In recent years, the number of new anthelmintics compounds introduced to the market to control the infections produced by gastrointestinal nematodes has been limited, mainly because of economic difficulties in the development and marketing of new drugs . In the last 2 decades, only four drugs have been introduced on the market: emodepside , monepantel , derquantel  and tribendimidine . Therefore, there is 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. Although BZ resistance is present in many gastrointestinal species infecting livestock, the synthesis of novel BZ derivatives may lead to compounds with improved properties such as better solubility and pharmacokinetic profile, resulting in increased effectiveness . Some promising compounds, such as tenvermectin , diisopropylphenyl-imidazole  and mebendazole hydrochloride , have been developed in recent years following this approach.
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 of T. muris and adult stages of T. muris and H. polygyrus. The anthelmintic activity of most of these compounds was previously tested in vitro against the gastrointestinal nematode infecting sheep Teladorsagia circumcincta [18, 21] and some of them were also tested against Leishmania spp. [31, 32], Trypanosoma spp. [17, 33] and Strongyloides venezuelensis .
The L1 assay has proven to be a good tool to screen new potential candidate compounds before carrying out adult motility assays, the in vitro assay of choice, which is more expensive, labour intensive and time-consuming, and it requires the use of live animals . Moreover, the results obtained with the motility assay based on L1 seem to correspond to the findings observed with adult T. muris . However, some studies showed that L1 appears to be more sensitive to drugs than older stages of T. muris [36, 37], which can facilitate the discarding compounds with no activity.
In the present study, 10 out of the 41 compounds tested showed activity > 90% against the L1 stage of T. muris at 100 µM, and only three, namely AO14, BZ12 and BZ6, reached an IC50 < 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 with the previous study carried out against T. circumcincta reveals that of the ten compounds screened at 100 µM that showed > 90% activity against T. muris L1, six (BZ1, BZ2, BZ6, AO11, AO15 and AA18) also showed ovicidal activity against T. circumcincta, but only BZ6 reached an IC50 value < 10 µM (IC50 = 6.54 µM). In the case of T. circumcincta L1, four of them (AO5, AO11, AA18 and BZ6) reached IC50 values < 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 < 10 µM (IC50 for AO5 = 5.55 µM, IC50 for AO11 = 4.58 µM) against T. circumcincta L3. Some of the compounds that did not show activity in the L1 T. muris assay had shown activity against other parasite models such as Trypanosoma brucei (compounds AO4 and AA19 with IC50 values close to 0.5 µM) and Leishmania spp. (compounds AA25 and AA26). This is also the case for the study carried out on S. venezuelensis L3, in which compounds AO6, AA18, AA19, AA24 and AA25 showed activity against this nematode (IC50 values ranging from 31.9 ± 0.5 μM to 39.1 ± 4.7 μM), but only compound AA18 showed activity against L1 of T. muris in the current study.
Thus, BZ6 seems to be the only compound reaching IC50 values < 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 an 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 benzimidazole derivatives, 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 such 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) and 5-NO2–4’-Cl/diMe (BZ12 and BZ13) produced a deadly effect > 90% on the initial screening of T. muris L1. Regarding the substituent present on the B-phenyl ring (R2), 4’-Cl− is required 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, such as 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 such as 5-NO2 (BZ13), gave good anthelmintic inhibitory activity in T. muris L1 (99.40 inhibition at 100 µM), although its IC50 was > 10 µM.
Comparing the results of the adult motility assay of the present study with previous experiments using the marketed human drugs (ABZ, MBZ, LEV and PYR) showed that the IC50 values obtained are much lower (8.1 µM for BZ12) since BZ compounds showed a lack of activity on T. muris adults and LEV and PYR displayed IC50 values around 68 and 57 µM, respectively .
All compounds tested against L1 had a possible toxic potential, as their SIs were very close to one, except BZ6 and AO14, which reached values > 4 in both cell lines. Regarding the SIs obtained in the adult assays, although they were in any case > 1, 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.