In recent years, the number of new anthelmintics compounds introduced into the market to control the infections produced by gastrointestinal nematodes has been limited, mainly due to economic difficulties in the development and marketing of new drugs . Only four drugs have entered the market the last two decades: emodepside , monepantel , derquantel  and tribendimidine . There is therefore a clear need to develop novel anthelmintic drugs for the control of these parasitic worms in humans and farm animals. One of the approaches proposed to alleviate the severe scarcity of anthelmintics is the synthesis of new derivatives of known drugs. This approach can lead to new compounds with improved properties such as better solubility and pharmacokinetic profile, resulting in increased effectiveness .
Based on these assumptions, in the present study, a total of 15 AO and 11 AA derivatives, both structurally related to sphingosine, and 15 benzimidazole derivatives, were tested against L1 and adult stages of T. muris and H. polygyrus. The anthelmintic activity of most of these compounds were previously tested in vitro against the gastrointestinal nematode infecting sheep Teladorsagia circumcincta [18,21] and some of them were also tested against Leishmania sp. [19,28], Trypanosoma sp. [17,29] and Strongyloides venezuelensis .
In the present study 10 out of the 41 compounds tested showed activity higher than 90% against the L1 stage of T. muris at 100 µM, and only three namely AO14, BZ12 and BZ6, reached an IC50 lower than 10 µM. The screening performed at a single final concentration of 10 µM on adults showed that only BZ12 and BZ6 had significant activity against the adult stage of T. muris and H. polygyrus, respectively.
Comparing the results obtained with these derivatives to the previous study carried out against T. circumcincta, it reveals that of the 10 compounds screened at 100 µM that showed more than 90% activity against T. muris L1, six (BZ1, BZ2, BZ6, AO11, AO15 and AA18) of them also showed ovicidal activity against T. circumcincta eggs, but only BZ6 reached an IC50 value lower than 10 µM (IC50= 6.54 µM). In the case of T. circumcincta L1, four of them (AO5, AO11, AA18 and BZ6) reached IC50 values below 10 µM (IC50 for AO5=2.87 µM, IC50 for AO11=1.21 µM, IC50 for AA18=6.29 µM and IC50 for BZ6=5.01 µM) and only AO5 and AO11 showed IC50 values below 10 µM (IC50 for AO5=5.55 µM, IC50 for AO11=4.58 µM) against T. circumcincta L3. Some of the compounds that have not shown activity in the L1 T. muris assay had shown activity against other parasite models such as Trypanosoma brucei (compounds AO4 and AA19 with IC50s close to 0.5 µM) and Leishmania sp. (compounds AA25 and AA26). This is also the case of the study carried out on S. venezuelensis L3, in which compounds AO6, AA18, AA19, AA24 and AA25 showed activity against this nematode (IC50s ranging from 31.9±0.5 µM to 39.1±4.7 µM), but only compound AA18 showed activity in the current study, against L1 of T. muris.
Thus, compared to previous studies, BZ6 seems to be the only compound reaching IC50 values below 10 µM in both eggs and L1 of T. circumcincta (IC50=6.54 µM in eggs and IC50=5.01 µM in L1) and also in L1 of T. muris (IC50= 4.17 µM), with values quite close to each other). However, BZ6 did not have any affect against the adult stage of T. muris at a concentration of 10 µM (17.2% of activity) but it was effective against H. polygyrus adults (100% of activity) displaying an IC50 of 5.3 µM. On the other hand, BZ12 did not produce effect against any of the stages of T. circumcincta, eggs, L1 or L3, but it showed activity against T muris L1 with an IC50 of 8.89 µM. Moreover, this BZ12 reached an efficacy of 53.3 and 81.7% on the adult stage of H. polygyrus and T. muris at 10 µM, respectively, presenting an IC50 of 8.1 µM in the latter.
In terms of the relationship between the structure and efficacy of the compounds and focusing on the benzimidazoles, the only group of compounds that has shown significant efficacy on the adult stage of the parasites in this study, we can observe that the presence of a mild basic group as the–NH2 group on R1 (BZ15) did not induce any measurable effect on the nematode viability, while the combinations of 5-Me–4’-OMe/Cl (BZ1 and BZ2), 5-Cl – 4’-Cl (BZ6), 5-NO2 – 4’-Cl/diMe (BZ12 and BZ13) produce a deadly effect higher than 90% on the initial screening of T. muris L1. Regarding to substituent present on the B-phenyl ring (R2), the 4’-Cl− is convenience for the anthelmintic effect since all compounds with this substituent at this position showed anthelmintic activity on T. muris L1 (BZ2, BZ6 and BZ12), including here the two most potent compounds (BZ6 and BZ12), while double substitutions on this ring, as those 3’-NO24’-OMe (BZ4, BZ9 and BZ14) or 3’-NH2 4’-OMe (BZ10), led to inactivity. However, a di-substitution in position 2´ and 6´ with electron donating groups such as 2´,6´-diMe in addition to a polar group in ring A as 5-NO2 (BZ13), gave good anthelmintic inhibitory activity in T. muris L1 (99.40 inhibition at 100 µM), although its IC50 was higher than 10 µM.
All compounds tested against L1 had a possible toxic potential as their SIs were very close to one, except for BZ6 and AO14, which reached values greater than four in both cell lines. Regarding to the SIs obtained in the adult assays, although in any case they were greater than one, BZ6 seems to be a safer candidate than BZ12, as it had SI values of 4.3 for Caco2 cells and 3.1 for HepG2 cells.