Hycanthone,1 a New Active Metabolite of ... - ACS Publications

May 1, 2002 - Hycanthone,1 a New Active Metabolite of Lucanthone2. D. Rosi, G. Peruzzotti, E. W. Dennis, D. A. Berberian, H. Freele, B. F. Tullar, and...
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HYCANTHONE, 9 METABOLITE OF LUCAXTHONE

September 1967

TIBLEI BIOLOGICAL RESULTS

NO.

1

3 4 2 8 7

9 12 1 14

:z

17

s

n

H (lidocaiiie) CHrCH=CHa CH~CECH CHZSH CH,S-] 2 CH,SH CH,S-]2 OOC2H6 CHaOH C H?OOCHy CONH?

CPH;

CzH: CrHs CHI CHI CnH, CaH5

C?H5 C2Hi

C?Ha C~H,

Potency ratio a t 1 hr

1 1 3 0 G 0 0 1 0 .5 0.4

0

(1%) (45-60 min). Compound 13 was active only at the 1G/c level and the effect was of shorter duration (30-60 min). In the irritancy test, 1% solutions of 13 and lidocaine gave no significant effects when compared with saline. Compound 3, however, showed pronounced wheal formation at 1%, but was without effect at 0.5%. I n summary, therefore, of the ten compounds examined, four were without local anesthetic activity. Four

Time (inin) to -50'; anpsllieva at concn of 0 55; 0 25% 1%

68 >240 205

41 45

52 10.5 180 0 >240 0 0 0 22 0 0

27 >240 0 0 60 140 36 0

1GO

0 >240 0 0 0 0 0 0

Tissue damage

Slight Severe a t 1% Severe a t all levels Slight Severe a t all levels Slight Slight Slight Slight Slight Slight

compounds gave prolonged effects in the Bulbring and Wajda test; in two this was associated with irreversible tissue damage and in the nerve block test the duration of action of the other two did not differ markedly from that of lidocaine. Acknowledgment.-We thank A h . B. Basil for some of the pharmacological results and A h . P. W. Tipton and A h . AI. de Bruin for skillful technical assistance.

Hycanthone,' a New Active Metabolite of Lucanthone2 D. ROSI,G. PERUZZOTTI, E. W. DEXXIS, D. A. BERBERIAS, H. FREELE, B. F. TULLAR, AKD S. ARCHER Sterling-Vinthrop Xesearch Inslitute, Rensselaer, L\7ew York

Received F e b m a i y 2, 1967

* *

lliciobiological ovidatioii of lucaiithoiie2 fiiriiished hycaiithone,' the 4-hydroaymethyl analog ab the maiii product. Hycaiithoiie is a highly active schistosomicidal ageiit wheii given orally or intraperitoiieally. It has been identified chromatographically i n the urines of man, monkey, and mouse followiiig medication with 1r:ca.nthone. I t s chemical, physical, and biological properties suggebt t h a t i t is the active metabolite of lucarithone.

hlmost thirty years ago AIauss3 synthesized a series of xanthenones, some of which were shown by Kiliuth and Gonnert4 to have schistosomicidal activity \Then administered orally to mice infected with a Liberian strain of Schistosonaa naansoni. This was a signal chemotherapeutic achievement because this was the first orally effective nonmetallic organic compound found to possess such biological activity. The most interesting members from the point of view of structure-activity relationships were miracil A (I), lucanthone (11),and the 6-chloro analog (111). On the basis of evaluation in mice it mas estabished that the thioxantheri-9-one I1 was more effective than

0

NHCHzCH2N(C2H5),

0

NHCH2CH2N(C2HS),

O q

CH3

I

0

CH, I1

NHCH2CH2N(C2HJ2

c1

CH,

III

( 1 ) F o r a preliminary communication see D. Rosi, G. Peruzzotti, E. IT-. Dennis, D. A . Berberian, H . Freele. a n d S.Archer, S a t u r e , 208, 1005 (1966). Hycanthone is t h e generic name for 1-[2-(diethylamino)ethylamin0]-4hydroxymethyl tiiioxanthen-9-one. (2) Lucanthone is t h e generic name for miracil D. ( 3 ) H. Maims, Chem. Ber.. 81, 19 (1948). hlthough t h e chemistry was reported a f t e r IVorld K a r 11, i t is well k n o x n t h a t t h e original v o r k was carried o u t in t h e late 1930's. ( 4 ) \V. Iiikuth and R. Gbnnert, Ann. T r o p . M e d . Parasitol., 42, 256 (ltc4U).

0 /I

NHCHzCH2N(CZHJz

I

CH3

Iv

the oxygeri isosterc I aiid that certain substituents ( e . g . , chlorine) in posit’ioii ( j of the thioxanthene nu(~1e~is iiicreased activity. I t was found arid later corifirnied by others; that the basic side chain (::in he varied ovcr ii limited range with little loss iu biological activity. However: replacement, of the 4-methyl group by ot’her substitueiits abolished t,he schistosoniioidal Iieduction of t’he urisubstituted carbocyclic8 ring of I1 furnished 11‘ which was an active schistosomiridal I;urther siniplicatioii of this ring systeni \vas arhicvcd when it was disvoveretl that t,he bicyclic, t~hiochromoncT’ was active.’ .\lnuss, et d.:* found that the iiioiiocyclic toluidine \?I linon-11 as mirasan was highly effective in mice. The unalogous piperazine T’II was also report’ed t o be :t ( *t,i vc .

V C1

VI1

The structural feature necessary for biological actioii (miinioii to all these classes of compounds is a dialkylamirioalkylamino group para to a niethyl group o n a11 aromatic ring. This conclusion is based on the schist’osoniicidal activity in mice. The results in 5’. ” m s o n i infected monkeys are quite different. For example, 111 whicsh is very effective in mire is essentially devoid of activity in t’hep r i n ~ a t , e .lIirasan ~ T’I is ineffective in nioiikeys arid hunians.l0 However, lucanthone, although oiily riioderately effective i n micej4 is highly effcrtive in the n1011lcey~~~~ and has enjoyed some csliriic*nlsuccesslI at doses which suggest that’ in mail the d i ~ i gis somewhat less active thsii in the nionliey. Uiifort,uiiately the poor human t,oleraiice of lucanthoiie militate;; against it8s being the drug of rhoice in the treatmentJ of S. haeulmtobiuiii and S . imnsoni infectioris.ll-’? Biotahemical aiid metabolic studies oil lucarit’horie ~veresooii initiated, owing to the vliiiiczll interest in the drug a i i d t,o t’he suspicioii. erigeiidered in part’ b y t’hc t . i l d. \ r v l i v r ani1 t‘. \ I . SiitPr. . I . .Im. C h e m . ,Soi- of tlic ~ i i o - t nbuudaiit iiicnihcr X (Chart I) of t h i s iiiixturc> 111dicated that i t r i oxygen atom n - a b iiitroducccl i i i t o t l r c x molecule. ‘l’he alcoholic nature of this iiew f u i i d i o i i mas demoiistrnted by the easy preparation of the mrresponding acetute XI. The nnir spectra of 11, X (Figure 1). and XI clearly showed that the l-nieth~.] signal in the spectrum of lucm~thonen-ah replaced 1)y a signal for n methylene group next to oxygcn. 111th(1 (35

869

September 1967

0 I1

NHCH,CH,N( C,HI),

4

~ H ~ O H

I

XVIII

0

NHCH,CH,N(C,H,)I

;..a 0.5 N H,PO,

NaBH,

I

CHO

0 II

NHCH2CH2N(C2H5),

I

x IV

X

XI1

0 CH,-

CH,OH

OH NHCH,CH,N( CzH,),

I

I

I

CHIOH XI11

>;VI

rimr spectrum of the acetate XI the signal at' 309 cps was assigned to the methylene protons in the 4 position and the 123-cps singlet was assigned to the methyl of the acetate radical. A chromatographically less polar companion of X was the aldehyde XII. The aldehydic nature of this conipoutid was demonstrated by the elemental analysis, the presence of a carbonyl band in the infrared spectrum, arid the iimr spectrum which showed a signal at 590 cps, rharacteristic of an aldehyde proton. Sodium bdrohydride reduction furnished the alcohol XI6 which was reoxidized to the aldehyde with ITnOz. The most polar microbial metabolit'e was the readily identified acid X I I I . There was no indication that microbiological oxidation orcurred on the sulfur atom of lucanthotie. The sulfoxide XVII was obtained by H2OX oxidation of x. In the course of isolating the alcohol X from fernientation broths it was noted that the substance was quite sensitive to acid. Brief exposure of niethylerie chloride solution of X to the action of 1 N phosphoric acid served to convert the metabolite to a new high-melt.ing substance. The elemental analysis and uv spectrum indicated little change in composition but there were no hydroxyl bands present in the spectrum of the new substance which was assigned structure XIV. Further exposure to acid of either X or XIV resulted in the formation of a water-soluble material showing a uv spectrum resembling that of the starting material. (16) This conversion proved to be a convenient preparative method for labelinn X. T h e siitistitiition of sodium borutritide in the rediiction afforded t l i e tritiated nlcoiiol containing n tritium a t o m on the carbon in the liydroxvii~etliylgroup.

CH,-

XV

Sincbe the new substance could not be removed from aqueous s'olution with an acidic Dowex ion-exchange column, it was felt that we were dealing with a polymeric quaternary ammonium salt as shown in strurture XV. Reduction of X with sodium amalgam gave the thioxanthen-9-01 XS'I. Biologically the most outstanding property of X was its high oral schistosomicidal activity in mire and hamsters. In the former spevies X wa5 about three to four times as active as 11. In contrast to lucanthone the new hydroxymethyl compound was active when administered intraperitoneally. This new metabolite of lucanthone has been given the generic name hycanthone. In view of the biological acativity of hycanthone considerable effort was expended 011 a study of the fermentation procedure in an attempt to augment the yield of the over-all conversion of lucanthone to hycanthone, to incarease the concbentration of substrate, and to simplify the isolation procedure. It was found that careful control of the sterility and composition of the medium, pH of the fermentation, and rate of substrate addition served to inwease the over-all conrw~sionof I1 to X and increase the efficiency of the process. One of the factors which limited high initial concentrations of the substrate was its inherent fungicidal activity. At levels of 1.5 g/l., growth inhibition of A . sclerotioruin occurred. Fortunately, hycanthone was much less potent in this respect and efficient conversions of substrate were achieved by the gradual addition of I1 to the fermentation medium a t such a rate that sublethal coriceritratioiis were niaititained a t all times.

I

.-N H

378

275

228 H

65

>

September 1967

HYCAXTHONE, A ~\IETABOLITE OF LUCANTHOKE

871

depended on its metabolic conversion to XYIII then complex urinary excretion pattern of metabolites, we the latter ought to be readily detected in the urine of decaided to reinvestigate this species first. Accordingly, mice and to be absent in the urine of medicated moIikeys were medicated with lucarithorie and the monkeys. urines were collected and carefully extracted in such a The hydroxyniethyl derivative XT’III was obtained manner t h a t exposure to the action of acidic media was by incubation of III with .4.scleyotiorum and served as avoided. Examination of t h e extract by nieans of thin the reference substance. I t s structure was confirmed layer chroniatography revealed the usual multipliclty by nmr arid uv spectroscopy. As in the case of hyof spots, the major one corresponded in Rf to the sulfoxcanthone, the 6-rhloro analog 111 was administered to itle T’III. There was a farrit spot corresponding to monkeys and mice, arid the urines were examined for hycanthone. However, when the urines were first the presence of the hydroxyiiiethyl metabolite by means incubated with the enzyme glururonidaie and the of thin layer chromatography arid uv spectroscopy. extraction procedure repeated, the spot corresponding X V I I I was readily detected in the mouse urine but to hycanthone increased markedly in intensity. h p could not be found in the urinary extracts of the pareritly the nelv metabolite was excreted as a glucuronmonkey. ide, a not unexpected finding if indeed lucanthone was tieing transformed to hycarithoiie by the monkey. 0 NHCHZCHZN(CZH5)i The ultraviolet spectrum of the new nietabolite wab the II I same a\ that of hycanthone and cochromatography with an authentic sample of X in several solvent systems c1 indicated that the two were identical. Lucanthone is generally thought to be niore effective ~H,OH in the monkey than in the mouse. I t s action in man XVIIl probably ocrupies an intermediate position between 0 NHCHZCHZNHC2HS 0 NHCH2CH2NHC2Hj these species. Although the intraspecies coniparisons are difficult to make, owing to differences of degree of infection and to differences in criteria for cure. nevertheless, a reaqonable estimate of species potency can be made. I n this laboratory the ED50( P O ) of lucanthone CH3 CH,OH in mice is regarded to be 40-50 mg/kg/day given for XIX xx 3 consecutive days. In the nionliey doses of 10 mg/kg/ day for 3 days can induce radical cures. The human The greater effectiveness of I11 in mice as compared dose is about 2 5 mg/kg/day for 3 or 4 days.g This to lucanthorie could be due to either of these factors: regimen effects cures in greater than .iOYGof the pa(a) the drug is metabolized to the hydroxyniethyl tients receiving medicaation. analog more efficiently then lucanthorie in the mouse, or We have compared the urinary excretion patterns of (b) X\XI is intrinsically niore active than hycanthone. these three species after medication with lucanthone. The last alternative was found to be incorrert by In contrast to Strufe n-e could not detect the sulfone comparison of the EDSO’sof the tn-o drugs (Table I ) . IX9 in the urine of mice. (Heterocyclic sulfides on I n our view the experimental observations discussed metabolic oxidation generally furnished the correspondabove support the hypothesis that lucanthone and some ing sulfoxide but exceptions are k n o N ” ) Hycanthone of its congeners are indeed being converted t o an active could be detected only in trace amounts even after metabolite which is the corresponding 4-hydroxymethyl glucuronidase treatment. The finding is in keeping analog. On the basis of an analysis of structurewith the relatively poor activity of lucanthone in this activity relationships Gonnert’o concluded that in the species. lucanthone and related series a methyl group p a ~ ato A volunteer took lucanthone arid his 24-hr urine the basic side chain is essential for activity. The sample was examined for hycanthone. Our procedure biological basis for this conclusion has now been did not permit us to detect Strufe’s “chromopeptide,” established. although the sulfoxide IX appeared to be present. During the course of this study of lucanthone Visual inspecxtion of the thin layer chromatography of metabolism in the monkey we attempted to identify the urine extracts after glucuronidase treatment insome of the other urinary excretion products. Owing dicated that the amount of hycarithone present was to the coniplexitp of the nietabolism of this drug in the greater than that noted in the mouse but less than that monkey20 we confined our attention to those compounds excreted by the monkey. Thus, the urinary excretion which were, first, fairly mobile in a thin layer chromaof hycanthone in these species is roughly parallel to the tography system in which the developing solution was potency of lucanthoiie in the same species. These ether-methanol-triethylamine (S : 1 : 1); second, soluble preliminary findings, which need confirmation b!, more in dilute niineral acid, indicating that the aliphatic quantitative work, are compatible with the hypothesis amine residue was still present; arid third, spectrothat lueanthone is metabolically converted to an active scopically unchanged by the presence of alltali, an metabolite. indication that no ring hydroxylation had owurred. Kiltuth and Goniiert4had found that in the niouie GI n one nionhey, fed a total of 150 mg of drug, oiily 57c c+hloro-1-[~-(dreth.irlaiiiiiio)eth~l]-~-niethylthioxaritliei~could be ac.caounted for as lucarithone or it5 truiisforma%one (111) was niore ac*tive than luraiithone but intion products. Of this about GOYGu a s directly exactive in the monkey. If the biological activity of 111

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(19) 11. T . \!‘illiarns, “l3etoxication Aleciianisrns.” 2nd ed, J o h n \Vile>, end Suns, Inc., K e w 1-urk, N. Y., 1Y59, p 863 ff.

(20) Visual inspection of a carefully prepared tliin layer hand cliroinatopram revealed at least twelve distinct and moliile hands u i t l i a great deal

of material still present a t the origin.

Vol. 10

ROSI,PERUZZOTI, DENNIS,BERBERIAN, FREELE,TULLAR, AND ARCHER

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HYCANTHONE, A 1 I E T A B O L I T E OF LUCANTHOKE

September 1967

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veredfrom the iiriiie. Of this aborit two-fifths was excreted :is gliic,iiroiiic acid cotijiigates. 111 the secwiid moiikey only aboiit : ! . 5 c xtrwtioii with vxtrarts %-ere exainirted by tlc iibiitg fiyhteiii A . 111 c:ic-lt viiw trace amoiitits of the origirial drug (Rf 0.70) was otihei,ved h i i t the c~~rrespotiding hydroxymethyl i~oinp~~iiiid STTI1 (I:, 0.30) c o i d d iiot, be detected. B. Mouse.~--l) n e hutidred niice were niediwted wit,h I1 I (500 mg/kg) iti the same matiiier described above for Iiiratithotie. Examination of the ext,racts by rlc revealed a cwmpotieiit whohe chromatogr:tphir arid spectroscopic properties were t.he s a t i i ~iiy t h a t of the aiitheritic hydroxymethyl compoitnd X\-III.