September 1967
ASTITRICHOMOKAL SITROIMII)AZOLER
891
Nitroimidazole Derivatives. Relationship between Structure and Antitrichomonal Activity
K. BUTLER,H. I,. HOWES,J. E. LYXCH,AND D. K. PIRIE Chas. Pfiter .Tfedical Research Laboratories, Groton, Connecticut Rerewed Januayy 1 1 , 1987 Rrzvsed Alfnniiwr?pfR e c m e d .llay 12, 1967
The i n Liivo trichomonacidal properties of some nitroimidazoles have been determined and are reported. Biological activity is associated with the 1-alkyl-3-nitroimidazole riricleiis and is strongly iiiflrieiiced by the partitioii coefficient. Steric effects and possible metabolic effects are considered. Follr iiew nitroimidazoles are descrihed which are at least five times as potent as metronidazole.
TABLE I Trichomoniasis is a parasitic infection of the genitoA , mfi ( 4urinary tract caused by Trichomonas vaginalis. Treat0.01 .V HCI 0.01 N N a O H Compd ment of clinical trichonloniasis was revolutionized by1 296 13060) 358-360 (5500) the discovery in 1959 of the antitrichomonal activity 2 278-280 (5800) 311 (9200) of certain nitroimidazole derivatives.' Since that time, 3 303 (6700) 303 (6700) metronidazole (1-P-hydroxyethyl-2-methyl-5-nitroim-1-pCyanoethyl- 3-ni troimidazole) has gained wide acceptance for the systemic idazoles 3d5 (6400) 309-211 (8820) treatment of trichomoniasis.3 We undertook a study of a series of nitroimidazole derivatives in order to Evaluation of the aiititrichomonal properties of determine the structural features which control bionitroimidazoles mas carried out ill mice inoculated logical activity, our ultimate goal beiug the design of a intraperitoneally with 500,000 viable Trichomonas superior antitrichomonal compound. foetus organisms. Commencing 24 hr after inoculation Imidazole derivatives are readily prepared according the infected animals were treated with three daily oral t o the llaquenne p r ~ c e d u r e . ~Kitration of imidazoles doses of the test compound. The delay in treatment is yields the 4- ( 5 )nitroimidazole~,~ which can be alto facilitate the establishment of the infecting organism kylated to produce either the l-alkyl-4-nitro or l-alkylwhich in untreated mice produces a significant and %nitro derivatives depending upon the conditions emconsistent pathological pattern. Infected mice develop ployed. Alkylation of a nitroimidazole in the presence a marked swelling of the abdomen within 3 days, and at of a base yields a 1-alkyl-4-nitro derivative (3))whereas autopsy, usually 24 hr after the final drug treatment, the alkylation under neutral or acidic conditions generally abdominal cavity of infected control animals is filled gives a 1-alkyl-5-nitroimidazole (2) as the major prodwith a viscous white fluid containing numerous trichouct. Substituents at C-2 of the 4- (5-) nitroimidazole monads and leukocytes. Activity of test compounds (1) exert a steric influence on the direction of K-alkylais based on their ability to prevent the described pation. When R 2is large (e.g., t-butyl), alkylation occurs thology and eliminate the parasite. The relative potency at the least hindered position so that even under neutral of a series of compounds after assessing the minimum or acidic conditions appreciable quantities of the 1effective dose (JIED) for each candidate is based on alkyl-4-nitroimidazole (3) derivative is produced. A results from several trials. The JIED is defined as the series of nitroimidazoles obtained by these methods has lowest oral dose of the test compound which clears lOOY, been described in the recent literature.6 The imof the infected mice when administered daily on 3 conidazoles 1-3 can be easily distinguished by their charsecutive days. Tables 11-V include physical, chemiacteristic ultraviolet absorption spectra shown in cal, and biological data from studies with various nitroTable 1. imidazole derivatives. Antitrichomonal activity was observed for those compounds listed in Tables I11 and IV; other nitroimidazole derivatives were devoid of in vivo antitrichomonal activity even at high dosage. Simple 1-alkyl-Z-nitroimidazoles are weak bases (conjugate acid, pK = 2.13).' Protonation occurs only in strong acid solutions so that at physiological p H these compounds will exist in the uncharged form. When R', R2,and R3 ( 5 ) are varied through a range for simple alkyl substituents, we would expect little modification of the pK of the nitroimidazole nucleus, and this is supported by the close similarity of the ultraviolet absorption spectra in acidic solutions. The (1) F l a g s l a . ( 2 ) C. Cosar, L. Julou, and M. Bonacet, A n n . Inst. Pasteur, 96, 238 1-p-hydroxyethyl-5-nitroimidazoles have the same ab(1959). sorption spectra as the simple 1-alkyl derivatives, al(3) H. Beckman, Yearbook of Drug Therapy 1963-1964. Year Book Publishers, Inc., Chicago, Ill., 1964, p 383. though it is possible t o differentiate these compounds ( 4 ) Rlaquenne, Ann. Chim. (Paris), [a] 24, 525 (1891); H . J. L u c h and in 2 M H2S04 (metronidazole conjugate acid, pK = E . R. Kennedy. Org. &in., 22, 6 5 (1942). 7
( 5 ) R. G. Fargher and F. L. Pyman, J . Chem. Sac., 115, 217 (1919). (6) C . Conar. C. Crisan, R. Horcloia, R. M. Jacob, .J. Robert, 8. Tehelitcheff, and R . Vauprl, Arzneiniiltel-6'orsch ., 1 6 , 23 (1966).
( 7 ) G. G. Gallo, C. R. Pasqualucci, P. Radaelli, and G . C. Lancini, J . O r g . Chem., 29, 862 (1964).
RJ
MP,
I1 I1 I1 If
oc 37.31 xi, 10
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I1 I€ TT IT
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CIT, CIIj
183-1s;
2.3.5) .7 The 1-p-cyanoethyl-3-nitroiniidazoles seem to 1 ) slightly ~ less basic than the simple I-alkyl derivntives, and they are not completely protonated in 0.01 N HC1. This is apparent from observations of the ultraviolet absorption spectra. 4- (3-) Sitroimidazoles (1)are considerably more acidic than the corresponding I-alkyl derivatives and form yellow sodium salts in aqueous alltali. This difference in pK may account for the lack of biological activity of the 4- ( 3 - ) nitroimidazoles (1j , but the various 1-alkly-3-nitromidazoles considered in this article fall within %: narrow range of pk' Yuch that we n-ould not be able to differentiate between them or correlate their biological properties with their pK values.
"0 .;. 1
2 7 . 1.7 27.28 24, .5(i 24.61 24 ,90 3 2 . !Is
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.
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-1plot of the rn z Z L O activity of 2 (R? = H ; R1 = lo\\ VIalkyl) presented as log (l/AIEI)) against log K ( i v i octane-ayueoui buffer pH 7.0) is h w n i i i l'igure 1 Similar plots for 2, 12* = CH3 arid Ill = CH(CH,)?,arc' .;how11in I'igurea 2 and 3. The ihapes of the curve\ i i i thehe case, are similar, passing through a peak of optimum activit) at a certain i m g e of K , followed ti) :i plateau of diniinizhed activit) and eventual lo*s of :dl biological poteriq when I200 >ZOO >ZOO >200
too lipophilic to have really good antitrichomoiial activity. All of these observations can be conveniently summarized by superimposing Figure 1-3 upon Figure -1, as shown in Figure 5 . The most obvious feature of this presentation is that l-methyl-2-isopropy1-5-nitroimidazole has the greatest biological potency and its partition coefficient is close t o 1.0, nhich suggests that this is the ideal partition coefficient for good biological activity in this particular system. If we consider the series where R 2 = methyl we observe that as K increases there is an increase in biological activity followed by a depression (when R1 = E t , Pr, Bu, and pentyl) and finally all activity is lost when R 1= octyl. The partition coefficient of l-propyl-2-methyl-5-nitroimidazole is very close ( K = 1.05) to the value we consider necessary for optimum in vzvo antitrichomorial activity ( K = 1.0, as for l-methyl-2-isoproppl-5-nitroimidazole). Nevert heless, 1-n-propyl-'-met h y l - h i troimidazole fails to shox good antitrichomonal activity R * i-C3H7 because the 1-propyl substituent imposes a severe steric effect which drastically reduces biological potency. If n . we reduce the size of R1but keep K close to 1.0, we would s expect an increase in biological potency and this is ob> -0.05 served for 1-ethyl-2-ethyl-j-nitroimidazole. Further reduction of the size of R1results in still better antitrichomonal activity as observed for l-methyl-2-nprop!-1-5-nitroimidazole. In the case of these three , , . p 5 , , ( ,0,l 02 1 , , p5 , , , 2.0 , , 5.0 compounds K was essentially constant and we changed Log K Ii-octane ) only the size of R1, and the increase in potency is thus Figure 4. attributed to the reduction in size of this group. The final point on the vertical line AX is 1-methyl-'->-is+ activity is associated with a branched side-chain a t propyl-5-nitroimidazole. and for this substaiice the C-2 and a K value of about 1.0, but for both the improved potency must be due to modification of straight-chain and the branched-chain homologs the metabolism or fit of the drug. X similar set of complots resemble bell-shaped, normal distribution curves. pounds n ould be l-butyl-%meth> 1-, l-propyl-2-ethyl-, From such data we conclude that partition coefficient l-ethyl-2-isopropyl-, and l-methyl-2-isobuty1-3-1iitrois of prime importance for good biological potency, but imidazole, but all of these compounds are too lipophilic that the size of R' must be kept small. The pronounced ( K = ea. 3.0) to have really good arititrichomonal difference between straight-chain and branched-chain properties. It is clear that lengthening of R' in most alkyl substituents a t C-2 is probably associated with cases introduced severe steric effects long before we were drug metabolism. I t is known that 1,2-dimethyl-5able to obtain the ideal K value for optimum biological nitroimidazole is metabolized by oxidation a t the C-2 activity (all compounds t o the left of the vertical line substituent to produce an alcohol, which is further AX). Compounds to the right of this line are too oxidized to the carboxylic acid.* Substitution at C-2 lipophilic and/or too sterically hindered to be good will modify this metabolic process and change the drug trichomonacides. half-life, thus l-methyl-2-isopropyl-5-nitroimidazole The line BX liiiks a series of derivatives with equal should have a longer half-life than the 2-n-propyl steric effects a t Rl, and potency iricreases with inanalog and this, in part, might account for the superior creased values of K . parallel line liiik. the I-ethyl in vivo activity of the %isopropyl-;i-nitroimidazole derivatives. derivatives. A similar relationship is observed for the The major purpo were obt ;iiiietl f r o i r i c~onimercialsour('es, or were synthesized according to priblishetl 111 mother series, the riitroimidnzoles (2, 1%' = procedures: P , ~3-methylimidazole,g 2-et hylimidazole,' @-cy:irioethyl)proved to be too iiisoluble iii iwoctane t o +methylinlid -rz-propylimidazole, ~-iisoprop2-lirnid:tzol(,.' ptunit riccurate meahureineiit:, of K i i i this holveiit. ",i-dime~hylimidazole,'L L'-ethyl-l-meth?-Iimitianole,'? 2-rA)1lt>-lI k t i t i o i i coefficieiits for thew coinpouiid> arc reported iiiiidazole,4 L'-isoliiit~lirnidilzl,le.' :iiitf L'-l-l)u1ylirnidazole. l o 1 I-oct:iriol- aqueous buffer pH 7.0, arid the relation4- ( 5 - ) Nitroimidazo1es.--Sitroinii(lazoles were prepareti l'i.oiiL the parelit imidazole b>- n i t r a i i o i i xvcordiiig to published pi'(i*liip bet\\ ecii Cl15,1(curative dose i b that level which ced~irea.j~'3~'~ Data for v:ii.itru:, nitroimidaaoles :ire showii i t i afforcls pvtectioii to 307, of the treated mice) mid I< Table 11. (1-1~ct:uioI)is shonri in Figurc 6. It was iiot possible 1-Methyl-5-nitroimidazoles were prepared from the :LIP t o differentiate bet\\ eeii the 2-niethj 1, %-ethyl, arid 2propriate 4- ( 3 - ) iiitroimidnzole arid dimet hgl sulfate usiiig the procedure of P y n ~ a i i . ~ ~ Other , ' ~ l-slkg1-3-iiitroiInidazoles wew isopropj 1 arialogs oii the basis of IIED (Table 111) but :I. plot of the CDjo of these compounds against K gave :i -ymmetricaI bell-shaped curv(', with the %ethyl derivatives at the apes. For comparison, a plot of C'Dbo,'K (octanol) for wine l-methyl-2-alkyl-.i-nitroiniidrtzoleb is iiicluded in l'igure 6. We observetl f o r tho 1-/?-e> ~t1i11eth~I-2-:t11~~1-5-tiitroiiiiidaxc~le~, that :t ~~i~:~ticlic~d-cliuiii dl;) I \ub*titueiit at C-2 afford-
Scpteniber 1907
ti97
ANTHELMINTIC rrHIACYANINES
prepared from the 4- (5-) 1iitroimidaAole and the appropriate alkyl p-tol~enesulfonate.1~~1~ General Procedure.-2-Isobutyl-4- ( 5 )nitroimidazole (2.9 g, 0.017 mole) and the 8-cyano-p-toluenesulfonate (7.72 g, 0.034 mole) were combined and heated to 135" for 12 hr. The cooled mixture was extracted with water, and the aqueous extract was washed once with a small volume of CHC13 which was discarded. Addition of alkali to p H 9.0 gave a precipitate of the l-~-cyanoethyl-2-isobutyl-5-nitroimidazole which was extracted
out with CHC13, washed once with water, dried (Na2S04),arid evaporated to yield 1.65 g (43%) of the desired compound which was then recrystallized as the p-toluenesulfonate salt (mp 21 1") from 2-propanol. 1-Alkyl-4-nitroimidazoles were obtained by essentially the same procedure described by Cosar, et aZ.* l-~-Hydroxyethyl-2-methyl-4-nitroimidazole (68) is best obtained by heating an ethanolic solution of 2-methyl-4- (5) nitroimidazole with an excess (2 M ) of ethylene oxide in the presence of a catalytic amount of NaOH. The desired product is obtained in quantitative yield by evaporation of the solvent, followed by recrystallization from ethyl acetate.
(17) R . S. Tipson, 11. .i. Clapp, and L. H. Cretcher, J . Org. Chem., 12, 133 (1947). (18) S. Tclielitcheff. U . S. Patent 3,065,133 (1562); R. M. Jacob, G . L. Regnier, and C . Cristan, U . S. Patent 2,944,061 (1960): Societe des Usines Chimique Rhone-Poulenc, British Patent 837,838 (1960).
Anthelmintic Quaternary Salts.
Acknowledgment.-We
are greatly indebted to Dr.
R. L. Wagner and his group for the microanalyses.
Thiacyanines and Hemithiacy anines
11. L. GARMAISE, C. H . CHAMBERS, $J. KOMLOSSY, Abbott Laboratories Lim;ted, Montreal, P . Q., Canada AND
R. C . RICCRAE
Department of Parasitology, Abbott Laboratories, Sorth Chicago, Illinois Received A p r i l 10, 1967 Comparison of the activities of analogs of dithiazinine (111) against gsstrointest,inal nemzto-lea of sheep showed that the pentamethine chain is essential. Activity may be retzined when the 6 position is sitbstitritel with alkyl or alkoxy groups, but substitution with electron-withdrawing groups produces inactjive conpoitii.ls. ilnalogs with activity comparable to dithiazinine tended to be more toxic to mice than dithiazina itsdf. Two members of a series of hemithiacyanines containing the 4-dimethylamino-1,3-butadienyl grorir, w-re very active iii inhibiting Ascaris s u u m larval migration in mice and in swine and were more effective than dithitzinine in preventing liver pathology due to migratory ascariasis.
Since the initial discovery of anthelmintic activity in cyanine dyes,' a number of compounds containing the conjugated amidinium ion characteristic of the cyanines have found use as both veterinary and clinical anthelmintics. Dithiazinine (111), the most potent member of this class of anthelnlintics, is effective against a wide range of gastrointestinal tie mat ode^,^^^ but its use is severely limited by its toxicity. Because of its gastrointestinal side effects, the clinical application of dithiazinine is now restricted to cases of strongyloidiasis and severe trichuriasis. The structure-activity-toxicity relationships of some structural analogs of dithiazinirie were examined with the purpose of discovering compounds with comparable anthelmintic potency but with reduced mammalian toxicity. The structural features which were varied included the length of the polymethine chain, the replacement of the 3-methyl group by ethyl, and the introduction of substituents on the G position of the benzothiazole ring. In addition, a group of compounds in which one of the benzothiazole residues was replaced by a dimethylamino group (hemithiacyanines) was also prepared. The most convenient method for the preparation of the thiadicarbocyanines (pentamethinethittcyanines) was the treatment of 2-methyl-3-alkylbenzothiazolium iodides with 1-methyl-1,2-dihydro-2-iminopyrimidine hydroiodide4in the presence of triethylamine. ( 1 ) L. G. S. Brooker and L. .\. Sweet, Science, 106, 496 (1947). (2) A. C. Cuckler and I
