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Studies on Enteric Anthelmintics: Impact of Single Point Structural Change on the Activity Profile A. M. Khan, Suman Gupta, and J. C. Katiyar* Division of Parasitology, Central Drug Research Institute, Lucknow. India M. Shamim Akhtar, V. L. Sharma, and A. P. Bhaduri Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow, India Z. Naturforsch. 43b, 233-237 (1988); received May 20, 1987 Anthelmintic Activity, Intestinal Worm. Benzimidazole Carbamates, Structural Change, Heteroatom

The impact of change only in one heteroatom in the substituent at position 5(6) of methyl benzimidazole-2-carbamates on the profile of anthelmintic activity against intestinal worms An-cylostoma ceylanicum (hookworm), Nippostrongylus brasiliensis (trichostrongylid), Syphacia obvelata (oxyurid) and Hymenolepis nana (cestode) has been studied. The observations made are likely to evoke interest for understanding the SAR in methyl benzimidazole-2-carbamates anthel-mintics at a molecular level.

Introduction

Benzimidazole carbamates as anthelmintics are ex-tremely sensitive to minor structural changes. Earlier work in this direction relates to optimization of the biological activity [1], but no attempt appears to have been made to ascertain the impact of minor structural changes in benzimidazole anthelmintics on the profile of anthelmintic activity. The present work is, there-fore, directed towards studying the influence of single point change in the molecular architecture on the spectrum of activity against experimental intestinal worms. The change as would be evident from molecu-lar structures of compounds, methyl[5-(3-methoxy-carbonyl-2,5-dimethylfur-4-yl)-lH-benzimidazol-2-yl]carbamate (1) and methyl[5-(3-methoxycarbamyl-2,5-dimethylpyrrol-4-yl)-l H-benzimidazol-2-yl]car-bamate (2) relates only to one heteroatom (Fig. 1).

Me02C N

* Reprint requests to Dr. J. C. Katiyar. Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0932 - 0776/88/0300 - 0261 /$ 01.00/0

The details of the observations made are recorded here.

Materials and Methods Test compounds: The test compounds 1 and 2,

were prepared in this Institute and the purity was checked by T .L .C. , elemental analyses and spectro-scopic data, which appeared elsewhere [3, 4],

Parasites: The compounds were evaluated against Ancylostoma ceylanicum (hookworm), Nippostron-gylus brasiliensis (trichostrongylid), Syphacia obve-lata (oxyurid), and Hymenolepis nana (cestode).

Assay method Both compounds were ground to fine particles and

suspended with the help of Tween 80 [5]. For each dose schedule 2—3 animals were used and 2—4 repli-cates were done. The experimental animals were autopsied under deep ether anaesthesia.

1. Ancylostoma ceylanicum: (a) Adulticidal action: The drug testing was done according to the technique of Katiyar et al. [6—8]. Briefly, golden hamsters of either sex (50—60 g) were intubated with 60 ± 5 in-fective larvae (L3) of A. ceylanicum. On day 17 p.i . , the hamsters found positive by ovoscopic examina-tion were used for drug therapy. The compounds were administered orally or intraperitoneally. The efficacy was expressed in terms of absolute clearance of parasites from the host and percent worm reduc-tion.

(b) Larvicidal action: The screening technique fol-lowed was that of Katiyar et al. [8]. The test com-pounds were either fed or injected intraperitoneally on day 1, 3 and 6 post infection [9, 10] for evaluating

234 A. M. Khan et al. • Studies on Enteric Anthelmintics 234

effect on L3, L4 and L5 stages respectively. Treated and untreated hamsters were sacrificed on day 20 post infection when L3 reached patency in the intes-tine. The efficacy assessment was same as described above.

(c) Chemoprophylactic action: For evaluating chemoprophylactic potential, naive hamsters were injected with the test compounds intramuscularly in the dose schedule of 100 and 50 mg/kg b.w. x 1 and were challenged with 60 ± 5 L3 after day 7 and day 15 of treatment. The hamsters were sacrificed on day 20 of challenge to assess the prophylactic effect. 2. Nippostrongylus brasiliensis: Male rats of UF

strain weighing 30—40 g and infected subcutaneously with 500 infective larvae (L3) served as the experi-mental host [11]. The compounds in suspension forms were given p. o. to rats infected 9 days before. The treated and untreated controls were autopsied on the third day of the last dose [6].

3. Syphacia obvelata: The compounds were evalu-ated in either sex of Swiss albino mice (20—25 g) with naturally acquired infection of S. obvelata [6, 7], The caecum and colon were isolated, incised longitudinally in a petri dish and examined under the dissecting microscope. The criterion of efficacy was the absolute clearance of parasites from the treated mice because of wide variation in worm burden.

4. Hymenolepis nana: The efficacy assessment was made against egg (200) induced infection of H. nana in male rats weighing 35—40 g [12, 13]. The basis of drug efficacy was the complete removal of worms with scolices from the treated animals.

Results

Adulticidal activity

The activity of compounds against adult parasites of hookworm, trichostrongylid, oxyurid and tape-worms has been presented in Table I.

Compound 1 is superior than compound 2 against all the parasites tried.

All the worms of A. ceylanicum from the treated hamsters were knocked out by compound 1 at a sin-gle oral dose of 12.5 mg/kg. Lower doses showed dose dependent efficacy. Compound 2 in simultane-ous trials showed inferior activity, as an equivalent amount (12.5 mg/kgxl) failed to cure any of the treated hamsters, although per cent reduction in worm burden was 69.5.

Parenteral administration of compounds also en-dorsed the superiority of compound 1 over com-pound 2 by both the criteria of evaluation.

Against N. brasiliensis, both the compounds were ineffective even at the initial oral dose of 250 mg/kg b.w. x 3.

Against Syphacia obvelata (oxyurid) also, com-pound 1 at 100 mg/kg x 1 dose level was 100% effec-tive, whereas similar amount of compound 2 could cure only 66% of infected mice. Similar difference in activity was also observed in multiple dose schedule.

Against the cestode, H. nana, compound 1 was found to have better activity than compound 2, how-ever, the relative efficacy was only marginal.

Larvicidal action

Efficacy data against developing parasites of A. cey-lanicum have been summarised in Table Ha and b.

The results demonstrated that the compounds were more efficacious against all the larval stages by oral route compared to intraperitoneal administration. Here also compound 1 proved to have better larvici-dal effect than compound 2 irrespective of the larval stages.

Chemoprophylactic action

Compound 1 at 100 mg/kg b.w. x 1 dose was found to protect hamsters (67.87% worm reduction) to subsequent challenge of L3 upto day 15 while com-pound 2 exerted no prophylactic effect (not shown in the Table).

Discussion

Despite the fact that the test compounds have activ-ity against all the helminth parasites tested, only the detailed investigations against Ancylostoma cey-lanicum were undertaken as both the chemical agents had reasonably good efficacy against this parasite. The development of Ancylostoma ceylanicum is con-fined totally to gastro-intestinal tract [9] and benz-imidazole carbamates anthelmintics being poorly ab-sorbed through G. I. tract [2, 14] are fully available to the parasites. This is why better effects of these com-pounds have been observed in adults and larvae by oral route of administration. It is likely that because of the binding of the test compounds to blood proteins during parenteral administration these are not avail-able at the site of infection in effective concentration.

Change in the heteroatom (with asterisk) in these two compounds (Fig. 1) made a significant effect on the profile of anthelmintic activity against intestinal

A. M. Khan et al. • Studies on Enteric Anthelmintics 235

Tab. I. Efficacy of compounds 1 and 2 against adult enteric helminth parasites.

Compound 1 Compound 2 irasites Route Animals Percent Worm Percent Animals Percent Worm Percent

dose cleared/ animals recovery worm cleared/ animals recovery worm mg/kg treated freed mean reduc- treated freed mean reduc-

(No. of from with range tion (No. of from with range tion experiments) parasites experiments) parasites

2 3 4 5 6 7 8 9 10

ncylostoma p.o. vlanicum 100 x l 4/4 (2) 100 0 100 6/6 (2) 100 0 100 amsters) 50 x l 4/4 (2) 100 0 100 9/11 (4) 81.81 0.18 (0-1) 99.39

25 x l 4/4 (2) 100 0 100 0/8 (3) 0 6.00 (4-9) 80.76 12.5 x l 6/6 (3) 100 0 100 0/8 (3) 0 8.37 (7-12) 69.49 6.25x1 0/6 (3) 0 18.33 (12-28) 43.46 0/9 (3) 0 19.88 (16-25) 27.55 3.12x1 0/3 (1) 0 22.33 (17-26) 31.81 0/6 (2) 0 22.33 (16-27) 26.33

l.p. 50 x l 5/5 (2) 100 0 100 3/5 (2) 60 0.60 (1-2) 97.85 25 x l 2/5 (2) 40 1.60 (1-4) 94.28 0/6 (2) 0 10.60 (5-16) 61.92 12.5 x l 0/4 (2) 0 10.80 (7-14) 61.42 0/6 (2) 0 18.16 (10-32) 35.14 6.25x1 0/6 (2) 0 20.50 (19-22) 26.78 0/4 (2) 0 25.30 (22-29) 5.34

ppostrongylus p.o. asiliensis (rats) 250 x3 0/6 (2) 0 80 (25-140) 0 0/6 (2) 0 85 (30-145) 0 phacia p.o. velata 250 x l 6/6 (2) 100 - - 6/6 (2) 100 - -

lice) 100 x l 9/9 (3) 100 - - 4/6 (2) 66.66 - -

50 x l 4/9 (3) 44.44 - - 2/6 (2) 33 - -

25 x l 0/6 (2) 0 - - 0/9 (3) 0 - -

50 x3 6/6 (2) 100 - - 7/9 (3) 77 - -

25 x3 6/9 (3) 66.66 - - 3/6 (2) 50 - -

12.5 x3 0/6 (2) 0 - - 0/6 (2) 0 - -

menolepis p.o. na (rats) 250 x l 4/4 (2) 100 - - 6/6 (2) 100 - -

100 x l 1/4 (2) 25 - - 0/5 (2) 0 - -

50 x l 0/4 (2) 0 - - 0/4 (2) 0 - -

50 x3 10/10 (4) 100 - - 8/8 (4) 100 - -

25 x3 5/6 (3) 83.33 - - 2/4 (3) 50 - -

12.5 x3 0/4 (2) 0 - - 0/4 (2) 0 - -

Note: No. of parasites recovered from untreated control No. of Worm recovery animals (pooled data). animals mean with range

A. ceylanicum 29 29.48 (19-43) S. obvelata 21 19.30(7-41) N. brasiliensis 6 94 (39-135) H. nana 15 18 (10-60)

parasites. Since, benzimidazole-2-carbamates are known to exhibit their biological efficacy by inhibit-ing tubulin polymerization [1,2] the binding studies of these two compounds with parasites tubulin would help to map the topography of parasite receptor for evolving the observed biological response. In view of the significant difference of activity against nematodes and cestode, these compounds would serve as better tool for understanding the difference

of tubulins of cestodes and nematodes. It would be also interesting to study the bioavailability of the test compounds to ascertain the effect of this minor struc-tural change.

Financial assistance to AMK from ICMR and to MSA & VLS from CSIR are gratefully acknowl-edged.

236 A. M. Khan et al. • Studies on Enteric Anthelmintics 236

Table IIa. Efficacy of compounds 1 and 2 against developing stages of Ancylostoma ceylanicum by ORAL route.

Compound 1 Compound 2 Animals Percent Worm Percent Animals Percent Worm Percent

Stage Dose cleared/ animals reduction worm cleared/ animals reduction worm mg/kg treated freed mean reduc- treated freed mean reduc-

(No. of from with range tion (No. of from with range tion experiments) parasites experiments) parasites

1 2 3 4 5 6 7 8 9 10

L3 100 x l 0/9 (3) 0 5.33 (2-8) 82.61 0/10 (4) 0 7.40 (4-10) 72.17 50 x l 0/6 (2) 0 9.00 (3-12) 72.72 0/6 (2) 0 9.00 (6-11) 73.78 25 x l 0/6 (2) 0 12.66 (7-18) 62.38 0/4 (2) 0 23.25 (8-39) 32.27 12.5 x l 0/5 (2) 0 18.60 (13-37) 44.74 0/6 (2) 0 29.50 (17-44) 14.06 6.25x1 0/5 (2) 0 16.20 (14-19) 38.07 0/6 (2) 0 40.34 (32-48) 0

L4 100 x l 6/6 (2) 100 0 100 4/6 (2) 66.66 0.55 (0-2) 97.20 50 x l 0/6 (2) 0 4.50 (3-6) 86.36 1/6 (2) 16.66 2.00 (0-5) 93.25 25 x l 0/6 (2) 0 6.50 (3-10) 80.69 0/5 (2) 0 8.00 (3-14) 77.24 12.5 x l 0/6 (2) 0 7.33 (4-12) 78.23 0/5 (2) 0 25.00 (14-34) 28.89 6.25x1 0/7 (3) 0 22.43 (3-46) 29.18 0/5 (2) 0 29.13 (21-35) 6.18

u 100 x l 6/6 (2) 100 0 100 6/6 (2) 100 0 100 50 x l 6/6 (2) 100 0 100 0/6 (2) 0 8.50 (6-11) 71.25 25 x l 2/6 (2) 33.33 8.66 (0-21) 67.72 0/5 (2) 0 15.20 (11-19) 57.77 12.5 x l 0/6 (2) 0 7.50 (2-12) 73.68 0/5 (2) 0 18.60 (14-22) 37.09 6.25x1 0/6 (2) 0 7.67 (5-12) 70.00 0/5 (2) 0 42.34 (36-47) 12.89 3.12x1 0/6 (2) 0 34.33 (20-49) 15.75 0/5 (2) 0 45.90 (39-49) 0

Note: No. of parasites recovered No. of Worm recovery from untreated control animals. animals (mean with range)

39 32 (15-39)

Table II b. Efficacy of compound 1 and 2 against developing stages of Ancylostoma ceylanicum by INTRAPERITONEAL route.

Compound 1 Compound 2 Animals Percent Worm Percent Animals Percent Worm Percent

Stage Dose cleared/ animals reduction worm cleared/ animals reduction worm mg/kg treated freed mean reduc- treated freed mean reduc-

(No. of from with range tion (No. of from with range tion experiments) parasites experiments) parasites

1 2 3 4 5 6 7 8 9 10

L3 100 x l 0/6 (2) 0 8.33 (6-12) 68.15 0/8 (3) 0 12.50 (9-16) 53.30 50 x l 0/6 (2) 0 12.50 (9-15) 52.21 0/5 (2) 0 13.40 (8-18) 60.96 25 x l 0/6 (2) 0 23.16 (15-26) 31.67 0/6 (2) 0 26.33 (11-45) 23.30 12.5 x l 0/6 (2) 0 23.00 (17-33) 31.67 0/6 (2) 0 31.60 (19-44) 9.23 6.25x1 0/6 (2) 0 28.16 (19-43) 16.34 0/6 (2) 0 42.67 (38-46) 0

L4 100 x l 3/6 (2) 50 0.66 (0-2) 97.47 3/6 (2) 50 1.00 (0-3) 97.20 50 x l 0/6 (2) 0 11.33 (10-14) 66.17 0/5 (2) 0 4.20 (1-6) 85.83 25 x l 0/6 (2) 0 10.17 (4-16) 69.80 0/5 (2) 0 14.20 (8-26) 59.89 12.5 x l 0/6 (2) 0 25.00 (7-46) 25.75 0/5 (2) 0 26.40 (22-30) 24.91 6.25x1 0/6 (2) 0 24.67 (3-49) 26.73 0/5 (2) 0 32.66 (24-38) 7.11

L5 100 x l 6/6 (2) 100 0 100 6/6 (2) 100 0 100 50 x l 6/6 (2) 100 0 100 0/6 (2) 0 12.16 (8-15) 66.22 25 x l 2/6 (2) 33.33 11.67 (0-28) 56.55 0/6 (2) 0 22.16 (15-27) 38.44 12.5 x l 0/6 (2) 0 10.17 (5-22) 64.32 0/6 (2) 0 31.00 (26-39) 13.88 6.25x1 0/6 (2) 0 13.33 (8-21) 60.40 0/6 (2) 0 42.33 (32-49) 0.77

Note: No. of parasites recovered from untreated control animals.

No. of Worm recovery animals mean with range 39 32 (15-39)

A. M. Khan et al. • Studies on Enteric Anthelmintics 237

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