Organophosphorus Compounds as Schistosotnicicles
\
/
January 1907
OI~GANOPHOSPHOECS SCHI~TOSOMICIDMS
33
TABLE I THERIPEUTIC EFFECI.S OF ORG.INOPHOSPHORUS COMPOVSDS .LG.~INST S. itiunsoni IS 111~s
______
]lose, NO.
Ia
ma:hr/
No. of
day
days
129
14 14 14 14 10 10 14 14 10 10 14 14 14 14 14 14 14 14 14 14 14 14 14 10 9 14
No. of lie~lnlen
mice
0 125yc diet 0 0GC; diet
9 11 15
Treated------% of vorms dead
100
------Con Mean no. of live xorins
trois----M e a n no. norms of live dead worms
L/o of
s o . of mice
0 0.4 9.6
16.1 15.8 1s 37 18.8 10 3 s.3 ;ti 24.9 c 20 i Gavage X 2 45 9.4 24.9 20 b 13 13.8 10.9 Ip x 1 100 72 3 0 0 06yCdiet 19.5 Ib ” 100 6 i 0 17 0 016cc diet 12.i 20 7 i t 3.7 i 12.7 Gavage X 2 7 20 79 2 .5 10 10.9 Ip x 1 ti 29 8.4 14.9 ‘184 0 25c; diet 13 Iu 70 ) 12 9.0 11 13.8 0 06c; diet !)S 72 0 0 ,2 13.0 13 0 06rL diet Id 1 16.0 ‘10 56 6.6 10 0 016cc diet h72 (i 11.2 0 11.2 7 9 0 So/; diet I1 13.9 ci 11 0 13.4 287 0 2Fc diet 1 3 n 8.5 8 0 11.8 71 0 059, diet r I 10 89 0.8 0 15.6 63 0 06wc diet Tb 111 3 2.0 1 18.3 10 60 57 0 125% diet T 6 2.3 16.1 14 58 54 0 0 06‘; diet T 8 2.1 10 0 11.3 18 69 0 016c> diet 17.5 7 6.4 10 0 9 0 0 0 S 5 diet 18 8 0 17.5 10 0 4 11.7 0 0 0 4 5 diet in 10.5 9 0 18.8 12 30 Gavage X 2 8.7 X 0 14.0 11 9 6 Ip x 1 5.0 1 0 74 7 28.6 tis 0 0ciCl,diet T 11’ 2 12 i 15.3 0 16.4 30 1 Gavage X 2 S5 ti 0.9 11 1 338 15.0 0 2 5 5 diet 14 Y 10 1 400 4.6 10 1s.1 76 Gavage X 2 7 2 14.7 8 10 1 100 18.1 5 sc x 2 4 0.2 13.0 205 98 13 0 0 25% diet 1-1 14 13 5 6 13.3 0 13.0 74 0 06% diet 14 4 8.3 48 11 0 13.8 324 0 25% diet VI1 14 93 0.8 9 0 9 12.8 554 IX 0 5% diet 14 30 18 7.0 2 17 212 14.8 0 1257, diet 14 95 10 3 0.6 0 800 11.1 4 Gavage X 2 T S 89 1.6 10 1 400 1s.1 J Gavage X 2 0 10 200 9 11 9.4 Gavage X 2 15.8 10 10 10.5 10 200 13 0 15,s Gavage X 2 .5 200 9 1 9.9 10 0 11.1 6 sc x 2 276 5 99 0.1 1 s 13 15.2 0 2 5 c L diet 14 7.6 5 15 200 3 2 Gavage X 2 12.7 5 6 95 0.6 10 SI1 82 0 16.0 14 0 06% diet 2 10.3 0 22 3 10 16,O 0 OIGCc diet 14 When the compuiiiid is giveii iii the diet, it is mixed with the food and comprbea the stated percentage of the mouse daily diet. When the compoiind is administered by gavage or parenterally, the stated daily dose i. given either all a t once ( x 1 ) or is divided in half and administered twice daily ( x 2). b T, toxic (caused mortality or weight loss). S .j
ing treatment. Two riiorikeys given 20 mg/kg/day showed a suppression of egg excretion but were not ( w e d ; one of these lost weight (167,) arid had diarrhea but the other did not. One monkey given 10 nig/lig/ (lay showed only a transient suppression of egg excretion :md another given 3 nig/kg/day showed no evidence of therapeutic effect; both of these monkeys tolerated their doses well. With the exception of tris(p-ethylphenyl) phosphate (IT’),” which showed slight activity at toxic doses in mice, the only other active structural type was 4-tbutyl-2-chlorophenyl methylphosphoramidate (V, Ruel e r ~ ) , which ’~ was quite effective in mice both in the
98
19 23 37 11 11 10 13
--
0.4 0.4 0.3 1.0 1.0 0 0 1 1 0 0 0 0 0
diet at 0.257, and by gavage. -Activity wab fairly Tvidebpread among this structural type. Compounds TI and T 7 1 , for example. exhibited fair activity when administered at high levels in the diet. Certain minor
‘c1 VI
\‘I1 (11) Supplied b y 1)r. H. 1;. Rondy of Coalite a n d Chemical Prodiicts. ( 1 2 ) Compounds V-X were supplied by U r . E. Monroe of t h e Do\r Cliem-
ical C o .
structural variations did, however, abolish the biological activitj. Compound VIII, for example, showed no
sheep arid cattle.2n~21 This material also proved i o be an effective schistosoiiiicidal agent in mice: it was curative in amounts as low as 0.016% in the diet and had significant effect by gavage when administered a t 20 mg/lcg for 10 days. The other alkyl esters showed variable degrees of activity. The methyl and propyl aildogs ICarid I (X = S ; It = Pr) showed only very weak activity even at 0.237, while I d showed quite good activity even at 0.06y0but was not superior to the ethyl ester. The 172-nitro analog XI1 also exhibited ?P(OI(OEt)2
I
o/!\Y/~\o
XI I
OP(O)(OEt)XI11
good activity at 0.0670 but was not superior to the parent compound while the bis compound XI11 was inactive at 0.06% in the diet. The aryl esters and the two amine derivatives described in Table I1 showed essentially no activity when administered in the diet. Further studies were then initiated with the parent rompounds Is and Ib. I a has been shown to be converted by mammals to the oxygen analog Ib.18t1g I n certain organophosphorus insecticides thiophosphates are metabolized to the more active phosphates. The in viti o activity of I a and I b against S.mansoni was studied t o deterniirie whether a similar relationship was required for schistosome activity. The test preparations consisted of three pairs of worms in 2 nil of medium [73% horse serum, goyc physiologic saline solution, and 5% aqueous solutions of penicillin G (100 units/nil~ arid streptomycin (100 pg/ml) ] arid were incubated at 37". Preparations containing 0, 2 , 4, 8, or 16 p g of drug/nil were inspected at frequent intervals during 5 days. The death rate of worms in the presence of I a was not higriificantly higher than the nonniedicated controls. Compound I b also was tested in iitw under the same conditions as (bornpound Ia. It also failed to kill the worms in concentrations of 2-16 pg/nil during 5 days of exposure. The lack of in viti-o activity suggests either an insufficient concentration of drug in the medium 01metabolism to an active component by the intact animal. Compound I a was tested orally twice daily for therapeutic action against 8. iizansoni in six rhesus monkeys. The following results were obtained. Two monkeys were given 200 mg/kg/day for 10 days. One of these had a light infection of several months duration; following treatment it stopped passing eggs arid was judged cured on the basis of inability to find live worms at autopsy. The other animal had a heavy, recently induced infection; after treatment it showed a moderate suppression of egg excretion and at autopsy it had 50 live worms arid about an equal number of dead worms. Two moiikeys given 200 mg/lcg/day for 5 days showed ( 2 0 ) RI Federmann, D w f . Tieraerzfl. T o c h s c h r . , 71, 6 2 (1964). (21) AI. Stuber a n d H. Ende, Bedkner illunchener Tzeruerztl. Il*ochschr., 100 (1964).
no discernible therapeutic response : both coiitiriued to excrete eggs in large numbers. Two others that had old, light infections were given 100 mg/kg/day for 10 days; they showed moderate suppression of egg excretion and at autopsy 18 and 19 live worms, respectively, which was close to the normal number for untreated infections of comparable duration. All doses were well tolerated by gross examination. Compound I b was tested for therapeutic cffect against S.iiiansoni in three rhesus monkeys. Orie animal was given the drug orally in doses of 50 nig/kg/day (23 nig/lig twice daily) for 10 days ( 5 days/week for 2 weeks). I t s weight declined 23% during treatment. Egg excretion was discernibly suppressed but 23 live worms were found at autopsy. Two others were treated intraperitoneally once daily 5 days/week. Orie received I-mg/kg doses for the first week and 2 . 5 - m g k g doses during the second wrek. This animal died the day after the last dose and had 24 live worms; it did not survive long enough to assess an effect on egg excretion. The other monkey was given 1-mg/kg doses during the first two weeks and 2.5-nig/kg doses during the third week. This course of treatment was tolerated well but did riot suppress egg excretion and left 63 live wornis at aut opsy . I n view of the prominence of cholinesterase inhibition in the general biological activity of organophosphorus compounds, it is logical to suspect that their antischistosomal action might be mediated via this mechanism. Early in the present studies, we tested three classical cholinesterase inhibitors against S. iizansoni in mice for general guidance. The substances arid test conditions were as follow: tetraethyl pyrophosphate (TEPP) in dosea of 0.031 mg%g by gavage twice daily for 10 days (near the maximum tolerated amount); &met hyl-1-phenylpyrazol-3-yl dimethylcarbaniate ( P y rolan) as a 0.5ycdiet for 14 days: 3,3-dimethyl-3-oxo-lcyclohexen-1-yl dimethylrarbamate (Dimetan) as a 0.5% diet for 14 days. ,411 were ineffective. Such results discourage the expectations of a direct relatioiiship between cholinesterase inhibition and antischistosoma1 activity. :\loreover, review of the results from the large number and diverse types of organophosphorus compounds tested by us has failed to suggest relationships between structure and aritischistosonial activity or host toxicity. To the extent that host toxicity is due to cholinesterase inhibition, we also have been unable to detect ubeful structure-cholinesteraie inhibition relationships. Vnderstandably, such relationships may be obscured by physiological factors: nietabolic disposition, membrane permeability, etc. The problem of selecting a promising organophosphorus drug for trial against schistosomiasis has been further complicated b y dissimilar therapeutic indices among experimental hosts. Whereas some of the compounds had a relatively promising margin of safety in mice, none so far has shoirn high activity in inonlieys at welltolerated dose levels.
Experimental Sectionzz N-Hydroxynaphthalimide 0,O-Diethyl 0-Phosphorothioate Ester (Ia).-To a aolutioii of 40.8 g (0.192 mole) of N-hydroxynaphthalimide in 1 1. of dimethylformamide (UAIF) was added (22) Melting points \\ere taken on a Thornas-Hoover inelting point apparatus a n d a r e corrected.
