Molecular drug design, synthesis and pharmacophore site identification of spiroheterocyclic compounds: Trypanosoma crusi inhibiting studies

Article abstract of DOI:10.1007/s00044-012-0010-5

Synthesis and evaluation of the bioactivity of spiroheterocycles (STC) against Trypanosoma cruzi are described. Selectivity indices were improved for two compounds versus the leads 17 and 20, the spiro-thiochromanone (STC) derivatives 17-26, thus increasi

Full text of DOI:10.1007/s00044-012-0010-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:57–69
DOI 10.1007/s00044-012-0010-5
ORIGINAL RESEARCH
Molecular drug design, synthesis and pharmacophore site
identification of spiroheterocyclic compounds: Trypanosoma crusi
inhibiting studies
•
Taibi Ben Hadda Abdelali Kerbal Brahim Bennani
•
•
•
•
Ghali Al Houari Maria Daoudi Ana C. L. Leite
•
•
Vijay H. Masand Rahul D. Jawarkar Zoubida Charrouf
•
Received: 17 January 2012 / Accepted: 25 February 2012 / Published online: 13 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Synthesis and evaluation of the bioactivity of
spiroheterocycles (STC) against Trypanosoma cruzi are
described. Selectivity indices were improved for two
compounds versus the leads 17 and 20, the spiro-thio-
chromanone (STC) derivatives 17–26, thus increasing the
therapeutic interest of our family. As our previous studies
conducted on the structure of pharmacophore sites of our
compounds made us hypothesize the existence of original
sites, STC can be considered as promising tools further
anti-trypanosoma studies, as probes for affinity chemo-
therapy. Compounds 17 and 20 are more potent and more
selective than benznidazole and nifurtimox.
Keywords Trypanosoma cruzi (TC) ꢀ
Spiro-heterocycles ꢀ Antiparasitic activity ꢀ
Drug design ꢀ Virtual screening ꢀ POM analyzes ꢀ
Docking studies
Introduction
Benznidazole (Bdz) and nifurtimox (Nfx) have been two of
the few most widely used anti-Trypanosoma cruzi (TC)
drugs (Fig. 1). The success of Bdz was mainly due to its
outstanding clinical efficacy, and the slow speed at which
resistance developed to this drug. But the side effects and
the final arrival of resistance and the alarming spread of
Bdz-resistant TC on a global scale created an urgent need
for the development of novel TC drugs (Viotti et al., 2009;
Pinazo et al., 2010; Moll et al., 2008; Castro and Diaz de
Toranzo, 1988; Diaz et al., 2004). So novel anti-trypano-
soma drugs were strongly and urgently needed in goal to be
used as new drugs without side effects and multi-drug
resistance.
T. Ben Hadda (&)
Laboratoire Chimie Materiaux, Faculte Sciences,
´
Universite Mohamed I, Oujda, Morocco
´
´
e-mail: taibi.ben.hadda@gmail.com; tbenhadda@yahoo.fr
A. Kerbal ꢀ B. Bennani ꢀ G. A. Houari ꢀ M. Daoudi
´
Laboratoire Chimie Organique, Universite Sidi Mohamed
`
Ben Abdellah, Fes, Morocco
Works in our laboratories focus on new spiro-oxazoline-
thiochromanones. Our library of spiroheterocycles (STC)
derivatives (Badri et al., 1999; Al Houari et al., 2008a, b;
Bennani et al., 2002, 2007a, b; Akkurt et al., 2006, 2009;
Orhan et al., 2009; Ben Hadda et al., 2008) provided
compounds with similar pharmacophore site of clinical
drugs Bdz and the Nfx (Fig. 1).
A. C. L. Leite
LabSINFA, DPS-CHS, Federal University of Pernambuco,
Recife, Brazil
V. H. Masand
Department of Chemistry, Vidya Bharati College,
Camp, Amravati, Maharashtra 444602, India
R. D. Jawarkar
More recently, we have reported a study concerning one
series of STC derivatives as antiviral drugs (Orhan et al.,
2009). Among them, ten spiro-compounds displayed less
toxicity than Bdz and the Nfx, and six of them, compounds
17–22 (Fig. 1), have two combined antiviral pharmaco-
phore sites; (O–C–C–O–N) and (S–C–O–N).
Department of Pharmaceutical Chemistry, Sahyadri College
of Pharmacy, Methawade, Sangola, Solapur 413307,
Maharashtra, India
Z. Charrouf
Laboratoire Chimie Plantes Synthese Organique
`
Et Bioorganique, FS, Rabat, Morocco
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Med Chem Res (2013) 22:57–69
O-
N
H3C
H2N
N+
CH3
N
N
N
- O
N
δ
S
N
δ-
O
O
N
S
δ−
N+
δ-
O-
H
O
HN
N
O
δ-
O
N+
O-
Megazol
−
O
δ
Benznidazole (Bdz)
Nifurtimox (Nfx)
δ−
N
O
δ−
O
O
N
O
R1
R1
S
δ−
S
δ−
17-22
23-26
R2
R2
Candidates 17-26
Fig. 1 Structure of candidates spiroheterocyclic compounds 17–26 and clinical antiparasitic agents and their hypothetical S, N and/or O,
N-pharmacophore site (Jarrahpour et al., 2010)
For all these reasons, compounds 17–22 could represent
potential antiparasitic activity for further evaluation as anti-
TC candidates. Pharmacological studies conducted on
compounds suggested a Bdz-like mode of action but also
the involvement of additional mechanisms (Anaflous et al.,
2004). Bioinformatics studies (Bennani et al., 2007a, b)
also suggest the existence of additional combined phar-
macophore sites, possibly involving an original mechanism
of drug/target interaction.
reference drugs by more constraint entities; candidates
17–26.
Chemistry
Synthesis of compounds 19–26
All synthesized compounds 17–26 were prepared by mix-
ing an equimolar ratio of 3-arylideneisothiochroman-4-
ones 11–13 or 3-arylidenethiochroman-4-ones 14–16 and
oximes 6–10 in chloroform containing sodium hypochlorite
solution (NaOCl, 18°) at 0°C (Scheme 1) which success-
fully lead to a new series of spiromolecules 22–26, con-
taining thiochomanone moiety in good yield (81–92%).
The reaction was rapid, and no observation supported any
kind of side product. All the products were obtained as
solids and their purities were checked by thin layer chro-
matography. All the new synthesized compounds 22–26
were characterized by spectroscopic techniques (IR, 1H
NMR, and 13C NMR) and their elemental analyzes. The
first series 17–22 is already well known and has been
Considering good pharmacological results obtained in
this family, and taking into account some previously
established structure-activity relationship, we decided to
further enlarge chemical diversity to get more SAR
information in our new spiro-oxazole-thiochromanone
(SOT) family and in the hope to improve activity/cyto-
toxicity ratio. First, considering compound Bdz as a ref-
erence drug, we decided to introduce chemical diversity at
the place of one of the two heterocyclic moieties (Fig. 1).
Second, taking into account the important rigidity of
spiranic structure in our candidates 17–26, a high anti-
parasitic activity is predicted; in our previous series, we
decided to replace both flexible pharmacophore sites of
123

Med Chem Res (2013) 22:57–69
59
O
H
O
O
N
R2
S
R1
(11)-(13)
S
OH
(17)-(22)
N
H
R2
NaOCl/CHCl3
(-10 to 0 °C)
R1
H2O
N
(6)-(10)
R1
(1)-(5)
O
S
O
N
R1
O
H
(23)-(26)
S
R2
(14)-(16)
R2
(R1)
(R2)
(R1 , R2 )
(R1 ,R2)
1, 6:
H
CH3
OCH3
11: CH3
12: OCH3
17:
18:
19:
20:
21:
22:
(CH3, CH3)
(CH3, OCH3)
(OCH3, CH3)
23: (H, OCH3)
24: (CH3, H)
2, 7:
3, 8:
4, 9:
5, 10:
NO2
(CH3, OCH3)
13:
14:
15:
25:
Cl
NO2
H
OCH3
(OCH3, OCH3 )
(Cl, NO2)
(NO2 , CH3 )
26: (CH3, NO2 )
16: NO2
Scheme 1 Synthesis of spiroheterocycles 17–26
previously described by our group (Bennani et al.,
2007a, b).
inhibitory effect on the growth of different tested strains. A
significant activity was observed by all known compounds
17–22 against trypomastigotes and epimastigotes. Com-
pounds 21 and 22 showed moderate activity. Whereas
compounds 17, 19, and 20 showed significant activity against
trypomastigotes. Compounds 17, 19, 21, and 22 showed
moderate activity whereas compounds 18 and 20 showed
significant activity against epimastigotes. In contrast to new
prepared series 23–26, all the compounds showed moderate
to significant activity against TC strains. Compound 20
against both trypomastigotes and epimastigotes showed
excellent activity. So although most of the compounds
showed moderate to significant activity against all parasite
strains but compound 20 was the most active one (Table 1).
Pharmacology
Biological activity
Antiprotozoan activity (in vitro)
The anti-TC activity of the first series of compounds 17–22 is
determined by their inhibition of parasite growth using the
Bdz-resistant strain. In parallel, all compounds were tested
for cytotoxicity upon a BALB/c mice splenocytes. The other
new molecules of the series 23–26 were also tested in this
same protocol, but none activity was observed at concen-
tration below 100 lg mL-1. Results are given in Table 1.
All compounds 17–26 were tested against TC using the
Bdz-resistant strain and they were tested for cytotoxicity
upon a BALB/c mice splenocytes. The results were com-
pared with those of the standard drugs Bdz and Nfx. All
the synthesized compounds exhibited varying degree of
Results and discussion
Spectroscopic characterizations of 23–26
In the IR spectra of compounds 23–26, a characteristic
strong band at 1,660–1,684 cm-1 is assigned to the
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Med Chem Res (2013) 22:57–69
Table 1 In vitro bioactivity (IC50 in lM) of spiro-heterocyclic compounds 17–26 against Trypanosoma cruzi
Compound
R1
R2
Cytotoxicity
(lg mL-1 a
IC50 (lM)b Trypanosoma cruzi, Y strain
)
Trypomastigotes
at 24 h
Epimastigotes
at 11 days
17
CH3
CH3
OCH3
OCH3
Cl
CH3
OCH3
CH3
OCH3
NO2
CH3
OCH3
H
50
3.78
19.1
18
5.0
19.0
5.5
19
10
1.9
62.5
20
50
1.5
1.95
21
5.0
22.0
20.5
22
NO2
H
50
45.4
27.9
23
Not active
Not active
Not active
Not active
25
Not active
Not active
Not active
Not active
5.0
Not active
Not active
Not active
Not active
6.6
24
CH3
CH3
CH3
25
OCH3
NO2
–
26
Benznidazole
Nifurtimox
a
–
1.0 (3.4)
8.5
1.9
Expressed as the highest concentration tested that was not cytotoxic for the BALB/c mice splenocytes. Values in lM are showed in parentheses
b
Calculated at five concentrations using data obtained from at least three independent experiments, with a SD less than 10% in all cases
1
Table 2 Selected IR, H and 13C spectral data of series 17–26
Compd.
Substituents
IR
m (cm-1
m (CO)
1H NMRa
Ha
13C NMRa
CH2
)
0
0
0
R1
R2
Hb
H4
CH
C3
C4
17
18
19
20
21
22
23
24
25
26
a
CH3
CH3
1,675
1,675
1,675
1,665
1,680
1,680
1,668
1,662
1,680
1,684
3.5
4.7
6.10
6.1
6.1
6.1
6.2
6.1
5.3
5.4
5.3
5.4
28.8
56.4
94.6
95.0
94.4
95.0
94.4
95.6
86.8
86.2
86.2
86.7
184.1
180.8
184.1
184.2
183.4
183.6
189.2
188.6
188.9
188.1
J = 16.8
3.5
CH3
OCH3
OCH3
CH3
NO2
H
OCH3
CH3
4.7
4.7
4.7
4.7
4.7
3.1
3.0
3.1
3.1
28.1
28.1
28.2
28.0
28.1
31.9
31.6
31.5
31.2
57.0
56.4
56.2
56.0
55.9
56.5
56.7
56.2
56.4
J = 16.8
3.7
J = 16.8
3.50
OCH3
NO2
CH3
J = 16.8
3.6
J = 16.8
3.6
J = 16.8
3.0
OCH3
H
J = 14.0
3.0
CH3
CH3
CH3
J = 14.0
3.0
OCH3
NO2
J = 14.0
3.0
J = 14.0
(CDCl3, d ppm/TMS, J in Hz)
stretching of carbonyl C=O group of thio and isothio-
chromanone moiety. Displacement of this band is an evi-
H-40 proton of oxazolyl ring appeared at 5.3–5.5 ppm as
singlet. A multiplet was observed at 6.9–7.3 ppm assigned
for aromatic protons of phenyl rings. The CH3 or OCH3
protons in all cases appeared as a singlet in the region at 2.3
and 3.8 ppm, respectively. Furthermore, the number of the
1
dence of the formation of condensation product. H NMR
spectra of the compounds 17–26 displayed thiomethine
(CH2S) peak at d 3.0 and 3.1 ppm as doublets for Ha/Hb.
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Med Chem Res (2013) 22:57–69
61
proton calculated from the integration curves and those
obtained from the values of the expected CHN analysis
agreed well with each other (Table 2). 13C NMR spectra of
compounds 17–26 appeared thus to be essentially due to
the presence of the sulfur atom in position-2, consistent
with prediction of hypothetic anti-TC pharmacophore site
(Fig. 1).
compounds 17–26 displayed thiomethine-C (S–CH2) peak
at 28–32 ppm and an azomethine-C (N=C–C4 H–) peak at
0
In order to assess the role of lipophilic of terminal aryls
and electronic impact on the antiparasitic activity we cal-
culated the partial charges of each heteroatom. Even if our
compounds display generally good inhibition of TC strain
and high intraparasite accumulation by weak-base charac-
ter, no direct correlation could be established between
these parameters and anti-TC activity. Exceptions such as
aromatic ketone derivatives made us suppose the existence
of additional mechanisms. The next step will consist in the
search of these putative biological targets by the affinity
docking and POM techniques, using the two hits of the
series. Compounds 17 or 20 can be considered as good
candidates for this technique, as their respective combined
pharmacophore sites enable fixation on various targets
while preserving significant antiparasitic activities
(respective IC50 3.78 and 1.5 lM).
180–189 ppm which further supported the results obtained
in 01H NMR. Compounds 23–26 displayed C2, C3/C3 , C4/
0
0
C4 , C3 ou C5 , C6, C7 and C8 carbons of thiochromanone
0
and oxazolyl moiety at 188 (C4=O), 86 (spiro-C3,5 ),
0
56 (C4 ), 31 (–SC2H2–), 21 (CH3), 125, 125, 128, 140, 141,
159, 160 (quaternary aromatic C) and 114; 125, 126, 127,
129, 130, 133 ppm (tertiary aromatic C).
Molecular properties calculations
In series 17–22 the introduction of a variety of fragments
by means of the two terminal aryl substituted on positions
para provided compounds with a broad range of anti-TC
activities. The highest anti-TC activities were obtained for
compounds 17 and 20 which exhibited low IC50 values
between 1.5 and 3.78 lM, up to 5.7-fold lower than Nfx or
up to 3.5-fold lower than Bdz.
Replacement of the electron attractor nitro moiety (R1)
in compound 22 by electron donor methyl moiety (R1) in
compounds 17 and 19 provided compounds with activities
three to sixfold better than reference drugs (Nfx and Bdz),
suggesting that rather bulky and hydrophobic substituents
can be introduced in this position without losing activity.
Change of sulfur atom’s position from S-2 (compounds 17–
22 to position S-1) compounds 23–26 of the central ring
decrease considerably the anti-TC activity when compared
to their isomers, compounds 17–22.
Introduction of R2 substituent by means of the second
aryl link at position C-40 led to more effective inhibition in
the case of derivatives containing electro-donor group as
like methoxy or methyl groups. Indeed, activities of the
amine compounds 18 and 25, having the same substituents
[(R1, R2)=(CH3, OCH3)] were found to be completely
different; if compound 18 is moderately bioactive
(IC50 = 19 lM), the later compound 25 and its analogous
23–26 are not active.
POM virtual screening
Trypanosoma cruzi is much more difficult to treat than
human African trypanosoma (HAT), since this trypanoso-
matid parasite is intracellular, and drugs used for the dis-
ease must pass through mammalian and parasite cell
membranes to be effective. Laboratory and clinical studies
conducted since 1969 have demonstrated that Nfx and Bdz
are the best agents for treating human TC infection,
although they are far from being ideal drugs. Nfx and Bdz
are indicated in the acute phase of the infection, the con-
genital form, reactivation of disease associated with
immunosuppression, and in transfusions and organ trans-
plants involving infected individuals. Both drugs are taken
orally and must be given divided into 2–3 fractions after
meals. They are generally well tolerated by children, par-
ticularly in the acute phase of the disease, but relatively
frequent and severe gastrointestinal or dermatological
adverse reactions may be observed. Recurrence of the
disease is a significant problem, and as such these drugs are
considered generally ineffective. The main limitations of
both drugs are their long courses of administration and the
occurrence of adverse side effects. The related compound
megazol has also been used for chagas disease, but its use
was discontinued because of severe mutagenic and cyto-
toxic effects (Pinazo et al., 2010).
Comparison of compounds 17–26 with clinical refer-
ence drugs concerning their pharmacophore sites showed
that rigidification in this region of our candidates yielded a
great win in activity but also a decrease in cytotoxicity,
which finally provided for candidates 17–22 important
increase in the selectivity index. Further in vivo studies are
going to be conducted on compounds 17 and 20 displaying
selectivity indices superior to that of reference compounds
(Nfx and Bdz). Displacement of the sulfur atom in these
compounds 17–26 from position S-2 to S-1 in series of
heterocycles 23–26 led to a loss of the ability to inhibit TC
(with no exception). Inhibition of TC strain by our
Petra calculations
PETRA is a program package comprising various empiri-
cal methods for the calculation of physicochemical
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Med Chem Res (2013) 22:57–69
Osiris calculations
properties in organic molecules. All methods are empirical
in nature and have been developed over the last 20 years in
the research group of Prof. J. Gasteiger. The following
chemical effects can be quantified: heats of formation,
bond dissociation energies, sigma charge distribution,
p-charge distribution, inductive effect, resonance effect and
delocalization energies and polarizability effect.
Structure based design is now fairly routine but many
potential drugs fail to reach the clinic because of ADME-
Tox liabilities. One very important class of enzymes,
responsible for many ADMET problems, is the cyto-
chromes P450. Inhibition of these or production of
unwanted metabolites can result in many adverse drug
reactions. Of the most important program, Osiris is already
available online (Rauf et al., 2010; Sheikh et al., 2011;
Jawarkar et al., 2010a, b, c; Parvez et al., 2010a, b, c, d;
Chohan et al., 2010a, b, c, d).
The series 17–26 of STC have been subjected to delo-
calised-charge calculations using Petra method of the
non-hydrogen common atoms, obtained from the partial
pi-charge of the heteroatoms, have been used to model the
bioactivity against TC.
The Osiris property explorer shown in this page is an
integral part of Actelion’s inhouse substance registration
system. It lets you draw chemical structures and calculates
on-the-fly various drug-relevant properties whenever a
structure is valid. Prediction results are valued and color
coded. Properties with high risks of undesired effects like
mutagenicity or a poor intestinal absorption are shown in
red. Whereas a green color indicates drug-conform
behavior (Table 3).
It is found that the negative charges of the sulfur of
isothiochromanone moiety and nitrogen atom of oxazol
ring contribute positively in favor of antitrypanosomal
activity, more, and this is in good agreement with the
hypotyhetic mode of antitrypanosomal action of the com-
pounds bearing (Xd-/Yd-) pharmacophore site (X, Y=O,
N, S), Fig. 1. It was previously hypothesized that differ-
ence in charges between two heteroatoms of the same
dipolar pharmacophore site (Xd-/Yd?) may facilitate the
inhibition of bacteria, more than viruses and funguses
(Bennani et al., 2007a, b); Anaflous et al., 2004; Ben
Hadda et al., 2003). It is further found that the activity
increases with increase in negative charge of one hetero-
atom of the common pharmacophore fragment of the hits.
The presence of pi-delocalisation phenomena in rigid
pharmacophore sites was presented previously since 2007
(Bennani et al., 2007a, b).
With our recent publications of the drug design com-
bination of various pharmacophore sites by using spiro-
heterocyclic structure, it is now possible to predict activity
and/or inhibition with increasing success in two targets
(bacteria and HIV), (Bennani et al., 2007a, b). This is done
using a combined electronic/structure docking procedure
and an example will be given here (Table 3). The
remarkably well behaved mutagenicity of divers synthetic
molecules classified in data base of CELERON company
of Swiss can be used to quantify the role played by various
organic groups in promoting or interfering with the way a
drug can associate with DNA.
On the basis of this pharmacophore analog system
described above, in compound 17, sets of distribution of
pharmacophore sites in series 17–22 and 23–26 could be
considered as probably active in the presence of TC and
bacteria. This synergistic and streamlined working proce-
dure led to highly active rigid/selective (Xd-/Yd-) receptor
ligands. However, a little difference in their respective
binding affinities was consistently found for the two iso-
meric series 17–22 and 23–26. The analysis of isomeric
differences due to S heteroatom position (C-1 or C-2) in
candidates revealed a favorable (Sd-/Nd-) interaction in
17–22, whereas 23–26 showed no activity against TC. So
the antitrypanosomal activity is related with possible sec-
ondary electronic interaction with the positively charged
side chains of the virus target(s). Attempt was made to
evaluate steric and indicator parameters which emerged as
important contributors from previous pharmacologic anal-
ysis. The present results support the previous observations
that thiochromanone ring in adjacent spiro-position of
Prediction results are valued and color coded. Properties
with high risks of undesired effects like mutagenicity or a
poor intestinal absorption are shown in red. Whereas a
green color indicates drug-conform behavior (http://www.
organic-chemistry.org/prog/peo). Note: the side effects
most commonly associated with Bdz therapy are rash and
gastrointestinal symptoms (nausea). Rarely, peripheral
neuropathy may present after prolonged treatment.
Molinspiration calculations
CLogP (octanol/water partition coefficient) is calculated by
the methodology developed by molinspiration as a sum of
fragment based contributions and correction factors
(Table 4). The method is very robust and is able to process
practically all organic and most organometallic molecules.
Molecular polar surface area TPSA is calculated based on
the methodology published by Ertl et al. (2000) as a sum of
O–N=C could generate two (Sd-/Nd-) and (Od-/Nd-
)
pharmacophore sites which are conducive to the activity to
anti-HIV (Od-/Nd-) (Bennani et al., 2007a, b) and anti-
trypanosomal activity (Sd-/Nd-).
123

Med Chem Res (2013) 22:57–69
63
Table 3 Osiris calculations of
compounds 17–22
Toxicity risks
MUT
Osiris calculations
Compd.
TUM
IRRIT
RE
MW
cLog P
Sol
DL
DS
17
18
399
415
415
431
450
430
260
301
7.5
7.0
7.0
6.6
7.3
7.0
0.6
0.92
−8.1
−7.8
−7.8
−7.5
0.7
1.0
1.0
2.3
0.21
0.22
0.22
0.25
0.12
0.12
0.18
0.47
19
20
21
−8.6 −6.6
−8.3 −9.2
−1.6 −3.3
−3.4 −2.6
22
MUT mutagenic, TUM
tumorigenic, IRRIT irritant, RE
reproductive effective, Sol
solubility, DL drug-likeness, DS
drug-score, Bdz benznidazole,
Nfx nifurtimox, the reference
drugs
Bdz
Nfx
Table 4 Molinspiration calculations of compounds 17–22
Compd.
(R1, R2)
Molinspiration calculations
Drug-likeness
GPCRL
cLog P
TPSA
OH/NH
N viol.
Vol.
ICM
KI
NRL
17
(CH3, CH3)
(CH3, OCH3)
(OCH3, CH3)
(OCH3, OCH3)
(Cl, NO2)
(NO2, CH3)
–
6
38
48
48
57
85
85
93
109
0
0
0
0
0
0
1
0
1
1
1
1
1
1
0
0
358
367
367
376
362
365
225
246
-0.21
-0.20
-0.20
-0.17
-0.28
-0.32
-0.39
-1.24
-0.37
-0.42
-0.42
-0.36
-0.39
-0.45
-0.69
-1.46
-0.72
-0.70
-0.70
-0.66
-0.77
-0.79
-0.84
-0.92
-0.57
-0.54
-0.54
-0.47
-0.64
-0.63
-1.55
-1.49
18
5.7
5.7
5.3
5.8
5.6
0.7
1.2
19
20
21
22
Bdz
Nfx
–
TPSA total molecular polar surface area, N viol. number of violation of five Lipinsky rules, Vol. volume, GPCRL GPCR ligand, ICM ion channel
modulator, KI kinase inhibitor, NRL nuclear receptor ligand
fragment contributions. O– and N– centered polar frag-
ments are considered. PSA has been shown to be a very
good descriptor characterizing drug absorption, including
intestinal absorption, bioavailability, Caco-2 permeability,
and blood–brain barrier penetration. Prediction results of
compounds 17–26 molecular properties (TPSA, GPCR
ligand and ICM) are valued (Table 4).
hydrophobic and arene–arene interactions. No H-bonding
is present between drug and receptor (Fig. 2). Figure 2d
shows blue—H-bonding donor, red—H-bond acceptor, and
white—hydrophobic region. This is receptor based contour
map of compound 17.
From above docking poses, it is clear that during the
interaction with cruzin, the six membered ring containing
suphur and five membered ring containing N and O are
almost perpendicular to each other. From the above anal-
ysis it is clear that in ring B, instead of keto group a H-bond
donor group like –OH is required (because blue region is
very close this group (Fig. 2). In ring D, at ortho or meta
position one hydrophobic group of small size like –CH3 is
required (Figs. 2, 3). In ring C, at ortho or meta or para at
least one H-bond donor group like –OH or –NH2 is
required.
Doking of compounds 17–22 in cruzin enzyme
A specific inhibitor of the neuraminidase of the protozoan
parasite TC was isolated since 1987 and named cruzin
(Prioli et al., 1987).
Below is the same diagrams showing interaction of
compound 17 with receptor of cruzin. Interestingly, the
prominent interactions between the drug and receptor are
123

64
Med Chem Res (2013) 22:57–69
Fig. 2 Docking poses (a–d) of hit 17 in receptor of cruzin inhibitor
Conclusion
Trypanosoma and Leishmania species (Changtam et al.,
2010).
This study provided us with additional structure-activity
and structure-cytotoxicity information in the SOT family.
Indeed, this study proved that a number of substitutions
lead to compounds with high activities and reduced
cytotoxicities.
Experimental
Materials and methods
General procedure
Synthesis and evaluation of the spiroheterocyclic com-
pounds 17–22 showed that lipophilic substituents bearing
electro-donor groups could be introduced in oxazolidine
moiety while maintaining a high antiparasitic activity.
Introduction of sulfur atom in position-1 instead of posi-
tion-2 on the SOT template provided four additional
compounds 23–26 with no anti-parasital activity. Taking
into account our first structure-activity relationship, these
libraries will include on the terminal aryls of the molecule
derivatives of para-positions of the two aryl substituents
(sugar and hydroxylated groups).
1H and 13C NMR spectra were recorded on a Bruker
spectrospin spectrometer operating at 80 MHz for 1H
´
(Universite Paul Sabatier, Toulouse, France), an AC 200
spectrometer (operating at 200.12 MHz for 1H, 50.32 MHz
l3
´
´
for C), (Universite de Franche-Compte, Besanc¸on,
France). Chemical shifts are listed in ppm and are reported
relative to tetramethylsilane (1H, 13C), residual solvent
peaks being used as internal standard. Complete assign-
ments of the 13C spectra required non-decoupled 13C-NMR
The spiro-molecules bearing (Sd-/Nd-) pharmacophore
site with dS—N = 3.5 A, constitute a potential antitrypan-
osomal drugs. A similar (Od-/Od-)-pharmacophore is
found in curcuminoid analogs with potent activity against
1
spectra with selective H decoupling. Spectrometers, mass
spectra on a Platform II Micro Mass spectrometer, Infrared
spectra were obtained on a BECKMAN 310 spectrometer.
123

Med Chem Res (2013) 22:57–69
65
Fig. 3 Docking poses (a–d) of hit 20 in receptor of cruzin inhibitor
30,40-Di-(4-methyl-phenyl)-40H-spiro[isothiochromene-
3,50-isoxazol]-4(1H)-one (17)
Mass spectra on a HEWLETT PACKARD 5989A Mass
´
spectrometer (70 eV) and elemental analysis (Universite
Paul Sabatier, Toulouse, France).
White powder M.p. = 179°C; yield = 85%, IR (KBr,
1
t cm-1): 1,675 (C=O). H NMR (CDCl3, d0 ppm, J Hz):
General synthesis of 30,40-diaryl-40H-
spiro[isothiochromene-3,50-isoxazol]-4(1H)-ones 17–22
and 23–26
8.1–7.1 (m, 12H, aromatic H); 6.1 (s, 1H, CH4 ); 4.7 (d, 1H,
2
2
H1a, JH1a–H1b = 16.8); 3.50 (d, 1H, H1b, JH1b–H1a
=
16.8); 2.5 (s, 3H, CH3);02.3 (s, 3H,0 CH3). 13C NMR: 184.1
(C4=O); 94.6 (spiro-C3,5 ); 56.4 (C4 ); 28.8 (–SC1H2–); 21.5
(2CH3); 125.30; 130.10; 131.40; 138.45; 140.60; 140.85;
160.25 (quaternary aromatic C); 127.50; 127.70; 127.75;
129.30; 129.35; 130.00; 130.45; 132.90 (tertiary aromatic
C). Masse spectrum (m/z): calc. for C25H21NSO2 (M?)
399.50; found 399.12. Anal. Calc. for C25H21NSO2:
C 75.16; H 5.30; N 3.51; found: C 74.95; H 5.27; N 3.54.
All starting material 1–5, intermediaries 6–10 and 11–16
have been previously prepared and described by our group
in literature (Al Houari et al., 2008a, b; Badri et al., 1999;
Ben Hadda et al., 2007, 2008; Bennani et al., 2002, 2007a,
b). We report here the synthesis of spiro-molecules 17–26:
in an erlenmeyer equipped with a bulb for addition, a
mixture of 10 mmol of the 3-arylideneisothiochroman-
4-one 11–13 or 10 mmol of the 3-arylidenethiochroman-
4-one 14–16 and 12 mmol of oxime 6–10 in 20 ml of
chloroform was placed in a ice-salt bath, and under mag-
netic agitation 10 ml of a sodium hypochlorite solution
(NaOCl, 18°) was added. Agitation was maintained for 1 h
after the addition. The organic phase was separated,
washed several times with water, and dried on sodium
sulfate. The residue obtained after evaporation of the sol-
vent was recrystallized from ethanol.
30-(4-Methyl-phenyl)-40-(4-methoxy-phenyl)-40H-
spiro[isothiochromene-3,50-isoxazol]-4(1H)-one (18)
White powder M.p. = 150°C; yield = 90%. IR (KBr;
1
t cm-1): 1,675 (C=O). H NMR (CDCl3, d ppm, J Hz):
0
8.2–6.8 (m, 12H, aromat. H); 6.1 (s, 1H, CH4 ); 4.7 (d, 1H,
2
H1a, JH1a–H1b = 16.8); 3.7 (s, 3H, OCH3); 3.5 (d, 1H,
H1b, 2JH1b–H1a = 16.8); 2.3 (s, 3H, CH3). 13C NMR: 180.8
123

66
Med Chem Res (2013) 22:57–69
0
0
(C4=O); 95.0 (spiro-C3,5 ); 57.0 (C4 ); 55.0 (OCH3); 28.1
(–SC1H2–); 21.1 (CH3); 120.50; 130.15; 131.35; 138.40;
140.90; 159.80; 161.20 (quaternary aromatic C); 114.10;
127.50; 127.80; 129.30; 129.35; 130.10; 131.40; 132.90
(tertiary aromatic C). Masse spectrum (m/z): calc. for
C25H21NSO3 (M?) 415.50; found 415.12. Anal. Calc. for
C25H21NSO3: C 72.27; H 5.09; N 3.37; found: C 70.08;
H 4.97; N 3.41.
(tertiary aromatic C). Masse spectrum (m/z): calc. for
C23H15N2O4ClS (M?) 450.89; found 450.05. Anal. Calc.
for C23H15N2O4ClS: C 61.27; H 3.35; N 6.21; found:
C 60.95; H 3.26; N 6.28.
30-(4-Nitro-phenyl)-40-(4-methyl-phenyl)-40H-
spiro[isothiochromene-3,50-isoxazol]-4(1H)-one (22)
White powder M.p. = 187–189°C; yield = 65%; IR (KBr;
1
30-(4-Methoxy-phenyl)-40-(4-methyl-phenyl)-40H-
t cm-1): 1,680 (C=O). H NMR (CDCl3, d0 ppm, J Hz):
spiro[isothiochromene-3,50-isoxazol]-4(1H)-one (19)
8.1–7.1 (m, 12H, aromatic H); 6.1 (s, 1H, CH4 ); 4.7 (d, 1H,
H1a, 2JH1a–H1b = 16.8); 3.6 (d, 1H, H1b, 2JH1b–
H1a = 16.8); 2.3 (s, 3H). 13C NMR: 183.6 (C4=O); 95.6
White powder M.p. = 153–155°C; yield = 90%; IR (KBr,
0
0
t cm-1): 1,675 (C=O). H NMR (CDCl3, d0 ppm, J Hz):
(spiro-C3,5 ); 55.9 (C4 ); 28.1 (–SC1H2–); 21.3 (CH3);
128.50; 130.00; 131.00; 139.15; 140.70; 148.60; 158.80
(quaternary aromatic C); 123.90; 127.75; 127.90; 128.10;
128.80; 129.05; 129.65; 130.90 (tertiary aromatic C).
Masse spectrum (m/z): calc. for C24H18N2SO4 (M?)
430.47; found 430.09. Anal. Calc. for C24H18N2SO4:
C 66.96; H 4.21; N 6.51; found: C 67.12; H 4.18; N 5.95.
1
8.1–6.7 (m, 12H, aromatic H); 6.1 (s, 1H, CH4 ); 4.7 (d, 1H,
2
H1a, JH1a–H1b = 16.8); 3.70 (s, 3H, OCH3); 3.5 (d, 1H,
H1b, 2JH1b–H1a = 16.8); 02.3 (s, 3H, CH3). 13C NMR: 184.1
0
(C4=O); 94.4 (spiro-C3,5 ); 56.4 (C4 ); 55.2 (OCH3); 28.1
(–SC1H2–); 21.3 (CH3); 120.50; 130.20; 131.35; 138.45;
140.90; 159.85; 161.20 (quaternary aromatic C); 114.10;
127.55; 127.80; 129.35; 130.05; 130.40; 132.95 (tertiary
aromatic C). Masse spectrum (m/z): calc. for C25H21NSO3
(M?) 415.50; found 415.12. Anal. Calc. for C25H21NSO2:
C 72.27; H 5.09; N 3.37; found: C 71.98; H 4.97; N 3.41.
30-Phenyl-40-(4-methoxy-phenyl)-40H-spiro[thiochromene-
3,50-isoxazol]-4(1H)-one (23)
White powder M.p. = 183–185°C; yield = 87%. IR (KBr;
1
30,40-Di-(4-methoxy-phenyl)-40H-spiro[isothiochromene-
t cm-1): 1,668 (C=O). H NMR (CDCl3, d ppm, J Hz):
0
3,50-isoxazol]-4(1H)-one (20)
8.13–6.9 (m, 13H, aromat. H); 5.36 (s, 1H, CH4 ); 3.8 (s,
3H, OCH3); 3.14 (d, 1H, H2b, 2JH2a–H2b = 14.01); 3.05 (d,
2
White powder M.p. = 154–156°C; yield = 85%; IR (KBr;
1H, H2a, JH2a–H2b = 14.01). 013C NMR: 189.3 (C4=O);
0
t cm-1): 1,665 (C=O). H NMR (CDCl3, d ppm, J Hz):
86.81 (spiro-C3,5 ); 56.5 (C4 ); 55.67 (OCH3); 31.91
(–SC2H2–); 128.73; 129.23; 130.60; 130.60; 141.75;
160.09; 160.56 (quaternary aromatic C); 115.03; 125.39;
125.47; 127.04; 127.95; 128.98; 131.37; 134.19 (tertiary
aromatic C). Masse spectrum (m/z): calc. for C24H19NO3S
(M?) 401.47; found 401.11. Anal. Calc. for C24H19NO3S:
C 71.91; H 4.73; N 6.51; found: C 67.12; H 4.18;
N 53; found: C 71.80; H 4.77; N 3.49.
1
0
8.1–6 (m, 12H, aromat. H); 6.1 (s, 1H, CH4 ); 4.7 (d, 1H,
2
H1a, JH1a–H1b = 16.8); 3.7 (s, 6H, 2 OCH3); 3.5 (d, 1H,
H1b, 2JH1b–H1a = 16.8). 13C NMR: 184.2 (C4=O); 95.0
0
0
(spiro-C3,5 ); 56.2 (C4 ); 55.3 (2OCH3); 28.2 (–SC1H2–);
120.50; 125.15; 130.40; 131.40; 140.90; 159.70; 159.80;
161.20 (quaternary aromatic C); tertiary aromatic C:
113.90; 114.10; 127.50; 127.75; 129.35; 130.40; 131.30;
132.90 (tertiary aromatic C). Masse spectrum (m/z): calc.
for C25H21NSO4 (M?) 431.50; found 431.11. Anal. Calc.
for C25H21NSO4: C 69.59; H 4.91; N 3.25; found: C 69.83;
H 5.13; N 3.15.
30-(4-Methyl-phenyl)-40-phenyl-40H-spiro[thiochromene-
3,50-isoxazol]-4(1H)-one (24)
White powder M.p. = 192–193°C; yield = 92%; IR (KBr;
1
30-(4-Chloro-phenyl)-40-(4-nitro-phenyl)-40H-
t cm-1): 1,662 (C=O). H NMR (CDCl3, d ppm, J Hz):
0
spiro[isothiochromene-3,50-isoxazol]-4(1H)-one (21)
8.13–6.9 (m, 13H, aromat. H); 5.4 (s, 1H, CH4 ); 3.14 (d,
1H, H2b, 2JH2a–H2b = 14.01); 3.05 (d, 1H, H2a, 2JH2a–
H2b = 14.01); 2.3 (s, 3H, CH3). 13C NMR: 186.2 (C4=O);
White powder M.p. = 149–151°C; yield = 60%; IR (KBr;
0
0
t cm-1): 1,665 (C=O). H NMR (CDCl3, d ppm, J Hz):
86.2 (spiro-C3,5 ); 56.7 (C4 ); 31.6 (–SC2H2–); 21.5 (CH3);
125.37; 128.82; 133.42; 140.55; 141.35; 160.12 (quater-
nary aromatic C); 125.07; 126.64; 127.51; 128.53; 129.26;
129.34; 130.99 (tertiary aromatic C). Masse spectrum
(m/z): calc. for C24H19NO2S (M?) 385.47; found 385.11.
Anal. Calc. for C24H19NO2S: C 74.78; H 4.97; N 3.63;
found: C 74.37; H 4.85; N 3.49.
1
8.2–7.2 (m, 12H, aromat. H); 6.2 (s, 1H, CH4); 4.7 (d, 1H,
H1a, 2JH1a–H1b = 16.8); 3.6 (d, 1H, H1b, 2JH1b–
0
H1a0 = 16.8); 13C NMR: 183.4 (C4=O); 94.4 (C3,5 ); 56.0
(C4 ); 28.0 (–SC1H2–); 125.90; 130.75; 137.60; 139.80;
140.40; 148.10; 158.55 (qquaternary aromatic C); 123.80;
127.80; 127.90; 128.80; 129.25; 130.50; 131.10; 133.40
123

Med Chem Res (2013) 22:57–69
67
30-(4-Methyl-phenyl)-40-(4-methoxy-phenyl)-40H-
´
(Novafarma, Anapolis, GO, Brazil). Each compound was
evaluated at five concentrations (1, 2, 5, 10, 25, 50, and
100 lg mL-1), in triplicate. Cultures were incubated in the
spiro[thiochromene-3,50-isoxazol]-4(1H)-one (25)
presence of H-thymidine (1 lCi well-1) for 24 h at 37°C
3
White powder M.p. = 172–174°; yield = 85%; IR (KBr;
1
t cm-1): 1,680 (C=O). H NMR (CDCl3, d0 ppm, J Hz):
and 5% CO2. After this period, the content of the plate was
harvested to determine 3H-thymidine incorporation using a
beta-radiation counter (LKB Wallac Rack Beta, Pharmacia
Biotech). The cytotoxicity of the compounds was deter-
mined by comparing the percentage of 3H-thymidine
incorporation (as an indicator of cell viability) in drug-
treated wells in comparison to untreated wells. Non-cyto-
toxic concentrations were defined as those causing a
reduction of 3H-thymidine incorporation below 10% in
relation to untreated controls.
8.1–6.9 (m, 12H, aromat. H); 5.4 (s, 1H, CH4 ); 3.8 (s, 3H,
OCH3); 3.13 (d, 1H, H2b, 2JH2a–H2b = 14.01); 3.04 (d, 1H,
H2a, 2JH2a–H2b = 14.01); 2.3 (s, 0 3H, CH3). 13C NMR:
0
188.96 (C4=O); 86.2 (spiro-C3,5 ); 56.24 (C4 ); 31.51
(–SC2H2–); 21.45 (CH3); 125.13; 125.43; 128.84; 140.48;
141.33; 159.68; 160.10 (quaternary aromatic C); 114.56;
125.05; 126.63; 127.49; 129.30; 130.96; 133.73 (tertiary
aromatic C). Masse spectrum (m/z): calc. for C25H21NO3S
(M?) 415.50; found 415.12. Anal. Calc. for C25H21NO3S:
C 72.27; H 5.09; N 3.37; found: C 71.98; H 4.98; N 3.41.
In vitro antiproliferative activity
30-(4-Methyl-phenyl)-40-(4-nitro-phenyl)-40H-
spiro[thiochromene-3,50-isoxazol]-4(1H)-one (26)
Epimastigotes of TC (Y strain) were cultivated at 26°C in
liver infusion tryptose medium (LIT) supplemented with
10% fetal calf serum, 1% hemin, 1% R9 medium, and
50 lg mL-1 gentamycin. Parasites (106 cells mL-1) were
cultured in a fresh medium in the absence or in the pres-
ence of the compounds being tested (from stock solution in
DMSO). Cell growth was determined after 11 days of
culture by counting viable forms in a hemacytometer, in
triplicate. The spiro-heterocycles compounds used were
from a stock solution in DMSO. To determine the IC50,
cultures of Y strain epimastigotes in the presence of dif-
ferent concentrations of the compounds were evaluated
after 11 days as described above. IC50 calculation was
carried out using non-linear regression on Prism 4.0
GraphPad software. Biological assays on Y strain TC try-
pomastigotes were obtained from culture supernatants of
vero cell line at 37°C and placed in 96-well plates
(4 9 105 well-1) in a DMEM medium supplemented with
10% FCS and 50 lg mL-1 gentamycin. Viable parasites
were counted in a hemacytometer 24 h after addition of
complexes by way of trypan blue exclusion. The percent-
age of inhibition was calculated in relation to untreated
cultures. The same procedure was performed for Bdz and
Nfx (reference drugs) and vehicle alone, DMSO as blank.
White powder M.p. = 194–196°C; yield = 81%; IR (KBr;
1
t cm-1): 1,684 (C=O). H NMR (CDCl3, d ppm, J Hz):
0
8.3–7.1 (m, 12H, aromat. H); 5.46 (s, 1H, CH4 ); 3.12 (d,
1H, H2b, 2JH2a–H2b = 14.01); 3.03 (d, 1H, H2a, 2JH2a–
H2b = 14.01); 2.31 (s, 3H, CH3). 13C NMR: 188.15
0
0
(C4=O); 86.77 (spiro-C3,5 ); 56.46 (C4 ); 31.24 (–SC2H2–);
21.45 (CH3); 124.58; 129.57; 134.48; 140.87; 147.97;
141.61; 159.36 (quaternary aromatic C); 124.37; 125.39;
126.72; 127.38; 129.57; 130.20; 131.14; 134.11 (tertiary
aromatic C). Masse spectrum (m/z): calc. for C24H18N2O4S
(M?) 430.47; found 430.09. Anal. Calc. for C24H18N2O4S:
C 66.96; H 4.21; N 6.51; found: C 67.02; H 4.17; N 6.48.
Biological properties
Antitypanosomal activity (in vitro)
The anti-TC activities of the compounds were determined
by their inhibition of parasite growth using the Bdz-resis-
tant strain. In parallel, all compounds were tested for
cytotoxicity upon a BALB/c mice splenocytes. All com-
pounds were tested against TC using the Bdz-resistant
strain and they were tested for cytotoxicity upon a BALB/c
mice splenocytes.
Acknowledgments We are grateful to staff of Federal University of
Pernambuco, Recife-Brazil for the help in biological assay. Prof.
T. Ben Hadda would like to thank the ACTELION; the Biopharma-
ceutical Company of Swiss, for the online molecular properties
calculations.
In vitro cytotoxicity
The cytotoxicity of compounds was determined using
BALB/c mice splenocytes (5 9 106 cells well-1) cultured
in 96-well plates in Dulbecco’s modified eagle’s medium
(DMEM, Sigma Chemical Co., St. Louis, MO) supple-
mented with 10% of fetal calf serum (FCS; Cultilab,
Campinas, SP, Brazil) and 50 lg mL-1 of gentamycin
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