Active anti-inflammatory and hypolipidemic derivatives of lorazepam

Article abstract of DOI:10.3390/molecules24183277

Novel derivatives of some non steroidal anti-inflammatory drugs, as well as of the antioxidants α-lipoic acid, trolox and (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid with lorazepam were synthesised by a straightforward method at satisfactory to

Full text of DOI:10.3390/molecules24183277

molecules
Article
Active Anti-Inflammatory and Hypolipidemic
Derivatives of Lorazepam
Panagiotis Theodosis-Nobelos 1,2, Georgios Papagiouvannis 1, Panos N. Kourounakis 1 and
Eleni A. Rekka 1,
*
1
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki,
54124 Thessaloniki, Greece
Department of Pharmacy, School of Health Sciences, Frederick University, Nicosia 1036, Cyprus
Correspondence: rekka@pharm.auth.gr; Tel.: +30-231-099-7614
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Athina Geronikaki
Received: 22 July 2019; Accepted: 2 September 2019; Published: 9 September 2019
Abstract: Novel derivatives of some non steroidal anti-inflammatory drugs, as well as of the
antioxidants -lipoic acid, trolox and (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid with
α
lorazepam were synthesised by a straightforward method at satisfactory to high yields (40%–93%).
All the tested derivatives strongly decreased lipidemic indices in rat plasma after Triton induced
hyperlipidaemia. They also reduced acute inflammation and a number of them demonstrated
lipoxygenase inhibitory activity. Those compounds acquiring antioxidant moiety were inhibitors
of lipid peroxidation and radical scavengers. Therefore, the synthesised compounds may add to
the current knowledge about multifunctional agents acting against various disorders implicating
inflammation, dyslipidaemia and oxidative stress.
Keywords: non steroidal anti-inflammatory drugs; lorazepam derivatives; antioxidants;
inflammation; hyperlipidemia; lipoxygenase inhibition
1. Introduction
Lorazepam (7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one) is a
benzodiazepine derivative, used as anxiolytic, sedative, in status epilepticus and in the treatment of
alcohol withdrawal. It is known that benzodiazepines act by potentiating the interaction of GABA
with GABAA receptors. Biologic stress has been described as the non-specific adaptive response of the
organism to various stimuli, physical, psychological or emotional, such as fear and anxiety [
and others [ ] have shown that biologic stress induces oxidative stress, and that GABAergic modulation
may offer protection against immobilization-induced stress and oxidative damage [ ]. Moreover, it
has been reported that the benzodiazepine midazolam protects against neuronal degeneration and
apoptosis induced by biological and oxidative stress [ ]. Alimentary dyslipidemia disturbed anxiety
level and cognitive processes in mice [ ] and a diet rich in saturated fat and fructose caused high
1]. We [1]
2
2
3
4
serum cholesterol and triglyceride concentrations and induced a condition in rats similar to the human
metabolic syndrome. This condition was accompanied by anxiety-related behaviour, which correlated
significantly with oxidative stress. In addition, the degree of lipid peroxidation correlated well with the
metabolic effects of the diet [5]. Finally, a number of benzodiazepine derivatives reduced hyperalgesia
in rats in a dose-dependent manner, using the carrageenan-induced method [6].
Taking the above evidence into consideration, in this investigation we report the synthesis and
biological evaluation of esters of lorazepam with five classical NSAIDs and the antioxidants α-lipoic acid
((R)-5-(1,2-dithiolan-3-yl)pentanoic acid), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) and (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid (BHA). The aim of this work was to
Molecules 2019, 24, 3277; doi:10.3390/molecules24183277
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3277
2 of 12
investigate whether such derivatives would combine hypolipidemic and anti-inflammatory activity
and to examine their potential lipoxygenase inhibitory, antioxidant and radical scavenging activities.
We have demonstrated that a number of lorazepam derivatives possess hypolipidemic action [
Furthermore, alpha-lipoic acid reduced inflammation and oxidative stress in patients with metabolic
syndrome [ ]. We have also reported that trolox and cinnamic acid derivatives demonstrated antioxidant,
7].
8
anti-inflammatory and hypolipidemic activities [
structure, was also used.
9]. Lastly, indole-3-acetic acid, part of the indomethacin
2. Results and Discussion
2.1. Synthesis
Compounds 1–6, 8, 9 were synthesised by direct esterification of the carboxylic group of the
respective acids with lorazepam, using N,N0-dicyclohexyl-carbodiimide (DCC), at room temperature
and high yields. For compound 7, carbonyldiimidazole (CDI) was used (Figure 1).
H
H
N
O
N
O
O
Cl
Cl
O
HO
R
O
A) DCC/DMAP/CH2Cl2/r.t.
B) CDI/THF/r.t.
+
N
N
R
OH
Cl
Cl
Yield
(%)
No.
RCOOH
O
1
Ibuprofen
Naproxen
74
OH
O
O
2
3
93
85
OH
O
Ketoprofen
OH
OH
O
O
O
4
Indomethacin
81
N
O
Cl
O
OH
NH
5
6
7
Tolfenamic acid
α-Lipoic acid
Trolox
41
71
85
Cl
O
S
OH
S
O
HO
OH
OH
O
O
8
9
BHA
40
40
HO
OH
Indole-3-acetic acid
O
N
H
Figure 1. Synthesis and structures of the examined compounds. r.t.

Molecules 2019, 24, 3277
3 of 12
2.2. Biological Activity
2.2.1. In Vivo Experiments
Effect of Compounds on Acute Inflammation in Rats
The effect of the synthesised compounds on acute inflammation, applying the carrageenan rat paw
oedema model, as well as the anti-inflammatory activity of the parent NSAIDs, are shown in Table 1.
Table 1. Effect of compounds 1–9, ibuprofen, naproxen, ketoprofen, indomethacin and tolfenamic acid
on carrageenan-induced rat paw oedema a.
Compound
% Oedema Reduction
1
68 **
36 *
Ibuprofen
2
46 **
11 *
Naproxen
3
65 **
47 *
Ketoprofen
4
67 **
42 **
55 **
24 **
39 *
56 *
37 *
43 **
Indomethacin
5
Tolfenamic acid
6
7
8
9
a The effect on oedema is expressed as percent inhibition of oedema in comparison to controls, 3.5 h post administration.
All compounds were administered intra peritonealy (i.p.) at 0.15 mmol/kg body weight. Significant difference from
control: * p < 0.01, ** p < 0.001 (Student’s t test).
The carrageenan-induced paw oedema is a commonly and widely used model of acute
inflammation. The early phase of carrageenan inflammation is characterised mainly by the release of
histamine, serotonin and bradykinin. In the late phase, more than two hours after administration, the
additional effects of neutrophil infiltration, prostaglandin production and pro-inflammatory cytokine
release develop [10]. In this investigation, oedema was estimated 3.5 h after carrageenan administration.
All compounds demonstrated increased anti-inflammatory activity. NSAID derivatives
more potent than their individual parent acids. Especially, compound was four times more active
than naproxen. The activity of and were about two fold higher than ibuprofen and tolfenamic acid,
1–5 were
2
1
5
respectively. Overall, these results indicate a further enhancement of the anti-inflammatory activity
by the performed molecular modification. It has been found that diazepam reduces the number of
inflammatory cells in the central nervous system [11], treatment with high diazepam doses decreases
paw oedema after carrageenan-induced injury [12] and that benzodiazepine derivatives with imide
substitution at the C3 of the benzodiazepine ring reduced up to 80% carrageenan rat paw hyperalgesia,
in a dose dependent manner, and this activity was at least partly attributed to bradykinin B1 receptor
antagonism [6].
Compounds
anti-inflammatory activity for trolox. Additionally, butylated hydroxytoluene (BHT), a well known
antioxidant structurally similar to the parent acid BHA, lacks such action either in vivo 13], or
in vitro 14]. Thus, the antioxidant activity alone does not seem to be entirely responsible for
the anti-inflammatory activity. Furthermore, compounds and also inhibited paw oedema,
7 and 8 showed considerable anti-inflammatory activity. There is not any reported
[
[
6
9
although they do not express any antioxidant activity (part 2.2.2.1.). Indole-3-acetic acid has no
reported anti-inflammatory action, while lipoic acid is only a weak anti-inflammatory agent at
similar doses [15]. Again, it seems convincing that the lorazepam moiety contributes to increased
anti-inflammatory activity.

Molecules 2019, 24, 3277
4 of 12
Effect of Compounds on Hyperlipidemia in Rats
The effect of the compounds under investigation on plasma total cholesterol, triglyceride and
LDL-cholesterol levels, 24 h post injection, determined in rats after Triton-induced hyperlipidaemia is
shown in Table 2. Simvastatin, ibuprofen and naproxen are included for comparison.
Table 2. Effect of the tested compounds, simvastatin, ibuprofen and naproxen on Triton WR1339
(tyloxapol) induced hyperlipidemia.
Dose i.p.
(µmol/kg)
% Reduction
TG b
Compound
TC a
LDL-C c
1
1
2
4
5
6
6
7
150
50
50
50
50
150
50
150
150
50
150
300
500
c
82 ***
44 ***
60 ***
59 ***
56 ***
82 ***
48 ***
72 ***
81 ***
59 ***
73 ***
41 ***
53 ***
65 ***
57 **
71 ***
59 ***
57 ***
41 **
39 ***
64 *
66 ***
52 ***
-
38 ***
44 ***
56 *
43 **
42 ***
60 ***
48 ***
60 *
47 ***
69 *
8
8
63 ***
44 **
70 ***
42 ***
26 ***
Simvastatin
Ibuprofen
Naproxen
b
a
TC: Total cholesterol; TG: Triglycerides; LDL-C: LDL cholesterol. Tyloxapol: 200 mg/kg, i.p. Significant
difference from hyperlipidemic control: * p < 0.01, ** p < 0.005, *** p < 0.001 (Student’s t test).
Tyloxapol (Triton WR1339) is a nonionic surfactant used to induce hyperlipidemia in experimental
animals if administered parenterally. It leads to accumulation of triglycerides, VLDL- and
LDL-cholesterol in plasma, reaching maximal effect 24 h after administration. These actions are due to
inhibition of lipoprotein lipase and to stimulation of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA)
reductase [9,16].
Compounds
1 and 6–8 were administered at 0.15mmol/kg and reduced greatly all lipidemic
indices, e.g., total cholesterol reduction was at the range of 80%, comparable to that of simvastatin.
For most compounds a lower dose, 0.05mmol/kg, was tested and found that still a very significant
reduction of lipidemia was achieved, further indicating a dose dependent action. Compounds
1 and 2
were more active than ibuprofen and naproxen at 6–10 times lower dose. These results may be related
to their high anti-inflammatory activity and partly may be related to a potential antioxidant effect.
2.2.2. In Vitro Experiments
Antioxidant Activity
Considering that free radicals are implicated in inflammatory processes, the synthesised
compounds were tested for antioxidant activity, expressed as inhibition of rat microsomal
membrane lipid peroxidation induced by ferrous ascorbate, as well as interaction with
1,1-diphenyl-2-picrylhydrazyl radical (DPPH). The percent interaction with DPPH and the IC50 values
of the active final compounds and trolox from rat hepatic microsomal membrane lipid peroxidation,
after 45 min of incubation are shown in Table 3.

Molecules 2019, 24, 3277
5 of 12
Table 3. Interaction of compounds 7, 8 and trolox, at various concentrations, with DPPH (200 µ
M) a
and their effect on lipid peroxidation b.
Percent Interaction with DPPH
Inhibition of Lipid
Peroxidation IC50 (µM)
Compound
200 µM
100 µM
50 µM
7
8
91
90
92
82
49
90
46
30
38
2.5
> 100
25
Trolox
a After 30 min of incubation. b After 45 min of incubation. Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid. All determinations were performed at least in triplicate and standard deviation is always within
mean value.
±
10% of the
The time course of lipid peroxidation inhibition, as affected by various concentrations of
shown in Figure 2.
7 is
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Compound 7
IC50 = 2.5 μΜ
Control
1 μΜ
2 μΜ
3 μΜ
0
5
10 15 20 25 30 35 40 45
Time (min)
Figure 2. Inhibition of lipid peroxidation, as affected by various concentrations of 7, in relation to time.
Most compounds, except for
peroxidation test, compound showed moderate activity. This may be due to the high lipophilicity of
compound (logP = 7.4) which reduced its solubility in the aqueous reaction environment. However, it
interacted strongly with DPPH, in a way comparable to that of trolox. Compound was almost tenfold
7 and 8, were practically inactive in these experiments. In the lipid
8
8
7
more potent inhibitor of lipid peroxidation than trolox, a reference antioxidant, and an effective radical
scavenger. It is possible that the hypolipidemic and anti-inflammatory effects of these compounds are
related, at least partly, to their antioxidant activity. The lipoic acid derivative
this is in accordance with the observation that lipoic acid can scavenge only very reactive radicals, while
the reduced form, 6,8-dimercaptooctanoic acid, is a strong antioxidant due to hydrogen transfer [17 18].
may contribute to the in vivo anti-inflammatory
6 was found inactive, and
,
Thus, it could be suggested that the reduced form of
and hypolipidemic activity of this compound.
6
Inhibition of Lipoxygenase
Lipoxygenases, involved in inflammation, are a family of enzymes that catalyse the dioxygenation
of polyunsaturated fatty acids which contain the cis-1,4-pentadiene structure. Although there are
several enzymes, they all catalyse the stereo- and regio-specific peroxidation of arachidonic or linoleic
acid in the presence of molecular oxygen. Soybean lipoxygenase-1 can use arachidonic acid as substrate,
with about 15% of activity for linoleic acid. It has been found that arachidonic acid binding sites in plant
lipoxygenases share almost the same similarity with animal 5-lipoxygenase [19]. 5-Lipoxygenase activity
contributes to atherosclerosis via oxidation of low-density lipoprotein. Furthermore, studies using

Molecules 2019, 24, 3277
6 of 12
5-lipoxygenase-deficient mice show that 5-lipoxygenase activity may contribute to stress and depression
behaviour [20].
The effect of the synthesised compounds on soybean lipoxygenase-1, using linoleic acid as
substrate, is demonstrated in Table 4. In this table, the inhibition offered by nordihydroguaiaretic acid
(NDGA), a potent lipoxygenase inhibitor, is included. BHA and trolox could not inhibit lipoxygenase
even at concentrations much higher than 300 µM. In addition, no inhibition was observed when linoleic
acid was used at 1 mM, a concentration higher than the saturating substrate concentration, under
the same experimental conditions. The decline of inhibition by increasing the concentration of the
substrate indicates a competitive inhibition of lipoxygenase.
Table 4. Effect of compounds
lipoxygenase a.
1–
9, BHT, ibuprofen, ketoprofen, tolfenamic acid and NDGA on
IC50 (µM) or %
Inhibition/µM
Compound
1
14%/50 µM
2
-
3
84
4
86
5
22%/100 µM
6
7
60
-
8
9
44
255
192
200
220
170
1.3
BHT
Ibuprofen
Ketoprofen
Tolfenamic acid
NDGA
a
After 7 min of incubation; BHT: 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene); NDGA:
nordihydroguaiaretic acid; -: inactive.
The time course of lipoxygenase inhibition by the most active of the synthesised compounds,
3 and 8, is shown in Figure 3.
1.0
Compound 3
control
IC50 = 84 μΜ
0.8
50μΜ
0.6
60μΜ
75μΜ
0.4
0.2
0.0
100μΜ
0
60
120
180
240
300
360
420
Τime (s)
Figure 3. Cont.

Molecules 2019, 24, 3277
7 of 12
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Compound 8
IC50 = 44 μΜ
control
10 mM
25 μΜ
50 μΜ
75 μΜ
0
60
120
180
240
300
360
420
Time (s)
Figure 3. Time course of lipoxygenase inhibition by various concentrations of compounds 3 and 8.
From the presented results it could be observed that, when lorazepam was esterified with rigid
acids, e.g., naproxen ( ), trolox ( ), indole-3-acetic acid ( ), inhibition was insignificant or absent,
2
7
9
whereas, less rigid substitution seems to contribute to stronger inhibition, e.g., ketoprofen (3), lipoic
acid (6), BHA (8).
3. Materials and Methods
3.1. General
All commercially available reagents were purchased from Merck (Kenilworth, NJ, USA) and used
without further purification. -Carrageenan and lipoxygenase type I from soybean were purchased
κ
from Sigma (St. Louis, MO, USA). The IR spectra were recorded on a Perkin Elmer Spectrum BX
FT-IR spectrometer (Waltham, MA, USA). The 1H-NMR spectra were recorded using an AGILENT
DD2-500 MHz spectrometer (Santa Clara, CA, USA). All chemical shifts are reported in δ (ppm) and
signals are given as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Melting points (m.p.) were
determined with a MEL-TEMPII Laboratory Devices, Sigma-Aldrich (Milwaukee WI, USA) apparatus
and are uncorrected. The microanalyses were performed on a Perkin-Elmer 2400 CHN elemental
analyser. Wistar rats (160–220 g, 3–4 months old) were kept in the Centre of the School of Veterinary
Medicine (EL54 BIO42), Aristotelian University of Thessaloniki, which is registered by the official state
veterinary authorities (presidential degree 56/2013, in harmonization with the European Directive
2010/63/EEC). The experimental protocols were approved by the Animal Ethics Committee of the
Prefecture of Central Macedonia (no. 270079/2500).
3.2. Synthesis
General Method for the Synthesis of Compounds 1–9
A) In a solution of the corresponding acid (1mmol) in dichloromethane (CH2Cl2) lorazepam is
suspended (1.05 mmol) and N,N0-dicyclohexylcarbodiimide (DCC, 1.3mmol) was added. The reaction
mixture was stirred for 4–12h. After filtration, the final compounds were isolated with flash
chromatography using petroleum ether and ethyl acetate as eluents.
B) For compound 7: Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 1mmol) was
dissolved in tetrahydrofuran (THF) and carbonyldiimidazole (CDI, 1.2 mmol) was added. After stirring
for 45 min, lorazepam (1.05 mmol) was added and the reaction was left for 12 h at room temperature.
The solvent was distilled off and the residue was dissolved in ethyl acetate and washed with water.

Molecules 2019, 24, 3277
8 of 12
The solution was dried over Na2SO4 and the final compound was isolated with flash chromatography
using petroleum ether and ethyl acetate as eluents.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(4-isobutylphenyl)
propanoate (
105–122 ◦C. IR (nujol): 3297 (N-H), 1738, 1714 (C=O ester diastereomers), 1621 (C=O amide), 1527 (C-C
aromatic) cm−1 1H-NMR (CDCl3)
: 0.92 (d, 6H, J = 6.4 Hz, CH3-CH-CH3), 1.65 (dd, 3H, J = 14.1,
1). Flash chromatography (petroleum ether/ethyl acetate, 4/1). White solid, yield 74%, m.p.
.
δ
7.1 Hz, -CO-CH-CH3), 1.95–1.78 (m, 1H, CH3-CH-CH3), 2.46 (d, 2H, J = 7.1 Hz, -CH2CH-(CH3)2), 4.05
(q, 1H, J = 7.1 Hz, -CO-CH-CH3), 6.02, 6.00 (s, 1H, -O-CH-C=O), 7.17–7.00 (m, 4H, aromatic ibuprofen),
7.65–7.30 (m, 7H, aromatic lorazepam), 9.21 (s, 1H, -NH). Anal. Calcd for C28H26Cl2N2O3: C, 66.02; H,
5.14; N, 5.50. Found: C, 65.82; H, 5.30; N, 5.33.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl
2-(6-methoxynaphthalen-2-yl)propanoate (
acetate, 3/1). White solid, yield 93%, m.p. 136–139 C. IR (nujol): 3220 (N-H), 1702 (C=O ester),
1627 (C=O amide), 1605, 1569 (C-C aromatic) cm−1
2
).
Flash chromatography (petroleum ether/ethyl
◦
.
1H-NMR (CDCl3)
δ: 1.75 (d, 3H, J = 7.7 Hz,
CH3-CH-C=O), 3.94 (s, 3H, -O-CH3), 4.28–4.10 (m, 1H, CH3-CH-C=O), 6.01 (s, 1H, -O-CH-C=O),
6.99 (d, 1H, J = 8.5 Hz, naphthyl C2), 7.04 (s, 1H, naphthyl C10), 7.18–7.08 (m, 2H, naphthyl C7,
C8), 7.45–7.25 (m, 3H, chlorophenyl C3, C4, C5 and naphthyl C5), 7.52 (d, 1H, J = 8.5 Hz, naphthyl
C3), 7.76–7.55 (m, 3H, chlorophenyl C6 and chlorophenylamino C3, C5), 7.83 (d, 1H, J = 8.1 Hz,
chlorophenylamino C6), 9.42 (s, 1H, O=C-NH-). Anal. Calcd for C29H22Cl2N2O4: C, 65.30; H, 4.16; N,
5.25. Found: C, 65.42; H, 4.26; N, 4.89.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl
2-(3-benzoylphenyl)propanoate (
3
).
Flash chromatography (petroleum ether/ethyl acetate,
3/1).White solid, yield 85%, m.p.195–201 ◦C. IR (nujol): 3203 (N-H), 1739 (C=O ketone), 1712, 1696 (C=O
ester diastereomers), 1650 (C=O amide), 1596 (C-C aromatic) cm−1. H-NMR (CDCl3 + DMSO-d6)
1
δ
: 1.73–1.67 (m, 3H, -CO-CH-CH3), 4.20–4.08 (m, 1H, -CO-CH-CH3), 6.04, 6.02 (s, 1H, -O-CH-C=O),
7.15–7.00 (m, 2H, isopropylphenyl C5, chlorophenyl C5), 7.93–7.33 (m, 14H, aromatic), 8.70, 8.73 (s, 1H,
NH-). Anal. Calcd for C31H22Cl2N2O4: C, 66.80; H, 3.98; N, 5.03. Found: C, 66.54; H, 3.91; N, 4.67.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(1-(4-chlorobenzoyl)-
5-methoxy-2-methyl-1H-indol-3-yl)acetate (4). Flash chromatography (petroleum ether/ethyl acetate,
2/1). White solid, yield 81%, m.p. 189–191 ◦C. IR (nujol): 3302 (N-H), 1748 (C=O ester), 1699 (C=O
1
amide indole), 1620 (C=O amide lorazepam), 1574 (C-C aromatic) cm−1. H-NMR (CDCl3 + DMSO-d6)
δ
: 2.25 (s, 3H, CH3-C-N-), 3.71 (s, 3H, -O-CH3), 3.83 (s, 2H, -CH2-C=O), 5.84 (s, 1H, -O-CH-C=O), 6.55
(d, 1H, J = 9.0 Hz, indole C6), 6.85 (d, 1H, J = 9.0 Hz, indole C7), 6.89 (s, 1H, indole C4), 6.98 (s, 1H,
chlorophenylamino C3), 7.13 (d, 1H, J = 8.7 Hz, chlorophenyl C3), 7.31–7.21 (m, 4H, chlorophenylamino
C5 and chlorophenyl C4, C5, C6), 7.33 (d, 2H, J = 8.4 Hz, aromatic chloro-benzoyl C3, C5), 7.41 (d, 1H,
J = 6.0 Hz, chlorophenylamino C6), 7.54 (d, 2H, J = 8.4 Hz, chlorobenzoyl C2, C6), 10.61 (s, 1H, -NH-).
Anal. Calcd for C34H24Cl3N3O5: C, 61.79; H, 4.56; N, 6.36. Found: C, 61.65; H, 4.22; N, 5.92.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl
2-((3-chloro-2-methylphenyl)amino)benzoate (
acetate, 2/1). White solid, yield 41%, m.p. 206–207 ◦C. IR (nujol): 3327 (N-H), 1721 (C=O ester), 1635
(C=O amide), 1583 (C-C aromatic) cm−1 1H-NMR (CDCl3 + DMSO-d6)
: 2.25 (s, 3H, -C 3), 6.14
(s, 1H, -O-C -C=O), 6.80–6.67 (m, 2H, aromatic amino methyl phenyl C6 and aromatic benzoic
5). Flash chromatography (petroleum ether/ethyl
.
δ
H
H
acid C5), 7.45–6.96 (m, 10H, aromatic chloro-methyl-phenylamino C4, C5, benzoic acid C3, C4, C6,
chlorophenylamino C3, C5 and chlorophenyl C3, C4, C5), 7.55 (d, 1H, J = 7.6 Hz, chlorophenyl C6),
8.25 (d, 1H, J = 8.1 Hz, chlorophenylamino C6), 9.09 (s, 1H, -NH-), 10.93 (s, 1H, O=C-NH-). Anal.
Calcd for C29H20Cl3N3O3: C, 61.66; H, 3.57; N, 7.44. Found: C, 62.03; H, 3.53; N, 7.81.

Molecules 2019, 24, 3277
9 of 12
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl
5-(1,2-dithiolan
-3-yl)pentanoate (6). Flash chromatography (petroleum ether/ethyl acetate, 1/1). White solid, yield
71%, m.p. 184–189 ◦C. IR (nujol): 3237 (N-H), 1713, 1682 (C=O ester), 1620 (C=O amide), 1592 (C-C
1
aromatic) cm−1. H-NMR (CDCl3 + DMSO-d6)
δ: 1.80–1.67 (m, 2H, C3 H), 1.62–1.47 (m, 4H, C4, C5),
1.93–1.84 (m, 1H, C7 axial), 2.33–2.21 (m, 1H, C7 equatorial), 2.43–2.35 (m, 2H, C2), 3.03–2.86 (m,
2H, C6, C8 axial), 3.42–3.32 (m, 1H, C8 equatorial), 5.75 (s, 1H, -O-CH-C=O), 6.83 (d, 1H, J = 2.3 Hz,
chlorophenyl C3), 7.09 (d, 1H, J = 8.7 Hz, chlorophenyl C6), 7.28–7.17 (m, 4H, chlorophenylamino C3,
C5 and chlorophenyl C4, C5), 7.39–7.34 (m, 1H, chlorophenylamino C6), 10.64 (s, 1H, O=C-NH-). Anal.
Calcd for C23H22Cl2N2O3S2: C, 54.22; H, 4.35; N, 5.50. Found: C, 54.41; H, 4.68; N, 5.33.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylate (7). Flash chromatography (petroleum ether/ethyl acetate, 3/2).
White solid, yield 85%, m.p. 138–144 ◦C. IR (nujol): 3400 (O-H), 3250 (N-H), 1680 (C=O ester), 1631
1
(C=O amide) cm−1. H-NMR (CDCl3)
δ: 1.78 (s, 3H, 2-CH3), 2.00 (s, 3H, 5-CH3), 2.10 (s, 3H, 7-CH3),
2,15 (s, 3H, 8–CH3), 2.20–2.65 (m, 4H, chromane), 5.10 (s, 1H, -OH), 6.10 (s, 1H, -O-CH-C=O), 7.20 - 7.82
(m, 7H, aromatic), 9.40 (s, 1H, -NH-). Anal. Calcd for C29H26Cl2N2O5: C, 62.94; H, 4.74; N, 5.06.
Found: C, 62.69; H, 4.96; N, 5.13.
(E)-7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 3-(3,5-di-tert-butyl-
4-hydroxyphenyl)acrylate (8). Flash chromatography (petroleum ether/ethyl acetate, 6/1 and then 3/1).
White solid, yield 40%, m.p. 274 ◦C. IR (nujol): 3611 (O-H), 3277 (N-H), 1710 (C=O ester), 1627 (C=O
1
amide), 1593 (C-C aromatic) cm−1. H-NMR (CDCl3 + DMSO-d6)
δ
: 1.49 (s, 18H, -CH3), 5.56 (s, 1H,
phenol OH), 6.00 (s, 1H, -O-CH-C=O), 6.61 (d, 1H, J = 15.9 Hz, CH=CH-C=O), 7.89 (d, 1H, J = 15.9 Hz,
CH=CH-C=O), 7.13 (d, 1H, J = 2.2 Hz, chlorophenyl C6), 7.21 (d, 1H, J = 8.7 Hz, chlorophenyl C3),
7.51–7.39 (m, 6H, chlorophenyl C4, C5, chlorophenylamino C3, C5 and di-tert-butylphenyl C2, C6),
7.64 (d, 1H, J = 7.4 Hz, chlorophenylamino C6), 7.89 (d, 1H, J = 15.9 Hz, CH=CH-C=O), 8.97 (s, 1H,
-NH-). Anal. Calcd for C32H32Cl2N2O4 x0.7CH2Cl2: C, 61.47; H, 5.27; N, 4.38. Found: C, 61.31; H, 5.58;
N, 4.06.
7-Chloro-5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl 2-(1H-indol-3-yl)acetate
(9
). Flash chromatography (petroleum ether/ethyl acetate, 2/1). White solid, yield 40%, m.p. 257–258 ◦C.
IR (nujol): 3360 (N-H), 1760 (C=O ester), 1713 (C=O amide), 1621 (C-C aromatic) cm−1
.
1H-NMR
(CDCl3 + DMSO-d6) : 3.96 (s, 1H, -CH2-C=O), 5.89 (s, 1H, -O-CH-C=O), 6.94 (d, 1H, J = 2.2 Hz,
δ
indole C7), 6.99 (d, 1H, J = 7.5 Hz, indole C6), 7.06 (t, 1H, J = 7.5 Hz, indole C6), 7.25–7.17 (m, 2H,
aromatic indole C2, C4), 7.59–7.28 (m, 7H, aromatic lorazepam), 10.16 (s, 1H, indole -NH-), 10.89 (s, 1H,
O=C-NH-). Anal. Calcd for C25H17Cl2N3O3 x2.4H2O: C, 57.57; H, 4.21; N, 8.06. Found: C, 57.53; H,
3.88; N, 8.12.
3.3. Effect on Carrageenan-Induced Rat Paw Oedema
An aqueous solution of carrageenan was prepared (1% w/v) and 0.1 mL of this was injected i.d. into
the right hind paw of female rats; the left paw served as control. The tested compounds (suspended in
water with a few drops of Tween 80) were given intra peritoneally (i.p.) (0.15 mmol/kg) 5 min prior to
the carrageenan injection. After 3.5 h the hind paws were weighed separately. The produced oedema
was estimated as paw weight increase [18].
3.4. Effect on Plasma Total Cholesterol, Triglyceride and LDL-cholesterol Levels
A solution of Triton WR 1339 (tyloxapol) in saline was administered i.p. (200 mg/kg) to male rats
and 1 h later the examined compound (0.15 and/or 0.05 mmol/kg, suspended in water with a few drops
of Tween 80) was given i.p. After 24 h, blood was taken from the aorta and used for the determination
of plasma total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C)
concentrations, using commercial kits, against standard solutions [21].

Molecules 2019, 24, 3277
10 of 12
3.5. Effect on Lipid Peroxidation
The incubation mixture contained heat-inactivated rat hepatic microsomal fraction, ascorbic acid
(0.2 mM) in Tris–HCl/KCl buffer (pH 7.4) and the test compounds dissolved in dimethylsulphoxide.
The reaction was initiated by FeSO4 (10 µ
M) and the mixture was incubated at 37 ◦C. Aliquots were
taken at various time intervals for 45 min. Lipid peroxidation was assessed spectrophotometrically
(535 against 600 nm) by the determination of 2-thiobarbituric acid (TBA) reactive material [22].
3.6. Interaction with the Stable Radical 1,1-diphenyl-2-picrylhydrazyl (DPPH)
Compounds (in absolute ethanol, final concentration 50–200
µ
M) were added to an equal volume
◦
of an ethanolic solution of DPPH (final concentration 200
Absorbance (517 nm) was recorded after 30 min [22].
µM) at room temperature (22
±
2 C).
3.7. Effect on Lipoxygenase Activity
The reaction mixture contained the test compounds dissolved in ethanol and soybean lipoxygenase
(250 U/mL) in Tris buffer (pH 9). The reaction was initiated by the addition of sodium linoleate (0.1 mM)
◦
and monitored for 7 min at 28 C, by recording the absorbance of a conjugated diene structure at
234 nm. For the estimation of the type of inhibition, the above experiments were repeated, using
sodium linoleate at 1 mM, which is higher than the saturating substrate concentration [18].
4. Conclusions
In conclusion, the synthesised compounds presented important hypolipidemic activity and
significant in vivo anti-inflammatory properties, being superior to the parent acids. It is reported that
anxiolytic therapy improves stress-induced inflammation [23]. Furthermore, inflammation, endothelial
dysfunction and platelet aggregation may connect anxiety disorders with cardiovascular diseases [24].
Finally, we have shown that molecules containing lorazepam and antioxidants, linked by a GABA
residue, reduced stress, caused by immobilization and fasting, as well as the subsequent oxidative
damage [7]. Complex stress-related disorders could be treated effectively with agents designed to act
at different stages of their pathogenesis. The present research may provide useful candidate molecules
towards this target.
Author Contributions: Investigation, P.T.-N. and G.P.; Supervision, E.A.R.; Writing—review & editing, P.N.K.
Funding: This research received no external funding
Acknowledgments: G.P. acknowledges the General Secretariat for Research and Technology (GSRT) of Greece
and the Hellenic Foundation for Research and Innovation (HFRI) for a grant supporting his PhD research.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Pantelidou, M.; Tsiakitzis, K.; Rekka, E.A.; Kourounakis, P.N. Biologic stress, oxidative stress, and resistance
to drugs: What is hidden behind. Molecules 2017, 22, 307. [CrossRef] [PubMed]
Kumar, A.; Goyal, R. Possible involvement of GABAergic modulation in the protective effect of
gabapentin against immobilization stress-induced behavior alterations and oxidative damage in mice.
Fundam. Clin. Pharmacol. 2007, 21, 575–581. [CrossRef] [PubMed]
2.
3.
4.
Liu, J.Y.; Guo, F.; Wu, H.L.; Wang, Y.; Liu, J.S. Midazolam anesthesia protects neuronal cells from oxidative
stress-induced death via activation of the JNK-ERK pathway. Mol. Med. Rep. 2017, 15, 169–179. [CrossRef]
[PubMed]
Apryatin, S.A.; Sidorova, Y.S.; Shipelin, V.A.; Balakina, A.; Trusov, N.V.; Mazo, V.K. Neuromotor activity,
anxiety and cognitive function in the in vivo model of alimentary hyperlipidemia and obesity. Bull. Exp.
Biol. Med. 2017, 163, 37–44. [CrossRef] [PubMed]

Molecules 2019, 24, 3277
11 of 12
5.
6.
Gancheva, S.; Galunska, B.; Zhelyazkova-Savova, M. Diets rich in saturated fat and fructose induce anxiety
and depression-like behaviours in the rat: Is there a role for lipid peroxidation? Int. J. Exp. Pathol. 2017, 98,
296–306. [CrossRef]
Wood, M.R.; Kim, J.J.; Han, W.; Dorsey, B.D.; Homnick, C.F.; DiPardo, R.M.; Kuduk, S.D.; MacNeil, T.;
Murphy, K.L.; Lis, E.V.; et al. Benzodiazepines as potent and selective bradykinin B1 antagonists. J. Med. Chem.
2003, 46, 1803–1806. [CrossRef]
7.
8.
Tsiakitzis, K.C.; Rekka, E.A.; Kourounakis, A.P.; Kourounakis, P.N. Novel compounds designed as antistress
agents. J. Med. Chem. 2009, 52, 7315–7318. [CrossRef]
Akbari, M.; Ostadmohammadi, V.; Tabrizi, R.; Mobini, M.; Lankarani, K.B.; Moosazadeh, M.; Heydari, S.T.;
Chamani, M.; Kolahdooz, F.; Asemi, Z. The effects of alpha-lipoic acid supplementation on inflammatory
markers among patients with metabolic syndrome and related disorders: A systematic review and
meta-analysis of randomized controlled trials. Nutr. Metab. (Lond) 2018, 15, 39. [CrossRef]
Theodosis-Nobelos, P.; Kourounakis, P.N.; Rekka, E.A. Anti-inflammatory and hypolipidemic effect of novel
conjugates with trolox and other antioxidant acids. Med. Chem. 2017, 13, 214–225. [CrossRef]
9.
10. Mansouri, M.T.; Hemmati, A.A.; Naghizadeh, B.; Mard, S.A.; Rezaie, A.; Ghorbanzadeh, B. A study of the
mechanisms underlying the anti-inflammatory effect of ellagic acid in carrageenan-induced paw edema in
rats. Indian J. Pharm. 2015, 47, 292–298.
11. Fernández Hurst, N.; Zanetti, S.R.; Báez, N.S.; Bibolini, M.J.; Bouzat, C.; Roth, G.A. Diazepam treatment
reduces inflammatory cells and mediators in the central nervous system of rats with experimental autoimmune
encephalomyelitis. J. Neuroimmunol. 2017, 313, 145–151. [CrossRef] [PubMed]
12. Lazzarini, R.; Paulino, C.A.; Malucelli, B.E.; Palermo-Neto, J. Effects of high doses of diazepam on
carrageenin-induced paw edema in rats. Braz J. Med. Biol. Res. 1996, 29, 1525–1529. [PubMed]
13. Ziakas, G.N.; Rekka, E.A.; Gavalas, A.M.; Eleftheriou, P.T.; Kourounakis, P.N. New analogues of butylated
hydroxytoluene as anti-inflammatory and antioxidant agents. Bioorg. Med. Chem. 2006, 14, 5616–5624.
[CrossRef] [PubMed]
14. Murakami, Y.; Kawata, A.; Katayama, T.; Fujisawa, S. Anti-inflammatory activity of the artificial antioxidants
2-tert-butyl-4-methoxyphenol (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT) and 2,4,6-tri-tert-butylphenol
(TBP), and their various combinations. In Vivo 2015, 29, 197–206. [PubMed]
15. El-Shitany, N.A.; El-Masry, S.A.; El-Ghareib, M.A.; El-Desoky, K. Thioctic acid protects against
carrageenan-induced acute inflammation in rats by reduction in oxidative stress, downregulation of
COX-2 mRNA and enhancement of IL-10 mRNA. Fundam. Clin. Pharm. 2010, 24, 91–99. [CrossRef] [PubMed]
16. Zarzecki, M.S.; Araujo, S.M.; Bortolotto, V.C.; de Paula, M.T.; Jesse, C.R.; Prigol, M. Hypolipidemic action of
chrysin on Triton WR-1339-induced hyperlipidemia in female C57BL/6 mice. Toxicol Rep. 2014, 1, 200–208.
[CrossRef] [PubMed]
17. Castañeda-Arriaga, R.; Alvarez-Idaboy, J.R. Lipoic acid and dihydrolipoic acid. A comprehensive theoretical
study of their antioxidant activity supported by available experimental kinetic data. J. Chem Inf Model. 2014
54, 1642–1652. [CrossRef] [PubMed]
,
18. Theodosis-Nobelos, P.; Kourti, M.; Gavalas, A.; Rekka, E.A. Amides of non-steroidal anti-inflammatory drugs
with thiomorpholine can yield hypolipidemic agents with improved anti-inflammatory activity. Bioorg Med.
Chem. Lett. 2016, 26, 910–913. [CrossRef]
19. Reddy, K.K.; Vidya Rajan, V.K.; Gupta, A.; Aparoy, P.; Reddanna, P. Exploration of binding site pattern in
arachidonic acid metabolizing enzymes, Cyclooxygenases and Lipoxygenases. BMC Res. Notes 2015, 8, 152.
[CrossRef]
20. Manev, R.; Manev, H. 5-Lipoxygenase as a putative link between cardiovascular and psychiatric disorders.
Crit. Rev. Neurobiol 2004, 16, 181–186. [CrossRef]
21. Tooulia, K.K.; Theodosis-Nobelos, P.; Rekka, E.A. Thiomorpholine derivatives with hypolipidemic and
antioxidant activity. Arch. Pharm (Weinh.) 2015, 348, 629–634. [CrossRef] [PubMed]
22. Tsiakitzis, K.C.; Papagiouvannis, G.; Theodosis-Nobelos, P.; Tziona, P.; Kourounakis, P.N.; Rekka, E.A.
Synthesis, antioxidant and anti-inflammatory effects of antioxidant acid amides with GABA and
n-acyl-pyrrolidin-2-ones. Curr. Chem. Biol. 2017, 11, 127–139. [CrossRef]

Molecules 2019, 24, 3277
12 of 12
23. Ramirez, K.; Sheridan, J.F. Antidepressant imipramine diminishes stress-induced inflammation in the
periphery and central nervous system and related anxiety- and depressive- like behaviors. Brain Behav. Immun.
2016, 57, 293–303. [CrossRef] [PubMed]
24. Celano, C.M.; Daunis, D.J.; Lokko, H.; Campbell, K.A.; Huffman, J.C. Anxiety disorders and cardiovascular
disease. Curr. Psychiatry Rep. 2016, 18, 101. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 1–9 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).