llarch 1969
CHEMOTHERAPEUTIC
.4t 3-.i weeks of age, the chicks were transferred to individiial cages with hardware cloth floors where the efficacy experiments were conducted. The Eimeria fenella cultures used in these experiments were aerially propagated in onr laboratory over a period of several years. These cultures were isolated by single oocyst inoculation of coccidiosis-free birds to ensure the purity of the cultures. Infection was accomplished by depositing a predetermined volume of calibrated oocyst suspension directly into the crop of each chick. The compounds tested in these trials were incorporated into a standard ration and fed to the birds for 2 days prior t,o infection, and continued for the duration of the test. T h e anticoccidial efficacy in these experiments was based on
&SITROTHIrlZOLES
303
three factors: (1) mortality, (2) weight gain or loss, and (3) droppings scores. The primary criterion of efficacy was the mortality produced in the medicated-infected chicks as compared to the nonmedicated-infected chicks. Droppings scores and ratios of mean weight gains, medicated-infected vs. nonmedicated-noninfected, were used as indicators of m ~ r b i d i t y . ~
Acknowledgment.-The authors wish to thank Professor Joseph Cannon of the University of Iowa for helpful discussions of this work. They are also indebted to l h Carol Barker and A h . Alarvin Carr for obtaining the analytical data arid assisting with the experiments.
Chemotherapeutic Nitroheterocycles. Derivatives of 5-Nitrothiazole-2-carboxaldehydeand 5-Nitrothiazole-2-carboxylic Acid’ DAVID W. HENRI Depai linent of Pharinat eutical Chemistry, Stanford Reseaxh Inslztute, Menlo Paik, Califomza Yj026
Receiced July 29, 1968 Revised Manuscript Received October 14, 1968 A series of new 5-nitrothiazoles bearing carbon substituents in the 2 position has been prepared. Treatment of 2-bromo-5-nitrothiazole with CuCX provided 5-nitrothiazole-2-carbonitrile, a keg intermediate for subsequent conversion to other derivatives of 5-nitrothiazole-2-carboxylic acid. The corresponding aldehyde was obtained by condensing 2-methyl-5-nitrothiazole with benzaldehyde and oxidatively cleaving the resulting styryl intermediate. The compounds prepared in this study were evaluated in vivo for antimalarial and antischistosomal activity and in citro for activity against bacteria, yeast, and a finigue. Little activity was noted in the malaria arid schistosomiasis tests, but broad-spectrum inhibitory effectswere widely evident in the in vitro assays. The most potent compound, 5-nitrothiazole-2-carboxaldehydeacethydrazone, was inhibitory a t 1 pg/ml or less in all but one of the latter tests.
Among the several classes of nitroheterocyclic drugs possessing useful properties in clinical or veterinary medicine, the 5-nitrothiazoles are of special recent interest. I n addition to the well-established use of 2-amino-5-nitrothiazole, and simple derivatives thereof (la-c), for the treatment of histomoniasis in turkey^,^ another closely related nitrothiazole, niridazole (la), has been found highly effective in human schistosomiasis4-6 and amebia~is.~Jt’ Favorable preliminary results against two other parasitic diseases, and strongyloidiasis,8,11 have also ( 1 ) This work was siipported hy t h e U. Y. Army Medical Researcli and Development Command under Contract No. DA-49-193-AID-2750. This is Contribution Numl>er 420 from t h e Army Research Program on Malaria. (2) IY. T. Colnell, .I. H. Lange, and D . TV. Henry [ J . ,Wed. Chem., 11, 282 (1968)l pro>-ideleading references in this area. (3) L. Joyner, S. Davies, and S. Kendall in “Experimental Cliemotherapy,” 1’01.I , R . J. Schnitzer and F. Havking, E d . , Academic Press, New T o r k , N. Y., 1963. pp 341-343. (4) Conference on t h e Pharmacological and Chemotherapeutic Properties of Kiridazole a n d Other Antischistosomal Compounds, New York, N. Y . , Oct IC-13, 1967; sponsored hy T h e New York Academy of Science, Section of 13iological and Medical Sciences and Division of Microbiology. ( 5 ) Actu T r o p . , S w p l 3 , 1 1966. This publication comprises the proceed11g5 oi a symposium entitled TherPpuetique Kouvelle de la BilharzioPe et de I‘amebiase that was held in Lisbon, J u n e 2-4, 1965, under the sponsorship of Professor F. S. d a Cruz Ferreira of the Institute de Medicine Tropicale. (6) (a) H. L. Wolfe, L a n c e t , 350 (1967); (b) J. E. hfchlahon and C. P. Kilala, B r i t . ,Wed. J . , 1047 (1966): ( e ) P. Schmidt a n d JI. \Vilhelm, Angeu. Chem. Intern. Ed. E n d . , 6, 857 (1966): (d) A. Ruas and L. T. hlmeido Franco, A n n . T r o p . ,Wed. Purasild., 60, 288 (1966). J. Wilmot, I. hlacLeod, and R. Elsdon-Dew, A m . J . T r o p . .Wed. Hug., 15, 300 (1966); (b) T. Kradolfer and R . Jarumilinta, A 7 ! n , I’rop. .Ired. I ’ o r i i s i t o l . , 69, 210 (1965). (8) G . Raffier, ref 4 . (9) G . Raffier, A c t u T r o p . . 22, 330 (1965). (10) .I0. . Lucas, ref 1. (11) G. Raffier, Ezpe,.ientiu. 22, 826 (1966).
been reported for this drug. I n a recent paper in which Avramoff, et a1.,I2 revealed a group of bis-5-nitrothiazoles with marked in vitro antiprotozoal activity,
0 la, X = NH, b, X = NHCOCH3 c, X = NHCONHEt
2
d, X = N -?H
0
3a, R1= CH,CH-OH; R2 = CH, b, Rl=&=CHj C, R1= CHj; &=p-C,H,F
they provided a brief survey of current developments in the nitrothiazole field. A characteristic feature of essentially all reported chemotherapeutic nitrothiazoles is the presence of a free or substituted amino group in the 2 position. I n contrast, the antiprotozoal nitrofuran^'^,'^ (e.g., furazol(12) AI. Avramoff, S. Adler, and A. Foner, J . .Wed. Chem., 10, 1138 (1967).
(13) H. Paul and I f . Paul In “Experimental Chemotherapy.” Vol. 11. R . J. Schnitzerand F. Hawking, Ed., Academic Press, Yen P o r k , N. Y.,1964, Chapter 7. (14) K. hliura. and Ii. Reckendrof, I ’ i o g ~ . .Ired. Chem., 6, 320 (1967).
I:
II I1 I1
JIarch 1969
CHEMOTHERAPETTIC ~I~ITROTHTAZOLES
30.5
5-Nitrothiazole-2-carboxaldehyde(8).-A mixtiire of 22.0 g (0.093 mole) of powdered 15, 42.8 g (0.20 mole) of XaI04, 300 nil of ethylene glycol dimethyl et,her (glyme), 100 ml of I€:(), and 1.0 g of Os04 (added last) was stirred mechanically for 12 hr a t room temperature.32 The reaction mixture was poured into ca. 1 1. of Ha0 and extracted with CHC13 (three times). After drying (XaSOI) solvents were largely removed in z"xo and the very dark residue was filtered through glass wool. The collected solids were waphed with a little CHCla and the combined filtrate aiid aaFhings were distilled. Benzaldehyde was collected at 2.5-3.5' (1-3 mm), followed by the desired product (4.4g, 2 9 5 ) at, 80-90" (1.0 mm). I t crystallized iipoii cooling below room temperature and melted at about 20". This compound, although nearly colorless, causes persistent dark brown to black stains on Experimental Section 30 the skiii. A pure sample of the aldehyde wap collected by glpc (conditions, 5-Nitrothiazole-2-carbonitrile(4).-A stirred mixtiire of 30 g 91.4 X 9.63 cm column packed with 7% S E 30 on Gas-pak F, mole) of CiiCK in 1,50 nil of I l l I F at, 140" was treated (30-80 mesh, 147", 100 ml of T€e/miti); retention time 2.7 min. 0.0 g (0.144 mole) of 2-hromo-3-iiitrothiazole.21 After 1 or Anal. (C,H,N&S) C , I T , E. 2 mill, the ieniperatrire (Jft,he mixtiire rose sporitaiieorisly to 1%' Derivatives of 5-nitrothiazole-2-carboxaldehydeformed rapidly without, exteriial heating a i d was then maiiitained a t 150" by in aqrieoua EtOH from equimolar amounts of reagents in the healiiig. Exactly 10 niin after the bromo compound had been presence of a few drops of 3 S HCI. They precipitated sponadded, the mixture was cooled under the tap and poured slowly taneorisly except for the oxime which reqliired dilution with into SO0 ml of vigorously stirred Et2O. The yellow supernatant HzO: semicarbazone (IS), mp 260-270" dec, 93yo, yellow was decanted from the dark viscous oil that precipitated. The crystals (Anal. ( C j H S L k S ) C, H, N); oxime (19), mp 153-163" Et20 phase was extracted four times with H20 and dried (Xa2dec, 837& off-white crystals (Anal. (CIHaY30aS)C, H, ?S); SO,). Evaporation in z'acuo left 13.3 g (59%) of an orange, acethydrazone (20), mp 238-244" dec, 67$',, yellow crystals (Anal. readily crystalline product suitable for further reactions. A (C,H,K,O,S) C, H, N ) ; 3-(5-nitro-2-thiazolylmethylideneamino)3.1-g sample was recrystallized from aqueous MeOH to give 1.2 g 2-oxazolidinone (21), mp 220-225", 8OYc,,yellow cryqtals (;Inal. of ycllow crystals, mp 48.349.5". Anal. (CIHN302S) C, H, K. IC?HRN,O,S)C. H , N). Ethyl 5-Nitrothiazole-2-carboximidate (5).-A slightly warm, ' Ethyl ~2~Methyl:5-nitrothiazole-4-carboxylate (IO).-Ethyl 2filtered, stirred solution of 6.0 g (39 mmoles) of crude 4 methylthiazole-4-carbo~ylate~~ (1.0 g, 3.8 mmoles) was iiit,rated in 23 ml of absolnte EtOH was placed in an ice bath and was with N20,-BFa in CH3r\102 by esseiitially the method w e d by treated quickly with a solution of 8 mmoles of XaOEt in 4 ml of Parent for 2 - n i t r o t h i a ~ o l e . ~Examination ~ of the crnde oily absolute EtOH. The mixture, which instantly turned very product (0.76 g, isolated by Et20 extraction) by glpc showed it to dark, was seeded immediately and stirred for 3-4 min longer. consist of about 60% of 10,2 0 5 of starting material, and 20% of Dilution with 30 ml of 1120 gave a dark, crystalline suspension of 9;the latter compound was apparently formed from the product, product (4.5 g). Recrystallization (charcoal) from 75 ml of during work-up. Pure nitro ester was isolated by preparative 90% EtOH (diluted while warm with 150 ml of H20) gave 3.4 g glpc. I t was an oil that crystallized upon chilling in the refrigerAnal. (C6H7s303s) C, H , S. ( 4 4 5 ) of tan material, mp 87-88'. ator. The ir spectrum was consistent, with the assigned striicEthyl 5-Nitrothiazole-2-carboxylate(6).-Hydrolysis of 189 ture. ilnal. (C,H8K204S)C , H, N. mg (0.94 mmole) of 5 in dilute HC1 (immediate reaction a t 25') 2-Methyl-5-nitrothiazole (9) from Ethyl 2-Methyl-5-nitroprovided IS%mg of crude prodiict, mp 38-43', analytical sample thiazole-4-carboxylate (10).--A solution of 40 mg (0.18 mmole) mp 48-49' (hexane). A n d . (C6H6X20,S)C , H, S. of 10 in 0.3 ml of EtOH was heated overnight with 1 ml of 33% 2-( 5-Nitro-2-thiazolyI)-2-thiazoline (7).--A solution of 0.69 g aqueous HZSOn. The mixture was diluted with 2.5 ml of HZO (3.4 nimoles) of 5 and 0.39 g 13.4 mmoles) of 2-mercaptoethyland extracted with E t 2 0 . Removal of the Et& left 13 mg amine hydrochloride in 6 ml oi absoliite EtOH was refluxed for (56%) of crystalline product, mp 63-68", whose ir spectrum was .tallhe mass that formed was crushed in identicalwith that of 9,lit.23mp 70-72". situ, and ea. 10-15 ml of IT20 TTas added. After cooling in ice, 2,4-Di(p-chloro-P-styryl)-5-nitrothiazole (13).-A mixtiire of the dark brown, crnde prodrict (0.56 g) was filtered off and washed 2.0 g (12.7 mmoles) of 2,4-dimethyl-5-nitrothia~ole,~~ 7.15 g of (H2O). The crude material was extracted three times a t 90-100" p-chlorobenzaldehyde (51 mmoles), 20 ml of absolute Et,OH, and with a total of 50 ml of heptane. The combined extracts were 1 ml of piperidine was heated a t 75' for 1.5 hr. The product, concentrated oti ihe steam bath under a stream of N2 until the which crystallized from the dark solution during the reaction, was prodiict. began to precipitate. Chilling netted 266 mg (36%~)of filtered off and washed with EtOH and HiO; crnde weight 1.8 g. reddish, flaky crystals, mp 142-144.,j0, analytical sample mp I t was crystallized from MerCO to give 1.0 g of product melting at 14.5-146.5O (EtOH). ;inn(. iCsH,Ka02Pz)C, H, N. 221-227", analytical specimen mp 223-227". .-lnal. (CIQH,~CI,2-Methyl-5-nitrothiazole (9) and 2-methyl-4-nitrothiazole (16) 920,s) C . H, X. were prepared from 2-methylt,hiazole31 by the met,hod of A ~ a t o . * ~ 2-(~-Chloro-p-styryl)-4-methyl-5-nitrothiazole (12).-A mix2-(p-Styryl)-5-nitrothiazole(15).--A misttire of 28.8 g (0.20 ture of 0.40 g (2.5 mmole?) of 2,4-dimethy1-5-nitrothia~ole,~5 0.3.; mole) of 9, SO ml of benzaldehyde (ea. 0.8 mole), 20 ml of piperig (2.5 mmoles) of p-chlorobenzaldehyde, and 2 drops of piperidiiie dine acetate catalyst solntion, and 50 ml of absolute EtOH was was heated a t 140" for 2 hr. The dark reaction mixture was heated at 70-80O for 10 hr. [The catalyst solntion was prepared cooled and triturated with 3.5 ml of absolute EtOH. The resnltk)y mixing 5.7 nil (6.0 g, 0.10 mole) of -4cOH and 9.9 ml (8.5 g, ing crystalline suspension was chilled and filtered, and the crude 0.10 mole) of piperidine in 30 nil of absolute EtOI-I.] A few product (416 mg) was washed with EtOH. Tlc (silica gel, Etr(>) ci;ystals of previoiisly obtained prodiict were added a t the beginshowed that the bi5 condensation product (Rr 0.85) and the dening of the reaction. Cooling in ic.e caused precipitation of the sired monocoiidensation product (Ri0.7.5) were preseiit in aboiit dark kjrown, crystalline prodiict. Purification was effected by equal quantities. Sublimation (l5O0, 0.1 mm) provided 196 mg washirig thoroughly with Et0I-I and sublimation at 153-16O0 (28%;) of nearly pure 12 (by tlc). Recrystallized from .\Ie?CO (0.1 m m ) ; yield of yellow-ot,ange cryatals, 23.4 g (50%);mp 163and resublimed it melted a t mp 170-172". .Inn/. (CI2H',CI?;,166'; analj-t,ical sample mp 164-166' (EtOH). Anal. (CIIHSNY- 02s)C , H ; N : calcd, 9.99; found, 10.45. OLS) C, H, N. 4-Methyl-5-nitrothiazole (14) from 2-(p-Chloro-P-styryl)-4methyl-5-nitrothiazole (12).-A mixtiire of 0.28 g (1.0 mmole) of 12 in 10 ml of l\le2C0was stirred in an ire bath and treated over ca. 20 min with 0.42 g (2.67 mmole-) of KAInO4. After
spectrum, growth-inhibitory properties are present in the type of nitrothiazole examined in this study. All of the compounds except 11, 13, and 15 blocked the growth of all test organisms in the 100-1000-pg/ml range and several (19-21) were uniformly active in the 0.1-10-pg/nil range. Furazolidone analog 21 was 0.1-0.01 times as active 3s the nitrofuran parent. Xcethydrazone 20, thc most potent nitrothinzole in this te>t, K L ~ only slightly less active than furazolidone.
~~
(30) Melting points were taken in a RIel-Temp apparatus and are corrected. Microanalyses were performed hy RIiw n e t t ? 1IcCarthy of t h e Stanford Research Institrite .\nalyrical Department. \There analyses are i n d i r a t d only 1)s t h e symliols of the elements, analytical resiilrp oiitainmi f o r tliose rlements were nitliin ~ 0 . 4 7 of ~the rheoretiral values. (31) R . fIantIley, E. T. G. IIrrington, 11. , 4 z z n r o , and .I. lIctzger, Bull. Sor. Shim. l,',,tiiire, 1!101 (1!+63).
