**5.1 Medium-throughput phenotypic screening results**

*In vitro* phenotypic screening of selected compound sets has provided several new drug leads for further optimization [8, 59]. Compound sets prepared by the Medicines for Malaria Venture (MMV) have proven particularly fruitful in this regard. The first of these sets to be assessed was the Malaria Box, which contained 400 diverse, commercially available compounds, 200 of which were "drug-like" according to Lipinski's Rule of Five, all with confirmed *in vitro* activity against the blood stage of *P. falciparum* [60]. *In vitro* screening of these compounds against newly transformed schistosomula (NTS) was followed by similar testing against adult parasites; the five most active of these compounds (**23**–**27**, **Figure 5**) were then tested *in vivo* for efficacy and pharmacokinetic properties [61]. While three of the five were ineffective *in vivo* (WBR <20%), compounds **26** and **27** were able to reduce worm burdens in infected mice by 52.5% and 40.8%, respectively, with a single 400 mg/kg dose [61].

The diarylurea MMV665852 (**26**) above stood out for its good *in vivo* activity and its ease of synthesis, so it was chosen for further development. A set of MMV665852 analogs, including bissulfonamide, oxalamide, thiourea, carbamate, imidazolidinone, and pyrazine central moieties, was assessed against *S. japonicum* [62]. The parent MMV665852, along with six urea analogs, demonstrated IC50's under 10 μM for both juvenile and adult parasites in *in vitro* testing, but none of them produced WBR values above 35% in mice harboring either a juvenile or an adult *S. japonicum* infection.

**Figure 5.** *Antischistosomal hits from the MMV malaria box (***23***–***27***).*

Commercially available analogs of **26**, including ureas (25), benzamides (17), and carbamates (4), were screened for activity against *S. mansoni* as above [63]. While nine of these compounds had IC90's of <10 μM against adult worms, only the salicylanilide **28** (**Figure 6**) demonstrated significant *in vivo* activity. While its worm burden reduction was greater than that of the lead compound **26**, its cytotoxicity (as measured against L6 cells), and the resulting poor selectivity index (4.9), may preclude its further development as antischistosomal lead.

Further exploration of the diarylurea chemotype resulted in the synthesis and testing of 20 new analogs designed with aqueous solubility and chemical diversity in mind. Seven of these analogs demonstrated sub-micromolar IC50's against adult *S. mansoni* with high antischistosomal selectivities [64]. Three of these (**29**–**31**), all bearing 4-fluoro-3-trifluoromethylaniline moieties, showed modest *in vivo* activity, with WBRs between 37% and 50%. Pharmacokinetic data suggest that **31** has significantly higher overall systemic exposure than the other two, perhaps due to the pyridine substituent. N,N′-diarylureas bearing pentafluorosulfanyl (∙SF5) groups, such as **32**, have also been synthesized and assessed; like the other ureas tested, they demonstrated excellent activity *in vitro* (IC50's as low as 0.6 μM against *S. mansoni* NTS) but marginal efficacy *in vivo* [65].

Another of the leads from the Malaria Box screening, the dianilinoquinoxaline MMV007204 (**27**), was also selected for further development. Quinoxaline compounds have previously demonstrated utility against other parasitic diseases such

**Figure 6.** *Antischistosomal analogs of diarylurea* **26** *and quinoxaline* **27** *(***28***–***38***).*

#### *Recent Advances in Anti-Schistosomiasis Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.103056*

as malaria, Chagas disease, leishmaniasis, amebiasis, giardiasis, and filariasis [66]. Analogs of quinoxaline **27** (47, including 12 triazoloquinoxalines) were screened as above; three nitroquinoxalines (**33**–**35**) showed IC50's of under 0.31 μM against adult *S. mansoni* worms. Again, the *in vivo* potency of these compounds was underwhelming, with highest WBR among them 46.3% for compound **35** [67]. In a separate contemporaneous study, other quinoxaline analogs of **36** bearing nitro, amine and amide functionalities were screened for both phenotypic and motility effects on schistosomula [68]. Compared to compound **27**, compounds **36**, **37** and **38** showed significantly greater efficacy against the adult worms; the latter two compounds also showed excellent activity against *S. japonicum* and *S. haematobium* adults [68].

The MMV Stasis Box, containing 400 compounds that whose development as drugs was stopped at an advanced stage for various reasons, was also explored as a source of new chemotypes for anti-schistosomiasis drug development [69]. Eleven of these compounds showed an *in vitro* effect against adults of least 75%, with four demonstrating complete lethality, but the only compound to have an *in vivo* effect on worm burden over 50% was MMV690534, (**39**, **Figure 7**) with a 51.4% WBR. Compound **39** is a TGF-β receptor I kinase inhibitor developed for cancer chemotherapy; [70] other kinase inhibitors with anti-schistosomiasis activity will be discussed later in this review.

The MMV also prepared a Pathogen Box containing 400 compounds with activity against various neglected diseases, including malaria, tuberculosis, toxoplasmosis, and schistosomiasis. Three institutions explored this compound set for anti-schistosomiasis activity; teams at the Swiss Tropical and Public Health (TPH) [71] and the University of California-San Diego (UCSD) conducted *in vitro* phenotypic assays of these compounds against *S. mansoni* NTS, while a team at the Fundação Oswaldo Cruz (FIOCRUZ) used a metabolic activity indicator to assess schistosomula viability [72]. The two phenotypic assays showed a strong 87% concordance, but the inclusion of the FIOCRUZ assay only lowered the overall concordance slightly, to 74%. At 72 h drug treatment, 35 compounds in the Pathogen Box, including the antimalarial mefloquine (**18**), registered as "active" on all three screens against schistosomula. Five of those common hits demonstrated moderate *in vivo* activity in mice infected with *S. mansoni*: MMV022478 (**40**, 70.7% WBR), MMV022029 (**41**, 67.8%), MMV688761 (**42**, 55.2%), MMV687273 (**43**, 22.4%), and MMV690102 (**44**, 32.8%) (**Figure 7**) [71].

Notably, **PZQ** was *not* one of those 35 common hits, showing only borderline activity in the Swiss TPH screen and no activity in the FIOCRUZ screen. This reminds us that overreliance on obvious phenotypic signs in screening might be keeping us from discovering anti-schistosomiasis compounds with more subtle modes of action, especially modes that rely on the host immune response. A recent essay by Zamanian and Chan recommends the further development of *in vitro* screens to more closely model *in vivo* environments [73].

The most recent MMV Box to be assessed for anti-schistosomiasis activity was the Pandemic Response Box, a set of compounds with antibacterial, antiviral and/ or antifungal activity [74]. Phenotypic screening found 17 of these 400 compounds to have at least moderate activity (>66%) against adult *S. mansoni in* vitro. The most promising of these compounds was found to be the isoquinoline MMV1581558 (**45**), with an EC50 of 0.18 ± 0.01 μM against adult *S. mansoni*, and a WBR of 42 ± 25% in *in vivo* testing.

Phenotypic screening of a set of 2160 compounds purchased from Microsource Discovery Systems, containing 821 FDA-approved drugs, against *S. mansoni* NTS yielded about 100 hits, which were narrowed by subsequent screening against adult worms as well as consideration of known compound toxicity and side effects [75]. The ionophoric antibiotic lasalocid sodium **46** (**Figure 8**) effected moderate reductions in worm burden (~40%) and egg burden as well as improvements in spleen and liver pathology in the same model [75]. The anthelminthic niclosamide (**47**) demonstrated excellent *in vitro* activity but no WBR in infected mice; among related salicylanilides that were tested, rafoxanide (**48**) reduced WBRs by half at a 50 mg/kg dose [75].

Recently, a set of 73 non-steroidal anti-inflammatory drugs (NSAIDs) was screened for activity against *S. mansoni* [76]; this was in part motivated by the reported antischistosomal activity of the NSAID diclofenac (**49**), which is structurally similar to PZQ [77]. The most active NSAID in the set proved to be mefenamic acid (**50**), with good activity *in vitro* (EC50 = 11.1 μM) and *in vivo* (at 400 mg/kg, >70% reduction in both worm and egg burden) [76].

#### **5.2 High-throughput screening results**

Development of reliable high-throughput screening (HTS) tools promises to accelerate the identification of novel anti-schistosomiasis chemotypes [78]. Using a previously developed high-throughput protocol for screening NTS [79], Mansour et al. tested over 294,000 compounds taken from MMV, Pfizer, European Screening

**Figure 8.** *Other hits from phenotypic screening (***46***–***50***).*

*Recent Advances in Anti-Schistosomiasis Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.103056*

#### **Figure 9.**

*Leads resulting from a large high-throughput screening experiment (***51***–***59***).*

Port, GSK (the Tres Cantos Antimalarial Set), and Enamine [80]. The compounds from this set selected for further development, compounds **51**–**57** (**Figure 9**) and the previously mentioned TRP channel ligand **4**, demonstrated EC50 values under 7 μM for NTS, and under 15 μM for juvenile and adult worms.

Several of these leads bear indole or azaindole (e.g., triazolopyridine) units; indoles similar to **57** have also demonstrated activity against *S. mansoni* peroxiredoxin Prx2 and TGR in other high-throughput screening assays [81, 82]. Further development of the lead compound **52** led to the development of a series of pyrazolopyrimidines and imidazopyrazines, the latter typified by compounds **58** and **59** [83]. Compound **58** combined exceptional potency in *in vitro* testing (EC50 27 nM against juvenile worms, and 46 nM against adult worms) with decent metabolic stability and good *in vivo* efficacy.

Another HTS strategy uses ATP quantitation to assess test compounds' effect on the number and viability of schistosomula in a sample [84]. Applying this screen to a 40,000-sample set, followed by clustering and retesting, led to compounds **60**–**62** (**Figure 10**) being identified as the most promising leads [85]. The latter of those, perhexiline maleate (**62**), is an anti-angina drug whose efficacy against schistosomiasis had been studied previously [86, 87]. Starting from those three hits, pharmacophore

#### **Figure 10.** *Leads resulting from a high throughput screen using ATP quantitation (***60***–***67***).*

modeling resulted in the selection of compounds **63**–**67** as novel scaffolds for potential development. All eight of these compounds not only proved efficacious, *in vitro* and *in vivo*, against both juvenile and adult worms at 10 μM, but strongly impaired egg production in *S. mansoni* at sub-lethal doses (2.5–5 μM) [85].
