Deciphering the enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using complementary computational tools

Article abstract of DOI:10.1080/14756366.2020.1712595

A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in v

Full text of DOI:10.1080/14756366.2020.1712595

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Deciphering the enzymatic target of a new family
of antischistosomal agents bearing a quinazoline
scaffold using complementary computational
tools
Victor Sebastian-Perez, Alfonso García-Rubia, Sayed H. Seif el-Din, Abdel-
Nasser A. Sabra, Naglaa M. El-Lakkany, Samia William, Tom L. Blundell, Louis
Maes, Ana Martinez, Nuria E. Campillo, Sanaa S. Botros & Carmen Gil
To cite this article: Victor Sebastian-Perez, Alfonso García-Rubia, Sayed H. Seif el-Din,
Abdel-Nasser A. Sabra, Naglaa M. El-Lakkany, Samia William, Tom L. Blundell, Louis Maes,
Ana Martinez, Nuria E. Campillo, Sanaa S. Botros & Carmen Gil (2020) Deciphering the
enzymatic target of a new family of antischistosomal agents bearing a quinazoline scaffold using
complementary computational tools, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
511-523, DOI: 10.1080/14756366.2020.1712595
To link to this article: https://doi.org/10.1080/14756366.2020.1712595
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 511–523
https://doi.org/10.1080/14756366.2020.1712595
RESEARCH PAPER
Deciphering the enzymatic target of a new family of antischistosomal agents
bearing a quinazoline scaffold using complementary computational tools
a
a
b
b
ꢀ
ꢀ
ꢀ
, Alfonso Garcıa-Rubia , Sayed H. Seif el-Din , Abdel-Nasser A. Sabra ,
Victor Sebastian-Perez
Naglaa M. El-Lakkanyb, Samia Williamc, Tom L. Blundelld, Louis Maese, Ana Martineza , Nuria E. Campilloa
Sanaa S. Botrosb and Carmen Gila
,
a
b
ꢀ
Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain; Pharmacology Department, Theodor Bilharz Research Institute, Giza, Egypt;
cParasitology Department, Theodor Bilharz Research Institute, Giza, Egypt; Department of Biochemistry, University of Cambridge, Cambridge,
UK; Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium
d
e
ABSTRACT
ARTICLE HISTORY
Received 3 October 2019
Revised 10 December 2019
Accepted 27 December 2019
A previous phenotypic screening campaign led to the identification of a quinazoline derivative with prom-
ising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of
this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant
reduction of total worms and a complete disappearance of immature eggs when administered concomi-
tantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost
importance because eggs are responsible for the pathology and transmission of the disease.
Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of
the parent compound was carried out leading to derivatives with improved drug-like properties.
Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using
computational tools and the binding mode to the target enzyme, aldose reductase, was proposed.
KEYWORDS
Drug discovery; quinazoline;
Schistosoma mansoni;
target deconvolution
GRAPHICAL ABSTRACT
Introduction
representing a serious health problem especially in poor commun-
ities. The disease has recently also reached Europe, demonstrating
the possibility to emerge in new geographical areas previously
unknown related to migration movements and parasite gen-
etic variants2.
Treatment and control of all forms of schistosomiasis fully rely
on mass drug administration with the only available antischistoso-
mal drug praziquantel (PZQ)3. Even considering it is safe, effective,
operationally convenient and low-cost, there is an increasing con-
Schistosomiasis is a parasitic infectious disease caused by a trema-
tode belonging to Schistosoma spp. Transmission occurs through
contact with freshwater that is contaminated with larval forms
(furcocercariae). Once in the human body, the larvae become
adults in the blood vessels where the females release eggs. Part
of the eggs is passed in the faeces or urine to continue the para-
site’s life cycle by contaminating water while most become
trapped in body tissues causing immune-inflammatory responses
and progressive damage to organs1. This neglected tropical dis- cern among the scientific community to anticipate PZQ thera-
ease is endemic in a number of tropical and subtropical countries peutic failure4. The massive use for many years has clearly
ꢀ
CONTACT Carmen Gil
carmen.gil@csic.es
Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain; Sanaa S. Botros
s.botros@tbri.gov.eg
Pharmacology Department, Theodor Bilharz Research Institute, Giza 12411, Egypt
ꢀ
These authors have contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

512
V. SEBASTIAN-PEREZ ET AL.
Chemical shifts for 13C NMR are reported in terms of chemical
shift (d ppm). Purity of assayed compounds was determined by
elemental analysis recorded on Heraeus CHN-O-rapid analyser per-
formed by the analytical department at CAI (UCM) and values
were within 0.4% of the theoretical values for all compounds.
Method A (R35Me). General procedure for the synthesis of
substituted 3-benzylquinazolin-2,4(1H,3H)-dione using anthranilate
methyl ester derivatives as starting material. In a round-bottomed
flask with a magnetic stir bar, the corresponding anthranilate
methyl ester is dissolved in toluene (5 mL/mmol) and cold at 0 ꢁC.
1.1 equiv. of the corresponding isocyanate is added to the mix-
ture, which is stirred overnight at rt. Then, 8 mL of toluene and
2 mL/mmol of NaOH (6 M) is added and the mixture is heated to
70 ꢁC until complete conversion to the 3-substituted quinazoline-
dione (6 h). The reaction mixture is cooled in an ice bath and
water (5 mL/mmol) is added, which causes a precipitate. This slurry
is stirred for 30 min, filtered, washed with water (5 mL/mmol), and
then heptanes (3 ꢂ 10 mL/mmol) before drying the solids under a
stream of nitrogen. Products are generally obtained as white or
off-white solids and were used in the next step without further
purification.
NPD-1246 (1)
- EC50 value of 50 mM in male and female Schistosoma mansoni.
- Uncoupling with absence of eggs at 100, 50 and 25 mM.
- Reduction in egg number at 25, 10 and 5 mM.
Figure 1. In vitro findings for NPD-1246 (1) previously reported14.
increased the risk of resistance development. This fact, together
with the lack of efficacy against immatures makes the develop-
ment of new drugs more urgent5.
Historically, drug discovery for schistosomiasis has been based
on phenotypic screening using whole-organism assays, however,
new chemotherapeutics with known mechanism-of-action (MOA)
are highly desirable to anticipate drug resistance6,7. Particular
advantages and disadvantages of phenotypic vs. target-based
approaches are well known, and the combination of both strat-
egies is logically the best way to move forward and optimise the
drug discovery process8,9. Recognising the pivotal role of target
identification and the challenging task of identifying the MOA for
bioactive small molecules, significant progress has been made to
develop a number of computational strategies to unveil the MOA
of phenotypic hits10. In silico target identification offers chances
for drug repurposing and for the detection of new links between
disease and known targets. Large datasets such as ChEMBL11 or
PubChem12 are now available and contain an impressive amount
of biological data related to the activity of millions of ligands in
multiple assays. As such, they provide an invaluable source of
information for the development of knowledge-based approaches
guided by computational techniques13.
In a previous in vitro phenotypic screening campaign using
Schistosoma mansoni with worm killing as primary outcome, com-
pounds from a selected library were successfully classified and pri-
oritised based on potency and selectivity14. The present work
expands the evaluation of the antischistosomal activity for the
quinazoline NPD-1246 (1) (Figure 1) which was one of the best
in vitro “hits”, involving (i) in vivo evaluation in the S. mansoni-
infected mouse model, (ii) a medicinal chemistry programme to
obtain better drug-like compounds and (iii) exploration of the
putative target using computational consensus methodology
applying ligand-based approaches.
Method B (R35H). General procedure for the synthesis of sub-
stituted 3-benzylquinazolin-2,4(1H,3H)-dione using anthranilic acid
derivatives as starting material. In a round-bottomed flask with a
magnetic stir bar, the corresponding anthranilic acid derivative is
dissolved in diethyl ether (8 mL/mmol) and 1.1 equiv. of the corre-
sponding isocyanate is added drop by drop to the mixture, which
is stirred overnight at rt. The solvent is rotary evaporated and
replaced with EtOH (10 mL/mmol). Then, 2 mL/mmol of concen-
trated HCl (12.1 M) is added and the mixture is heated to 70 ꢁC
until complete conversion to the 3-substituted quinazolinedione
(3 h). The reaction mixture is cooled in an ice bath and water
(5 mL/mmol) is added, which causes a precipitate. This slurry is
stirred for 30 min, filtered, washed with water (5 mL/mmol), and
then heptanes (3 ꢂ 10 mL/mmol) before drying the solids under a
stream of nitrogen. Products are generally obtained as white or
off-white solids and were used in the next step without further
purification.
General procedure for the synthesis of substituted 3-ben-
zyl-1-(4-(trifluoromethyl)benzyl)quinazolin-2,4(1H,3H)-dione. In
a round-bottomed flask with a magnetic stir bar, substituted
3-benzylquinazolinedione previously obtained by method A or B
is dissolved in DMF (3mL/mmol) and 1.5 equiv. of 4-(trifluorome-
thyl)benzyl chloride and 3 equiv. of NaHCO3 are added. The mix-
ture is heated to 130 ꢁC overnight. The reaction mixture is cooled
and water (5mL/mmol) and EtOAc (15 mL/mmol) are added. Upon
separation of the layers, the aqueous phase was extracted with
EtOAc (3 ꢂ 10 mL). The organic layer was washed sequentially with
sat. aq. NH4Cl, brine, then dried over Na2SO4 anhydrous. The des-
iccant was filtered and solvent removed under vacuum. The resi-
due was purified by flash column chromatography using as
eluents mixtures of solvents (EtOAc/hexane, 1:9–6:4) as indicated
in each case to obtain the desired products.
Materials and methods
Chemical procedures
Substrates were purchased from commercial sources and used
without further purification. Melting points were determined with
a Mettler Toledo MP70 apparatus. Flash column chromatography
was carried out with automated silica gel column chromatography
at medium pressure using silica gel (E. Merck, Grade 60, particle
size 0.040–0.063 mm, 230–240 mesh ASTM) with the indicated
solvent as eluent. Compounds were detected with UV light
(254 nm). 1H NMR or 13C NMR experiments were obtained on the
Bruker AVANCE-300 or Bruker AVANCE-500 spectrometers. 1H and
13C spectra were calibrated using residual chloroform or DMSO as
an internal reference (CHCl3: d 7.26 ppm and d 77.3 ppm, respect-
ively. DMSO: 2.54 ppm and 40.4 ppm, respectively). Chemical shifts
3-Benzyl-1-(4-(trifluoromethyl)benzyl)quinazoline-2,4(1H,3H)-
dione
(1.2 mmol) obtained by method A from methyl 2-aminobenzoate
and 1-(isocyanatomethyl)benzene), 4-(trifluoromethyl)benzyl
(1).
Reagents:
3-benzylquinazolin-2,4(1H,3H)-dione
chloride (266 mL, 1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF
(6.0 mL). Purification: EtOAc/Hex (1:4). Yield: 207 mg, 42%. Whitish
solid. mp: 160–162 ꢁC. mp lit.15: 160–161 ꢁC.
1
for H NMR are reported as follows: chemical shift (d ppm), multi-
plicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, dd ¼ double
3-(4-Fluorobenzyl)-1-(4-(trifluoromethyl)benzyl)quinazolin-2,
of doublets, ddd ¼ doublet of double of doublets, td ¼ triplet of 4(1H,3H)-dione (2). Reagents: 3-(4-fluorobenzyl)quinazolin-
doublets, m ¼ multiplet), coupling constant (Hz), and integration. 2,4(1H,3H)-dione (1.2 mmol) obtained by method A from methyl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
513
2-aminobenzoate and 1-fluoro-4-(isocyanatomethyl)benzene), from 2-amino-4-bromobenzoic acid and 1-fluoro-4-(isocyanatome-
4-(trifluoromethyl)benzyl chloride (266 mL, 1.8 mmol), NaHCO3
(302 mg, 3.6 mmol) and DMF (6.0 mL). Purification: EtOAc/Hex
(1:4). Yield: 102 mg, 20%. Whitish solid. mp: 142–144 ꢁC. 1H NMR
(300 MHz, CDCl3) d 8.18 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.57–7.39 (m,
5H), 7.26 (d, J ¼ 8.0 Hz, 2H), 7.20–7.09 (m, 1H), 6.92 (t, J ¼ 8.7 Hz,
3H), 5.34 (s, 2H), 5.21 (s, 2H). 13C NMR (75 MHz, CDCl3) d 162.4
(d, JC-F¼247 Hz), 161.5, 151.3, 139.7 (q, JC-F¼2 Hz), 139.6, 135.3,
132.6 (d, JC-F ¼ 3 Hz), 131.1 (d, JC-F¼ 8 Hz), 130.0 (q, JC-F ¼ 32 Hz),
129.4, 126.7, 126.0 (q, JC-F ¼ 4 Hz), 124.1 (q, JC-F ¼ 272 Hz), 123.4,
115.7, 115.3 (d, JC-F ¼ 22 Hz), 114.0, 47.0, 44.4. Anal.
(C23H16F4N2O2) Calculated: C 64.49%, H 3.76%, N 6.54%. Found:
C 64.19%, H 3.80%, N 6.49%.
thyl)benzene),
4-(trifluoromethyl)benzyl
chloride
(266 mL,
1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF (6.0 mL).
Purification: EtOAc/Hex (1:4). Yield: 140 mg, 22%. Whitish solid.
mp: 153–155 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.13 (d, J ¼ 8.4 Hz,
1H), 7.64 (d, J ¼ 8.0 Hz, 2H), 7.50–7.41 (m, 2H), 7.44–7.32 (m, 3H),
7.22 (d, J ¼ 1.6 Hz, 1H), 7.02 (t, J ¼ 8.7 Hz, 2H), 5.39 (s, 2H), 5.28 (s,
2H). 13C NMR (75 MHz, CDCl3) d 162.5 (d, JC-F ¼ 247 Hz), 160.9,
151.0, 140.5, 139.0 (q, JC-F ¼ 2 Hz), 132.3 (d, JC-F ¼ 2 Hz), 131.1 (d, JC-
¼ 8 Hz), 130.7, 130.3 (q, JC-F ¼ 32 Hz), 130.3, 126.9, 126.7, 126.2 (q,
F
JC-F ¼ 4 Hz), 123.8 (q, JC-F ¼ 272 Hz), 117.0, 115.3 (d, JC-F ¼ 22 Hz),
114.5, 47.14, 44.59. Anal. (C23H15BrF4N2O2) Calculated: C 54.46%, H
2.98%, N 5.52%. Found: C 54.43%, H 3.01%, N 5.50%.
1-(4-(Trifluoromethyl)benzyl)3-(4-methoxybenzyl)quinazolin-
2,4(1H,3H)-dione (3). Reagents: 3-(4-methoxybenzyl)quinazolin-
2,4(1H,3H)-dione (1.2 mmol) obtained by method A from methyl
2-aminobenzoate and 1-(isocyanatomethyl)-4-methoxybenzene),
4-(trifluoromethyl)benzyl chloride (266 mL, 1.8 mmol), NaHCO3
(302 mg, 3.6 mmol) and DMF (6.0 mL). Purification: EtOAc/Hex
(1:4). Yield: 111 mg, 21%. Whitish solid. mp: 130–132 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 8.21–8.11 (m, 1H), 7.80–7.66 (m, 3H),
7.62–7.52 (m, 2H), 7.42–7.25 (m, 4H), 6.98–6.87 (m, 2H), 5.53 (s,
2H), 5.18 (s, 2H), 3.77 (s, 3H). 13C NMR (75 MHz, DMSO-d6) d
161.5, 158.9, 151.3, 141.7, 139.9, 135.9, 129.8, 129.5, 128.6, 128.3
(d, JC-F¼32.1 Hz), 127.6, 126.0 (q, J ¼ 3.8 Hz), 124.5 (q, J ¼ 272 Hz),
123.6, 122.8, 115.4 (d, JC-F¼21.4 Hz), 114.1, 55.5, 46.6, 44.4. Anal.
(C24H19F3N2O3) Calculated: C 65.45%, H 4.35%, N 6.36%. Found: C
65.26%, H 4.47%, N 5.98%.
7-Bromo-1-(4-(trifluoromethyl)benzyl)-3-(4-methoxybenzyl)-
quinazolin-2,4(1H,3H)-dione (7). Reagents: 7-bromo-3-(4-
methoxybenzyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained
by method B from 2-amino-4-bromobenzoic acid and 1-(isocya-
natomethyl)-4-methoxybenzene),
4-(trifluoromethyl)benzyl
chloride (266 mL, 1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and
DMF (6.0mL). Purification: EtOAc/Hex (1:4). Yield: 218 mg, 35%.
Whitish solid. mp: 138–140 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.39
(d, J ¼ 2.4 Hz, 1H), 7.62 (dd, J ¼ 8.2, 4.5 Hz, 3H), 7.56–7.47 (m,
2H), 7.34 (d, J ¼ 8.0 Hz, 2H), 6.88 (m, 3H), 5.40 (s, 2H), 5.28 (s,
2H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3) d 160.4, 159.2, 151.0,
139.3 (q, JC-F ¼ 2 Hz), 138.5, 137.9, 131.8, 130.7, 130.2 (q,
JC-F ¼ 32 Hz), 128.7, 126.6, 126.1 (q, JC-F ¼ 4 Hz), 123.8 (q,
JC-F ¼ 272 Hz), 117.4, 116.3, 115.8, 113.8, 55.2, 47.1, 44.8. Anal.
(C24H18BrF3N2O3) Calculated: C 55.51%, H 3.49%, N 5.39%.
Found: C 55.31%, H 3.54%, N 5.30.
6-Chloro-3-(4-fluorobenzyl)-1-(4-(trifluoromethyl)benzyl)qui-
nazolin-2,4(1H,3H)-dione (8). Reagents: 6-chloro-3-(4-fluoroben-
zyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained by method A
from methyl 2-amino-5-chlorobenzoate and 1-fluoro-4-(isocyana-
tomethyl)benzene), 4-(trifluoromethyl)benzyl chloride (266 mL,
1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF (6.0 mL).
Purification: EtOAc/Hex (1:4). Yield: 55 mg, 10%. Whitish solid.
mp: 160–162 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.19 (d, J ¼ 8.5 Hz,
1H), 7.62 (d, J ¼ 8.1 Hz, 2H), 7.54 (dd, J ¼ 8.6, 5.5 Hz, 2H), 7.34 (d,
J ¼ 8.0 Hz, 2H), 7.21 (dd, J ¼ 8.5, 1.7 Hz, 1H), 7.05–6.95 (m, 3H),
5.37 (s, 2H), 5.26 (s, 2H). 13C NMR (75 MHz, CDCl3) d 163.0 (d,
JC-F ¼ 247 Hz), 161.2, 151.5, 142.2, 140.9, 139.4 (q, JC-F ¼ 2 Hz),
132.7 (d, JC-F ¼ 3 Hz), 131.5 (d, JC-F ¼ 8 Hz), 131.1, 130.7 (q,
JC-F ¼ 32 Hz), 127.1, 126.5 (q, JC-F ¼ 4 Hz), 124.4, 124.1 (q,
JC-F ¼ 272 Hz), 115.7 (d, JC-F ¼ 22 Hz), 114.5, 114.5, 47.5, 44.9. Anal.
(C23H15ClF4N2O2) Calculated: C 59.69%, H 3.27%, N 6.05%. Found:
C 59.62%, H 3.30%, N 6.01%.
6-Chloro-1-(4-(trifluoromethyl)benzyl)-3-(4-methoxybenzyl)-
quinazolin-2,4(1H,3H)-dione (9). Reagents: 6-chloro-3-(4-methox-
ybenzyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained by
method A from methyl 2-amino-5-chlorobenzoate and 1-(isocyana-
tomethyl)-4-methoxybenzene), 4-(trifluoromethyl)benzyl chloride
(266 mL, 1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF (6.0 mL).
Purification: EtOAc/Hex (1:4). Yield: 387 mg, 71%. Whitish solid.
mp: 145–147 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.21 (d, J ¼ 8.4 Hz,
1H), 7.64 (d, J ¼ 8.1 Hz, 2H), 7.58–7.47 (m, 2H), 7.37 (d, J ¼ 8.0 Hz,
2H), 7.22 (dd, J ¼ 8.5, 1.7 Hz, 1H), 7.02 (d, J ¼ 1.7 Hz, 1H), 6.91–6.83
(m, 2H), 5.39 (s, 2H), 5.27 (s, 2H), 3.81 (s, 3H). 13C NMR (75 MHz,
CDCl3) d 160.8, 159.2, 151.2, 141.6, 140.5, 139.1 (q, JC-F ¼ 2 Hz),
130.7, 130.2 (q, JC-F ¼ 32 Hz), 128.8, 126.7, 126.1 (q, JC-F ¼ 4 Hz),
124.0 (q, JC-F ¼ 272 Hz), 123.9, 114.3, 114.0, 113.8, 55.2, 47.1, 44.7.
Anal. (C24H18ClF3N2O3) Calculated: C 60.70%, H 3.82%, N 5.90%.
6-Bromo-3-(4-fluorobenzyl)-1-(4-(trifluoromethyl)benzyl)qui-
nazolin-2,4(1H,3H)-dione (4). Reagents: 6-bromo-3-(4-fluoroben-
zyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained by method B
from 2-amino-5-bromobenzoic acid and 1-fluoro-4-(isocyanato-
methyl)benzene), 4-(trifluoromethyl)benzyl chloride (191 mL,
1.5 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF (6.0 mL).
Purification: EtOAc/Hex (1:4). Yield: 176 mg, 29%. Whitish solid.
mp: 186–188 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.29 (d, J ¼ 2.4 Hz,
1H), 7.59–7.37 (m, 5H), 7.24 (d, J ¼ 8.0 Hz, 2H), 6.92 (t, J ¼ 8.7 Hz,
2H), 6.81 (d, J ¼ 8.9 Hz, 1H), 5.31 (s, 2H), 5.19 (s, 2H). 13C NMR
(75 MHz, CDCl3) d 162.3 (d, JC-F ¼ 247 Hz), 160.4, 150.9, 139.2 (q,
JC-F ¼ 2 Hz), 138.5, 138.1, 132.3 (d, JC-F ¼ 2 Hz), 131.8, 131.2 (d, JC-
¼ 8 Hz), 130.3 (q, JC-F ¼ 32 Hz), 126.6, 126.1 (q, JC-F ¼ 4 Hz), 123.7
F
(q, JC-F ¼ 272 Hz), 117.3, 116.4, 115.9, 115.3 (d, JC-F ¼ 22 Hz), 47.2,
44.6. Anal. (C23H15BrF4N2O2) Calculated: C 54.46%, H 2.98%, N
5.52%. Found: C 54.50%, H 2.94%, N 5.51%.
6-Bromo-1-(4-(trifluoromethyl)benzyl)-3-(4-methoxybenzyl)-
quinazolin-2,4(1H,3H)-dione (5). Reagents: 6-bromo-3-(4-
methoxybenzyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained by
method B from 2-amino-5-bromobenzoic acid and 1-(isocyanato-
methyl)-4-methoxybenzene), 4-(trifluoromethyl)benzyl chloride
(266 mL, 1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF (6.0 mL).
Purification: EtOAc/Hex (1:4). Yield: 405 mg, 65%. Whitish solid.
mp: 147–149 ꢁC. 1H NMR (300 MHz, CDCl3) d 8.10 (d, J ¼ 8.4 Hz,
1H), 7.61 (d, J ¼ 8.1 Hz, 2H), 7.50 (d, J ¼ 8.7 Hz, 2H), 7.42–7.29 (m,
3H), 7.18 (d, J ¼ 1.6 Hz, 1H), 6.91–6.80 (m, 2H), 5.36 (s, 2H), 5.24 (s,
2H), 3.78 (s, 3H). 13C NMR (75 MHz, CDCl3) d 161.3, 159.6, 151.5,
140.8, 139.5 (q, JC-F ¼ 2 Hz), 131.1, 131.1, 130.6 (q, JC-F ¼ 32 Hz),
130.5, 129.1, 127.1, 127.1, 126.5 (q, JC-F ¼ 4 Hz), 124.3 (q,
JC-F ¼ 272 Hz), 117.3, 115.0, 114.2, 55.6, 47.5, 45.1. Anal.
(C24H18BrF3N2O3) Calculated: C 55.51%, H 3.49%, N 5.39%. Found:
C 55.63%, H 3.55%, N 5.31%.
7-Bromo-3-(4-fluorobenzyl)-1-(4-(trifluoromethyl)benzyl)qui-
nazolin-2,4(1H,3H)-dione (6). Reagents: 7-bromo-3-(4-fluoroben-
zyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained by method B Found: C 60.72%, H 3.80%, N 5.86%.

514
V. SEBASTIAN-PEREZ ET AL.
3-(4-Fluorobenzyl)-1-(4-(trifluoromethyl)benzyl)-6,7-dimethox- stirred for 30 min, filtered, washed with water (10 mL/mmol), and
yquinazolin-2,4(1H,3H)-dione (10). Reagents: 3-(4-fluorobenzyl)-
6,7-dimethoxyquinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained by
method A from methyl 2-amino-4,5-dimethoxibenzoate and 1-flu-
oro-4-(isocyanatomethyl)benzene), 4-(trifluoromethyl)benzyl chlor-
ide (266 mL, 1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF
(6.0 mL). Purification: EtOAc/Hex (1:2). Yield: 73 mg, 15%. Whitish
solid. mp: 180–182 ꢁC. 1H NMR (300 MHz, CDCl3) d 7.62 (m, 3H),
7.57 (dd, J ¼ 8.6, 5.5 Hz, 2H), 7.38 (d, J ¼ 8.0 Hz, 2H), 7.02 (t,
J ¼ 8.7 Hz, 2H), 6.43 (s, 1H), 5.42 (s, 2H), 5.30 (s, 2H), 3.93 (s, 3H),
3.77 (s, 3H). 13C NMR (75 MHz, CDCl3) d 162.7 (d, JC-F ¼ 246 Hz),
161.5, 155.6, 151.9, 146.2, 140.3 (q, JC-F ¼ 2 Hz), 135.6, 133.2 (d,
JC-F ¼ 3 Hz), 131.4 (d, JC-F ¼ 8 Hz), 130.6 (q, JC-F ¼ 32 Hz), 127.1,
126.4 (q, JC-F ¼ 4 Hz), 124.0 (q, JC-F ¼ 272 Hz), 115.6 (d, JC-F ¼ 22 Hz),
109.6, 108.5, 97.4, 56.7, 56.5, 47.7, 44.8. Anal. (C25H20F4N2O4)
Calculated: C 61.48%, H 4.13%, N 5.74%. Found: C 60.67%, H
4.11%, N 5.68%.
then heptanes (3 ꢂ 10 mL/mmol) before drying the solids under a
stream of nitrogen); 4-(trifluoromethyl)benzyl chloride (266 mL,
1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF (6.0 mL).
Purification: EtOAc/Hex (1:4). Yield: 204 mg, 32%. Whitish solid.
mp: 158–160 ꢁC. 1H NMR (500 MHz, CDCl3) d 8.10 (dd, J ¼ 2.3,
0.9 Hz, 1H), 7.71 (dd, J ¼ 2.3, 0.8 Hz, 1H), 7.57 (d, J ¼ 8.1 Hz, 2H),
7.50–7.40 (m, 2H), 7.34–7.23 (m, 2H), 6.84 (d, J ¼ 8.7 Hz, 2H), 5.76
(s, 2H), 5.19 (s, 2H), 3.80 (s, 3H), 2.39 (s, 3H). 13C NMR (125 MHz,
CDCl3) d 161.1, 159.6, 152.6, 143.2, 142.2, 137.6, 135.5, 131.0, 129.6
(q, JC-F ¼ 32 Hz), 129.6, 129.1, 126.9, 125.8 (q, JC-F ¼ 4 Hz), 124.4 (q,
JC-F ¼ 272 Hz), 120.0, 114.1, 107.8, 55.6, 51.6, 45.2, 20.4. Anal.
(C25H20BrF3N2O3) Calculated: C 56.30%, H 3.78%, N 5.25%. Found:
C 56.20%, H 3.76%, N 5.18%.
Microsomal stability assays
1-(4-(Trifluoromethyl)benzyl)-6,7-dimethoxy-3-(4-methoxyben-
zyl)quinazolin-2,4(1H,3H)-dione (11). Reagents: 6-7-dimethoxy-3-
(4-methoxybenzyl)quinazolin-2,4(1H,3H)-dione (1.2 mmol) obtained
by method A from methyl 2-amino-4,5-dimethoxibenzoate and
1-(isocyanatomethyl)-4-methoxybenzene), 4-(trifluoromethyl)benzyl
chloride (266 mL, 1.8 mmol), NaHCO3 (302 mg, 3.6 mmol) and DMF
(6.0 mL). Purification: EtOAc/Hex (1:2). Yield: 72 mg, 12%. Whitish
solid. mp: 171–173 ꢁC. 1H NMR (300 MHz, CDCl3) d 7.55 (s, 1H),
7.52 (d, J ¼ 8.0 Hz, 2H), 7.44 (d, J ¼ 8.6 Hz, 2H), 7.28 (d, J ¼ 8.0 Hz,
2H), 6.78 (d, J ¼ 8.7 Hz, 2H), 6.32 (s, 1H), 5.32 (s, 2H), 5.19 (s, 2H),
Mouse or human liver microsomes (S9), reduced nicotinamide
adenine dinucleotide phosphate (NADPH) generating system solu-
tions and uridine glucuronosyltransferase (UGT) reaction mix (BD
Biosciences) were kept at ꢃ80 ꢁC. The test compounds and refer-
ence compound diclofenac were formulated in DMSO at 10 mM.
The assay was carried out based on the BD Biosciences Guidelines
for Use (TF000017 Rev1.0) with minor adaptations. The metabolic
stability of the compounds was studied through the CYP450 super-
family (Phase-I metabolism) by fortification with NADPH and
3.83 (s, 3H), 3.70 (s, 3H), 3.67 (s, 3H). 13C NMR (75 MHz, CDCl3) d through UGT enzymes (Phase-II metabolism) by fortification with
161.6, 159.5, 155.5, 151.9, 146.2, 140.5 (q, JC-F ¼ 2 Hz), 135.6, 131.0,
uridine diphosphate glucuronic acid (UDPGA). For CYP450 and
other NADPH dependent enzymes, the compounds were incu-
bated at 5 mM together with 0.5 mg/mL S9 in potassium phos-
130.5 (q, JC-F ¼ 32 Hz), 129.7, 127.1, 126.4 (q, JC-F ¼ 4 Hz), 124.3 (q,
JC-F ¼ 272 Hz), 114.1, 109.6, 108.6, 97.4, 56.6, 56.5, 55.6, 47.6, 45.0.
Anal. (C26H23F3N2O5) Calculated: C 62.40%, H 4.63%, N: 5.60%.
Found: C 62.09%, H 4.56%, N: 5.52%.
phate buffer in a reaction started by the addition of 1 mM NADP.
At defined time points, 20 mL was withdrawn from the reaction
mixture and 80 mL cold acetonitrile (ACN) was added to inactivate
the enzymes and precipitate the protein. The mixture was vor-
texed for 30 s and centrifuged at 4 ꢁC for 5 min to collect the
supernatant. For UGT enzymes, the compounds were incubated at
5 mM together with 0.5 mg/mL S9 in a reaction started by the add-
ition of 2 mM UDPGA cofactor. The loss of parent compound was
8-Bromo-3-(4-fluorobenzyl)-1-(4-(trifluoromethyl)benzyl)-6-
methylquinazolin-2,4(1H,3H)-dione (12). Reagents: 8-bromo-3-
(4-fluorobenzyl)-6-methylquinazolin-2,4(1H,3H)-dione
(1.2 mmol)
obtained by method B from 2-amino-3-bromo-6-methylbenzoic
acid and 1-fluoro-4-(isocyanatomethyl)benzene), 4-(trifluorome-
thyl)benzyl chloride (266 mL, 1.8 mmol), NaHCO3 (302 mg,
3.6 mmol) and DMF (6.0 mL). Purification: EtOAc/Hex (1:4). Yield:
106 mg, 17%. Whitish solid. mp: 183–185 ꢁC. 1H NMR (300 MHz,
CDCl3) d 8.00 (d, J ¼ 2.2 Hz, 1H), 7.62 (d, J ¼ 2.2 Hz, 1H), 7.47 (d,
J ¼ 8.1 Hz, 2H), 7.38 (dd, J ¼ 8.6, 5.5 Hz, 2H), 7.17 (d, J ¼ 8.0 Hz, 2H),
6.88 (t, J ¼ 8.7 Hz, 2H), 5.66 (s, 2H), 5.11 (s, 2H), 2.29 (s, 3H). 13C
NMR (75 MHz, CDCl3) d 162.3 (d, JC-F ¼ 247 Hz), 160.7, 152.2, 142.9,
141.7 (q, JC-F ¼ 2 Hz), 137.2, 135.2, 132.2 (d, JC-F ¼ 2 Hz), 131.1 (d, JC-
determined using liquid chromatography (UPLC) (Waters AquityTM
)
coupled with tandem quadrupole mass spectrometry (MS2)
(Waters XevoTM), equipped with an electrospray ionisation (ESI)
interface and operated in multiple reaction monitoring
(MRM) mode.
¼ 8 Hz), 129.5 (q, JC-F ¼ 32 Hz), 129.2, 126.5, 125.4 (q, JC-F ¼ 4 Hz),
F
In vivo antischistosomal efficacy
124.0 (q, JC-F ¼ 272 Hz), 119.5, 115.2 (d, JC-F ¼ 22 Hz), 107.5, 51.3,
44.6, 20.0. Anal. (C24H17BrF4N2O2) Calculated: C 55.35%, H 3.29%,
N 5.37%. Found: C 55.35%, H 3.35%, N 5.35%.
PZQ was obtained from Egyptian International Pharmaceutical
Industries Company (EIPICO) and aminobenzotriazole (ABT) from
Fluorochem Ltd.
8-Bromo-1-(4-(trifluoromethyl)benzyl)-3-(4-methoxybenzyl)-6-
methylquinazolin-2,4(1H,3H)-dione (13). Reagents: 8-bromo-3-(4-
methoxybenzyl)-6-methylquinazolin-2,4(1H,3H)-dione
(1.2 mmol)
Experimental animals
obtained by this method: In round-bottomed flask with a mag-
netic stir bar, 2-amino-3-bromo-5-methylbenzoic acid (1.0 equiv.)
is dissolved in THF (5 mL/mmol) and 1.0 equiv. of 1-(isocyanato-
methyl)-4-methoxybenzene is added to the mixture, which is
stirred in an oil bath at 80 ꢁC overnight. The solvent is rotary
evaporated and replaced with EtOH (20 mL/mmol). Then, 2 mL/g
of concentrated HCl (12.1 M) is added and the mixture is reheated
to 70 ꢁC until complete conversion to the 3-substituted quinazoli-
nedione (3 h). The reaction mixture is cooled in an ice bath and
Male Swiss albino mice (CD-1) obtained from SBSC and weigh-
ing 18–20 g were housed under environmentally controlled
room temperature of 20–22 ꢁC, 12 h light/dark cycle and
50–60% humidity with food and water ad libitum throughout
the acclimatisation and experimental periods. All the animal
experiments were conducted in accordance with the Guide for
Care and Use of Laboratory Animals and were approved by the
Institutional Review Board of Theodor Bilharz Research
water (20 mL/g) is added, which causes a precipitate. This slurry is Institute (TBRI).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
515
Infection and experimental design
S. mansoni ovipositing capacity
Mice were infected with S. mansoni cercariae [provided by 12-well tissue culture plates were used, each well containing 6–8
Schistosome Biology Supply Center (SBSC)] using body immer-
sion16 by exposure to 80 10 cercariae/mouse. Infected mice were
divided into six groups: groups 1 and 2 were treated the vehicle
and the CYP450 inhibitor ABT respectively; groups 3 and 4 were
dosed for 5 days with NPD-1246 at 20 and 10 mg/kg, respectively,
while group 5 was treated with PZQ at 10 mg/kg/day for 5 days.
Group 6 was treated with NPD-1246 combined with PZQ, each at
10 mg/kg/day for 5 days starting from week 7 post-infection. To
minimise first-pass elimination, ABT was administered at 100 mg/
kg/day for 5 days 2 h prior to each compound administration.
NPD-1246 and PZQ were freshly suspended in 2% Cremophore-EL
(Sigma-Aldrich). All drug administrations were performed orally.
worms with at least one worm couple. Worms were incubated
overnight in a CO2 incubator at 37 ꢁC. Each concentration was
tested in duplicate wells. On day 4, eggs were counted and dis-
carded and the medium was changed. On day 5 (end of observa-
tion period) newly deposited eggs were counted. The final egg
number is the total count of days 4 and 5 for each concentration
tested and the final recording of the percentage of egg reduction
was determined as [the total number of eggs on days 4 and 5 of
control – the total number of eggs on days 4 and 5 of treated]/
the total number of eggs on days 4 and 5 of control ꢂ 100.
Metabolic stability prediction
To determine the most likely sites of CYP450 mediated metabolism
of NPD-1246, SMARTCyp22 was applied using the default settings.
This tool determines the sites in a molecule that are susceptible
to be metabolised using the 2D structure of the compound. It is
based on a model that predicts the reactivity at C, S, N and P
positions in a given ligand based on a series of over 40 rules
derived from quantum chemical and calculations of energies
required for oxidation using density functional theory (DFT).
Finally, the atoms in the molecule are ranked according to
these results.
Parasitological criteria for cure
Ten days post-treatment, all mice were sacrificed and perfused,
and the number of worms recovered (worm burden) was quanti-
fied and sexed17. The number of eggs per gram of liver or intes-
tinal tissue was counted18. The percentage of egg developmental
stages (oogram pattern) was studied19 in which eggs at different
stages of maturity (from I to IV) were identified and counted.
Mature eggs and dead eggs (granular, dark, and semi-transparent)
were also counted in three fragments of intestine and the mean
number of each stage was calculated.
Target prediction
Statistical analysis
The percentage reduction of worm or egg burden in each treated
group was calculated. The 50% effective concentration (EC50) was
calculated using Prism (GraphPad; Version 5.0) software using a
variable slope for the sigmoidal curve with an upper limit of
100%. Results are expressed as mean SEM. A two-tailed, unpaired
Student’s t-test was used to detect the significance of difference
between the means of different groups. Results are considered
significant if p value is <0.05.
With the objective of searching a potential MOA for this family of
compounds, a target prediction study was performed. A consen-
sus methodology was applied considering several approaches that
involve ligand-based target prediction. Three different strategies
were selected as representatives of the different tools available.
Among them, polypharmacology browser (PPB)23 was used using
all the fingerprints combinations available in the tool and default
parameters. This technique was selected because it applies an
important number of fingerprints alone and in combination. Also,
similarity ensemble approach (SEA) was selected24,25 since both
tools search in databases such as ChEMBL with an impressive
amount of biological data to retrieve the most accurate results.
These methods were used using default settings. From the list of
potential targets, the first 35 were further considered. Finally, a
search on the PDB using the main scaffold of the compounds was
carried out to look for already crystallised structures.
In vitro S. mansoni worm killing
Stock solutions of 5 mM PZQ and the quinazoline derivatives 2–13
were prepared in DMSO. Concentrations of 100 mM, 50 mM, 25 mM,
10 mM and 5 mM were freshly prepared on the day of experiment
in RPMI-1640 medium. All compounds were initially tested at 100
and 50 mM; those showing worm killing were further tested at 25,
10 and 5 mM.
Worms were obtained from SBSC of TBRI. Six to eight worms
were placed in 12-well tissue culture plates and fresh RPMI-1640
medium (supplemented with glutamine, 20% newborn calf serum
and streptomycin, penicillin, and gentamicin), containing the indi-
cated concentration of the test compound was added20,21. Worms
were incubated overnight in a CO2 incubator at 37 ꢁC. On the 2nd
day, worms were examined by microscopy, washed three times
with normal saline, fresh medium was added and the incubation
was continued. On the 3rd day, worm motility was observed and
on the 4th day, medium was changed again. On day 5 (end of
the observation period), worms were microscopically examined for
their motility and appearance. Each concentration was tested in
duplicate, and the final recording of percent worm mortality was
determined as the number of dead worms [contracted and opa-
que] divided by the total number of worms ꢂ 100. Negative con-
trols used medium without additions or medium with 2% DMSO;
positive control media containing identical concentrations of PZQ
were tested in parallel.
Homology modelling
In order to produce the most accurate model for S. mansoni
aldose reductase (G4LXS0), a template search was performed
using the Swiss-Model server26. In the next step, the sequential
alignment of the target protein was carried out with the different
templates selected, the human aldose reductase (P15121) and
S. japonicum Q5DD64 sequences. The templates and target
sequences were retrieved from Uniprot database27.
Due to the high identity with the S. japonicum protein, this
template was selected to further build the target model using the
Swiss-Model server. Once the model was built, the estimation of
the protein model accuracy is required. For that purpose, model
quality assessment was performed using different metrics. The
local composite scoring function QMEAN (Qualitative Model
Energy Analysis) allows discriminating good from bad models
assessing geometrical aspects of the protein structure using

516
V. SEBASTIAN-PEREZ ET AL.
Table 1. In vitro metabolic stability of NPD-1246: percentage of parent com-
pound remaining over time in the presence of mouse liver microsomes.
several statistical descriptors. Energetic and geometric quality
assessment was also performed using Ramachandran plot28,
ProSA29, ERRAT30 or VERIFY3D31 tools.
% Parent compound remaining upon incubation
NPD-1246
Average
Diclofenac
Average
Phase I/II
Time (min)
SD
SD
Docking studies
CYP450-NADPH
0
15
30
60
100
66
27
8
ꢃ
100
87
70
ꢃ
5
2
Ligand preparation
9
2
6
1
The preparation of the test compounds and the 2D-to-3D conver-
48
sion was carried out using the LigPrep tool32, a module of the
(n ¼ 2)
(n ¼ 2)
€
Schrodinger software package. This tool allows the preparation of
UGT enzymes
0
15
30
60
100
111
111
106
ꢃ
4
2
100
41
ꢃ
1
8
molecules including different steps such as the calculation of the
ionisation state of the molecules at a pH range, the addition of
hydrogen atoms, and a final energy minimisation using the OPLS-
2005 force field33,34. To perform the studies, physiological pH con-
ditions were used to prepare the molecules, all of them were
desalted and in the last step, the compounds were minimised
as default.
44
34
5
2
(n ¼ 2)
(n ¼ 2)
Results
In vitro metabolic stability
NPD-1246 was exposed to mouse S9 microsomal fractions to
investigate the in vitro metabolic stability through phase-I and
phase-II metabolism (Table 1). The results indicate that the com-
pound becomes extensively metabolised through phase-I but not
through phase-II metabolism. After 30 min, only 27% of parent
compound is left indicating poor metabolic stability. The reference
drug diclofenac showed extensive phase-I and phase-II metabol-
ism, validating the performance of the assay.
Protein preparation
The structures of the proteins used in this study were prepro-
cessed and refined using the Protein Preparation Wizard tool35,36
included on Maestro37. H-bond assignment and calculation of the
protonation state of the residues at physiological pH with a final
restraint minimisation were carried out.
Docking studies
Automated docking protocol was used to assess the appropriate
binding mode and suitable poses of the reference compound and
the ligand. A Lamarckian genetic algorithm38 method imple-
mented in the programme AutoDock 4.239 was applied. For dock-
ing calculations, Gasteiger charges were added, rotatable bonds
were set by AutoDock tools (ADT) and all torsions were allowed
to rotate for the ligand. In all the cases, we used grid maps with a
grid box size of 60 ꢂ 60 ꢂ 60 Å3 points and a grid-point spacing of
0.375 Å, using as centroid of the grid the key Trp111 presents in
the catalytic site. The docking protocol consisted of 200 independ-
ent genetic algorithm runs, population size of 150 and maximum
number of evaluation 250,000, while the remaining parameters
were conserved as default. Final best-docked poses were grouped
into clusters, within the default 2.0 Å RMSD. Best energetic and
most representative docking clusters were analysed by visual
inspection according to the binding energies and relative popula-
tion provided by the software. Best poses in these clusters were
considered as most reliable representatives of the ligand-binding
mode and were further studied.
Activity in the S. mansoni-infected mouse model
NPD-1246 was previously shown to have relevant schistosomicidal
activity potential in vitro14. Since NPD-1246 proved to be metabol-
ically unstable (Table 1), the compound was administered orally
concomitantly with the CYP450 inhibitor ABT to counter metabolic
degradation43. Infected mice were treated either alone with NPD-
1246 or in combination with PZQ. Control groups receiving ABT
or ABT þ PZQ were included as well.
The pooled data of two experiments (number of mice ranged
from 6 to 10/group in each experiment) upon a 5-day treatment
with NPD-1246 at 20 mg/kg revealed a reduction in total worm
burdens and intestinal tissue egg load by 24% and 18%, respect-
ively (Figure 2(A,B)), accompanied with significant increase in the
percentage of dead eggs when compared to the infected
untreated group (Figure 2(C)). Administration of NPD-1246 at
10 mg/kg did not produce any significant change in these param-
eters with respect to the group dosed at 20 mg/kg (Figure 2).
Treatment with PZQ at 10 mg/kg significantly reduced total worms
by 63% and hepatic and intestinal tissue egg loads by 38% and
70%. Total immature and mature eggs were also reduced with a
significant increase in dead eggs. Finally, 5-day co-treatment of
NPD-1246 and PZQ at 10 mg/kg/day revealed an enhanced reduc-
tion in total worms (80% vs. 63% for PZQ alone), intestinal tissue
egg load (79% vs. 70% for PZQ alone) with complete disappear-
ance of immature eggs and increase of dead eggs (84% vs. 66%
In the case of S. mansoni aldose reductase an additional strat-
egy was carried out to optimise the structure of the model built.
This strategy is called induced fit docking (IFD)40,41 and is based
on fitting the ligand to the protein binding sites allowing changes
in the residues geometry, mainly in their side chain orientations.
First, Prime42 predicts the active site structure using the pose of
compound NPD-1246 to rearrange nearby side chains of the pro- for PZQ alone).
tein and minimising the overall energy of the protein. Finally,
each ligand is re-docked into its corresponding low energy protein
Metabolic stability prediction
structures and the resulting complexes are ranked according to
docking score. Extra precision (XP) mode was used in a standard
protocol and no constraints were set, residues were optimised to
5.0 Å of the ligand poses and the rest of the parameters were set
as default.
NPD-1246 shows promising antischistosomal activity potential
in vivo, but was found metabolically unstable. A medicinal chemis-
try programme was therefore designed with the aim of improving
its drug-like properties that would enable further development.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
517
Worm burden
(A)
35
30
25
20
15
10
5
(+13)
*
(11)
(24)
*
(63)
*#
(80)
0
Figure 3. Metabolic site prediction using SMARTCyp web server for NPD-1246.
Top-ranked sites (red circles) and minor sites (blue circles) predicted to be metab-
olised by (A) CYP2C9, (B) CYP2D6 and (C) CYP3A4
.
p-substituents for the benzyl tail attached to N3, while the ben-
zene fused ring from the quinazoline remained without substitu-
ents or with halogen or alkyl groups attached to different
positions. The synthesis was accomplished in two stages following
previously described procedures15. In the first step, the reaction
between the corresponding benzyl isocyanate and the anthranilic
acid derivative yielded the substituted 3-benzylquinazolin-
2,4(1H,3H)-dione used in the next step without further purification.
In a second step, the mono-substituted quinazoline reacts with 4-
(trifluoromethyl)benzyl chloride in basic media to obtain the
desired di-substituted quinazolines 1–13 with moderate yields
(Scheme 1). The compounds were characterised based on the ana-
lytical data detailed in the chemical procedures on the experimen-
tal part.
(B)
Inf. Control
ABT
ABT+NPD1246 (10 mg)
ABT+NPD1246 (20 mg)
ABT+PZQ (10 mg)
ABT+NPD1246 (10 mg)+PZQ (10 mg)
(9)
30
*
(7)
(+9)
(18)
25
(+5)
20
(7)
*
15
10
5
*
*
(38)
(49)
*#
(70)
(79)
0
Hepatic
Intestinal
(C)
100
In vitro activity against S. mansoni
*
% Dead
90
80
70
60
50
40
30
20
10
0
The new quinazolines 2–13 were initially tested at 100 and 50 mM
against male and female worms and compared with the previ-
ously reported data for parent compound NPD-124614. The param-
eters for antischistosomal activity were worm mortality, motor
activity alterations (sluggish worm movement or spastic contrac-
tions), unpairing and absence or reduction in egg numbers (Table
2). While almost all the new compounds are able to reduce the
egg numbers and to separate or insult the coupling at both con-
centrations, 9 and 10 revealed 100% worm killing at 100 mM and
29% and 93% at 50 mM, respectively. Based on these data, both
compounds were selected for further dose-titration. The EC50 for 9
was comparable to NPD-1246 (1) (47 mM vs. 50 mM) with 10 being
slightly more active (EC50 25 mM) (Table 3). These values were
obtained taking both males and females into account. When ana-
lysing each sex separately, the results remain similar pointing out
that there are no obvious sex differences (Table 4). As for coupling
and ova production, both compounds present a similar behaviour
to NPD-1246 showing a significant reduction in egg numbers at
5 mM (i.e. the lowest concentration tested).
*
% Mature
*#
*
% Total
immature
*
*
Figure 2. Effect of NPD-1246 alone (in a dose of 20 or 10 mg/kg/day) or in com-
bination with PZQ (10mg/kg/day each) for 5 days treatment on (A) worm burden,
(B) tissue egg load and (C) oogram pattern in S. mansoni-infected mice sacrificed
10 days post end of treatment. Significantly different from infected control at
p < 0.05. Significantly different from PZQ group at p < 0.05. Numbers above col-
ꢀ
#
umns and between parentheses represent percentage change from infected con-
trol group.
Computational studies using SmartCyp22 were performed to iden-
tify the positions potentially susceptible for metabolic degrad-
ation. This software tool predicts CYP3A4, CYP2D6 and CYP2C9 effect
on the target molecule and ranks atoms according to their prob-
ability to be modified by metabolism (Supplementary Figures
S1–S3). Several potential sites were identified (Figure 3). The most
important of which are C6 in the quinazoline scaffold and the
meta- and para-positions of the benzyl substituent in the N1.
In vitro metabolic stability
The stability of 9 and 10 through phase-I and phase-II metabolism
by mouse microsomes was investigated in a similar way than for
NPD-1246 (Table 5). Both compounds presented a better profile
than NPD-1246 with 51% and 39% of 9 and 10 remaining after
30 min, hence indicating acceptable stability. With human micro-
somes, the stability was even better with 61% and 83% of 9 and
10, respectively remaining after 30 min. None of the compounds
was affected by phase-II metabolism.
Design and synthesis of NPD-1246 derivatives
With the aim of increasing the metabolic stability of 3-benzyl-1-
((4-trifluoromethyl)benzyl)quinazolin-2,4(1H,3H)-dione (NPD-1246,
1), different chemical modifications were prioritised to block
Computational target deconvolution
Based on the confirmed in vitro and in vivo activity potential of
the predicted positions. Methoxy and fluor were chosen as the quinazoline family against S. mansoni, in silico studies were

518
V. SEBASTIAN-PEREZ ET AL.
Scheme 1. Synthesis of 3-benzyl-1-(4-(trifluoromethyl)benzyl)quinazolin-2,4(1H,3H)-diones 1–13.
Table 2. Mature worm killing and ovipositing at 100 mM and 50 mM of new quinazolines (2–13) in comparison with previously reported data for NPD-1246 (1)14
.
Worm killinga (% of total)
Uncoupling
Reduction in number of eggs (%)
R1
R2
100 mM
50 mM
100 mM
50 mM
100 mM
50 mM
H
H
H
6-Br
6-Br
7-Br
7-Br
6-Cl
H
F
OMe
F
OMe
F
OMe
F
OMe
F
OMe
F
NPD-1246 (1)
100
100
75
100
100
88
53
0
0
13
0
0
0
0
29
93
0
0
0
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
No
100
100
100
100
100
100
50
100
100
100
100
100
100
100
100
100
100
31
100
35
100
100
100
100
50
2
3
4
5
6
7
8
9
10
11
12
13
0
100
100
100
86
0
33
6-Cl
6,7-diOMe
6,7-diOMe
6-Me,8-Br
6-Me,8-Br
OMe
Yes
100
aThe final recording of worm killing was determined on day 5 (end of the observation period).
Table 3. Mature (6 weeks old) worm killing and ovipositing under different concentrations of selected compounds in comparison with NPD-1246 (1) data14 previ-
ously reported.
Worm killinga (% of total)
Uncoupling
Reduction in number of eggs (%)
EC50
100 mM 50 mM 25 mM 10 mM 5 mM (mM) 100 mM 50 mM 25 mM 10 mM 5 mM 100 mM 50 mM 25 mM 10 mM 5 mM
NPD-1246 (1)
9
10
100
100
100
53
88
100
0
0
50
0
0
8
0
0
0
50
47
25
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
100
100
100
100
100
100
100
100
100
20
73
80
10
67
77
aThe final recording of worm killing was determined on day 5 (end of the observation period).
Table 4. Mature (male & female) worm killing under different concentrations of selected compounds in comparison with NPD-1246 (1) data14 previously reported.
Worm killinga (male worms)
Worm killinga (female worms)
EC50
EC50
100 mM
50 mM
25 mM
10 mM
5 mM
(mM)
100 mM
50 mM
25 mM
10 mM
5 mM
(mM)
NPD-1246 (1)
9
10
100
100
100
50
100
100
0
0
63
0
0
14
0
0
0
50
41
20
100
100
100
57
75
100
0
0
33
0
0
0
0
0
0
50
49
26
aThe final recording of worm killing was determined on day 5 (end of the observation period).
conducted to obtain additional information about their putative that had already been crystallised as protein inhibitors was per-
MOA. A consensus in silico methodology using tools based on lig- formed using the protein data bank (PDB)44. The three methodol-
and similarity was used, relying on the general assumption that ogies were sequentially applied to the reference compound
related molecules will have similar activity and interaction pat- NPD-1246.
terns. Firstly, the PPB23 was used as an exhaustive method in
The PPB search provides a list of potential targets, organisms
terms of fingerprint similarity. Secondly, the SEA24,25 was selected and cell lines where a compound may have biological activity. In
to identify targets based on a group of known compounds rather this study, the cell lines and organisms were discarded and only
than a single compound. As a third strategy, a structural compari- targets involving proteins and/or enzymes were conserved. The 35
son searching for similar structures in terms of chemical scaffold top-ranked results are collected in Supplementary Table S1. The

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
519
Table 5. In vitro metabolic stability of 9 and 10: percentage of parent compound remaining over time in the presence of mouse and human
liver microsomes.
% Parent compound remaining upon incubation
9
10
Diclofenac
Average
Microsomes
Mouse
Phase I/II
Time (min)
Average
SD
Average
SD
SD
CYP450-NADPH
0
15
30
60
100
91
51
–
3
3.3
0.45
100
85
39
–
100
61
34
–
5.3
0.55
0.43
44
32
27
(n ¼ 2)
(n ¼ 2)
(n ¼ 2)
(n ¼ 2)
(n ¼ 1)
(n ¼ 1)
(n ¼ 1)
(n ¼ 1)
UGT enzymes
CYP450-NADPH
UGT enzymes
0
15
30
60
100
101
103
105
–
100
89
83
–
8.3
8.7
100
34
32
2.6
3.6
0.8
91
18.3
30
(n ¼ 2)
(n ¼ 2)
(n ¼ 2)
Human
0
15
30
60
100
104
61
–
100
108
83
–
100
27
5
–
4.9
2.4
3.2
3.7
18.8
1.3
67
65
1
0
15
30
60
100
94
95
–
27.0
12.9
14.1
100
102
106
98
100
11
9
2.4
4.5
1.8
97
9
second approach using the SEA tool produced a ranking of tar-
gets, from which the top 35 were taken into account
(Supplementary Table S2). Finally, the chemical scaffold search in
the PDB retrieved five different results of crystallised quinazoline
derivatives with different enzymes (Figure 4).
With the three methodologies combined one common target
was revealed, namely the human aldose reductase. Moreover, one
compound crystallised with this enzyme, zenarestat (PDB code
1IEI)45 (Figure 4) retrieved in the search is a di-substituted quina-
zoline similar to NPD-1246.
Homology modelling
As the crystal structure of aldose reductase in S. mansoni was not
available at the time of the study, a search for homologous struc-
tures was done to build an accurate model that would guide us
in the computational studies on the potential binding mode of
our compounds in the enzyme. The closest homologue with a
crystal structure available is the aldose reductase of S. japonicum
(PDB code 4HBK)46 and a sequence alignment was carried out
(Supplementary Figure S4). The sequences of the Schistosoma spp
are almost identical showing an identity of 83.23% but the iden-
tity between the human and the S. mansoni proteins is 50.32%.
The similarity between the Schistosoma enzymes is even higher
considering that most of the mutations are related amino acids
with similar properties. For this reason, the structure of aldose
reductase from S. japonicum was chosen as the most accurate
template to obtain a reliable model of our target protein using
the Swiss-Model server26. The final structure was validated from a
geometric and energetic point of view using several widely used
metrics (Supplementary Table S3). More than 98% of the torsion
angles are in allowed regions of the Ramachandran plot28. It also
presents an overall quality factor over 93.3% for ERRAT30 param-
eter and 92.9% according to the verify analysis47. Superposition of
the crystal structures of human aldose reductase (PDB code 1IEI),
S. japonicum aldose reductase (PDB code 4HBK) and the homology
model of S. mansoni aldose reductase is depicted in Figure 5.
Figure 4. Quinazoline-related structures crystallised with different target proteins
according to the scaffold search in the PDB (access codes included).
Docking studies
Docking studies were performed to assess the potential binding
mode of NPD-1246 to S. mansoni aldose reductase. To validate
our docking protocol, binding mode studies were first carried out
with zenarestat, an inhibitor of the human aldose reductase crys-
tallised with the enzyme (PDB code 1IEI)45. It was observed that
the majority of conformations for zenarestat were grouped in a
single cluster with very good binding energy profile around
ꢃ9.75 kcal/mol. This cluster was visually analysed and the best
pose was compared to the crystal structure already available. The
docking binding pose of zenarestat (depicted in purple) is almost
identical to the one that was previously crystallised (shown in
cyan) (Figure 6(A)). In a similar way to the crystal structure, the
main interactions found are with aromatic residues from the cata-
lytic site: hydrogen bonds with Tyr48 and Trp111, and several
important aromatic interactions mainly critical with Trp20 and
Trp111 (Figure 6(B)).

520
V. SEBASTIAN-PEREZ ET AL.
Figure 5. Superposition of the crystal structures of human aldose reductase 1IEI, depicted in cyan, S. japonicum aldose reductase 4HBK in magenta and the homology
model of S. mansoni aldose reductase in purple, (A) front view and (B) back view.
Figure 6. (A) Superimposition of zenarestat in the crystal structure 1IEI depicted in cyan and validation docking results depicted in purple show the high similarity
between both poses in the human enzyme. (B) Detail of the zenarestat binding mode together with the main interactions found in the catalytic site of
aldose reductase.
Once the protocol was validated, the potential binding mode face-to-face P interactions with the quinazoline scaffold, while
His110 and Trp111 are important making face-to-edge p interac-
tions. As expected, due to the high similarity in terms of chemical
structure between NPD-1246 and the novel derivatives, 9 and 10,
the main interactions with the enzyme are maintained
(Supplementary Figure S5).
of NPD-1246 in the aldose reductase of S. mansoni model was
studied. Due to the fact that the model was based on 4HBK crys-
tal, an apo structure of the S. japonicum aldose reductase, IFD
studies40,41 were performed. Once the model was optimised, a
final docking with NPD-1246 was carried out using the previously
validated protocol. A clear defined cluster was obtained in which
the vast majority of the docking poses were grouped with very
similar conformations. The best-ranked pose of this cluster, with a
binding energy around ꢃ9.5 kcal/mol, was selected as representa-
tive of the binding mode of NPD-1246 in the protein. As shown in
Figure 7, the aromatic interactions are responsible for the stability
of the ligand in the catalytic binding site, driving the ligand-bind-
Discussion
A previous phenotypic screening allowed us to successfully clas-
sify and identify promising compounds for further development
based on antischistosomal in vitro potency. The selected quinazo-
line NPD-1246 showed an EC50 value of 50 mM in both males and
ing process. Tyr207 and Phe293 are key to the stability, making females and a significant reduction in egg numbers at lower

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
521
concentrations (Figure 1)14. Because metabolic stability is an
important drug-like property to be considered for in vivo studies,
the stability of NPD-1246 in the presence of mouse S9 microsomal
fraction was checked. Unfortunately, the compound was exten-
sively metabolised through phase-I metabolism (Table 1).
Nevertheless, to check the in vivo activity potential, concomitant
dosing with the CYP450 inhibitor ABT was carried out in the
experimental S. mansoni-infected mouse model (control, NPD-
1246, PZQ and combination of both).
NPD-1246 at 20 mg/kg orally showed a modest but significant
reduction in total worm and intestinal egg loads together with an
increase in the percentage of dead eggs. No significant differences
were observed after administration at 10 mg/kg. More relevant to
note is the synergy with PZQ when administered concomitantly
with NPD-1246 at 10 mg/kg each (Figure 2). Reduction of total
worms was 80% vs. 63% for PZQ alone with a complete dis-
appearance of immature eggs and significantly increased percen-
tages of dead eggs (84% vs. 66% for PZQ). This fact is of pivotal
importance because eggs are responsible for the pathology and
transmission of the disease1.
Figure 7. Detail of the binding mode of NPD-01246 in the S. mansoni aldose
reductase binding site.
These results encouraged us to design NPD-1246 derivatives
with improved metabolic stability. SMARTCyp22 software allowed
us to identify position C6 in the quinazoline scaffold and meta-
and para-positions of the benzyl substituents in the N1 as the
most susceptible sites for metabolism by CYP450 (Figure 3). Taking
these predictions into account, a series of chemically related com-
pounds with these positions blocked were synthesised following a
two-step synthetic procedure (Scheme 1). The new quinazolines
2–13 were tested in vitro against S. mansoni and their activity
compared with the parent compound NPD-1246 (Tables 2–4). Two
sequence alignment (Supplementary Figure S3). Docking calcula-
tions indicated that NPD-1246 and new quinazolines 9 and 10 are
able to bind the catalytic site of the enzyme through important
interactions with aromatic residues, such as Tyr207, His110, Trp111
and Phe293 (Figure 7 and Supplementary Figure S5).
In conclusion, this study showed the antischistosomal potential
of a new series of quinazoline derivatives through in vitro
and in vivo studies and successful design of new derivatives to
of them, 9 and 10 showed similar potency results as NPD-1246 overcome the metabolic stability issues of the parent compound
but with an improved metabolic stability (Table 5), representing a
significant improvement with respect to the original compounds.
The established antischistosomal potential of the quinazoline
derivatives prompted us to investigate their MOA since target
identification is becoming increasingly important to avoid/over-
come drug resistance. The analysis of results obtained separately
with three complementary computational methodologies
(Supplementary Tables S1, S2 and Figure 4) led us to propose
aldose reductase as the potential drug target for the quinazo-
line compounds.
NPD-1246. The putative molecular target aldose reductase was
identified by using complementary computational tools. This
enzyme emerges as a new potential target to develop antischisto-
somal agents while the new quinazolines 9 and 10 represent
improved candidates for further evaluation and development.
Disclosure statement
No potential conflict of interest was reported by the authors.
S. japonicum aldose reductase was previously crystallised and
antischistosomal activity of an inhibitor bearing two linked anthra-
quinone scaffolds was reported46. Although the role of S. japoni-
cum aldose reductase is not fully understood, aldose reductase is
believed to be an important antioxidant component in other
Funding
This study received funding from the EC 7th Framework
Programme (FP7-HEALTH-2013-INNOVATION-1, PDE4NPD no.
602666), RICET (RD16/0027/0010), FEDER funds and MECD [Grant
FPU15/1465 to V. S.-P.].
organisms48,49
. Moreover, antioxidant defense is an essential
mechanism for schistosomes to face the damage from host
immune and self-generated reactive oxygen species (ROS) and a
number of redox-associated proteins have been already consid-
ered as key enzymes for drug development50–53. Based on these
evidences, and after the cloning and characterisation of S. japoni-
cum aldose reductase, it was proposed as a potential drug target
for schistosomiasis due to its possible role in the worm antioxi-
dant mechanism54.
All in all, these facts increased our interest in the quinazoline
compounds as a new chemical class of inhibitors of this enzyme
and a homology model of the S. mansoni aldose reductase was
built based on the crystal structure of the S. japonicum counter-
part46 for checking the binding mode of NPD-1246. The catalytic
site of aldose reductase in S. mansoni is highly hydrophobic with
a high number of aromatic residues such as Trp20, Tyr48, Trp79,
Trp111, Phe122 or Tyr207 among others, highlighted in the
ORCID
Victor Sebastian-Perez
http://orcid.org/0000-0002-8248-4496
Ana Martinez
Nuria E. Campillo
http://orcid.org/0000-0002-2707-8110
http://orcid.org/0000-0002-9948-2665
Carmen Gil
http://orcid.org/0000-0002-3882-6081
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