CA1256861A - 5-pyrimidinecarboxamides and treatment of leukemia and tumors therewith - Google Patents

Abstract

Case 5993 KON 1A
ABSTRACT
Novel 5-pyimidinecarboxamides useful for regressing or inhibiting the growth of leukemia and tumors in mammals. The compounds have the formula:

wherein:
R is hydrogen, 2- or 3- halo, 4-fluoro, 2-methyl, 4-(C1-C6 alkoxyl), 4-trifluoromethyl, or hydroxyl, and R1 is hydrogen; or R is 2-fluoro and R1 is 4-fluoro; or R is 2-methoxy and R1 is 5-methyl; and R2 and R3 are hydrogen atoms or carbohydrate residues; and the pharmacologically acceptable acid -addition salts thereof.

Description

12~ D~ case 5993 KON lA

1 5-PYRIMIDINECARBOXAMIDES AND TREATMEN~ OF
LEUKEMIA AND TUMORS THEREWITH

Technic~l Fi-1~3 s This invention relates to new 5-pyrimidine-carboxamides, and the pharmacologically acceptable addi-tion salts and nucleosides thereof. ~ore particularly, the invention relates to new 5-pyrimidinecarboxamide 10 derivatives which have anti-leukem;ia and anti-tumor activity, to pharmaceutical compositions containing such derivative~ as the therapeutically effective constituents thereof, and to a method utilizing the same for inducing the regression of leukemia and/or the inhibition of growth of tumors in mammals.

1 Back~lround Art 5-pyrimidinecarboxamides, and particularly 5-carboxamides of barbituric acid, have previously been described as potential anti-cancer agents D Thus, Takeda Pharmaceutical Industries' Japanese Patent Publication No. 1,445/64, published on February 14, 1964, suggests the use of compounds of the formula:

(I) ~ ON

R

i.e., 5-phenylcarbamoylbarbituric ~cid (whereln R ls hydrogen) and l-substltuted-phenylcarbamoylbarbiturlc aclds (wherein R is alkyl or phenyl), for such purpose.
20 When subjected to ln vivo testing on Ehrlich Ascites car-cinoma in mice the unsubstituted compound, but neither of its l-methyl or l-phenyl-substituted derivatives, exhibited anti-tumor activity. Chem. ~ Pharm. Bull.
~Tokyo) 8, 1021-1028 (1960~.
Analogs of similar barbituric acid derivatives have also been described in the literature. Thus, N-sub-stituted-2-amidocarbonylthiobarbituric acids of the formula:

O
Il X
R2_~ ~ C - N~ R
(II3 ~ ~ H

R~

1 wherein Rl is alkyl, alkenyl, various substituted alkyl, alkenyl or carbonyl, or optionally substituted aryl or aralkyl, R2 and R3 each independen~ly is alkyl, alkenyl, cycloalkyl, aryl, aralkyl or hydrogen, provided that not more than one of R2 and R3 is hydrocJen, and X is oxygen or ~ulfur, are di~closed in Bayer AG German Offen. 24 05 732 and in Kramer et al., U.S. Patent No. 3,961,061 granted on June 1, 1976. These thiobarbituric acid derivatives are described as possessing insecticidal, acaricidal, 10 fungicidal and bactericidal properties.
Other 5-carboxamido-substituted thiobarbituric acids such as:

~ ~3 R ~ C
~III) OH

~2 wherein X is oxygen or sulfur, Rl and R2 may each be alkyl, alkenyl, benzyl or unsubstituted or s~bstituted phenyl, R3 may be halogen, nitro or trihalomethyl, R4 is hydrogen, halogen or trihalomethyl, and ~5 is hydrogen, halogen, methyl or methoxy, are also described in the patent literature. Such compounds are disclosed in Ciba-Geigy European Patent Publication No. 74,335 and in De Sousa et al~, V.S. Patent No. 4l283,444 granted on August 11, 1981, as useful for protecting keratinous mate-rial, especially wool, from insect attack.
It is among the objects of the present invention to provide a new class of 5-pyrimidinecarboxamides, in particular a new group of 5-carboxamide-2-thiobarbituric acid derivatives, which are useful anti-leukemia and anti-tumor agents, as well as pharmaceutical compositions and therapeutic methods for utilizing the same. Other objects and advantages of the invention will be apparent from the ~2~;6~
~ - 5 -following detailed description of preferred embodiments th~ r eof .

~'~S6~

1 Summary of the Invention The novel 5-pyrimidinecarboxamides of the pre-sert invention are 5-carboxamide 2-thiobarbituric acid derivatives of the formula:

lû (;i;V) l~t CON~Rl ~ OH

15 wherein R is hydrogen, 2 or 3-halo, 2-methyl, 4-fluoro, 4-(Cl-C6 alkoxyl), 2 or 4-trlfluoromethyl, or hydroxyl, and Rl is hydrogen;
or R is 2-fluoro and ~1 is 4-fluoro;
or R is 2-methoxy and Rl is 5-methyl; and R2 and R3 are hydrogen atoms or carbohydrate residues; and the pharmacologically acceptable addi~ion salts thereof.
When R2 is hydrogen, addition salts may be formed with a variety of pharmacologically acceptable organic and invrganic salt-forming reagents. Useful addi-tion salts may thus be formed by admixture of the organic acid with one equivalent of a base, e.g., an organic amine such as triethylamine or N-methyl glucamine, and inorganic cations such as sodium, potassium or the like. The addi-tion salts of the organic acids of the invention are, in general, crystalline solids which are rela~ively insoluble in both polar solvents such as water, methanol and ethanol and non-polar organic solvents such as diethyl ether, benzene, ~oluene and the like~ ~hey are somewhat soluble in aprotic solvents such as dimethylformamide and dimethylsulfoxide.

~l256~
1 On the other hand, when R2 is a carbohydrate residue it may be furano~yl (e.gO, arabinofuranosyl or ribofuranosyl), pyranosyl (e.g~, arabinopyranosyl, glucopyranosyl, rhamnopyranosyl, or xylopyranosyl), their deoxy derivatives, or their aliphatic analogs ~e.g., hydroxyalkoxyalkyl or polyhydroxyalkyl groups having from

2 to 12 carbon atoms in each of the alkoxy and alkyl moieties thereof, such as 2-hydroxyethoxymethyl or 2,3-di-hydroxypropyl. As used herein, the term ~carbohydrate 10 residuen is intended to refer to those cyclic and acyclic groups which form pyrimidine nucleosides or the pseudo nucleosides, e.g., materials including both the cyclic and acyclic groups specified hereinabove.
The 5-carboxamide-2-thiobarbituric acid 15 derivatives of the invention can exist in the form illus-trated ln Formula IV or in any of its tautomeric forms.
For ease o~ understandlng, the compounds of the invention ~ill only be illustrated herein in the form shown in Formula IV but will be understood to embrace the tautomers thereof, or tautomeric mixtures.
The novel 5-carboxamide-2-thiobarbituric acid derivatives of the invention may be readily prepared by reacting 2-thiobarbituric acid with phenylisocyanate or an appropriate substituted phenylisocyanate, in the presence of a solvent or dispersing medium such as dimethyl sulfoxide, pyridine, dimethylformamide, N-methylpyr-rolidone, dimethylacetamide, sulfolane, tetrahydrothio-phene oxide, acetonitrile, or a tertiary amine such as triethylamine. The molar proportions of the 2-thiobarbi-

3~ turic acid to the phenylisocyanate reactant may range fromabout 2:1 to 1:2, and are preferably from about 1.1:1 to 1:1.1, stoichiometric proportions generally sufficing.
The reaction may be carried out at temperatures varying from about 0 to 200C, usually at from about 24 to 1~0C;
in most cases, the reaction proceeds quite well at temper-atures of from about 80 to 100C. Formation of the 5-carboxamide derivatives is substantially complete within reaction periods varying from about 1/2 to 6, and usually from ahout 2 to 4, hours.

:L2~6~

1 Alternatively, the carboxamides may be prepared by other routes. For example, thiourea may be reacted with appropria~ely substituted 2-benzoylamino propanedioic diesters, and the resulting products separa~ed and 5 recovered. O~her syntheses of the 5-carboxamide-2-thio-barbituric acid deriva~ives will readily occur to those ~ skilled in the art.
The novel compounds of the invention are cytotoxic agents useful to induce the regression of blood 10 malignancies such as leukemia, as well as to înhibit the yrowth of solid and non-solid tumors. They may be used alone or in combination with other chemotherapeutic agents active for these purposes. As used herein, the terms nregression~ and ~inhibition" comprehend arresting or 15 retarding the growth of the malignancy or other manifestatlon of the disease, as compared with the course of the disease ln the absence of treatment.
Admlnistration of the novel 5-carboxamido-2-thiobarbituric acid derivatives to mice in amounts ranging 2~ from about 12-~00 mg./kg., preferably from about 25-100 mg./kg., of body weight has been found effective to ind~ce the regression of leukemia and to inhibit the gro~th of tumors. The interrelationship of dosages for mammals of other sizes and species is described by Freireich, E.J., 25 et al., ~uantitative Comparison of Toxicity of Anti-cancer Agents in ~ouse, Rat, Hamster, Dog. Monkey and Man, Cancer Chemotherapy, Reg. 50, No. 4,219-244, May 1966.
The dosage level may, of course, be adjusted to provide optimum therapeutic response. For example, sev-30 eral divided doses may be administered daily, or the dosemay be proportionally reduced, as indicated ky the exigen-cies of the therapeutic situation.
~ he active compounds may suitably be adminis-tered parenterally, intraperitoneally, intravenously or 35 orally. Solutions or dispersions of the active compounds can be prepared in water, suitably mixed with a sur~actant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and ~ 25~
g 1 mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preserva-tive to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable 5 use include sterile aqueous solutions or di~persi~ns and sterile powders for the extemporaneous prep~ration of sterile injectable solutions or dispersions. For such uses the form must be sterile and must be fluid to the extent necessary to provide easy syringability. It must lO be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microor~anisms such as bacteria and fungi.
The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, a polyol 15 (for example, glycerol, propylene glycol, and liquid poly-ethylene glycol, or the like), suita~le mixtures thereof, and vegetable oils. The proper fluidity can be maln-tained, for example, by the use o~ a coatlng ~uch as lecithln, by the malntenance of the required particle size ~ in the case of a dispersion, and by the use of surfac-tants. Prevention of the action of microorganisms can be insured by various anti-bacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, thimerosal, or the like. In many cases it may be prefer-25 able to include isotonic agents, for example sugars or sodium chloride, in the dosage form. Prolonged absorption of the injectable formulations can be brought about by incorporating agents delaying absorption, for examFle, aluminum monostearate and gelatin, therein.
Sterile injectable solutions are prepared by incorporating the active compound in the appropriatesolvent, in admixture with various of the other ingredi-ents enumerated above, as reguired, followed by filtered sterilization. Generally, dispersions are prepared by 35 incorporating the sterilized active ingredient in a ster-ile vehicle which contains the dispersing medium and any other required ingredients. When, on the other hand, sterile powders are used to prepare sterile injectable 1 ~olutions, it is preferred to subject a sterile, filtered solution of the ~esired ingredients to vacuum drying or freeze-drying, yielding a powder of the active ingredient plus any additional desired ingredien~s.
As used herein, ~pharmaceutically acceptable, substantially nontoxic carrier or excipientn includes sol-~ vents, di~persing media, eoatinys, antibacterial and anti-fungal agents, isotonic and absorption delaying agents and the likeO The use of such media and agents as carriers or excipients for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active ingredient or toxic, its use in the therapeutic formulations of the invention is contemplated. Supplementary active ingredi-ents can also be incorporated in the therapeuticcompositions.
It may be advantageous to formulate the compositions of the lnvention ln unlt dosage forms for ease of administration and uniformity of dosage. A unit dosage form, as used herein, refers to a physically discrete unit suitable for use as a unitary dosage for the mammalian subjects to be treated; each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier.
Specifications for unit dosage forms are dictated by and directly depend on (a) the unique characteristics of the active material and the particular thera~eutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition, without excessive cytotoxic side effects.
Regression of leukemia and inhibition of tumor growth ~ay be attained, for example, by the use of daily dosing for up to 5 or 10 days, or longer. Multiple dosing, or dosin~ on any desired periodic basis, may also be utilized. The therapeutically active ingredient is thus administered in amounts sufficient to aid regression ~l256~

l and inhibition of furthcr growth of the leukemia or tumor, in the absence of excessive deleterious side effects of a cytotoxic nature.

1 Brlef Description of the Drawin~
The accompanying drawing is a graph ~howing the effects of treatment schedule and route of administration of one of the compounds of the invention in the regression of L1210 lymphoid leukemia.

1 Best Mode For Carryln~ Out The Invention Preferred amony the 5~arboxamido-2-thiobarbituric acid derivatives hereof are compounds within the scope of Formula IV above, wherein R and Rl are hydrogen (Example 1 below~, R is 2-chloro and Rl is hydrogen (Example 2)l R is 2-methyl and Rl is hydrogen (Example 3), R i6 3-fluoro and Rl is hydrogen (Example 4), R is 4-.fluoro and Rl is hydrogen ~E~ample 5), R is 4-methoxy and Rl is hydrogen (Example 6), R is 4-ethoxy amd Rl is hydrogen (Example 7), 10 R is 2-fluoro and Rl is hydrogen (Example 8~, R is 2-fluoro and Rl is 4-fluoro ~Example 9), and R is 2-methoxy and Rl is 5-methyl (Example 10). Particularly preferred is the compound of Example 1, viz., 1,2,3,4-tet-rahydro-6-hydroxy-4-oxo- N-phenyl-2-thioxo~5-pyrimidine-carboxamide:

.

The invention will be described in greaterdetail in connection with the following specific examples illustrating the preparation and pharmacological testing of preferred embodiments of the compounds of the inven-tion:

Preearation of 1,2,3,4-tetrahydro-6-hxd~y

-4-oxo-N-phenyl-2-thioxv-5-Fyrimid~r~c~r~c~ride A. Reaction of Thiobarbituric Acid with Phenylisocyanate 14.4 9 of 2-thiobarbituric acid (which may, alternatively, be named dihydro-2-thioxo-4,6-(lH,5H)-pyrimidinedione or 4,6-dihydroxy-2-mercaptopyrimi~ine) and 11~9 g of phenylisocyanate were dissolved in dry pyridine 1 (100 ml.). The solution was heated with stirring, and maintained at 7S-85~C for about 4 hours. Upon cooling, an orange-colored solid precipi~atled out which was isolated, washed with about 25 ml dimethylformamide and dried.
Yield: 16.B g (64~) NMR (DMSO) 7.1-8.0 d~(multiplet;integral 5); 11.4 ~ (sin-glet, 1); 12.0-13.7 ~ (broad diffuse peak, 3).
An elemental analysis for CllHgN303S gave the 10 ~ollowing results:
Calculated Found(~) C 50.19 50.30 H 3.45 4.02 N 15.96 15.75 Mass spectrometric analysis was as follows:
Calculated Found .. .. _ .
M/E ~ 263 263 The compound decomposed at 310C~. q'he structure was further confirmed by an X-ray crystallographic study of 2Q the triethylammonium salt.
B. Reaction of Thiourea with _arboxanilidomalonate Thiourea (1.5g) was intimately mixed with carboxanilidomalonate, viz., (C2H502C)2 CHCONH ~ ~
(3.6g), and very gently heated in a small flask in an oil bathO At about 155C the reaction mixture became semi-liquid with a solid residue remaining in the bo~tom of the flask. At about l50~C the reaction ~ixture began to thicken, a volatile material being evolved. The reaction mixture was heated to 180, left at that tempera-ture for 1/2 hour, and then cooled. A khaki, ochrecolored powder product was thus produced.
The product was washed with ethanol and dried (1.89). The mass spectrum was consistent with the prod~ct of Example lA. Molecular Weight - 263; 171 lpyrimidine fragment); 93 (anilide fragment).

~ ~ 5 6 8 Prepafation of N-(2-chlorophenyl)-1,2,3,4-tetrahydro-6-h,ydroxy-4-oxo-2-thioxo-5-eyrimidinecarboxamide 2-thio~arbituric acid (14.4 9) was carefully dried, finely powdered and suspencled in dry pyridine (100 ml). The cuspension was warmed with ~tirring to about - 50, and 2-chlorophenylisocyanate (15.35 g) added. Much of the suspension went into solution. The mixture was stirred at 75-85 for 4 hours and left overnight at room 10 temperature.
The pyrimidinecarboxamide was collected as a purple powder; it was washed with a small quantity of pyridine, which removed most of the color, resuspended and triturated in 100% ethanol, collected and dried. Yield 23 g (77%), off-white powder, no sharp melting point ~decomposes above 250C). NMR ~DMS0) 7.1-~.3 ~ ~mul-tlplet; lntegral 4); 11. ~singlet, 1); 11.7-13.0 ~broad dlffuse peak, 3).
Mass Spectrum 299-297 ~molecular ion, chlorine 2~ isotopes); 171 (pyrimidine carbonyl fragment); 129-127 (o-chloroaniline, chlorine isotopes).

Preparation of 1,2~3 4-tetrahvdro-6-hYdroxY-N
(2-methylphenyl)-4-- ~ -5-pyr mldine-carboxamide The procedure described in Example 2 was repeated, reacting 2-thiobarbituric acid and 2-methyl~
phenylisocyanate to give the pyrimidinecarboxamide as a tan powder, mp 250 (dec.); NMR (DMSO), 2.30~, singlet, integral 3; 7.15-B.00 ~ multiplet, 4; 11.4 ~ singlet, 1;
12.0-13.7 ~, broad diffuse peak, 3. Mass Spectrum 277, 171,107.

6~

aration of N-(3-fluoro~enyl?-lL~L_,4-tetrahydro -6-hydeoxv-4-oxo-2-thioxo-5-~Yrimidinecarboxamide The procedure described in Example 2 was repeated, reacting 3-fluorophenyl isocyanate to give the pyrimidinecarboxamide as a pinkish powder, mp ~ 250 (dec.) NMR tDMSO); 6.7-7.7 ~ ~ultiplet, integral 4; 11.4 singlet, l; 12-13 3, broad diffuse peak, 3. Mass Spectrum 2~1,171,111.

Pre~aration of_N-!4-fluoropheny tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide 2-thiobarbituric acid (14.4 g) was suspended in pyridine and 4-fluorophenyl isocyanate ~13.7 g) was added thereto. The reaction mixture was maintained at 90C for one hour, and thereafter let overnight a~ room tempera-ture. The solids formed were collected, washed with 2~ pyridine, re-suspended in ethanol, and again collected and dried. A pale pink powder product was thereby obtained, mp ~ 250C (dec.), NMR (DMSO) 7.0-7.7 ~ (multiplet, integral 4); 10.7-11.4 ~ (overlapping broad singlets, combined integral 4). MS, M/e 281 (Calc., 2Bl~.

Preparation of 1L2, 3~4-tetrahydro-6-hydroxy-N-(4-methoxyphenyl?-4-oxo-2-thiox~-~-pyri~idinecarboxamide The procedure described in Example 2 was repeated, reacting 4-methoxyphenylisocyanate to give the pyrimidinecarboxamide as a yellow powder, mp 330 (dec.), NMR (DMSO) 3.81 ~ (singlet, integral 3); 6.9-7.6 ~ (two symmetrical near-doublets, 4); 11.4 ~ (singlet, 1) 11~7-1203 ~ broad diffuse peak, 3. MS, 293,171,1~3.

Preparation of N-(4-ethoxyphenyl~-1,2,3,4-tetrahydro-6-hydroxy-4-oxo 2-thioxo~Ryrimidinecarboxamide The procedure described in Example 2 was ~ s~

1 repeated, reacting 4-ethoxyphenylisocyanate ~o give the pyrimidine as a yellowish-pink powder, mp ~ 250 ~dec.), NMR (~MSO) 1.35 ~ (triplet, integral 3; 4.1 ~ (~uartet, 2) 6.9-7.6 ~ (two symme~rical near-do~blets, 4); 11.4 (singlet, 1) 12-13 ~, low broad diffuse peak. MS
307~171,137.

Preparation of N-(2-fluorophenYl)-1,2,3,4-tetra-hydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidine-carboxamide .
The procedure described in Example 2 wasrepeated, reacting 2 fluorophenyl isocyanate to give the pyrimidine as a pale pinkish-purple po~der, m.p. ~ 250 tdec.). NMR (DMSO) 7.2-8.4 ~ (complex multiplets), 11.8 (singlet). MS 281, 171, 111.

Preparatlon of N-~2 4-diEluoro~henYl)-1 2 3 4-The procedure described in Example 2 wasrepeated, eeacting 2,4-difluorophenyl isocyanate to give the pyrimidine as a pale pinkish-purple powder, m.p.>
250 (dec.). NMR (DMSO) 7.0-8.3 ~ (complex m~ltiplet), 11.8 ~ (singlet); broad diffuse multiplet ca. 10.7-11.8 MS, 299, 171, 129.

Preparation of 1,2,3,~ ~e~hv~ ~ xy-N-(2-methoxY-5-methylphenyl)-4-oxo-2-thioxo-_ 5-pyri~idinecar~o-xamide 3~
The procedure described in Example 2 was repeated, reacting 2-methoxy-5-methylphenyl isocyanate to give the pyrimidine as a pink powder, m.p. ~ 2B0 (decO).
NMR (DMSO), 2.3 ~ (singlet, integral 3); 3.9 ~ (singlet, 35 ~integral 3); 7.0 ~ (broad singlet, integral 2); 7.9 (broad singlet, integral 1); 11.6 ~ (kroad singlet, integral 1). MS 307, 171, 137.

~ 5 6 1 Antitumor Activity Of The Compound of Example 1 Tbe spectrum of antitumor activity of the compound of Example 1 was determin~ed employing a number of ~tandard National Cancer In~titute (NCI) protocols. The antitumor activity was determined in vivo ~gainst several different tumors, employing various treatment schedules and routes of administration. The results ob~ained~
expressed in percent increases in :life-span (% ILS) of the test animals, are summarized in the following table.

~Lr2~i6 SUMMARY OF ANTITUMOR ACTIVITY OF COMPOUND

Tumor Treatmen~ Activity Rating2 Schedule (% ILS, cures/total) Murine tumors:

10 i,p. B16 melanoma Days 1~9 +~ (93,85) s.c. CD8Fl mammary tumor Staging Day sOc. Colon 38 tumor Days 2-9 i.p. L1210 leukemia Days 1-9 ~+ (~ 275, 4/6;
~2~, 6/6 s.c. L1210 leukemia Days 1-9 ~ 200, 5/6, ~154,3/6) i.c. L1210 leukemia Days 1-9 ~ ~34,28) i.v. Lewis lung carcinoma Days 1-9 i.p. M5076 sarcoma Days 1-13 ++ (72,2/10;
72) i.p. P388 leukemia Days 1-5 ~+ (101,94) Human tumor x~ Lraft:
25 s.r.c. MX-l mammary Days 1-9 tumor 1. The test compound was administered i.p. (intraperi-toneally) once a day on the days indicated, except in the case of the test for inhibition of the colon 38 tumor in which it was administered seven times per day, and the tests for inhibition of the M5076 sarcoma and MX-l mammary tumors ln which it was administered four times daily.
2. Activity: +~ Reproduced activity: ~ 50~ ILS for i.p. and i.v. (intravenous) im~lanted tumors (> 75% ILS for P388), ~90% inhibition of tumor growth for s.c.
~subcutaneous) and s.r,c. (su~renal ~256~6~L
.

1 capsule), implanted tumors (~ 100~
inhibition for the staged CD8Fl tumor).

+ Reproduced activity: 25-~9~ ~LS for B16, L1210 and M5076t 20 74% ILS for P388, 40-49 ILS for Lewis lung, 58-89~ 80 8~ and 80-99% inhibition of tumor growth for the colon 38, xenografts and the staged CD8F
mammary tumor, respectively.
1~
- Inactive.
In both the i.p. and s.c. L1210 leukemia systems, a 100 mg/kg dose of the test compound administered i.p. daily for 9 days was curative in at least 50% of the test mice. The 100 mg/kg dose occa-sionally demonstrated some toxicity ln the i.p. system.
Employing a 50 mg/kg dose in these systems, Inaximum lncreased llfe spans of 87-190% were obtained.
Using the same treatment regimen (100 mg./kg.
administered i.p. daily for 9 days), marginal activity (ILS = 28-34~) was observed against the i.c.
(intracranially) implanted L1210, indicative of a systemic rather than an i.c. effect.
In the B16 melanoma Cystem~ optimal ILS values ~5 of 93 and 85% were observed following daily i.p. treatment of 100 mg/kg for 9 days. Activity (ILS = 25%) was observed over at least a 4~fold dosage range.
In three experiments involving the i.p.
implanted M5076 sarcoma, maximum ILS values of 72, 72 and 48% were achieved after i.p. treatment on days 1, 5, 9 and 13.
- The compound of Example 1 also demonstrated good activity in the standard NCI lymphocytic leukemia P388 preliminary screen, producing maximum ILS values of 101%, 94% and 62% following i.p. administration of a 50 mg/kg dose daily during a five day test.
The test compound was ineffective against the s.c. implanted CD8Fl mammary and colon 38 carcinoma~, the ~L2~r.~68 1 i.v. implan~ed Lewis lung carcinoma and the s.r.c. human MX-l mammary carcinoma xenograft under the experimental conditions employed.
The data obtained in the various tests summa-rized above are tabulated in Table ][I below. The ratio of - the survival ~ime for the treated animals tTJ to the sur-vival time for the control animals ~C~ deter~ined at Yarying dosages in the respective n vivo tests is set orth in the table:

~ ~ 5 IN VIVO TEST DATA

,, _ NCI TEST DOSE
PROTOCOL ~9~ TREATED/CONTROL PERCENT _ (i~po~ 50 1~2 1~9 implanted 25 137 158 10 melanoma) 6 108 3CDJ2 900 (-) ~-) (s.c.- 450 (-) (-) implanted 225 (-) (-) staged112 85 80 mammary56 85 61 adeno-carcinoma CD8Fl) 3C872 900 (_ . .
(s.c.- 450 (-) implanted 225 51 colon 38112 (~) 68 25 carcinoma) 56 70 120 _ (i.p.-implanted leukemia) 3LE32 200 (~) (~) (s.c.- 100 3~0(5~ 254(3) imPlanted5~ 140 127 leukemia) 12.5 109 104 i68~i1 1 TABLE II (Cont'd) IN VIVO TEST DATA

NCI TEST DOSE
~ROTOCOL mq/kg TREATED/CONTROL PERCENT _ 3LE37 200 (-) (-) (i.c.- 100 128 134 implanted 50 113 108 g leukemia) 12.5 94 106 3MBG5600 (-) ~s.~.c.300 (-) human150 (-) 15 mammary75 98 carcinoma MX-l xeno-graft) _ 20 3M531 200 (-) (-) (i.p.- 100 148 172 implanted 50 124 147 sarcoma) 12.5 117 - - _ _ _ _ 3PS31 200 (~
(i.p.- 100 ~-) (-) implanted 50 162 194 201 30leukemia) 12.5 120 6.25 116 3.13 110 35 (i.v.- 50 115 Lewis 25 106 lung 12.5 103 carcinoma) S~8~L

* (~) ~ cures in 'cest at specified dose, (-~=toxic dose, blank - no test ~2~86~iL

1 Effects Of Treatment Schedule And Route Of Administration On The Activity Of The Compound of ~xample 1 A~jainst s.c.-__ Implanted L 1210 Leukemia _ _ The influences of ~reatment schedule and routeof administration on the antitumor activity of the compound of Example 1 were evaluated using the s.c.
implan~ed L1210 leukemia system. The drug was tested in the form of a freeze dried dosage form containing 50 mg.
of the compound and 100 mg. N-methylglucamine, reconstituted with 5 ml. of sterile water to yield a 10 mg/ml. solution at about pH 9.5.
The percentage increases in life span ~%ILSI are shown in the accompanying drawing for different treatment regimens and routes of administration. As illustrated, increases in life span were noted using all treatments ~d routes o~ admlnistratlon save ~or the l.p. and l.v. one day, one injectlon treatments (tests A and G in the drawinq). The highest percent ILS was 471 obtained by daily i.p. injections of the active material on a five day schedule of 45 mg/kg/injection, corresponding to a total dose of 225 mg/kg/duration of thR treatment (test C in the drawing). This dosage also gave 5 cures. Administration of the drug by oral injection daily for nine days also resulted in a high percent ILS of 45~, employing a dosage of 124 mg/kg~injection, and a c~mulative doqe of 1116 mg/kg/duration of treatment (test I in the drawing). Use of this regimen resulted in 4 cures.
In the studies illustrated in the drawing toxic-ity was noted with the highest dose in each treatment schedule except for that involving i.p. administration every three hours on days one, five and nine of a nine day test (test F).
It may be seen that, under the experimental con-ditions utilized, substantial increases in life span ~by definition, in excess of 25% ILS) were obtained utilizing each of the routes of administration and treatment sched-ules save for the single treatment i.p. and ioV~ routes.

6~
-26~
1 Comparison Of The Antitumor Activities Of Variety Of Test Compounds In The Regression Of i.p~-Implanted Lymphoicl Leukemia L1210 Samples of the te~t compounds of E~amples 1 - 10 and a number of structurally~related control compounds were tested in accordance with National Cancer Institute test protocol 3LE31 (NCI Protocol 1.100, Cancer Chemotherapy Reports Part 3, Vol. 3, No. 2, September 1972) to determine the effects of the several compounds on i.p.- implanted L1210 leuke~ia (J. Nat'1. Cancer Inst.
13(5):1328, 1953). Each test involved implantation of the leukemia cells in six DBA/2 mice, one sex per experiment, the male mice weighing a minimum of 18 grams and the female mice weighing a minimum of 17 grams, and all of the test animals being within a three gram weight range. ~he test compound was admlnlstered by i.p. injections, ln 0.1 ml. doses o~ dllu~ed ascltlc fluid (105 cells per dose), commencing one day after the tumor implant and continuing daily for nine days.
The test animals were weighed and survivors recorded on a regular basis during a thirty day test period. The ra~io of survival time for the treated and control animals (T/C) was determined as a percentage.
The tests were carried out at varying dosage levels and with varying numbers of repetitions, depending upon the results obtained with each test compoud. It has been statistically determined in the 3LE31 test system that an initial T/C value at least equal to 125% is necessary to demonstrate activity, while a reproducible T/C equal to or greater than 125% warrants further study.
A reproducible T/C of 150% or higher i5 considered signif-icant activity.
The number of mice "cured~, viz., those surviving from each animal test group after the thirty day test period, is indicated in parenthesis following the ~/C
percentage data in Table III below:

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1 As may be ~een from Table III, the compound of Example 1 exhibited significant activity in the i.p.-implanted lymphoid leukemia test at dosage levels of both 50 mg/kg. and 100 mg/kg., and produced a number of cures at 100 mg/kg. With the exceFtion of the compound of Example 5, the materials of the oth~er examples similarly exhibited significant activity in the test. On the other hand, only two control compounds, the barbituric acid derivatives of Controls B and Ct exhibited significant 10 activity in the test. The compound of Control Ay previously believed to exhibit moderate activity in the 3LE31 test protocol, was found upon further testing to exhibit a T/C less than 125~ and thus to be inactive.
From the preceding, it will be seen that, in 15 accordance wlth the present lnvention, a class of novel

5 pyrimidlnecarboxamldes ls provided, the members of whlch exhlblt substantlal cytotoxlc activity and induce regres--sion and/or inhibit growth of leukemia and various malig-nant tumors in mammals. It will be apparent that various changes may be made in the method of preparation and use, as well as in the particular substitution, of the thera-peutically active compounds of the invention. Accord-ingly, the preceding disclosure should be construed as illustrative only, and the scope of the invention should be inter~reted in accorclance with the claims appended hereto.

3~

Claims (48)

-39-

1. A process for the production of a compound of the formula:

wherein R is hydrogen, 2- or 3-halo, 2-methyl, 4-fluoro, 2- or 4-alkoxy having from 1 to 6 carbon atoms, 2- or 4-trifluoromethyl, and R1 is hydrogen; or R is 2-fluoro and R1 is 4-fluoro; or R is 2-methoxy and R1 is 5 methyl;
and R2 and R3 are hydrogen atoms or carbohydrate residues; and, with the exception of the compound wherein R is 4-methoxy and R1 is hydrogen, the pharmacologically acceptable acid addition salts thereof, which process comprises a process chosen from the group consisting of (a) reacting a 2-thiobarbituric acid compound:
with a phenylisocyanate in the presence of a solvent or dispersing medium;
(b) reacting a thiourea of formula with an arylaminocarbonylpropanedioic acid diester wherein R7 is lower alkyl;
(c) reacting an S-substituted pseudo thiourea wherein R' is lower alkyl,with an arylaminocarbonylpropanedioic acid diester to form the corresponding 2-substituted thiopyrimidinyl compound:

wherein R' is lower alkyl, and reacting the 2-substituted thiopyrimidinyl compound with hydrogen sulfide or an alkali metal or ammonium salt thereof:
(d) alkylating a compound of the formula:

to produce a 2-(alkylthio)-substituted compound of the formula reacting the resulting product with a phenylisocyanate to form a compound incorporating the desired 5-carboxamido moiety, having the formula:

;and reacting the compound thus formed with hydrogen sulfide or an alkali metal or ammonium salt thereof; and (e) reacting the corresponding 2-aminopyrimidinecarboxamide:

with hydrogen sulfide or an alkali metal or ammonlum salt thereof to displace the amino group therefrom.

2. A compound of the formula wherein:
R is hydrogen, 2- or 3-halo, 2-methyl, 4-fluoro, 2- or 4-alkoxy having from 1 to 6 carbon atoms, 2 or 4-trifluoromethyl, and R1 is hydrogen; or R is 2-fluoro and R1 is 4-fluoro; or R is 2-methoxy and R1 is 5-methyl;
and R2 and R3 are hydrogen atoms or carbohydrate residues; and, with the exception of the compound wherein R is 4-methoxy and R1 is hydrogen,the pharmacologically acceptable acid addition salts thereof .

3. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 2 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

4. The process as in Claim 1, wherein R is hydrogen or a 2-chloro, 2-methyl, 3-fluoro, 4-fluoro, 4-methoxy or a 4-ethoxy group.

5. A compound of the formula wherein:
R is hydrogen or a 2-chloro, 2-methyl, 3-fluoro, 4-fluoro, 4-methoxy or a 4-ethoxy group, and Re is hydrogen;
and R2 and R3 are hydrogen atoms or carbohydrate residues; and, with the exception of the compound wherein R is 4-methoxy and R1 is hydrogen, the pharmacologically acceptable acid-addition salts thereof.

6. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 5 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

7. The process as in Claim 1 for preparing 1,2,3,4-Tetrahydro-6-hydroxy-4-oxo-N-phenyl-2-thioxo-5-pyrimidinecarboxamide.

8. 1,2,3,4-Tetrahydro-6-hydroxy-4-oxo-N-phenyl-2-thioxo-5-pyrimidinecarboxamide.

9. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 8 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

10. The process as in Claim 1 for preparing N-(2-Chloro-phenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

11. N-(2-Chlorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4 oxo-2-thioxo-5-pyrimidinecarboxamide.

12. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 11 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

13. The process as in Claim 1 for preparing 1,2,3,4-Tetrahydro-6-hydroxy-N-(2-methylphenyl)-4-oxo-2-thioxo 5-pyrimi-dinecarboxamide.

14. 1,2,3,4-Tetrahydro-6-hydroxy-N-(2-methylphenyl)--4-oxo-2-thioxo-5-pyrimidinecarboxamide.

15. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 14 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

16. The process as in Claim 1 for preparing N-(3-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

17. N-(3-Fluorophenyl)-1,2,3,4 tetrahydro-6-hydroxy 4-oxo-2-thioxo-5-pyrimidinecarboxamide,

18. A pharmaceuticsl composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 17 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

19. The process as in Claim 1 for preparing N-(4-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

20. N-(4-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

21. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 20 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

22. The process as in Claim 1 for preparing 1,2,3,4-Tetrahydro-6-hydroxy-N-(4-methoxyphenyl)-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

23. 1,2,3,4-Tetrahydro-6-hydroxy-N-(4-methoxyphenyl)-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

24. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 23 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

25. The process as in Claim 1 for preparing N-(4-Ethoxyphenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

26. N-(4-Ethoxyphenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

27. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 26 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

28. The process as in Claim 1 for preparing N (2-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

29. N-(2-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

30. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors in mammals, which comprises a therapeutically effective amount of the compound of Claim 29 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

31. The process as in Claim 1 for preparing N-(2,4-Difluorophenyl)-1,2,3,4-tetrahydro-6 hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

32. N-(2,4-Difluorophenyl) 1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

33. A pharmaceutical composition gor inducing regression of leukemia and inhibition of the qrowth of tumors is mammals, which comprises a therapeutically effective amount of the compound of Claim 32 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

34. The process as in Claim 1 for preparing 1,2,3,4-Tetrahydro-6-hydroxy-N-(2-methoxy-5-methyl-phenyl)-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

35. 1,2,3,4-Tetrahydro-6-hydroxy-N-(2-methoxy-5-phenyl)--4-oxo-2-thioxo-5-pyrimidinecarboxamide.

36. A pharmaceutical composition for inducing regression of leukemia and inhibition of the growth of tumors mammals, which comprises a therapeutically effective amount of the compound of Claim 35 in admixture with a pharmaceutically acceptable, substantially nontoxic carrier or excipient.

37. A process for the production of a compound of the formula:

wherein R is hydrogen, 2- or 3-halo, 2-methyl, 4-fluoro, 2-or 4-alkoxy having from 1 to 6 carbon atoms, 2 or 4-trifluoromethyl, and R1 is hydrogen; or R is 2-fluoro and R1 is 4-fluoro; or R is 2-methoxy and R1 is 5-methyl:
and R2 and R3 are hydrogen atoms or carbohydrate residues: and, with the exception of the compound wherein R is 4-methoxy and R1 is hydrogen, the pharmacologically acceptable acid addition salts thereof, which process comprises reacting a 2-thiobarbituric acid compound:

with a phenylisocyanate in the presence of a solvent or dispersing medium.

38. The process as in Claim 37, wherein R is hydrogen or a 2-chloro, 2 methyl, 3-fluoro, 4-fluoro, 4-methoxy or a 4-ethoxy group.

39. The process as in Claim 37 for preparing 1,2,3,4-Tetrahydro-6-hydroxy-4-oxo-N-phenyl-2-thioxo-5-pyrimidinecarboxamide.

40. The process as in Claim 37 for preparing N-(2-Chloro-phenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

41. The process as in Claim 37 for preparing 1,2,3,4-Tetrahydro-6-hydroxy-N-(2-methylphenyl)-4-oxo-2-thioxo-5-pyrimidinenecarboxamide.

42. The process as in Claim 37 for preparing N-(3-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

43. The process as in Claim 37 for preparing N-(4-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

44. The process as in Claim 37 for preparing 1,2,3,4 Tetrahydro-6-hydroxy-N-(4-methoxyphenyl)-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

45. The process as in Claim 37 for preparing N-(4-Ethoxyphenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

46. The process as in Claim 37 for preparing N-(2-Fluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thioxo-5-pyrimidinecarboxamide.

47. The process as in Claim 37 for preparing N-(2,4-Difluorophenyl)-1,2,3,4-tetrahydro-6-hydroxy-4-oxo-2-thi-oxo-5-pyrimidinecarboxamide.

48. The process as in Claim 37 for preparing 1,2,3,4-tetrahydro-6-hydroxy-N-(2-methoxy-5-methylphenyl)-4-oxo-2-thioxo-5-pyrimidinecarboxamide.