Active Anti-Inflammatory and Hypolipidemic Derivatives of Lorazepam

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 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.


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 GABA A 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 [1]. We [1] and others [2] have shown that biologic stress induces oxidative stress, and that GABAergic modulation may offer protection against immobilization-induced stress and oxidative damage [2]. Moreover, it has been reported that the benzodiazepine midazolam protects against neuronal degeneration and apoptosis induced by biological and oxidative stress [3]. Alimentary dyslipidemia disturbed anxiety level and cognitive processes in mice [4] and a diet rich in saturated fat and fructose caused high 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].

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. 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 1-5 were more potent than their individual parent acids. Especially, compound 2 was four times more active than naproxen. The activity of 1 and 5 were about two fold higher than ibuprofen and tolfenamic acid, 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 B 1 receptor antagonism [6].
Compounds 7 and 8 showed considerable anti-inflammatory activity. There is not any reported 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 6 and 9 also inhibited paw oedema, 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.

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. 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.

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 IC 50 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. The time course of lipid peroxidation inhibition, as affected by various concentrations of 7 is shown in Figure 2.  The time course of lipid peroxidation inhibition, as affected by various concentrations of 7 is shown in Figure 2. Most compounds, except for 7 and 8, were practically inactive in these experiments. In the lipid peroxidation test, compound 8 showed moderate activity. This may be due to the high lipophilicity of compound 8 (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 7 was almost tenfold 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 6 was found inactive, and 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]. Thus, it could be suggested that the reduced form of 6 may contribute to the in vivo anti-inflammatory and hypolipidemic activity of this compound.

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-  Most compounds, except for 7 and 8, were practically inactive in these experiments. In the lipid peroxidation test, compound 8 showed moderate activity. This may be due to the high lipophilicity of compound 8 (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 7 was almost tenfold 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 6 was found inactive, and 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]. Thus, it could be suggested that the reduced form of 6 may contribute to the in vivo anti-inflammatory and hypolipidemic activity of this compound.

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 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. The time course of lipoxygenase inhibition by the most active of the synthesised compounds, 3 and 8, is shown in Figure 3. lipoxygenase [19]. 5-Lipoxygenase activity contributes to atherosclerosis via oxidation of low-density lipoprotein. Furthermore, studies using 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 µΜ. 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. The time course of lipoxygenase inhibition by the most active of the synthesised compounds, 3 and 8, is shown in Figure 3.  From the presented results it could be observed that, when lorazepam was esterified with rigid acids, e.g., naproxen (2), trolox (7), indole-3-acetic acid (9), inhibition was insignificant or absent, whereas, less rigid substitution seems to contribute to stronger inhibition, e.g., ketoprofen (3), lipoic acid (6), BHA (8).

General
All commercially available reagents were purchased from Merck (Kenilworth, NJ, USA) and used without further purification. κ-Carrageenan and lipoxygenase type I from soybean were purchased from Sigma (St. Louis, MO, USA). The IR spectra were recorded on a Perkin Elmer Spectrum BX FT-IR spectrometer (Waltham, MA, USA). The 1 H-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).

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,N′-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.

General
All commercially available reagents were purchased from Merck (Kenilworth, NJ, USA) and used without further purification. κ-Carrageenan and lipoxygenase type I from soybean were purchased from Sigma (St. Louis, MO, USA). The IR spectra were recorded on a Perkin Elmer Spectrum BX FT-IR spectrometer (Waltham, MA, USA). The 1 H-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).

Synthesis
General Method for the Synthesis of Compounds 1-9 A) In a solution of the corresponding acid (1mmol) in dichloromethane (CH 2 Cl 2 ) lorazepam is suspended (1.05 mmol) and N,N -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.
The solution was dried over Na 2 SO 4 and the final compound was isolated with flash chromatography using petroleum ether and ethyl acetate as eluents.

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 FeSO 4 (10 µM) 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]. Absorbance (517 nm) was recorded after 30 min [22].

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].

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.