Design, synthesis and in vivo evaluation of sodium 2-benzyl-chloromalonates as new central nervous system depressants

Article abstract of DOI:10.1039/c5md00187k

This work describes the design, synthesis and in vivo evaluation of new central nervous system depressing agents that show low levels of acute toxicity as well as high solubility in water and exhibit anxiolytic and hypnotic effects. These new compounds are sodium 2-benzyl-2-chloromalonates, which were designed using molecular modelling techniques and synthesized in four steps, with an overall yield between 41% and 65%. In vivo tests with mice, including pentobarbital-induced sleep, rotarod, open field and elevated plus maze tests, were carried out, and the results indicated that some of these agents induce activities similar to those induced by diazepam but with lower hypnotic potency and greater anxiolytic potency. These compounds were also orally administered to mice in doses of 2 g kg^-1, and their effects were evaluated for 14 days. The mice did not show signs of intoxication, confirming that sodium 2-benzyl-2-chloromalonates exhibit low levels of acute toxicity. These observations indicate that sodium 2-benzyl-2-chloromalonates and their analogues are efficient and safer central nervous system depressing drugs relative to existing standards of care.

Full text of DOI:10.1039/c5md00187k

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Design, synthesis and in vivo evaluation of sodium 2-benzyl-
chloromalonates as new central nervous system depressants
Andreia Aguiar Vieira,a Bruno Guimarães Marinho,b Luana Gonçalves de Souza,a
Patricia Dias Fernandes,c and José D. FigueroaꢀVillara
a
Medicinal Chemistry Group, Department of Chemistry, Military Institute of
Engineering, Praça General Tibúrcio 80, Urca, Rio de JaneiroꢀRJ, Brazil.
b
Laboratory of Pharmacology, Department of Physiological Sciences, Federal
Rural University of Rio de Janeiro, BR465, Km07, Seropédica, RJ, Brazil
c Laboratory of Pharmacology of Pain and Inflammation, Institute of Biomedical
Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373,
CCS, Rio de JaneiroꢀRJ, Brazil.
Abstract
This work describes the design, synthesis and in vivo evaluation of new central
nervous system depressing agents that show low levels of acute toxicity, high
solubility in water, and exhibit anxiolytic and hypnotic effects. These new
compounds are sodium 2ꢀbenzylꢀ2ꢀchloromalonates, which were designed using
molecular modelling techniques and synthesized in four steps, with an overall
yield between 41% and 65%. In vivo tests with mice, including pentobarbitalꢀ
induced sleep, rotarod, open field and elevated plus maze tests, were executed,
and the results indicated that some of these agents induce activities similar to
those induced by diazepam but with lower hypnotic potency and with greater
anxiolytic potency. These compounds were also orally administered to mice in
doses of 2 g/kg, and their effects were evaluated for 14 days. The mice did not
show signs of intoxication, confirming that sodium 2ꢀbenzylꢀ2ꢀchloromalonates
exhibit low levels of acute toxicity. These observations indicate that sodium 2ꢀ
benzylꢀ2ꢀchloromalonates and their analogues are efficient and safer central
nervous system depressing drugs relative to the existing standard of care.
Keywords
2ꢀbenzylꢀ2ꢀchloromalonates, rotarod, open field, elevated plus maze, anxiolytics.

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1. Introduction
Alteration of the central nervous system leads to anxiety disorders,1 which are
among the most common classes of mental problems present in the population,
with prevalence rates of approximately 15%.2 There are several drugs available
for the treatment of anxiety disorders, such as benzodiazepines and serotonin
reuptake inhibitors (SSRIs). Benzodiazepines are the most commonly used
pharmacological agents for the treatment of anxiety, but they are certainly over
prescribed, and their abuse potential leads to significant problems, generating
great concern among the general public and prescribing practitioners.3 One of
the other problems with benzodiazepines is that they may also induce insomnia,4
an effect that is sometimes undesirable. SSRIs have been shown to be effective
for treating of anxiety disorders.5,6 However, despite their therapeutic effects,
SSRIs are also associated with a wide variety of side effects, including weight
changes, insomnia, drowsiness or sedation, agitation, fatigue, dry mouth,
gastrointestinal disturbances, headache and sexual dysfunction.7 Therefore,
because of the disadvantages presented by conventional pharmacological
agents for the treatment of anxiety disorders, the development of alternative
treatments, particularly ones with less hypnotic effects, is important. The aim of
this work is to report the design and synthesis of sodium 2ꢀbenzylꢀ2ꢀ
chloromalonates, which are new compounds, and evaluate their toxicity and their
pharmacological effects on the central nervous system.
2. Results and Discussion
2.1 Design of New Tranquilizers
In 2011 we discovered new compounds that were effective in the treatment of
anxiety without causing drowsiness.8 However, because these compounds were
mildly toxic, they were used only as tranquilizers.9 To design new nonꢀtoxic
central nervous systemꢀdepressing agents, it was necessary to select new
different compounds without barbituric acid rings, with appropriate solubility in
water and with ability to interact with the GABAꢀB receptor. For this reason, we

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selected 2ꢀbenzylꢀ2ꢀchloromalonates, which are new molecules that may interact
with the GABA B receptor, in contrast to phenobarbital (as shown in Fig. 1).
Fig. 1: Comparison of phenobarbital (A) with 2ꢀbenzylꢀ2ꢀchloromalonates (B)
with respect to GABA receptor activity, and the list of selected compounds (C).
These new agents possess two parallel carboxylate groups and one aromatic
ring for interacting with cationic and πꢀstacking regions, as well as forming
hydrogen bonds. Their conformations were calculated using molecular modelling
by density functional theory calculations at the 6ꢀ311++G** level, as depicted in
Fig. 2. Their conformational preferences depend on the position and type of R
group bonded to the aromatic ring, which could affect their capacity to interact
with the GABA receptor.

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Fig. 2: Example of conformational variation from two of the designed compounds
(7E and 7F). The calculated dihedral angle rotation is C8ꢀC7ꢀC1ꢀC2 (Fig. 1).
The results from Fig. 2 indicate that, although that the rotation energy of 7E and
7F are basically similar, but 7F exhibits lower rotation energy than 7E; this
inequality could differentiate their pharmacological activity. Additionally, it was
found that the PSAs (Polar Surface Area) of 7E and 7F were lower than 60 Å2, a
condition that is very important for permeating the Blood Brain Barrier (BBB).
Before their conformational analysis it was necessary to test the propensities of
7E and 7F to interact with the GABAꢀB receptor by docking. Docking studies of 2ꢀ
benzylꢀ2ꢀchloromalonates 7A to 7F were performed using Autodock 4.210 and the
threeꢀdimensional structure of the GABAꢀB receptor from the Protein Data Bank
(PDB).11 The threeꢀdimensional structure of all compounds were designed with
the Spartan'6 program, and the docking method used by the program was
validated by reꢀdocking with the inhibitor in the crystallographic structure of
GABAꢀB, as reported in the PDB.11 The box for the docking calculation was
collocated on the GABAꢀB receptor with centralization of its active site, and the
maps for all of the types of atoms present on these molecules were calculated
with Autogrid. The parameters used for docking were the program standards and
50 structure conformations that were grouped in the clusters. These conditions
were used to evaluate the conformations and binding energies of all compounds

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with the GABAꢀB receptor. The results, as shown in Fig. 3 and Table 1,
demonstrate that all of these compounds interact with the same region of the
GABAꢀB receptor.
Fig. 3: Docking of sodium 2ꢀbenzylꢀ2ꢀchloromalonates with the GABAꢀB
receptor.
The docking results suggested that sodium 2ꢀbenzylꢀ2ꢀchloromalonates bind
strongly to the GABAꢀB receptor. Fig. 3 depicted the molecule docked at the
ligandꢀbinding site in the first subunit (GBR1), using PyMOL software to visualize

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the main amino acid residues that interact with the compounds. These
compounds interact with several of the ligandꢀbinding residues, including Ser130,
Gly151, Ser153 and Glu349. Hydrogen bond interactions between the carbonyl
groups and the amino groups of Ser130 and Ser153 were observed, and only 7D
presented a hydrogen bond interaction between the oxygen group and the amino
group of Ser131. The presence of Trp65 above the compounds may indicate the
presence of πꢀπ stacking interactions in all of the docked compounds. The
binding energy, inhibition constant and intermolecular energy of each compound
is displayed in Table 1. These energy results suggest the presence of strong
interactions with all the compounds with the receptor. The chlorine atom bonded
to C2 is important for to stabilizing the dicarboxylate group, avoiding enolization,
decarboxylation and preventing the benzyl group from being planar with the
dicarboxylate group, conditions that are important for their potential CNS activity.
Table 1: Docking results of sodium 2ꢀbenzylꢀ2ꢀchloromalonates with the GABAꢀB
receptor.
Binding
Energy
(kJ/mol)
ꢀ4.77
Inhibition
Constant
((M)
Intermol.
Energy
(kJ/mol)
ꢀ5.97
Hydrogen
Bonding
HꢀꢀꢀBond
Comp.
Length (Å)
7A
7B
7C
7D
317.48
Ser130 (OꢀꢀꢀHN)
Ser153 (OꢀꢀꢀHN)
Ser130 (OꢀꢀꢀHN)
Ser153 (OꢀꢀꢀHN)
Ser130 (OꢀꢀꢀHN)
Ser153 (OꢀꢀꢀHN)
Ser130 (OꢀꢀꢀHN)
Ser131 (OꢀꢀꢀHN)
Ser153 (OꢀꢀꢀHN)
Ser130 (OꢀꢀꢀHN)
Ser153 (OꢀꢀꢀHN)
Ser130 (OꢀꢀꢀHN)
Ser153 (OꢀꢀꢀHN)
1.965
1.726
2.070
1.980
1.954
2.073
1.900
2.005
2.140
1.977
1.855
2.017
1.819
ꢀ4.21
ꢀ4.80
ꢀ4.21
823.62
304.96
814.93
ꢀ5.40
ꢀ6.29
ꢀ6.00
7E
7F
ꢀ4.39
ꢀ4.34
609.69
654.41
ꢀ5.58
ꢀ5.54

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2.2 Synthesis
These new central nervous systemꢀdepressing agents were obtained in four
synthetic steps, with overall yields from 41% to 65%, as described in Fig. 4. In
the first step, selected benzaldehydes (1) were condensed with barbituric acid (2)
to obtain the respective benzylidene barbiturates (3), and in the second step the
polarized carbonꢀcarbon double bond was reduced with sodium borohydride.12
On the third step the reduced compounds (4) were treated with 3ꢀ
chloroisocyanuric acid (5) to obtain the respective 5ꢀchloroꢀ5ꢀbenzylbarbiturates
(6). The last step was the conversion of the 5ꢀchloroꢀ5ꢀbenzylbarbiturates to the
respective sodium 2ꢀbenzylꢀ2ꢀchloromalonates by hydrolysis with sodium
hydroxide in water at 25 °C. These products were purified by extraction from the
aqueous reaction mixture with ethyl ether and recrystallized in methanol. The
solubility of these compounds in water and ethyl ether indicates sufficient
lipophilicity for BBB permeation, suggesting that they could be effective in the
brain by oral administration. The structures of these new products were
determined by NMR, infrared spectroscopy and elemental analysis (CHN).
Fig. 4: Synthetic procedure for the preparation of sodium 2ꢀbenzylꢀ2ꢀ
chloromalonates (7) from the respective benzaldehydes (1).
The reactions of the reduced benzylidene barbiturates (4) with
trichloroisocyanuric acid sometimes lead to chlorination of the aromatic ring.8 For

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example, the benzaldehydes 1A (benzo[d][1,3]dioxoleꢀ5ꢀcarbaldehyde, R1=H),
1C (4ꢀmethoxyꢀbenzaldehyde, R3=H) and D(2ꢀethoxyꢀbenzaldehyde, R3=H),
yielded chlorination of C5 and the aromatic rings, leading to 3A with R1=Cl, 3C
with R3=Cl and 3D with R1=Cl. The other aldehydes lead to chlorination
1
exclusively on C2. The structure of the final products was confirmed by H and
13C NMR signal assignment (see experimental results and supplementary
material).
2.3 In vivo evaluation
The six obtained products were evaluated using pentobarbitalꢀinduced sleep,18
rotarod,13,14 elevated plusꢀmaze (EPM)15 and openꢀfield tests in mice.16,17 In all
the experiments, water was used as the negative control (A) and diazepam as
the positive control (B).
Oral administration of diazepam and sodium 2ꢀbenzylꢀ2ꢀchloromalonates,
excluding compound 7B, induced hypnosis in all of the mice a few minutes after
administration, as measured using the Pentobarbitalꢀinduced sleep test.18 Loss of
righting reflex, observed in this test, was defined as the inability of a mouse to
right itself within 30 s. Return of the righting response was defined as the
mouse’s ability to right itself twice in 1 min. Sleeping time, or duration of loss of
righting reflex, was defined as the time between loss and return of the righting
response, while onset of action is the time between injection of the compound
and loss of righting reflex.18 There was no reduction in the time of action onset
from any of the chloromalonate compounds and diazepam, whereas diazepam
and all sodium 2ꢀbenzylꢀ2ꢀchloromalonates except compound 7B potentiated the
hypnotic effect produced by pentobarbital (45 mg/kg; i.p.) and increased the
sleeping time (Fig. 5).

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Fig. 5: Pentobarbitalꢀinduced sleep test of compounds 7A to 7F, indicating the
onset of action (A) and sleeping time (B) in mice. Control group (water ꢀ A),
positive control group (Diazepam – B, 8 mg/kg) and compounds (30 mg/kg) were
administered orally. The results are expressed as the mean ± SEM (n=6). The
statistical significance was calculated using oneꢀway ANOVA followed by
Bonferroni’s test. * p < 0.05 when comparing the chloromalonate- and
Diazepamꢀtreated groups with the control group.
Crescent doses (5–150 mg/kg) of all the chloromalonates and diazepam were
administered orally to calculate the ED50 in the pentobarbitalꢀinduced sleep test
(Table 2). The approximate values of ED50 were used in subsequent methods.
Table 2 ꢀ Calculated ED50 values from the pentobarbitalꢀinduced sleep test in
mice. The ED50 value was obtained by fitting the data points representing the
hypnotic effect and demonstrated in the pentobarbitalꢀinduced sleep test by
nonlinear regression (sigmoidal dose response) using the GraphPad Prism
software version 5.0.
Compounds
ED50 values (95% confidence limits) (mg/kg)
8.17 (5.57 – 11.98)
Diazepam
7A
7B
7C
7D
7E
7F
26.43 (9.99 – 69.92)
90.12 (18.82 – 231.5)
33.81 (13.23 – 86.36)
30.73 (11.13 – 84.85)
29.45 (11.46 – 75.69)
23.45 (10.75 – 51.15)

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The rotarod test is an established method for evaluating motor impairment and
ataxia.14 The rotarod test results are displayed in Fig. 6. With the exception of 7B,
all of the chloromalonates showed an increase in the number of falls, indicating
motor impairment. The results obtained were (number of falls): Control (7.6 ±
1.3); Diazepam (34.0 ± 4.0); compound 7A (22.4 ± 4.0); compound 7B (11.8 ±
2.8); compound 7C (22.0 ± 3.8); compound 7D (19.2 ± 3.3); compound 7E (17.2
± 2.7); compound 7F (17.5 ± 0.9). These results suggest that compounds 7A and
7C are the most effective compounds. Both compounds increased the number of
falls by approximately 190%.
Fig. 6: Rotarod performance test of compounds 7A to 7F, indicating the number
of falls. Control group (water ꢀ A), positive control group (Diazepam – B, 8 mg/kg)
and compounds (30 mg/kg) were administered orally. The results are expressed
as the mean ± SEM (n=6). The statistical significance was calculated using oneꢀ
way ANOVA followed by Bonferroni’s test. * p < 0.05 when comparing the
chloromalonate- and Diazepamꢀtreated groups with the control group.
The open field test19 is considered an indicator of the animal’s emotional state20
and is currently used for the screening of drugs acting on the central nervous
system. All of the chloromalonates, except 7B, decreased ambulation and
rearing rates (Fig. 7). The results obtained for ambulation (walked squares) and
rearing (time of rearing), respectively, were: Control (299.8 ± 15.9; 70.5 ± 6.8s);
Diazepam (79.9 ± 6.7; 35.2 ± 8.0s); compound 7A (172.2 ± 16.7; 45.4 ± 8.2s);
compound 7B (250.8 ± 20.0; 58.7 ± 5.3s); compound 7C (143.8 ± 18.8; 49.0 ±
7.3s); compound 7D (209.4 ± 12.2; 48.4 ± 9.7s); compound 7E (143.5 ± 14.5;

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39.5 ± 7.7s); compound 7F (141.3 ± 14.9; 39.4 ± 5.3s). Compounds 7E and 7F
were the most effective for reducing the number of walked squares
(approximately 50%) and the rearing time (approximately 55%). Ambulation and
rearing can be regarded as indicators of locomotor activity and exploratory
behaviour.12 Therefore, it is suggested that these compounds decrease the
locomotor and exploratory activities of mice.
Fig. 7: Open field test of compounds 7A to 7F, indicating the number of walked
squares (A), time of rearing (B) and time of grooming (C) in mice. Control group
(water ꢀ A), positive control group (Diazepam – B, 8 mg/kg) and compounds (30
mg/kg) were administered orally. The results are expressed as the mean ± SEM
(n=6). The statistical significance was calculated using oneꢀway ANOVA followed
by Bonferroni’s test. * p < 0.05 when comparing the chloromalonate- and
Diazepamꢀtreated groups with the control group.
With the exception of 7B, all tested chloromalonates demonstrate negative
influence on exploratory behaviour (Fig. 7). It has been reported that high
emotionality inhibits exploration;19 the diminished exploratory behaviour could
therefore be a consequence of the emotional state. However, in the
pentobarbitalꢀinduced sleep test, the compounds affected an increase in the
sleeping time induced by pentobarbital, and, in the rotarod test, an increase in
the number of falls was observed (Fig. 6). Both results suggest that the reduced
exploratory behaviour is related to an impaired motor performance induced by
hypnotic activity and not by the emotional state.
Grooming is a “maintenance” behaviour that is specifically elicited in situations in
which an animal is in stressꢀinduced conflict or frustration. In such a situation,
grooming may play a deactivating role in restoring homeostasis.20 Grooming is
also positively correlated with emotional state.21 In Fig. 7, compounds 7E and 7F
showed a reduction in grooming time (58% and 37%, respectively), which

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indicate an influence on the emotional state of the animals, e.g., reduction of
anxiety and fear.
The elevated plus maze (EPM) test was first developed for rats,22 and later
adapted for mice.23 This test reflects a conflict between the rodents’ preference
for protected areas and their natural motivation to explore new environments.24 It
is based on the anxietyꢀrelated behaviour of rodents, the animals’ predisposition
towards dark and enclosed spaces and their unconditioned fear of heights and
open spaces.25 Fig. 8 shows the results obtained in the EPM test. Compounds
7E, 7F and Diazepam (B) induced the highest percentages of entry into the open
arms (48%, 44% and 44%, respectively) and increased the time spent in the
open arms compared with the control group (92%, 64% and 56%, respectively).
These results are consistent with the results obtained during the analysis of
grooming time by the open field test, indicating an anxiolytic activity produced by
compounds 7E and 7F.
Fig. 8: Elevated plus maze test of compounds 7A to 7F, indicating the number of
entries into open arms (A) and time in open arms (B) by mice. Control group
(water ꢀ A), positive control group (Diazepam – B, 30 mg/kg) and compounds (20
mg/kg) were administered orally. The results are expressed as the mean ± SEM
(n=6). The statistical significance was calculated by oneꢀway ANOVA followed by
Bonferroni’s test. * p < 0.05 when comparing the compounds- and Diazepamꢀ
treated groups with the control group.
In fact, an anxiolytic effect is suggested when the drug increases the number of
times that the animal enters into the open arms without altering the total number
of entries into the maze.26 In this work, the treated mice displayed a significant
increase in the percentage of entries and the time spent inside the plusꢀmaze

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open arms. Therefore, compounds 7E and 7F possess a similar profile to
anxiolytic drugs such as diazepam. The ED50 was calculated from the plusꢀmaze
test results. The ED50 values for compounds 7E and 7F were less than those of
diazepam, showing that compounds 7E and 7F have a greater anxiolytic potency
(Table 3).
Table 3 ꢀ Calculated ED50 values from the elevated plus maze test in mice. The
ED50 value was obtained by fitting the data points representing the anxiolytic
effect demonstrated in elevated plus maze test by nonlinear regression
(sigmoidal dose response) using the GraphPad Prism software version 5.0
Compounds
Diazepam
7E
ED50 values (95% confidence limits) (mg/kg)
32.39 (16.56 – 63.37)
18.58 (10.43 – 33.10)
7F
23.40 (13.11 – 41.76)
The mechanism of action of the chloromalonates may involve potentiation of the
inhibitory pathways (i.e., GABA, glycine) or inhibition of the excitatory pathways
(i.e., glutamate, noradrenaline, acetylcholine). Neuropeptides, dopamine, GABA,
endorphins and acetylcholine are implicated in rearing, grooming and locomotion
in mice and other small animals.27
All compounds described in this work were also evaluated for their acute toxicity
in mice. The acute toxicity test was performed according to the World Health
Organization (WHO)28 and the Organization of Economic Coꢀoperation and
Development (OECD) guidelines for testing chemicals.29 The investigated
compounds were administered orally in increasing doses up to 2000 mg/kg. The
animal behaviour was observed from 5 h after a single administration of each
compound and subsequently monitored daily until the 14th day. Acute toxicity
was expressed as the required dose in g/kg body weight to cause death in 50%
of tested animals (LD50). Any symptom of intoxication was recorded (i.e.,
disorientation, hyperactivity, piloerection and hyperventilation). Therefore, it was
confirmed that the investigated compounds were not toxic after acute oral

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administration (LD50>2000 mg/kg). Thus, the compounds may be classified as
nonꢀtoxic.30
The NMRꢀcalculated relaxation times (T1 and T2) and diffusion coefficient (D) of
these compounds were compared in pure conditions and in the presence of the
GABAꢀB receptor using the DOSY methodology, indicating their interaction (see
Table 4).
Table 4: Results of T1, T2 and coefficient density (D) from the interaction of 7D
and 7F with GABA receptor by NMR.
T1
T1+G
T2
T2+G
D
D+G
δH
ꢀT1
ꢀT2
ꢀD
H
7F
2
3
5
6
7
7.27 2.00 0.78 ꢀ1.22 2.70 0.80 ꢀ1.90 4.73 2.36 ꢀ2.37
7.35 3.15 1.58 ꢀ1.57 3.95 1.52 ꢀ2.43 4.70 2.33 ꢀ2.37
7.35 3.15 1.58 ꢀ1.57 3.95 1.52 ꢀ2.43 4.70 2.33 ꢀ2.37
7.27 2.00 0.78 ꢀ1.22 2.70 0.80 ꢀ1.90 4.73 2.36 ꢀ2.37
3.24 0.73 0.51 ꢀ0.22 0.69 0.37 ꢀ0.32 5.65 2.45 ꢀ3.20
7D
3
4
6
7
6.98 1.20 0.87 ꢀ0.33 1.70 0.42 ꢀ1.28 2.18 1.23 ꢀ0.95
7.21 1.60 1.12 ꢀ0.48 2.45 1.41 ꢀ1.04 2.38 1.29 ꢀ1.09
7.27 2.80 1.79 ꢀ1.11 2.98 1.23 ꢀ1.75 2.41 1.14 ꢀ1.27
3.31 0.65 0.39 ꢀ0.26 0.63 0.43 ꢀ0.20 2.09 1.19 ꢀ0.90
CH2 4.07 1.16 0.57 ꢀ0.59 1.05 0.24 ꢀ0.81 2.52 1.32 ꢀ1.20
CH3 1.37 1,52 0.61 ꢀ0.61 1.81 0.46 ꢀ1.35 3.23 1.26 ꢀ1.97
Information: H = Hydrogen; δH = signal H (in Hertz); T1 = spin lattice relaxation time of compound;
T1+G = spin lattice relaxation time of compound with GABA; ꢀT1 = difference between the spin
lattice relaxation time; T2 = spinꢀspin relaxation time of compound; T2+G = spinꢀspin relaxation
time of compound with GABA; D = diffusion coefficient of compound; D+G = diffusion coefficient
of compound with GABA; ꢀD = difference between the diffusion coefficient of compound and with
GABA.

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NMR assignments for the GABAꢀB receptor are not known due to its very high
mass; it was therefore not possible to confirm the site of interaction for these
agents by NMR. However, this type of interaction was previously calculated by
docking (Figure 3). These results indicate that all of these compounds interact
with the GABAꢀB receptor, and the information from Table 4 indicates that 7F
interacts more than 7D, a result that is consistent with the obtained in vivo
results. Figure 9 shows one example of DOSY, indicating the diffusion coefficient
of compound 7D pure (black signals) and mixture with the GABAꢀB receptor
(blue signals). Evidently, 7D interacts with the GABAꢀB receptor, but the
decrease of their diffusion coefficients with the receptor is lower than from 7F,
confirming that displays less efficiency that 7F, as it was observed by the in vivo
tests.
Fig. 9: Example of DOSY of 7D pure (black) and with the presence of GABAꢀB
receptor (blue).

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Sodium 2ꢀbenzylꢀ2ꢀchloromalonates exhibit good solubility in water and other
solvents such as ethyl ether and ethyl acetate, which suggests appropriate
properties for membrane and bloodꢀbrain barrier (BBB) permeation. The
pentobarbitalꢀinduced sleep, rotarod, elevated plusꢀmaze and openꢀfield tests
confirm that these compounds (7E and 7F) show anxiolytic activity as well as a
hypnotic effect. However, these compounds exhibit greater anxiolytic potency
and lower hypnotic potency than diazepam. Notably, the tested compounds were
not toxic after acute oral administrations even at a dose of 2000 mg/kg,
suggesting that these compounds are promising as new candidates for
psychotropic agent prototypes.
3. Conclusion
Sodium 2ꢀbenzylꢀ2ꢀchloromalonate compounds were prepared in four synthetic
steps, with 45% to 70% overall yields. The compounds exhibited good solubility
in water and in other solvents such as ethyl ether, suggesting that these
compounds may possess the right properties for bloodꢀbrain barrier (BBB)
permeation. These nonꢀtoxic agents, when tested by pentobarbitalꢀinduced
sleep, rotarod performance, elevated plusꢀmaze and openꢀfield tests, showed
anxiolytic and hypnotic effects, indicating that these compounds have the
potential to be used as therapeutic agents in psychological disorders.
The obtained experimental results indicated that compounds 7E and 7F were the
most effective (hypnotic and anxiolytic effects), whereas compounds 7A, 7C and
7D showed an intermediate capacity (hypnotic effect) and 7B was ineffective.
These results suggest that chlorination of the aromatic ring is important for
anxiolytic activity, especially when the Cl group is present at the ortho position,
suggesting that other electronegative groups on the aromatic ring could increase
their activity. The docking results indicate that all of the compounds interact with
the GABA receptor and suggest that 7A and 7C could be the most effective, but
the experimental tests confirm that 7E and 7F are the most effective, and 7A and
7C exhibit intermediate activity. Docking indicates that 7B is the least effective
compound, which was consistent with experimental results. The differences

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between the docking and the experimental tests suggest that the most active
compounds could also be interacting with other regions of the GABA receptor.
In general terms, the compounds 7E and 7F exhibited hypnotic and anxiolytic
effects with very low acute toxicity, showing greater anxiolytic potency and lower
hypnotic potency than diazepam, thus presenting a lower capacity to develop
hypnotic activity (an undesirable side effect). The fact that the oral administration
of these agents on mice afforded good results suggests that they could penetrate
the blood brain barrier (BBB). These compounds may be used for the design and
synthesis of new and more effective anxiolytic drugs.
4. Experimental Section
4.1 Materials and Equipment. All reagents and solvents for reactions were
obtained from SigmaꢀAldrich, St. Louis, MO, USA. The deuterated solvents were
acquired from Cambridge Isotope Laboratories (CIL). All compounds were
characterized by NMR and IR spectroscopy, as well as elemental analysis.
1
4.1.2 Nuclear Magnetic Resonance Spectroscopy. The H and 13C NMR
spectra were obtained using a Varian UNITYꢀ600 MHz spectrometer. All samples
of compounds 7A to 7F were prepared by dissolving 10 mg in 0.6 mL of 99%
D2O in 5 mm NMR tubes. The spectra were obtained at room temperature (20
1
°C) using 32 scans for H and 5000 scans for 13C. The determination of the
variation of T1 (Inversion Recuperation), T2 (CarrꢀPurcellꢀMeiboomꢀGill) and the
coefficient density (D, DOSY) were obtained from 10 mg of the agents and 10 ꢁg
of GABAꢀB receptor in 600 ꢁL of solvent (D2O) for the NMR tubes. The tests from
interaction of agents with the GABAꢀB receptor were performed initially with the
pure agent and after compared with the presence of the GABAꢀB receptor. The
GABAꢀB receptor was obtained from HEK (human embryonic kidney cells) cells
lines, as reported by AssadiꢀPorter et col., 2010.31

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4.1.3 Infrared Spectroscopy. The IR spectra were obtained on a Spectrum 100,
manufactured by PerkinꢀElmer. The samples were prepared by grinding 1.0 mg
of analyte in 100 mg of potassium bromide.
4.1.4 Elemental Analysis (CHN). The elemental analyses (CHN) were obtained
using an elemental analyser LCPB008ÉA 1112 and balance LCPB 006ꢀAT21.
4.2 Molecular Modelling and Docking
Molecular Modelling was executed with the program Spartan’6, using B3LYP with
6ꢀ311++G**. The effected calculations were structure conformation and benzyl
group rotation of each molecule, the polar surface area (PSA) of each
compound, and the electronic density of all atoms.
Docking studies were performed using Autodock 4.2.10 The Xꢀray
crystallographic structure of GABAꢀB complexed with 2ꢀhydroxysaclofen (PDB
code 4MQF) was obtained from the Protein Data Bank.11 All water molecules in
PDB files were removed, and hydrogen atoms were subsequently added to the
protein using SwissꢀPdbViewer software.32 For all the docking calculations the
parameters were set as follow: Center_x = ꢀ48.224, Center_y = 21.434, Center_z
= ꢀ25.85, grid box size of 60 x 46 x 50, exhaustiveness = 50. Other parameters
were retained unchanged. To validate our docking procedure, the coꢀcrystallized
ligand was reꢀdocking with this parameters and obtained the acceptable RMSD
value of 1,89 Å. Finally, the best conformation of the ligand with the lowest
binding energy was selected to analyze the interaction with the receptor. All the
figures were generated using the PyMOL software.33
4.2 Synthesis
4.2.1 Preparation of benzyl-5-chlorobarbiturates. All of the benzylꢀ5ꢀ
chlorobarbiturates (compounds 6) were prepared by the method described in a
previous article.8 The first step was the reaction of barbituric acid (2) with the
respective aldehydes (1) in ethanol/water 50:50 by refluxing for 30 minutes,

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leading to benzylidene barbiturates with yields of 93% to 98%. The second step
was the reduction of the polarized carbonꢀcarbon double bond with sodium
borohydride in 95% ethanol with reflux for 30 min. The reaction mixture was
treated with 6 N HCl to give a pH 2.0 solution, followed by the evaporation of
ethanol. The reaction mixture was then dissolved in water and the reduced
compounds 4 were extracted with ethyl acetate in 75% to 87% yields. The third
step was the reaction of trichloroisocyanuric acid (5) with the reduced
intermediates (4) in acetone under reflux for 2 h, providing the respective benzylꢀ
5ꢀchlorobarbiturates (6) in 82% to 94% yields.
4.2.2 General procedure for synthesis of sodium 2-benzyl-chloromalonates
(7). A mixture of 2.0 g of the benzylꢀ5ꢀchlorobarbiturate and 30 mL of distilled
water was prepared in a 100 mL roundꢀbottom flask. It was then rigorously stirred
at room temperature (25 °C) with a continuous dropwise addition of 0.02 M
NaOH until complete dissolution of the reaction mixture was obtained. The
respective sodium benzylꢀ2ꢀchloromalonates (7) were extracted with ethyl ether
(3x20 mL). Evaporation of ethyl ether afforded the pure solid products. All of the
final products have melting points higher than 400 °C.
4.2.2.1 Sodium 2-chloro-2-[(4-chlorobenzo[d][1,3]dioxol-5-
yl)methyl]malonate 7A. Compound 7A was obtained as a white solid, 1.85 g in
85% yield. IR: νmax 3678, 3266, 1620, 1647, 1482, 1135, 1126, 815 and 606 cmꢀ
1. 1H NMR (D2O): δ 3.38 (2H, s), 6.95 (1H, s), 6.92 (1H, s), 5.96 (2H, s). 13C NMR
(D2O): δ 178.2, 146.3, 146.2, 128.8, 126.4, 110.5, 109.9, 101.9, 82.2 and 37.8.
Elemental analysis: Calculated for C11H6O6Cl2Na2; Calculated; C=37.65, H=1.72,
found; C=38.78, H=1.78.
4.2.2.2 Sodium 2-benzyl-2-chloromalonate 7B. Compound 7B was obtained
as a white solid, 1.85 g, in 85% yield. IR: νmax 3422, 3037, 1602, 1427, 1218,
1
1117, 834 and 669. H NMR (D2O): δ 3.26 (2H, s), 7.35 (2H, d), 7.31 (2H, t),
13
7.27(1H, t). C NMR (D2O): δ 178.4, 138.0, 130.5, 128.1, 126.3, 82.1 and 41.3.

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Elemental analysis: Calculated for C10H7O4ClNa2; Calculated; C=44.07, H=2.54,
found; C=43.83, H=2.62.
4.2.2.3
Sodium
2-(3-chloro-4-methoxybenzyl)-2-chloromalonate
7C.
Compound 7C was obtained as a white solid, 1.70 g, in 78% yield. IR: νmax 3459,
3047, 1654, 1427, 1264, 1061, 842 and 806. 1H NMR (D2O): δ 3.17 (2H, s), 3.89
(3H, s), 7.33 (1H, d), 7.20 (1H, dd), 7.05 (1H, d). 13C NMR (D2O): δ 178.1, 152.6,
131.7, 131.6, 129.9, 120.5, 112.8, 82.2, 56.0 and 40.5. Elemental analysis:
Calculated for C11H8O5Cl2Na2; Calculated; C=39.21, H=2.37, found; C=39.43,
H=2.34.
4.2.2.4
Sodium
2-(5-chloro-2-ethoxybenzyl)-2-chloromalonate
7D.
Compound 7D was obtained as a white solid, 1.85 g, in 83% yield. IR: νmax 3497,
1
3339, 1675, 1574, 1236, 1135, 888 and 796. H NMR (D2O): δ 1.37 (3H, t), 3.31
(2H, s), 4.07 (2H, q), 6.98 (1H, d), 7.21 (1H, dd) and 7.27 (1H, s). 13C NMR
(D2O): δ 178.4, 156.0, 130.5, 129.2, 127.4, 125.1, 114.8, 82.1, 65.7, 34.5 and
14.2. Elemental analysis: Calculated for C12H10O5Cl2Na2; Calculated; C=41.06,
H=2.87, found; C=41.81, H=2.92.
4.2.2.5 Sodium 2-(2-chlorobenzyl)-2-chloromalonate 7E. Compound 7E was
obtained as a white solid, 1.85 g, in 87% yield. IR: νmax 3485; 3431; 1602; 1427;
1
1144; 1061; 851 and 751. H NMR (D2O): δ 3.49 (2H, s); 7.46 (1H, d); 7.43 (1H,
13
t); 7.26 (1H, t) and 7.24 (1H, d). C NMR (D2O): δ 178.3; 135.7; 134.9; 131.2;
129.5; 128,0; 126,9; 82.1 and 38.0. Elemental analysis: Calculated for
C10H6O4Cl2Na2; Calculated; C=39.13, H=1.97, found; C=39.76, H=2.04.
4.2.2.6 Sodium 2-(4-chlorobenzyl)-2-chloromalonate 7F. Compound 7F was
obtained as a white solid, 1.85 g, in 87% yield. IR: νmax 3440; 1602; 1492; 1208;
1098; 842 and 760. 1H NMR (D2O): δ 3.24 (2H, s); 7.27 (2H, d) and 7.35 (2H, d).
13C NMR (D2O): δ 180.7; 139.2; 134.3; 130.8; 84.8 and 43.6. Elemental analysis:
Elemental analysis: Calculated for C10H6O4Cl2Na2; Calculated; C=39.13, H=1.97,
found; C=39.74, H=2.02.

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4.3 Biological Tests
The experiments were carried out on male Swiss Webster mice (body weight 20ꢀ
24 g). The animals were housed in wire mesh cages at room temperature and
exposed to a 12 h light, 12 h dark cycle. The animals had free access to a
standard pellet diet, and tap water was given ad libitum. The protocol for this
study was approved by the ethics committee for Animal Research of the Federal
University of Rio de Janeiro under number DFBCICBꢀ015. The control and
experimental groups consisted of 6 animals each. The investigated compounds
were administered orally (p.o.), and Diazepam was administered subcutaneously
(s.c.) as a suspension in PBS (phosphate buffer saline).
4.3.1. Statistical analysis. Each of the experimental groups included 6 mice.
The results are presented as the mean ± SEM (standard error of mean) of the
rotarod, open field and elevated plus maze tests. Statistical significance between
groups was calculated by the application of one way analyses of variance
(ANOVA) followed by Bonferroni’s test. P< 0.05 was considered as statistically
significant. The estimated LD50 was obtained by fitting the data points
representing the percentage of deaths at increasing doses of the compounds up
to 2000 mg/kg, and the fits were calculated by a nonlinear regression method
using the Graph Pad Prism software version 5.0 (San Diego, CA, USA).
4.3.2. Pentobarbital-induced sleep test. All experiments were carried out
between 1:00 and 5:00 P.M. At 30 min after the oral administration of diazepam
and sodium 2ꢀbenzylꢀ2ꢀchloromalonates, sodium pentobarbital was injected
intraperitoneally (45 mg/kg) according to the method of Boehm et al., (2004)18.
Sleep latency was defined as the elapsed time between the pentobarbital
injection and sleep onset, and sleep duration was recorded as the time of the
righting reflex loss to the time of recovery.
4.3.3 Rotarod Test. The rotarod test is an established method for evaluating
motor impairment and ataxia.14 On the day before the test, the animals were
trained twice to maintain equilibrium for 5 min on a roller apparatus (‘Rotarod for

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mice’, Insight Equipments, Brazil). The speed selector was set to 10 rev/min.
Twentyꢀfour hours later, the mice were treated orally with the chloromalonates
(30 mg/kg, p.o.) and Diazepam (5 mg/kg, s.c.). The mice were placed on the
roller for 5 min, after administration of the chloromalonates (60 min) and
Diazepam (30 min). The degree of neurological deficit was evaluated by the
inability of the animal to remain on the roller for the test period and was reported
as the number of falls by the animals.
4.3.4 Open Field Test. The open field test was similar to the method described
by Barros et al., (1991).17 The mice received chloromalonates (30 mg/kg, p.o.) or
Diazepam (5 mg/kg, s.c.). The mice were placed individually in an observation
chamber, 60 min after oral administration (chloromalonates) and 30 min after
subcutaneous administration (Diazepam), whose floor was divided into 50
squares (5 cm x 5 cm). The total locomotion (number of floor units entered,
where the squares were crossed with all paws), time of grooming (time that the
mice spent cleaning their bodies with their paws by the picking of the body and
pubis with mouth and faceꢀwashing actions) and time of rearing (time the animal
stood on its hind legs, with its forearm against the wall of the observation cage or
in the free air) were quantified for 5 minutes.
4.3.5 Elevated Plus Maze Test. The elevated plus maze (EPM) test was first
developed for rats23 and later adapted for mice.24 The test uses openness
combined with elevation to generate behavioural changes predominantly related
to anxiety.25 It is based on the anxietyꢀrelated behaviour of rodents, the animals’
predisposition towards dark and enclosed spaces and an unconditioned fear of
heights and open spaces.26 The apparatus was made of grey acrylic material
(Insight®, São Paulo, Brazil) and consisted of two open arms (30 cm × 5 cm × 15
cm) and two enclosed arms (30 cm × 5 cm × 15 cm) radiating from a central
platform (5 × 5 cm) to form a plus (+) sign. The apparatus was elevated to a
height of 38.5 cm and placed inside a soundꢀattenuated room. The trial began by
placing an animal on the central platform of the maze facing an open arm 60 min
after p.o. administration of compounds and 30 min after s.c. administration of

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Diazepam. The percentage of entries into open arms and the time spent in the
open arms of the device were recorded during a 5ꢀminutes test period. The
animal was considered to have entered into an arm only when all four paws were
in that arm.34 The percentage of entries into open arms was calculated according
to the following formula, where NEOA is the number of entries into open arms
and TNE is the total number of entries:
NEOA
Percentage of entries in open arms =
X100
TNE
4.3.6 Toxicity Test. The acute toxicity test was performed according to the
World Health Organization (WHO)28 and the Organization of Economic Coꢀ
operation and Development (OECD) guidelines for testing chemicals.29 The
investigated compounds were administered orally in increasing doses up to 2000
mg/kg. The animal behaviour was observed from 5 h after a single administration
of the compounds and subsequently monitored daily until the 14th day. Acute
toxicity was expressed as the required dose in g/kg bodyweight to cause death in
50% of the animals tested (LD50).
Acknowledgement
We appreciate the economic support provided by INBEB (Instituto Nacional de
Ciência e Tecnologia de Biologia Estrutural e Bioimagen), CAPES (Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior), FAPERJ (Fundação Carlos
Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), and CNPq
(Conselho Nacional de Pesquisa).
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520x214mm (96 x 96 DPI)