Organophosphorus Compounds as Schistosotnicicles

\ /

January 1907 OI~GANOPHOSPHOECS SCHI~TOSOMICIDMS 33

NO.

Ia

Ib

I u

Id

I1

111

11’

Y

1-1

VI1 IX

s SI1

]lose, ma:hr/

day

129 S .j 1s 10 20 20 7 2 17 2 0 20

‘184 7 0 72 ‘10

h72 287

71 63 60 54 18 9 4

12 6

tis 3 0

338 400 100 205 74

324 554 212 800 400 200 200 200 276 200

82 22

TABLE I THERIPEUTIC EFFECI.S OF ORG.INOPHOSPHORUS COMPOVSDS .LG.~INST S. itiunsoni IS 111~s

No. of days

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

14

5 14 14 14 14 14 4

10 .5 6

14 5

14 14

1

7

J

l i e ~ l n l e n

0 125yc diet 0 0GC; diet

Gavage X 2 I p x 1 0 06yC diet 0 016cc diet Gavage X 2 I p x 1 0 25c; diet 0 06c; diet 0 06rL diet 0 016cc diet 0 So/; diet 0 2Fc diet 0 059, diet 0 06wc diet Tb 0 125% diet T 0 06‘; diet T 0 016c> diet 0 0 0 S 5 diet 0 0 0 4 5 diet Gavage X 2 I p x 1 0 0ciCl, diet T Gavage X 2 0 2 5 5 diet Gavage X 2 sc x 2 0 25% diet 0 06% diet 0 25% diet 0 5% diet 0 1257, diet Gavage X 2 T Gavage X 2 Gavage X 2 Gavage X 2 s c x 2 0 2 5 c L diet Gavage X 2 0 06% diet 0 OIGCc diet

______ Treated-------

No. of mice

9 11 15 3

i c

b

6 7 7 ti

0

(i ci

3

)

1

3

I r

3 6 8 7 8

X 1 i ti

10 8 4 5 4 9

18 3 S 9

10 9 5 5 6

in

3

% of v o r m s dead

100 98 37 ;ti 45 13

100 100

79 29 12 !)S 56 7

-- i t

1

n 89 57 58 69 18 0

30 11 74 2 S5 76 2 98 6 48 93 30 95 89 11 13 1

99 3

95 2

Mean no. of live xorins

0 0 . 4 9 . 6 s.3 9 . 4

13 .8 0 0 3 . 7 2 . 5 8 . 4 9 . 0 0 , 2 6 . 6

11.2 13.9 8 . 5 0 . 8 2 . 0 2 . 3 2 . 1 6 . 4

11.7 10.5 8 .7 5 . 0

15 .3 0 . 9 4 . 6

14.7 0 . 2

13 .3 8 . 3 0 . 8 7 . 0 0 . 6 1 . 6 9 . 4

10.5 9 . 9 0 . 1 7 . 6 0 . 6

10 .3

------Con trois-----

s o . of mice

19 23 37 11 11 10 13

i 10 13 11 13 10 9

11 8

10 10 14 10 10 10 9 9 7

12 11 10 10 13 13 11 9

17 10 10 10 10 10 13 15 10 10

” i

L/o of norms dead

0 . 4 0 . 4 0 . 3 1 .0 1 . 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 2 0 1 0 0 0 1 2 0 0

Mean no. of live worms

16.1 15 .8 18.8 24.9 24.9 10.9 19.5 1 2 . i 12.7 10.9 14.9 13 .8 13.0 16.0 11.2 13 .4 11.8 15.6 18 .3 16 .1 11.3 17.5 17.5 18 .8 14.0 28.6 16.4 15 .0 1s . 1 18 .1 13.0 13.0 13 .8 12.8 14.8 11.1 1s. 1 15.8 1 5 , s 11.1 15.2 12.7 16 .0 16,O

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).

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-t- butyl-2-chlorophenyl methylphosphoramidate (V, Rue- l e r ~ ) , ’ ~ which was quite effective in mice both in the

(11) Supplied b y 1) r . H. 1;. Rondy of Coalite and Chemical Prodiicts. (12) Compounds V-X were supplied by Ur . E. Monroe of t he Do\r Cliem-

ical Co.

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

structural variations did, however, abolish the biologi- cal 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: i t was cura- tive 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 vari- able degrees of activity. The methyl and propyl aildogs IC arid I (X = S; It = Pr) showed only very weak activity even at 0.237, while Id showed quite good activity even at 0.06y0 but was not superior to the ethyl ester. The 172-nitro analog XI1 also exhibited

?P(OI(OEt)2

o/!\Y/~\o I 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 in- active 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 con- verted by mammals to the oxygen analog Ib.18t1g I n certain organophosphorus insecticides thiophosphates are metabolized to the more active phosphates. The in vi t i o activity of I a and Ib against S. mansoni was studied to deterniirie whether a similar relationship was required for schistosome activity. The test preparations con- sisted 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 Ib 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 01-

metabolism to an active component by the intact ani- mal.

Compound I a was tested orally twice daily for thera- peutic 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; fol- lowing 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

(20 ) RI Federmann, D w f . Tieraerzf l . T o c h s c h r . , 71, 62 (1964). (21) AI . Stuber and H. Ende, Bedkner i l lunchener 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 excre- tion 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 ani- mal 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 anti- schistosomal action might be mediated via this mech- anism. 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 (Py - rolan) as a 0.5yc diet for 14 days: 3,3-dimethyl-3-oxo-l- cyclohexen-1-yl dimethylrarbamate (Dimetan) as a 0.5% diet for 14 days. ,411 were ineffective. Such re- sults discourage the expectations of a direct relatioii- ship between cholinesterase inhibition and antischisto- soma1 activity. :\loreover, review of the results from the large number and diverse types of organophos- phorus compounds tested by us has failed to suggest relationships between structure and aritischistosonial activity or host toxicity. To the extent that host toxic- ity is due to cholinesterase inhibition, we also have been unable to detect ubeful structure-cholinesteraie in- hibition relationships. Vnderstandably, such relation- ships may be obscured by physiological factors: nieta- bolic disposition, membrane permeability, etc. The problem of selecting a promising organophosphorus drug for trial against schistosomiasis has been further complicated by 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 well- tolerated 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-hydroxy- naphthalimide in 1 1. of dimethylformamide (UAIF) was added

(22) Melting points \ \ere taken on a Thornas-Hoover inelting point ap- paratus and are corrected.