J. Pharm. Sci., 57:1044 (1968). Following is a modified 
procedure. 
A stirred mixture of 18 g (0.103 mole) of finely powdered IV in 180 ml of 
water was cooled at 0-5 degrees. To this suspension was added, with 
vigorous stirring, 8 g (0.116 mole) of sodium nitrite. The mixture was 
stirred at 0-5 degrees for 20 minutes and filtered through a sintered 
glass funnel. The solid was thoroughly washed with 2.times.20 ml of cold 
water, 20 ml of ethanol, and 50 ml of ether. Agitation of the solid during 
washing is very important in this operation. The remaining light brown 
crystalline solid was then carefully transferred to a crystalline dish and 
dried at room temperature in vacuo to give 6.9 g (49% yield) of V. Its IR 
showed a characteristic triple bond absorption peak at 2235 cm.sup.-1. The 
product decomposed in a melting point tube without melting on slow 
heating, but on rapid heating, it decomposed violently with a sharp sound 
at ca. 160 degrees C. Care, therefore, should be exercised for the 
handling of this compound. Rapid, repeated scratching of the dried powder 
with a spatula may cause its rapid decomposition. 
From the filtrate there was obtained, on standing, 4.2 g (30% yield) of the 
triazine VI as light yellow shining platelets. This compound, on slow 
heating, did not melt below 300 degrees C. and, on rapid heating, 
decomposed with a muffled sound at ca. 170 degrees C. Unlike compound V, 
its IR did not show the triple bond absorption. 
EXAMPLE 4 
3-(2-Chloroethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide 
(a) To a mechanically stirred suspension of 2 g (0.0146 mole) of finely 
powdered V in 420 ml of dry ethyl acetate (caution: do not powder compound 
V alone, powder it in the ethyl acetate suspension) at room temperature 
was added 10.5 g (0.1 mole) of 2-chloroethylisocyanate. The mixture was 
stirred at room temperature in the absence of moisture for 30 min, then 
heated at 50-60 degrees C. with continuous stirring for 72 hrs. At the end 
of the period the suspension became a solution. A small amount of brown 
impurity was removed by filtration and the filtrate concentrated under 
reduced pressure at room temperature to 100 ml. The resulting light yellow 
solid was collected by filtration, washed with 200 ml of ether and dried 
at room temperature in vacuo to give 1.6 g (45% yield) of the desired 
product in analytically pure form, m.p. 193-194 degrees C. dec. From the 
filtrate there was obtained, on further concentration, 0.9 g of product, 
m.p. 167-169 degrees C. dec. Its IR spectrum was comparable to that of the 
first crop but tlc (EtOAc) indicated the presence of two spots. 
Anal. for C.sub.7 H.sub.7 ClN.sub.6 O.sub.2.1/2H.sub.2 O: C, 33.41; H, 
3.20; N, 33.40. Found: C, 33,10; H, 2.98; N, 33.20. 
Mass spectrum of the product: 242 (M.sup.+), 226 (M.sup.30 --NH.sub.2), 193 
(M.sup.+ --CH.sub.2 Cl), 137 (M.sup.+ --ClCH.sub.2 CH.sub.2 N.sub.3). 
.lambda.max CHCl.sub.3 312 nm (E 8500). 
The product is stable in chloroform solution but unstable in methanol. The 
UV absorption maximum of the compound in methanol, on standing, gradually 
shifted from 310 nm to 256 nm. 
(b) By the same procedure, but replacing the chloroethylisocyanate with a 
different, but equivalent amount of, haloethylisocyanate such as 
2-bromoethylisocyanate, 2-fluoroethylisocyanate, or 2-iodoethylisocyanate, 
there are obtaind: 
3-(2-bromoethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, 
3-(2-fluoroethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxamide, or 
3-(2-iodoethyl)-4-oxopyrazolo[5,1-d]-1,2,3,5-tetrazine-8-carboxmide.