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H. Yao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
Table 3
Worm burden and worm burden reductions of tested compounds in mice harboring a
Worm burden and worm burden reductions of tested compounds in mice harboring
juvenile S. japonicum infection
an adult S. japonicum infection
Compound
Dosage
(mg/kg)
No. of mice
investigated
Mean number
of worms (SD)
Worm burden
reduction (%)
Compound
Dosage
(mg/kg)
No. of mice
investigated
Mean number
of worms (SD)
Worm burden
reduction (%)
Control
MMV665852 400
15
16
17
18
37
38
5
6
5
5
5
6
4
6
5
39.8 (5.2)
27.0 (4.1)
28.8 (7.3)
35.2 (5.4)
34.0 (8.7)
32.3 (2.2)
37.3 (3.1)
26.5 (0.7)
11.0 (4.0)
—
Control
MMV665852 400
15
16
17
18
37
38
5
5
5
5
5
5
5
6
5
5
38.6 (2.3)
35.0 (4.1)
38.8 (5.3)
28.3 (5.3)
34.2 (3.7)
37.3 (4.0)
37.7 (3.1)
26.0 (3.7)
33.0 (4.8)
3.6 (1.1)
—
9.3
0
26.6
11.4
3.4
32.2⁄⁄
27.8⁄
11.6
14.6
18.8
6.4
400
200
400
400
400
200
400
400
200
400
400
400
200
200
400
2.3
33.4⁄
72.4⁄⁄
32.6⁄⁄
14.4
90.7⁄⁄
Artesunate
HP-b-CD-38
Praziquantel
SD, standard deviation. T-test was used to compare each compounds with the
control group (*p <0.05, **p <0.01).
SD, standard deviation. T-test was used to compare each compounds with the
control group (**p <0.01).
summarized in Table 2. Compound 38 exhibited the highest worm
reduction activity, with a worm burden reduction (WBR) of 33.4%
(P <0.05) at 200 mg/kg single oral dose. By comparison, the positive
control drug artesunate at 400 mg/kg characterized by a WBR of
72.4%. Other compounds resulted in WBRs as follows: compound
MMV665852, 32.2%; compound 15, 27.8%; compound 16, 11.6%;
compound 17, 14.6%; compound 18, 18.8% and compound 37,
6.4%. Toxicity was observed for compounds 16 and 38 at 400 mg/
kg: five out of six mice died after 1–2 d post-treatment, especially
for compound 38, two out of six tested mice died at day 1 and day
2 post-treatment at the dosage of 200 mg/kg,.
In mice harboring an adult S. japonicum infection, compound 38
also revealed the highest activity with worm burden reductions
(WBR) of 32.6% (P <0.01) (Table 3). Although compounds
MMV665852, 15, 17, 18 and 37 displayed high efficacy against
the adult worms in vitro, these compounds had a low in vivo
activity (WBR 0–11.4%) and no statistically significances.
Reasons, for example: low compound aqueous solubility, for the
in vitro/in vivo discrepancy were suggested. Therefore, hydrox-
ypropyl-b-cyclodextrin packed compound 38 was prepared and
the same dosage as free drug was administrated to the infected
mice. Unfortunately, a slight worm burden reduction rate decrease
was recorded compared to the free compound.
Schistosomiasis is listed as one of the neglected tropical dis-
eases by WHO. Despite the large number of people that are con-
taminated or being exposed to circumstance of infection in
tropical countries, research efforts toward the development of
novel antischistosomal drugs are slow over a half century. The bio-
logical knowledge in the parasite research field are far from rich in
the science community, resulting in the dependence on phenotype
screening as the main strategy for parasite drug discovery. Still, the
scope of reference molecules as lead candidates is inadequate.
MMV665852 was discovered and reported from screening over
400 commercially available malaria-active compounds by the
Keiser group. A single oral dose of 400 mg/kg to mice infected by
S. mansoni revealed high worm burden reduction of 52.5%. Due
to the low molecular weight and structural simplicity of
MMV665852, we figured that further investigation based on this
scaffold might reveal candidates with improved antischistosomal
activity as well as drug like property.
The MMV665852 compound is characterized by good copla-
narity and high rigidity, resulting in its low aqueous solubility and
probably consequent low in vivo activity. Therefore, our initial mod-
ification efforts focus on urea linker modification, with the aim of
increasing the linker flexibility. Our designed linkers are commonly
observed structural units and bioisosteres in medicinal chemistry.
Disappointingly, introduction of sp3 hybridized carbon to improve
the molecular flexibility failed to achieve the mission of activity
enhancement. Only the sulfamide 1 and triazine linker analog 10
displayed moderate in vitro worm killing capability. We then
resorted to installation of sp3 flexible carbons to the phenyl ring of
the urea analogs (compounds 18 and 39). Again, decreased worm
killing activity was observed for these two compounds in vitro.
An extensive structural activity relationship was investigated
on the substituents at the phenyl rings. We revealed that electron
withdrawing groups favored the compound antischistosomal
activity, while electron donating groups would do the contrary.
At the same time, the substitute position at the phenyl ring also
influenced the compound activity, with meta- or para- other than
ortho-position being helpful, as demonstrated by seven low IC50
value compounds: halogen or electron withdrawing trifluo-
romethyl group at meta- or para-positions (compounds 15, 16,
17, 18, 19, 37 and 38).
As mentioned by another research group,19 low aqueous solu-
bility was observed for these urea analogs. Efforts were spent to
optimize the solvent system in order to make a homogenous solu-
tion. Our tested solvents include: polyethylene glycol with H2O at
different ratio, glycerol–H2O at different ratio, even high liposolu-
bility corn oil couldn’t fully dissolve these compounds. Then, a sus-
pension of compound in corn oil facilitated by ultrasound was
administrated to the infected mice. For comparison, a complex of
hydroxypropyl-b-cyclodextrin (HP-b-CD) with compound 38 was
prepared and solution of this complex was tested in infected mice.
Unfortunately, the CD complex resulted in reduced worm burden
reduction. The results from corn oil suspensions were provided
in Tables 2 and 3.
Finally, the discrepancy for in vivo worm burden reduction
between reported data for S. mansoni and our tested S. japonicum
caught our attentions, although there was precedent that certain
type of compound would display different activity on S. mansoni
vs S. japonicum.20 The lead compound MMV665852 could achieve
worm burden reduction of 52.5% at single oral dose 400 mg/kg
treatment of mice harboring S. mansoni, while this number was
only 9.3% when applying to S. japonicum. Whether or not this sig-
nificant difference was related to the drug working mechanism
would be worthy of being studied further.
In summary, 45 N,N0-arylurea analogs were prepared and evalu-
ated as antischistosomal agents against S. japonicum. Although
some compounds exhibited strong in vitro worm killing potency,
their worm burden reduction activity was not promising in vivo.
Interestingly, the observed antischistosomal activity difference
between S. mansoni and S. japonicum might imply these compounds
inhibition mechanism being related to worm species specificity.
Acknowledgements
The Project is sponsored by the Scientific Research Foundation
for the Returned Overseas Chinese Scholars, State Education Min-
istry, Jiangsu province 333 project funding to Chunhua Qiao.