Synthesis, Biological Evaluation, and Structure-activity Relationship of Clonazepam, Meclonazepam, and 1,4-Benzodiazepine Compounds with Schistosomicidal Activity

Article abstract of DOI:10.1111/j.1747-0285.2012.01354.x

The inherent morbidity and mortality caused by schistosomiasis is a serious public health problem in developing countries. Praziquantel is the only drug in therapeutic use, leading to a permanent risk of parasite resistance. In search for new schistosomicidal drugs, meclonazepam, the 3-methyl-derivative of clonazepam, is still considered an interesting lead-candidate because it has a proven schistosomicidal effect in humans but adverse effects on the central nervous system did not allow its clinical use. Herein, the synthesis, in vitro biological evaluation, and molecular modeling of clonazepam, meclonazepam, and analogues are reported to establish the first structure-activity relationship for schistosomicidal benzodiazepines. Our findings indicate that the amide moiety [N₁H-C₂(=O)] is the principal pharmacophoric unit of 1,4-benzodiazepine schistosomicidal compounds and that substitution on the amide nitrogen atom (N₁ position) is not tolerated.

Full text of DOI:10.1111/j.1747-0285.2012.01354.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01354.x
Chem Biol Drug Des 2012; 79: 943–949
Research Article
Synthesis, Biological Evaluation, and
Structure–activity Relationship of Clonazepam,
Meclonazepam, and 1,4-Benzodiazepine
Compounds with Schistosomicidal Activity
Carla M. S. Menezes1, Gildardo Rivera1,†
Marina A. Alves1, Daniel N. do Amaral1,
,
occupied molecular orbital; ELUMO, energy of the lowest unoccupied
molecular orbital; l, dipole moment; MEP, molecular electrostatic
potential; ADMET, absorption, distribution, metabolism, elimination, and
toxicity.
2
2,3
¨
Jean Pierre B. Thibaut , Franc¸ois Noel
,
Eliezer J. Barreiro1,3 and Lı´dia M. Lima1,3,
*
Received 17 November 2009, revised 4 August 2011 and accepted for
publication 21 January 2012
1Laboratꢀrio de Avaliażo e Sꢁntese de Substꢂncias Bioativas
(LASSBio, http://www.farmacia.ufrj.br/lassbio/), Faculdade de
Farmꢃcia, Universidade Federal do Rio de Janeiro, P O Box 68024,
21944-971, Rio de Janeiro, RJ, Brazil
2Laboratꢀrio de Farmacologia Bioquꢁmica e Molecular, Instituto de
CiÞncias Biomꢄdicas, Universidade Federal do Rio de Janeiro,
Cidade Universitꢃria, 21941-912, Rio de Janeiro, RJ, Brazil
3Programa de Desenvolvimento de Fꢃrmacos, Instituto de CiÞncias
Biomꢄdicas, Universidade Federal do Rio de Janeiro, Cidade
Universitꢃria, 21941-912, Rio de Janeiro, Brazil
*Corresponding author: Lꢁdia M. Lima, lidia@pharma.ufrj.br
Actual address: Centro de Biotecnologia Genomica, Instituto
Politecnico Nacional, Boulevard del Maestro, s/n, 88710, Reynosa,
Mexico
Schistosomiasis is a neglected disease whose death rate has been
estimated at 280 000 a year, with other 200 millions infected and
800 millions at risk, especially in tropical and subtropical areas (1–
3). Praziquantel (1, Figure 1) is the only drug in therapeutic use,
leading to a permanent risk of parasite resistance (4). The schistos-
omicidal effect observed with 1,4-benzodiazepine compounds like
the anxiolytic ⁄ antiepileptic drug clonazepam (2, Figure 1) led to an
extensive screening that resulted in the identification of meclonaze-
pam (3 or Ro 11-3128, Figure 1) as an interesting schistosomicidal
lead-candidate ([5, Stçhler, apud Pica-Mattoccia et al. 6]). However,
adverse effects such as impairment of psychomotor functions, seda-
tion, and ataxia prevented the therapeutic use of this 3-methyl-
derivative of clonazepam (7).
The inherent morbidity and mortality caused by
schistosomiasis is a serious public health problem
in developing countries. Praziquantel is the only
drug in therapeutic use, leading to a permanent
risk of parasite resistance. In search for new
schistosomicidal drugs, meclonazepam, the 3-
methyl-derivative of clonazepam, is still consid-
ered an interesting lead-candidate because it has a
proven schistosomicidal effect in humans but
adverse effects on the central nervous system did
not allow its clinical use. Herein, the synthesis, in
vitro biological evaluation, and molecular model-
ing of clonazepam, meclonazepam, and analogues
are reported to establish the first structure–activ-
ity relationship for schistosomicidal benzodiaze-
pines. Our findings indicate that the amide moiety
[N1H-C2(=O)] is the principal pharmacophoric unit
of 1,4-benzodiazepine schistosomicidal com-
pounds and that substitution on the amide nitro-
gen atom (N1 position) is not tolerated.
As clonazepam (2) and meclonazepam (3) are effective against
Schistosoma mansoni and S. haematobium, like praziquantel (1),
but not against S. japonicum (Stçhler, apud Bennett [8]), they have
been proposed to act by a similar mode of action as praziquantel,
even though their exact molecular target is still a matter of
debate and could be different (6,9). Although the large intra-worm
calcium influx caused by praziquantel (1) has been associated with
muscular contraction and surface disruption of the worm, there is
now controversy regarding its role in the parasite death (10). The
benzodiazepine binding sites present in adult S. mansoni have also
been discarded as putative molecular targets for meclonazepam
and praziquantel, at least for their effects on worm musculature
(11).
In view of the urgent need for alternative schistosomicidal agents
and taken into account the schistosomicidal activity of clonazepam
(2) and meclonazepam (3), the objective of present work was to
establish a structure–activity relationship for 1,4-benzodiazepine
compounds with schistosomicidal activity. Six N1-modified deriva-
tives of clonazepam were synthesized [Figure 1: methyl (4), allyl (5),
benzyl (6), ethyloxycarbonylmethyl (7), methyloxycarbonylmethyl (8),
Key words: 1,4-benzodiazepine, clonazepam, meclonazepam,
pharmacophoric unit, schistosomiasis, structure–activity relationship
Abbreviations: IC50, concentration to kill half the population of
worms; M, concave 1,4-benzodiazepine boat conformation; P, convex 1,4-
benzodiazepine boat conformation; EHOMO, energy of the highest
943

Menezes et al.
R
O
*
N
1
3
R'
7
R
H
H
CH3
CH3
R'
H
CH3
H
X
O
O
O
O
W
W'
NO2 Cl
NO2 Cl Meclonazepam (3)
N
O
5
*
N
W
Clonazepam (2)
6
2
4
N
W'
Cl
NO2
H
F
Diazepam (10)
Flunitrazepam (11)
2'
H
O
Praziquantel (1)
R
O
R
CH3
CH2CH=CH2
CH2Ph
N
LASSBio-1252 (N-metilclonazepam) (4)
LASSBio-1253 (5)
1
3
7
5
N
O2N
LASSBio-1254 (6)
CH2CO2Et
CH2CO2Me
CH2CO2H
LASSBio-1117 (7)
LASSBio-1255 (8)
LASSBio-1132 (9)
Cl
2'
H
H
H
O
*
S
S
N
N
1
N
1
1
*
*
CH3
3
CH3
3
3
CH3
7
7
7
5
5
*
5
*
N
NH
NH
O2N
O2N
O2N
Cl
Cl
Cl
2'
2'
2'
(13)
H
(14)
(12)
H
H
O
*
O
*
O
*
N
1
N
1
N
1
3
CH3
3
CH3
3
CH3
7
7
7
5
5
5
N
N
N
N
N
H2N
N
N
Cl
Cl
Cl
F
F
OH
2'
2'
2'
OH
(17)
(15)
(16)
Figure 1: Praziquantel (1), clonazepam (2), meclonazepam (3), and other 1,4-benzodiazepine compounds (4–17).
and respective carboxymethyl (9)] and incorporated in our theoreti-
cal studies together with two classical 1,4-benzodiazepine drugs
[diazepam (10) and flunitrazepam (11)] and some analogues of me-
clonazepam reported by Mahajan et al. (12), (12–17; Figure 1).
3.17 mmol of clonazepam (2) and 0.5 g of KF-Al2O3 (40% w ⁄ t) in
100 mL of acetone were added to 3.8 mmol of the appropriated
alkyl halides. The suspension was stirred at reflux for 24 h. The clo-
nazepam N-1-alkylated analogues (4–9) were isolated by filtration
followed by addition of 50 mL water and extraction with dichlorom-
ethane (5 · 50 mL). The organic layer was dried (Na2SO4), filtered,
and evaporated to dryness. The residue was purified by column
chromatography on silica gel (CH2Cl2; CH2Cl2: MeOH 0.5%; and
CH2Cl2: MeOH 1%). Yields 26–36%. The products were identified
Methods and Materials
Chemistry
1
by H and 13C nuclear magnetic resonance (NMR) spectra, infrared
General procedure for the preparation of (E)-5-(2-chlorophenyl)-7-nitro-
1H-benzo[e](1,4)diazepin-2(3H)-oneN-1-alkylatedderivatives(4–9)
(IR) spectra, melting point, and elemental microanalyses.
944
Chem Biol Drug Des 2012; 79: 943–949

Synthesis, Biological Evaluation, and Structure–activity Relationship
Biological assays
The increase in the size of the N1 substituent in the 1,4-benzodiaze-
Clonazepam (2), meclonazepam (3, S-(+)-enantiomer), diazepam
(10), flunitrazepam (11), and praziquantel (1, R,S-(€)-praziquantel)
were evaluated according to their short-term effect on worm motil-
ity (13) and schistosomicidal effect estimated using the standard
operating procedures proposed by TDR-WHO (14).
pine system will increase the inversion barrier, while smaller sub-
stituents prevent resolution of these compounds at room
temperature because of the lower racemization barrier (20,22).
The analysis of 1H NMR spectra (300 MHz, DMSOd6 or CDCl3,
25 ꢀC) of N1-modified clonazepam analogues, where the hydrogen
present in the N1 atom of clonazepam (2) was substituted by
methyl (4), allyl (5), benzyl (6), and carboxymethylene groups (7–9),
revealed the presence of two doublet signals (JHeqHax = 10.8 Hz)
relative to the methylene (C3) hydrogens in the equatorial and axial
positions of the 1,4-benzodiazepine system, indicating that these
compounds exist as conformational enantiomers. On the other hand,
the 1H NMR spectra (300 MHz, DMSOd6 or CDCl3, 25 ꢀC) of clo-
nazepam revealed the presence of one broad signal at 4.31 ppm
relative to C3-protons (Hax and Heq), suggesting that the inversion
barrier is not high enough to resolve the methylene in the equato-
rial and axial positions, so that the interconversion of the seven-
membered boat, through the ring flipping, is very fast on the NMR
time scale at room temperature.
Molecular modeling studies
Calculation of the physicochemical properties and stereoelectronic
surfaces was performed using the SPARTAN'08 software.a The
MMFF94 force field (15) was employed to construct the molecular
structures of R-())-praziquantel (1), clonazepam (2), R-())-meclo-
nazepam and S-(+)-meclonazepam (3), the N1-modified clonazepam
analogues (4–9), diazepam (10), flunitrazepam (11), and meclo-
nazepam-related compounds (12–17) (12). Similar results were
obtained using the semiempirical methods PM3 (16) and AM1 (17),
being this last one chosen for further procedures. The P (convex)
conformations and M (concave) conformations were considered.
Pharmacokinetics and toxicological properties (ADMET) were esti-
mated using the VOLSURF+ (version 1.0.4) software.b
Biological assays
Results and Discussion
Short-term effect on worm motility
Chemistry
In control conditions, the worms exhibited a variety of spontaneous
body movements, including small and fast generalized shortening
and lengthening of the body and propagating body waves along the
anterior–posterior axis, similar to peristaltic waves (13). In this
model, praziquantel (1), clonazepam (2), and meclonazepam (3) lead
to a spastic paralysis followed by a rapid reduction in worm's body
area: approximately 30% after 1-min exposure to 1 lM praziquantel
and 10 lM meclonazepam and 20% with 10 lM clonazepam (11).
On the other hand, the N1-modified clonazepam analogues (4–9)
and the benzodiazepine drugs, diazepam (10) and flunitrazepam
(11), did not alter the motility and general aspect of the worms
during the 30-min period of observation after exposition to 10 lM
of the compounds.
The synthesis of derivatives 4–9 was performed using clonazepam
(2) as starting material and exploring a chemoselective N-alkylation
using KF ⁄ Al2O3 in acetone, at room temperature, in the presence of
the appropriate alkyl halides (18,19). Ester 7 was used as interme-
diate for the synthesis of compounds 8 and 9 in a two-step
sequence: transesterification and hydrolysis by general base cataly-
sis.
Usually, the 1,4-benzodiazepine system exists in a 7-membered boat
conformation with the hydrogens at C3 in axial and equatorial posi-
tions so that conformational enantiomers (M and P conformers) can
exist, as reported for diazepam and analogues (Figure 2) (20,21).
NO2
Cl
Hax
R
O
N
Heq
Cl
N
3
eqH
N
1
3
1
N
Hax
R
O
NO2
M-conformation
P-conformation
R = H (2, Clonazepam)
R = CH3 (4, LASSBio-1252)
R = CH2CH = CH2 (5, LASSBio-1253)
R = CH2Ph (6, LASSBio-1254)
R = CH2CO2Et (7, LASSBio-1117)
R = CH2CO2Me (8, LASSBio-1255)
R = CH2CO2H (9, LASSBio-1132)
Figure 2: 7-membered
conformation of the 1,4-benzodiaz-
epine system.
boat
Chem Biol Drug Des 2012; 79: 943–949
945

Menezes et al.
Schistosomicidal effect
effect, we analyzed the influence of the chemical functions and
substituent groups present in the 1,4-benzodiazepine system of clo-
nazepam (2), meclonazepam (3), their analogue and derivative com-
pounds (4–17). Only the M conformation will be considered from
here, based on preliminary results.
The in vitro schistosomicidal effect of praziquantel (1), clonazepam
(2), and meclonazepam (3) was confirmed because all worms died
after 5-day exposition at 1 lM, 20, and 5 lM, respectively. As
shown in Figure 3, the concentrations killing half the population of
worms (IC50) after the standard 5-day period of observation were
estimated at 0.4, 10, and 3 lM, for praziquantel (1), clonazepam
(2), and meclonazepam (3), respectively. In the same conditions, the
N1-modified clonazepam analogues (4–9) had no effect, even after
a 5-day exposition to 50 lM (Table 1).
Amide moiety [N1H-C2(=O)]
1 Identical HOMO distribution (predominance in the chlorophenyl
substituent group at 5-position) and molecular electrostatic potential
surfaces [composed by four negative potential regions located on
the amide nitrogen atom (N1), carbonyl oxygen atom (C2(=O)), imine
nitrogen atom (N4), and oxygen atoms of the nitro group (7-posi-
tion)] (data not shown) are observed in clonazepam (2) and meclo-
nazepam (3). Analyses of the correspondent MEPs also show
similar electron distribution such as a low electron density on the
region of the amide carbonyl carbon (2, Figure 4B) and (3, Fig-
ure 4C). Likewise, a low electron density is observed on the amide
region of R-())-praziquantel, especially at the amide carbonyl car-
bon C2 (1, Figure 4A). The identification of electron density regions
in a molecule allows proposing specific points able to establish
important electrostatic interactions in the process of drug-receptor
recognition therefore contributing to elucidate structure–activity
relationships and to recognize pharmacophoric features (26–28);
Molecular modeling studies
The conformational analysis employing the AM1 semiempirical
method (17) revealed that all 1,4-benzodiazepine compounds present
preferentially the M conformation, although the relatively low-
energy barrier between M and P conformers (ca. 1–5 kcal ⁄ mol)
should allow the free interconversion among them (23). In addition,
the methyl group (3-position) of meclonazepam (3) exhibits a lower
steric effect on the amide [N1H-C2(=O)] and imine (N4=C5) moieties
of the 1,4-diazepine ring when it is located at the equatorial posi-
tion (i.e., S-(+)-enantiomer) in the M conformation, the result that
might explain the major antiparasitic effect reported for this enan-
tiomer (4,6) but also its sedative effect (24). In addition, we have
previously reported (11) an atomic overlapping between the amide
moiety of the M conformation of S-(+)-meclonazepam (3) and the
most active R-())-praziquantel (1), in which the carbonyl carbon of
the isoquinoline-4-one (2-position, Figure 1) was considered to be
essential for the schistosomicidal effect (25). This observation
claims our attention in reason of experimental data, indicating a
similar mode of action between these compounds (6,9).
2 The analysis of the six inactive N1-modified clonazepam ana-
logues (4–9) showed increase in the HOMO distribution and the
size and number of negative potential regions on the 1,4-benzodi-
azepine system (data not shown). The enrichment of the electron
density occurred especially on the amide subunit and was inde-
pendent of the nature (size and poorer or richer inductive effect)
of the alkyl substituent. This is illustrated by the decrease in the
intensity and extension of the blue color (partially replaced by a
weak green color) in the correspondent region of the MEP of
LASSBio-1252 (4, Figure 4D), in comparison with the same region
of clonazepam (2, Figure 4B). Similar electron density distributions
were observed with diazepam (10) and flunitrazepam (11, Fig-
ure 4E), two N1-methyl 1,4-benzodiazepine drugs with no schistos-
omicidal effect (Table 1);
To challenge the importance of the amide moiety [N1H-C2(=O)] as a
pharmacophoric unit as well as to establish a structure–activity
relationship for 1,4-benzodiazepine compounds with schistosomicidal
100
75
50
25
0
3 The replacement of the carbonyl oxygen atom of meclonazepam
(3) by a more electropositive, polarizable, and isoster sulfur atom,
generating its 2-thiocarbonyl analogue (12) (12), produced a compa-
rable electrophilic profile (12, Figure 4F) of the amide moiety (3,
Figure 4C), compatible with the reported similar schistosomicidal
activities (IC50 of 1.12 and 1.01 lM, respectively) (Table 1). However,
such replacement (O fi S) could modify pharmacokinetics proper-
ties that are putatively unfavorable for the druggability. Indeed, pre-
dictions using VolSurf+ indicated: (i) decrease in metabolic stability
(MetStab = 57.48 (3) to 46.97% (12), suggesting a higher sensibil-
ity to CYP3A4, a major enzyme in clonazepam metabolism [29]); (ii)
decrease in water solubility [LgS7.5 = )4.28 (3) to )4.63 (12)]; (iii)
increase in lipophilicity [LOGP n-Oct = LgD7.5 = 3.063 (3) to 3.553
(12)]; and (iv) increase in the ability to cross the blood brain barrier
[LgBB = )0.19 (3) versus )0.30 (12)], a key drawback for the clini-
cal use of meclonazepam as a schistosomicidal drug;
0
1
10
100
Drug concentration (
M
)
Figure 3: Concentration-effect curve for the schistosomicidal
effect of praziquantel (1), clonazepam (2), and meclonazepam (3).
Adult male worms were exposed to different concentrations of R,S-
(€)-praziquantel (.), clonazepam ( ), or S-(+)-meclonazepam (•) dur-
ing 5 days in culture medium. At the end of this period, the worms
were observed individually with an inverted microscope to deter-
mine the number of dead parasites. Mortality (%) corresponds here
to the mean values (percent of dead worms) obtained from 3–6 dif-
ferent experiments with about 9–12 worms each.
946
Chem Biol Drug Des 2012; 79: 943–949

Synthesis, Biological Evaluation, and Structure–activity Relationship
Table 1: Schistosomicidal activity and physicochemical properties of 1,4-benzodiazepine compounds
R
X
*
N
1
R'
3
7
5
Z
Y
W
W'
2'
Compound
R
X
R'
Y-Z
W
W'
IC50 (lM)
IC50 (lM)a
V (ꢀ3)
l (D)
EHOMO (ev)
ELUMO (ev)
2
H
H
O
O
O
O
O
O
O
O
O
O
S
O
S
S
S
H
CH3
H
H
H
H
H
H
H
N=C
N=C
N=C
N=C
N=C
N=C
N=C
N=C
N=C
N=C
N=C
NH-CH2
NH-CH2
N=C
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
Cl
NO2
NO2
NO2
NO2
NH2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
10
3
281.87
300.28
300.96
333.38
384.09
368.33
349.79
329.15
279.35
292.77
309.08
304.55
312.84
288.97
399.88
0.86
1.15
1.10
1.31
1.45
2.18
2.15
2.00
4.88
0.86
0.45
4.43
3.68
5.86
5.51
)9.87
)9.85
)9.84
)9.82
)9.82
)9.90
)9.91
)9.41
)9.16
)9.81
)9.09
)9.52
)9.01
)8.52
)9.05
)1.43
)1.40
)1.43
)1.38
)1.37
)1.44
)1.45
)1.49
)0.37
)1.42
)1.67
)1.08
)1.58
)0.31
)1.19
3
1.12
4
CH3
CH2CH=CH2
CH2Ph
CH2CO2Et
CH2CO2Me
CH2CO2H
CH3
CH3
H
H
H
H
H
>50
>50
>50
>50
>50
>50
>50
>50
5
6
7
8
9
10
11
12
13
14
15
16
H
F
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
1.01
3.92
13.67
>29
N=C
N=N-(2¢¢,6¢¢-
8.63
Di-F-4¢¢-Phenol)
N=N-(b-Naphtol)
17
H
S
CH3
N=C
Cl
>22
440.65
4.69
)8.46
)1.33
The physicochemical properties were calculated using the AM1 semiempirical method.
IC50, Concentration to kill half the population of worms (avalues determined by Mahajan et al. [12], expressed originally in lg ⁄ mL); V, Molecular volume; l,
Dipole moment; EHOMO, Energy of the highest occupied molecular orbital; ELUMO, Energy of the lowest unoccupied molecular orbital.
The M conformation with the 2¢-halogem atom position oriented to the lower face of the plane of the 1,4-benzodiazepine system has been considered, except
for compounds 13 and 14.
B
A
C
F
E
D
G
J
H
I
Figure 4: Molecular electrostatic potential maps (MEPs) of the minimum energy conformations of (A) R-())-praziquantel (1), (B) clonaze-
pam (2), (C) S-(+)-meclonazepam (3), (D) LASSBio-1252 (4), (E) flunitrazepam (11), and the S-(+)-meclonazepam-related compounds: (F) 2-thio-
carbonyl analogue (12), (G) imine-reduced bond derivative (13), (H) amine analogue (15), and the 7-azo derivatives: (I) 2¢¢,6¢¢-difluoro-4¢¢-
phenol (16) and (J) b-naphtol (17) calculated using the AM1 semiempirical method (Appendix S3).
Chem Biol Drug Des 2012; 79: 943–949
947

Menezes et al.
4 All these results (1 and 3) indicate that the nitrogen atom of the
amide moiety [N1H-C2(=O)] should not be substituted and the
respective carbonyl carbon region of 1,4-benzodiazepines should
exhibit a low electron density to confer a schistosomicidal activity.
This could suggest that the interaction of the amide moiety with
the putative receptor might occur through electrostatic interaction
(e.g., hydrogen bond) or covalent interaction (i.e., electrophylic cen-
ter), perhaps with residues of specific parasite enzymes.
sity at the amide carbonyl carbon (2-position) (3, Figure 4C) in
comparison with the higher electron density effect promoted by
the electron donor amine group (15, Figure 4H). This comparison
was reinforced by a calculated meclonazepam analogue in which
the nitro group was removed, resulting in an intermediary electron
density, i.e., higher than in 3 but lower than in 12 (data not
shown). In addition, an electronic distribution similar to the one
of meclonazepam (3) was observed when the nitro group was
positioned at the 8-position, whereas a decrease in the electron
density and steric effect was verified when moving to the 6- or
9-positions; Finally, the subsequent conversion of the amine (15)
into diazo derivatives 16 and 17 (12) resulted in decrease in the
schistosomicidal activity in relation to meclonazepam (3) (Table 1)
and extension of the distribution of HOMO and increase in the
electron density at the 1,4-benzodiazepine system, including that
on the amide carbonyl carbon [C2(=O)] region [(16, Figure 4I) and
(17, Figure 4J)].
3-position
In apparent contradiction to this view, meclonazepam (3) was more
potent (lower IC50) than clonazepam (2) with respect to their schist-
osomicidal activity in vitro (Table 1), albeit the electron density at
the carbonyl carbon of the amide moiety [C2(=O)] is somewhat
lower in clonazepam (Figure 4B) than in meclonazepam (Figure 4C).
However, considering that there are no other steric or electronic dif-
ferences between these two compounds, the higher antiparasitic
effect of meclonazepam (3) might be attributed to the increment of
a hydrophobic interaction point with the putative receptor.
The physicochemical properties analyzed (V, l, EHOMO and ELUMO
)
(Table 1) varied according to the atom ⁄ group substitution, although
no correlation with the schistosomicidal properties was observed.
As a conclusion, we believe that the findings reported herein may
be helpful for researchers interested to the design of new schistos-
omicidal compounds, an important but neglected topic.
Imine bond (N4=C5)
Reduction of the meclonazepam imine bond resulted in two com-
pounds (13 and 14) (12) with a new chiral center at the carbon C5
and a higher flexibility of the 1,4-diazepine ring. Increase in and
delocalization of the negative potential on the nitrogen atom (4-
position) (not shown) and increase in the electron density at the 2-
oxo [C2(=O)] (13, Figure 4G) and, in particular, at the 2-thio [C2(=S)]
carbon atoms were also observed. Both structural features may
explain the decreased schistosomicidal activity reported for these 2-
carbonyl (13; IC50 = 3.92 lM) and 2-thiocarbonyl (14;
IC50 = 13.67 lM) dihydro derivatives in relation to their prototypes
(IC50 of 1.12 and 1.01 lM, respectively, for 3 and 12) (Table 1) and
indicate that the imine bond should be preserved.
Acknowledgments
This work was supported by CNPq INCT-INOFAR # 573.564 ⁄ 2008-6)
and FAPERJ (# E ⁄ 26-171.532 ⁄ 2006, E-26 ⁄ 100.488 ⁄ 2007, E-
26 ⁄ 150.732 ⁄ 2007, E-26 ⁄ 111.591 ⁄ 2008, and # 150.944 ⁄ 2007). We
thank Mr. Eduardo Miguez (IMA, UFRJ) for the NMR spectra.
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Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
Appendix S1. Chemistry.
Appendix S2. Biological assays (13).
Appendix S3. Molecular modeling studies.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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