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W. Duan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1587–1590
CH3
extensive SAR study at other positions or with different substituent
groups. Significantly, PZQ analogs with potent antischistosomal
activity against both juvenile and adult stage worm are particularly
needed.
In this Letter, we report our exploration of hydroxylation at po-
sition 10 in PZQ aromatic ring, and the biological evaluation of this
compound as well as its peroxide hybrid conjugates against Schis-
tosoma J.
H
O
H3C
O
O
O
O
O
O
O
O
H
COOH
H
O
O
COOH
CH3
OH
11
12
10
O
Figure 3. Structures of intermediates 10–12.
A well-known antimalarial drug artemisinin, and its derivatives
both artemether and artesunate (Fig. 1) are reported to show antis-
chistosomal activity to some extent. Especially, their worm killing
ability against juvenile schistosomes is superior to PZQ.11 Later on,
some synthetic compounds harboring the artemisinin’s pharmoco-
phore endoperoxide bridge, that is, 1,2,3,4-trioxane/1,2,4-trioxane
motif, were discovered to show high in vitro and in vivo activity
against both juvenile and adult stage of schistosomes.12,13 We
imagine that a hybrid molecular of PZQ and the endoperoxide moi-
ety in a proper manner should generate compounds that are sensi-
tive to both stage of worms. This conjugation strategy is also
utilized by a successful antimalarial drug trioxaquinesÒ, a dual
molecules containing a quinoline and a 1,2,4-trioxane unit.14,15
Herein, we designed eight hybrid molecules (Fig. 2). Compounds
2–5 contain two covalently linked pharmacophores: an endoper-
oxide bridge as in artemisinin derivative and a hydroxyl/amino-
praziquentel as in praziquentel. In compounds 6–9, the bulky
artemisinin derivative was replaced by small synthetic moieties
containing the endoperoxide bridge: 1,2,4,5-tetraoxane or 1,2,
4-trioxane.
10-Hydroxy-PZQ (compound 1, not reported in open patent or
literature) was prepared from 10-amino-PZQ, which was prepared
according to a reported procedure.9 Treatment of 10-amino-PZQ
with NaNO2, and subsequent hydrolysis under strong acidic condi-
tion allowed transformation of the amino group to hydroxyl. This
one-pot reaction provided compound 1 in 50% yield. Coupling of
the 10-amino/hydroxyl-PZQ with artesunate through amide or es-
ter linkage gave compound 2 and 3. Accordingly, to synthesize
compound 4–9, intermediates 10–12 (Fig. 3) were prepared
according to the reported method.16–18 In all cases, amide/ester
bond formation employed 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) and N-hydroxybenzotriazole (HOBt) as the
general coupling reagents, compounds were obtained in 45–85%
yield in this step.
amphotericin B. Cultures were kept at 37 °C in an atmosphere of
5% CO2 in air and were observed under a Leica MZ 12.5 stereomi-
croscope. Worms of S. japonicum were exposed overnight (16 h)
to various drugs, washed and subsequently cultured in drug-free
medium. Juvenile worms were obtained after day 16 infection. In
contrast, adult worms were obtained at day 42 infection. 10-Hy-
droxy-PZQ and other hybrid compounds were then added from
200 mg/mL DMSO stock solutions to achieve a series of diluted
concentrations 10–50 lM. Compound activity was assessed by
mortality and motility disturbances within 72 h. The score of worm
vitality is illustrated as: 3 scores: the highest score, as observed in
the control group during the observation period. Worms moved
more actively and softly, and the body was transparent. 2 scores:
worms acted all over the body, but stiffly and slowly, with the body
translucent.
1 score: parasites moved partially with opaque
appearance. 0 score: the worm remained contracted, did not re-
sume movements, we could deem it ‘dead’. The average score of
10–12 worms was counted for in vitro test. Data representative
of repeated experiments was shown in Table 1 and Table 2.
For the in vivo screen, mice were infected with ca. 50 schistoso-
miasis. J cercariae on day 0 followed by administration of 200 mg/
kg oral doses of test compounds suspended in distilled water to
groups of ten mice on day 14 post-infection (juvenile stage) and
21 post-infection (adult stage). At certain days post-treatment, ani-
mals were sacrificed and dissected to assess total worm reduction
as described in detail.12
For all compounds tested against adult schistosomiasis. J (Table
1), compounds 1–6 show worm killing activity superior to PZQ.
The result of particular significance is for 1–3. At concentration
as low as 15–25
By comparison, worm mortality induced by 50
in 72 h. Compound 8 had the weakest activity, no worm was killed
at concentration up to 50 M, but the worm vitality score reduced
to 3. The potency of compound 7 is marginally higher than com-
pound 8. However, the worm mortality is only 60% at 100 M,
l
M, these three compounds killed 100% worm.
lM PZQ is 62.9%
All prepared analogs were evaluated for their ability against
both adult and juvenile schistosomes J. in vitro. According to previ-
ously described method,19,20 8–12 worms obtained from single-sex
male infections were distributed in duplicate tissue culture dishes
(3.5 cm) in Dulbecco’s modified minimum Eagle’s medium (bicar-
bonate buffered) supplemented with 20% newborn calf serum,
l
l
which is obviously much lower compared to PZQ at the same con-
centration. Compound 9 displayed similar potency to PZQ.
Based on the adult worm killing ability, compounds displayed
100 U mLꢀ1 penicillin, 100 g mLꢀ1 streptomycin and 0.5 g mLꢀ1
l l
significant potency at 50 lM were further evaluated for their abil-
ity against juvenile worm (Table 2). Again, compounds 1–3 demon-
strated significant worm killing activity, reduced worm vitality to
1: R = H, X = O
100% in 72 h at 15 lM. Compounds 5–7, although less potent than
1–3, also displayed considerable effect against juvenile worm.
Among these, compound 5 is slightly less effective compared to
H
O
H
2: R' = OCOCH2CH2CO, X = NH
3: R' = OCOCH2CH2CO, X = O
R
X
O
O
H
4: R '= CH2CO,
5: R' = CH2CO,
X = NH
X = O
1–3, killed 100% worm at 25
modest activity against juvenile worms, reduced worm motility
by 100% and 14.3% at 50 M.
For those compounds shown significant effect against both
adult and juvenile worm at 50 M, the worm reduction activity
was evaluated in vivo (Table 3). All tested compounds 1–3, 5, 6 dis-
played modest to high worm reduction ability, total worm reduc-
tion rate is higher than 39%. Compound 1 was able to reduce 40%
worm load in vivo, which is in sharp contrast to other PZQ simple
derivatives showing low level of activities,9,10 suggesting variation
in the aromatic ring may serve a good choice for development
lM in 72 h. Compounds 6 and 7 had
O
O
R'
N
l
R =
N
O
O
O
O
O
O
6: X = NH
7: X = O
l
O
X = NH2
OH
O
O
8: X =NH
9: X = O
O
Figure 2. Target PZQ analogs 1–9.
Duan, Wen-Wen
