A method of treatment of tumors is provided based upon a compound of the formula ##STR1## Some aspects of the invention were supported in part by U.S. Public Health Service Grant CA-02817 from the National Cancer Institute and support from the Northeast NMR Facility at Yale University insofar as the use of high resolution NMR spectra is concerned that was made possible by a grant from the Chemical Division of the National Science Foundation (Grant No. CHE-7916210).

INFORMATION DISCLOSURE UNDER 37 CFR .sctn. 1.97 
5-Hydroxy-2-formylpyridine thiosemicarbazone is a well known compound that 
has been proposed as an anti-neoplastic agent and has received a Phase I 
trial in cancer patients. This agent was not chose for further 
development. Representative literature includes DeConti et al., "Clinical 
and Pharmacological Studies with 5-Hydroxy-2-formylpyridine 
Thiosemicarbazone", Cancer Res., 1972, 32, 1455-62. Similarly, 
4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone is also well known 
as manifested by Agrawal et al., "Potential Antitumor Agents. 13. 
4-Methyl-5-amino-1-formylisoquinoline thiosemicarbazone", J. Med. Chem., 
1976, 19, 970-72. Subsequent to this research, others have tried a variety 
of compounds which may be considered to be analogs of 2-formylpyridine 
thiosemicarbazones, as shown by the numerous compounds which have been 
synthesized and tested by French et al., ".alpha.-(N)-Formylheteroaromatic 
Thiosemicarbazones. * * *", J. Med. Chem., 1974, 17, 172-81. Among the 
various compounds reported by French et al. in Table I (page 174) may be 
mentioned 5-acetylamino-2-formylpyridine thiosemicarbazone (compound 49). 
A variety of compounds were synthesized with 4-position substitutions, as 
reported by Agrawal et al., "Potential Antitumor Agents. 14. 4-Substituted 
2-Formylpyridine Thiosemicarbazones", J. Med. Chem., 1976, 19, 1209-14. 
This Agrawal et al. research reports on compounds which include 
4-dimethylamino-2-formylpyridine thiosemicarbazone (page 1210, compound 5) 
and 4-piperidino-2-formylpyridine thiosemicarbazone (id., compound 20), 
4-pyrrolidinoamino-2-formylpyridine thiosemicarbazone (page 1211, compound 
28), 4-bis(hydroxyethyl)amino-2-formylpyridine thiosemicarbazone (id., 
compound 30) and structurally more remote forms; in no case is there a 
disclosure of a 2-formylpyridine thiosemicarbazone in this research of 
Agrawal et al. that has both one of the 4-position substituents and any 
substituent on the ring other than one example with a 3-methyl group (id., 
compound 37). French et al. have also made attempts to work in the field 
with 4-substitution. French et al. disclose only one compound which may be 
considered to be a (3 or 5)-substituted-4-methyl-2-formylpyridine 
thiosemicarbazone, i.e., the 3-species, 
3-hydroxy-4-methyl-2-formylpyridine thiosemicarbazone, which is compound 
51 and which is found to lack "significant activity." Most of the 
compounds among the 61 tabulated 2-formylpyridine thiosemicarbazones are 
indicated as possessing "significant activity," which is designated by an 
asterisk, as explained on page 175. An isomeric form is disclosed, namely, 
3-hydroxy-6-methyl-2-formylpyridine thiosemicarbazone, which is compound 
52 and which is also found to lack "significant activity." 
SUMMARY OF THE INVENTION 
In accordance with a first aspect of the invention there are provided 
compounds of the formula: 
##STR2## 
wherein one of R.sup.1 is NHR.sup.4 or NR.sup.4 R.sup.5 or R.sup.3 is 
NHR.sup.4, NR.sup.4 R.sup.5 or OH, and the other is hydrogen; 
R.sup.2 is hydrogen or C.sub.1-4 lower alkyl; 
R.sup.4 is hydrogen, hydroxyl, or C.sub.1-4 lower alkyl; and 
R.sup.5 is C.sub.1-4 lower alkyl. 
The term "C.sub.1-4 lower alkyl" refers to alkyl groups of up to four 
carbon atoms, methyl, ethyl, propyl and butyl; in accordance with a 
preferred embodiment, C.sub.1-4 lower alkyl represents methyl. In one 
embodiment there are provided compounds wherein R.sup.4 is hydrogen. In a 
further embodiment there are provided compounds wherein R.sup.2 is 
hydrogen. In a further embodiment there are provided compounds wherein 
R.sup.2 is lower alkyl preferably methyl. In accordance with a preferred 
embodiment, R.sup.4 is hydrogen, i.e., the compounds are 3- and 
5-amino-2-formylpyridine thiosemicarbazones. The compounds may be free 
from further substituents in accordance with one embodiment, i.e., R.sup.2 
is hydrogen, or in a second and also preferred embodiment the compounds of 
the invention are 3- and 5-amino-4-methyl-2-formylpyridine 
thiosemicarbazones, i.e., R.sup.2 is methyl. Representative compounds of 
the invention include 3-amino-2-formylpyridine thiosemicarbazone, 
5-amino-2-formylpyridine thiosemicarbazone, 
3-amino-4-methyl-2-formylpyridine thiosemicarbazone, 
5-amino-4-methyl-2-formylpyridine thiosemicarbazone, and 
5-hydroxyamino-4-methyl- 2-formylpyridine thiosemicarbazone. It is to be 
understood that any compound of the invention above or any other aspect 
should be understood as contemplating any pharmaceutically acceptable 
salts or hydrates thereof. 
A method is provided for the treatment of tumors in mammals, e.g., cats, 
dogs, rats, mice, monkey and man. All of the compounds of the 
aforementioned first aspect of the invention are specifically considered 
to be useful in the treatment of tumors. For example, all compounds of the 
first aspect of the invention are useful in the treatment of the L1210 
leukemia in mice. Dosages that are contemplated within the scope of the 
invention are from about 40 to about 100 mg/kg/day. 
In accordance with a second aspect of the invention there is provided a 
method for the treatment of tumors in mammals, e.g., cats, dogs, rats, 
mice, monkey and man, which comprises administration of the compounds 
3-hydroxy-4-methyl-2-formylpyridine thiosemicarbazone or 
5-hydroxy-4-methyl-2-formylpyridine thiosemicarbazone. For example, among 
tumors which may be treated in accordance with the second aspect of the 
invention may be mentioned the treatment of the L1210 leukemia in mice. 
Dosages that are contemplated within the scope of the invention are from 
about 4 to about 600 mg/kg/day. As part of this second aspect of the 
invention there is also provided the novel compound 
5-hydroxy-4-methyl-2-formylpyridine thiosemicarbazone. 
DETAILED DESCRIPTION 
The synthesis of the compounds of the first aspect of the invention is 
described in greater detail in the examples which follow. In general 
terms, there is first described the synthesis of various 3-amino, 5-amino- 
and 5-nitro-substituted 2-formylpyridine thiosemicarbazones. Oxidation of 
3-nitro-, 5-nitro-, 3-nitro-4-methyl- and 5-nitro-4-methyl-2-picolines 
with selenium dioxide in refluxing dioxane yielded the corresponding 
2-formylpyridines. To reduce the nitro groups to amino functions, the 
aldehydes were protected by conversion to the cyclic ethylene acetals, 
which were then reduced by catalytic hydrogenation using Pd/C as a 
catalyst to give the corresponding amino acetals. The resulting compounds 
were then reacted with thiosemicarbazide in ethanol containing 10% 
concentrated hydrochloric acid to form the desired thiosemicarbazone 
hydrochlorides; the free bases were liberated by treatment with aqueous 
sodium bicarbonate solution. Condensation of 5-nitro-2-formylpyridine and 
5-nitro-4-methyl-2-formylpyridine, with thiosemicarbazide in the presence 
of hydrochloric acid, followed by treatment with sodium bicarbonate, 
yielded the corresponding 5-nitro-substituted thiosemicarbazones. 
The acetamide and alkylsulfonamide derivatives of 3-amino- and 
5-amino-2-formylpyridine thiosemicarbazone were prepared. Acetylation with 
acetic anhydride in anhydrous pyridine gave acetamide derivatives which 
were then condensed with thiosemicarbazide to produce 
5-acetylamino-2-formylpyridine thiosemicarbazone and 3- and 
5-acetylamino-4-methyl-2-formylpyridine thiosemicarbazones. During the 
process of acidic hydrolysis of the ethylene acetal groups, some 
hydrolysis of the acetamide functions occurred even though reaction 
conditions were carefully controlled. The desired pure compounds were 
obtained by recrystallization from ethanol or by silica gel 
chromatography. Treatment of 2-(1,3-dioxolanyl)-4-methyl-5-aminopyridine 
with methanesulfonyl chloride or p-toluenesulfonyl chloride in anhydrous 
pyridine afforded the corresponding 5-methanesulfonylamino and 
p-toluenesulfonylamino derivatives, 
2-(1,3-dioxolanyl)-4-methyl-5-methanesulfonylaminopyridine and 
2-(1,3-dioxolanyl)-4-methyl-5-p-toluenesulfonylaminopyridine, 
respectively, which were then treated with thiosemicarbazide in the 
presence of concentrated hydrochloric acid to afford the corresponding 
5-methanesulfonylamino- and 
5-p-toluenesulfonylamino-4-methyl-2-formylpyridine thiosemicarbazones, 
5-methanesulfonylamino-4-methyl-2-formylpyridine thiosemicarbazone and 
5-toluenesulfonylamino-4-methyl-2-formylpyridine thiosemicarbazone. 
5-Hydroxyamino-4-methyl-2-formylpyridine thiosemicarbazone was synthesized 
by hydrogenation of 2-(1,3-dioxolanyl)-4-methyl-5-nitropyridine in ethanol 
using Pd(OH).sub.2 /C as a catalyst under 50 psi of hydrogen to yield the 
5-hydroxyamino derivative contaminated with about 10-15% of the 
corresponding 5-amino derivative. 
2-(1,3-Dioxolanyl)-4-methyl-5-hydroxyaminopyridine was easily purified by 
recrystallization from ethanol. The structure was assigned by NMR, mass 
spectroscopy and elemental analysis. During the reduction process, the 
rate of absorption of hydrogen decreased considerably after the formation 
of the 5-hydroxyamino derivative, 
2-(1,3-dioxolanyl)-4-methyl-5-hydroxyaminopyridine, and the reaction was 
terminated at this stage. When the reaction was allowed to proceed until 
the absorption of hydrogen was complete (about 24 h), however, the 5-amino 
derivative,2-(1,3-dioxolanyl)-4-methyl-5-aminopyridine, was obtained in 
nearly quantitative yield. 
Condensation of 2-(1,3-dioxolanyl)-4-methyl-5-hydroxyaminopyridine with 
thiosemicarbazide in the presence of concentrated hydrochloric acid, 
followed by treatment with sodium bicarbonate afforded the desired 
5-hydroxyamino-4-methyl-2-formylpyridine thiosemicarbazone. 
Melting points were determined with a Thomas-Hoover Unimelt apparatus and 
are uncorrected. .sup.1 H NMR spectra were recorded on a Varian EM-390 90 
MHz NMR spectrometer or a Bruker WM-500 500 MHz spectrometer with Me.sub.4 
Si as the internal reference. The mass spectra (at 70 eV) were provided by 
the Yale University Chemical Instrumentation Center. TLC was performed on 
EM precoated silica gel sheets containing a fluorescent indicator. 
Elemental analyses were carried out by the Baron Consulting Co., Orange, 
Conn. Where analyses are indicated only by symbols of the elements, the 
analytical results for those elements were within .+-.0.4% of the 
theoretical value. 
Use of the compound of the present invention is preferably carried out when 
the compound is in a pharmaceutically acceptable salt form. Acceptable 
salts include, for example, inorganic acid salts such as hydrochloride and 
hydrobromide, organic salts such as acetate, tartrate, citrate, fumarate, 
maleate, toluenesulfonate, methanesulfonate, ethanesulfonate, 
hydroxymethanesulfonate, and hydroxyethanesulfonate, metal salts such as 
sodium salt, potassium salt, calcium salt, and aluminum salt, and salts 
with a base such as triethylamine salt, guanidine salt, ammonium salt, 
hydrazine salt, quinine salt, and cinchonine salt. The salts are made 
using procedures that will be readily apparent to those skilled in the 
art. Hydrates, which are also contemplated within the scope of the 
presently claimed invention, can be formulated using principles well known 
to those of ordinary skill in the art. 
The presently claimed invention can be formulated as a pharmaceutical 
composition in accordance with procedures that will be readily apparent to 
those of ordinary skill in the art. Preferred pharmaceutical compositions 
are, for example, tablets, including lozenges and granules, caplets, 
dragees, pills, gelatin capsules, ampuls, and suppositories comprising the 
active ingredient together with a) diluents, e.g., lactose, dextrose, 
sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, 
e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or 
polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum 
silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium 
carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) 
disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or 
effervescent mixtures; and/or e) absorbents, colorants, flavors and 
sweeteners. Injectable compositions are preferably aqueous isotonic 
solutions or suspensions, and suppositories are advantageously prepared 
from fatty emulsions or suspensions. Said compositions may be sterilized 
and/or contain adjuvants, such as preserving, stabilizing, wetting or 
emulsifying agents, solution promoters, salts for regulating the osmotic 
pressure and/or buffers. In addition, they may also contain other 
therapeutically valuable substances. Said compositions are prepared 
according to conventional mixing, granulating or coating methods, 
respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of 
the active ingredient. 
Suitable formulations for transdermal application include an effective 
amount of a compound of the invention with a carrier. Advantageous 
carriers include absorbable pharmacologically acceptable solvents to 
assist passage through the skin of the host. Characteristically, 
transdermal devices are in the form of a bandage comprising a backing 
member, a reservoir containing the compound optionally with carriers, 
optionally a rate controlling barrier to deliver the compound to the skin 
of the host at a controlled and predetermined rate over a prolonged period 
of time, and means to secure the device to the skin.