Antioxidant Serine-(NSAID) Hybrids with Anti-Inflammatory and Hypolipidemic Potency

A series of L-serine amides of antioxidant acids, such as Trolox, (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic acid (phenolic derivative of cinnamic acid) and 3,5-di-tert-butyl-4-hydroxybenzoic acid (structurally similar to butylated hydroxytoluene), was synthesized. The hydroxy group of serine was esterified with two classical NSAIDs, ibuprofen and ketoprofen. The Trolox derivatives with ibuprofen (7) and ketoprofen (10) were the most potent inhibitors of lipid peroxidation (IC50 3.4 μΜ and 2.8 μΜ), several times more potent than the reference Trolox (IC50 25 μΜ). Most of the compounds decreased carrageenan-induced rat paw edema (37–67% at 150 μmol/kg). They were moderate inhibitors of soybean lipoxygenase, with the exception of ibuprofen derivative 8 (IC50 13 μΜ). The most active anti-inflammatory compounds exhibited a significant decrease in lipidemic indices in the plasma of Triton-induced hyperlipidemic rats, e.g., the most active compound 9 decreased triglycerides, total cholesterol and low-density lipoprotein cholesterol by 52%, 61% and 70%, respectively, at 150 μmol/kg (i.p.), similar to that of simvastatin, a well-known hypocholesterolemic drug. Since the designed compounds seem to exhibit multiple pharmacological actions, they may be of use for the development of agents against inflammatory and degenerative conditions.


Introduction
Inflammation is a key factor in various metabolic and cardiovascular disorders. Inflammatory and metabolic diseases are interconnected, e.g., NF-κB signaling is associated with insulin resistance and atherosclerosis. Furthermore, increased circulating factors such as glucose and fatty acids may account for the propagation of inflammatory signals [1,2]. In addition, neurodegenerative disorders are characterized by inflammatory stimuli and the activation of the innate immune system response in the central nervous system (CNS) [3].
The imbalance between oxidative versus antioxidant processes in favor of the former may result in the interruption of cellular homeostasis and an increase in cellular dysfunction, affecting many pathobiochemical markers and paths [4]. Inflammatory agonists such as IL-1β utilize reactive oxygen species (ROS) as part of their signaling, and factors such as NF-κB may be affected by oxidative stress [5,6], making the interrelation between oxidative stress and inflammation obvious.
ROS may deteriorate the vascular tone in response to metabolic conditions such as hyperglycemia, insulin resistance or hyperlipidemia. They may also disturb endothelial nitrogen monoxide synthetase (eNOS), leading to apoptosis and NOS decoupling. Thus, increased production of superoxide anion radical, peroxynitrite and deficiency of tetrahydrobiopterin impair the vascular relaxation and function [7]. In parallel, atherosclerotic plaque enhances cellular oxidation by macrophages, via the consumption of blood lipids

Synthesis
Compounds 4-6 were synthesized by direct amidation of L-serine hydrochloride with the respective acid (R1-COOH, R2-COOH and R3-COOH), via the formation of anhydride with DCC (N,N′-dicyclohexyl-carbodiimide) or the intermediate imidazole derivative using CDI (carbonyldiimidazole), with excellent yields (82-91%). In the case of Trolox (R1COOH), usage of DCC resulted in a bulky intermediate anhydride, leading to lower yields. The corresponding products were hydrolyzed with sodium hydroxide, yielding compounds 1-3, or esterified with ibuprofen (R4-COOH) and ketoprofen (R5-COOH), yielding compounds 7-9 and 10-12, respectively. The esterification reaction was carried out using DCC and DMAP with yields varying from 70 to 91%. In total, the lower yield was obtained with the Trolox derivatives, possibly due to steric hindrance.

Antioxidant and Radical Scavenging Activity
The effect of the new compounds and Trolox on rat hepatic microsomal membrane lipid peroxidation, expressed as IC50 values after 45 min of incubation, their clogP and total polar surface area (TPSA) values are shown in Table 1. For the acids 1, 2 and 3, logD values were calculated.

Synthesis
Compounds 4-6 were synthesized by direct amidation of L-serine hydrochloride with the respective acid (R 1 -COOH, R 2 -COOH and R 3 -COOH), via the formation of anhydride with DCC (N,N -dicyclohexyl-carbodiimide) or the intermediate imidazole derivative using CDI (carbonyldiimidazole), with excellent yields (82-91%). In the case of Trolox (R 1 COOH), usage of DCC resulted in a bulky intermediate anhydride, leading to lower yields. The corresponding products were hydrolyzed with sodium hydroxide, yielding compounds 1-3, or esterified with ibuprofen (R 4 -COOH) and ketoprofen (R 5 -COOH), yielding compounds 7-9 and 10-12, respectively. The esterification reaction was carried out using DCC and DMAP with yields varying from 70 to 91%. In total, the lower yield was obtained with the Trolox derivatives, possibly due to steric hindrance.

Antioxidant and Radical Scavenging Activity
The effect of the new compounds and Trolox on rat hepatic microsomal membrane lipid peroxidation, expressed as IC 50 values after 45 min of incubation, their clogP and total polar surface area (TPSA) values are shown in Table 1. For the acids 1, 2 and 3, logD values were calculated. The time course of lipid peroxidation, as affected by various concentrations of compounds 4 and 7, is shown in Figure 3.
The interaction of compounds with the lipophilic N-centered 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable free radical is another assay for evaluation of the antioxidant and reducing activity of compounds. The interaction of compounds 1, 3, 4, 6, 7, 8, 9, 10, 12 and Trolox, at various concentrations, with DPPH is presented in Table 2.  The time course of lipid peroxidation, as affected by various concentrations of compounds 4 and 7, is shown in Figure 3.

In Vivo Anti-Inflammatory Activity
The effect of the synthesized compounds on acute inflammation, applying the carrageenan paw edema model, as well as the anti-inflammatory activity of ibuprofen and ketoprofen, used as reference compounds, is shown in Table 3.

Lipoxygenase Inhibitory Activity
The ability of compounds 1-12 to inhibit lipoxygenase, presented as IC 50 values or percent inhibition at 100 µM, towards soybean lipoxygenase 1-B, using linoleic acid as a substrate, after 7 min of incubation, is demonstrated in Table 4. The IC 50 of nordihydroguaiaretic acid (NDGA), an antioxidant compound acting as a nonspecific inhibitor of lipoxygenase, together with ibuprofen, ketoprofen and BHT, is also included as a reference.

Hypolipidemic Effect
The anti-dyslipidemic activity of compound 4, the most active non-NSAID containing antioxidant derivative, and compounds 5, 9, 11 and 12, the most active anti-inflammatory compounds, was tested on Triton-induced hyperlipidemia in rats. Simvastatin was used as a reference compound. Results are shown in Table 5.

Antioxidant and Radical Scavenging Activity
Reactive nitrogen and oxygen species participate in the promotion of atherogenesis, mitochondrial dysfunction and protein aggregation, resulting in vascular and neural degeneration [27,28]. The most active antioxidants were the Trolox derivatives. Compound 10 is almost nine times more active than the parent antioxidant acid. The increased activity could be explained by the extended conjugation of the chroman ring, resulting in radical stabilization, after the phenolic hydrogen abstraction. This effect may be further enhanced by the high lipophilicity of compounds 4, 7 and 10, compared with Trolox, which permits an effective approach to the lipid phase. For the carboxylic acids 1, 2 and 3, logD (the octanol/water partition coefficient at pH 7.4) rather than logP is preferred for the correlation with their antioxidant activity. Thus, although Trolox derivative 4 is more active than 6, compound 1 is considerably less active than 3, probably due to the pronounced difference in their logD values. The low LogD value and the less extended conjugation may be the reason for the inactivity of compound 2.
Ibuprofen and ketoprofen derivatives seem to offer similar antioxidant activity depending on the relevant antioxidant moiety of the compound. The ethylated serine structures 5 and 6 had the highest activity in comparison to the other 3,5-di-tert-butyl-4-hydroxybenzoic acid and (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl) acrylic acid derivatives.
Compounds 8, 9 and 12 exhibited the same level of antioxidant activity with increasing concentrations. This may be due to their high lipophilicity leading to poor solubility in the experimental medium, and to difficulty in the IC 50 determination. However, both (E)-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylic derivatives 9 and 12 seem to be more potent than 8 or 11. Additionally, this is the case for derivatives 5 and 6. The antioxidant activity of 6 was almost seven times higher, compared with the 3,5-di-tert-butyl-4-hydroxybenzoic acid derivative 5. This difference in the activity is confirmed by our previous results of compounds bearing these acids, and the equal inhibition in various concentrations of the compounds seems to be related to their physicochemical properties and not only to their antioxidant capacity [26,29].
In the DPPH radical scavenging experiment, performed in a homogenous reaction mixture, the reducing activity of Trolox derivatives (1, 4, 7, 10) is about the same as with Trolox. Similarly, compounds 3, 6, 9 and 12 were potent reducing agents, whereas, in the lipid peroxidation assay, their high lipophilicity intervened with the assay performed in an aqueous medium. 3,5-Di-tert-butyl-4-hydroxybenzoic acid derivatives 2, 5, 8 and 11 were of low activity, partly in agreement (with the exception of compound 5) with their weak activity in the lipid peroxidation experiments.

In Vivo Anti-Inflammatory Activity
Carrageenan-induced paw edema is a well-known and widely used biphasic model of acute inflammation. In the delayed phase, more than one hour after administration, neutrophil infiltration, prostaglandin production and release of pro-inflammatory cytokines are involved [30].
All compounds could reduce paw edema from about 30 to 67%, at 150 µmol/kg, i.p., 3.5 h after carrageenan administration. Hydrolyzed and ethylated serine derivatives 1-6 were of similar activity according to their corresponding antioxidant moiety, with the exception of compound 6 that shows a remarkable and statistically significant improvement in inhibition compared with compound 3. Ibuprofen derivatives showed partly better (but not statistically significant) activity than the non-NSAID esterified compounds but were of lower potency than those of ketoprofen. However, with the exception of compound 10, all the NSAID derivatives were more active than the parent NSAID alone. This may indicate that the compounds act without prior hydrolysis to the parent NSAID. Apart from the antioxidant and reducing potential of the compounds, it seems that amidation with serine may add to the appearance of anti-inflammatory activity in the antioxidant acids. We have previously reported that esters or amides of ibuprofen and ketoprofen enhanced the anti-inflammatory activity of the parent molecules [31,32], and that antioxidant acids such as Trolox amidated with L-cysteine or cysteamine yielded potent anti-inflammatory agents [29]. Unlike butylated hydroxytoluene which is devoid of anti-inflammatory activity [23], 3,5-di-tert-butyl-4-hydroxybenzoic acid derivatives possess significant anti-inflammatory activity, not always related to the antioxidant activity of the compounds, a finding that may partly be related to the lipoxygenase inhibitory activity of the compounds [23,26,29,31,33]. Additionally, since oxidative stress plays a critical role in inflammatory processes, compounds bearing the 3,5-di-tert-butyl-4-hydroxybenzyl moiety may offer considerable anti-inflammatory activity [34,35]. Furthermore, this moiety has been shown to mitigate the production of various pro-inflammatory mediators such as IL-1b and TNF-a by decreasing the amount of various activating oxidant factors such as myeloperoxidase in neutrophils and various other immune cells [24]. Similarly, in this report, 3,5-di-tert-butyl-4-hydroxyphenyl derivatives, and especially compound 11, demonstrate an interestingly increased anti-inflammatory activity, especially if the low antioxidant capacity of these compounds is considered.

Lipoxygenase Inhibitory Activity
Lipoxygenases form the second major path of arachidonic acid metabolism, involved in the synthesis of leukotrienes that are implicated in various disorders, e.g., asthma, arthritis, atherosclerosis and neurodegenerative and autoimmune diseases. Furthermore, there is a lack of clinical LOX inhibitors, with the drawback of adverse effects restricting their approval [36]. Soybean lipoxygenase, having structural and functional similarities with mammalian lipoxygenases, is often used for the study of anti-inflammatory agents [37].
The majority of the compounds presented moderate activity in this experiment. The IC 50 values of some compounds could not be calculated, due to their low solubility in the reaction mixture. Trolox derivatives showed very low potency, as expected, considering that Trolox itself has an IC 50 higher than 300 µM, in combination with the increased rigidity and bulk of the corresponding compounds, especially that of 7 and 10. Di-tert-butylphenol derivatives 2, 3, 5 and 6 had similar activity to butylated hydroxytoluene. Ibuprofen and ketoprofen derivatives 8 and 11 were more active than the NSAID or BHT alone. It has been previously shown [26,38] that this structural 3,5-di-tert-butyl-4-hydroxyphenyl moiety may contribute to increased lipoxygenase inhibitory activity. Compound 8 demonstrated surprisingly high activity, several times higher than the parent compounds ibuprofen and compound 5, pointing out that they act as a whole molecular entity in LOX inhibition. Ibuprofen derivatives 8 and 9 were more active than ketoprofen derivatives 11 and 12. This may partly be related to the less rigid structure of the former NSAID, since we have shown that rigid molecules seem to offer diminished activity [39,40]. 4-Hydroxy cinnamic acid shows low potency in LOX inhibition; however, compounds bearing this structure or the (E)-phenyl-propene moiety show considerable activity on LOX inhibition [41,42].
When linoleic acid was used at 1 mM, a concentration higher than the saturating substrate concentration, no inhibition was observed, under the same experimental conditions, indicating competitive inhibition of lipoxygenase by the compounds.

Hypolipidemic Effect
Administration of Triton-WR1339 to rats leads to a sharp increase in plasma cholesterol and especially triglyceride levels, after 24 h, and a decrease in the above indices seems to partly interfere with cholesterol biosynthesis [43]. Simvastatin was used as a reference compound, since statins, apart from highly active hypolipidemic agents via 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibition, possess multiple actions such as antioxidant ability and eNOS activity [44].
Inflammation and oxidative stress are closely related to hyperlipidemia. Hypercholesterolemia and oxidized LDL cholesterol are the main contributors to the development of atherosclerosis and the progression of neurodegenerative disorders such as Alzheimer's disease [27,45]. Furthermore, we have shown that amide derivatives of NSAIDs and antioxidants possess hypolipidemic properties [23,26,29,32]. Compounds with dual antiinflammatory and antioxidant potential may improve the cardiovascular state of the patient via various mechanisms such as NF-κB reduction and Nrf2 amplification, together with the reduction in ox-LDL levels [46] All compounds reduced lipidemic indices considerably, although triglyceride reduction was less pronounced, whilst there were not significant differences between our tested compounds with the exception of the effect of compound 9 on LDL cholesterol levels. The relatively decreased activity on triglycerides may be related to the more profound increase in triglycerides by Triton administration. Compound 9 was the most active with similar results in total and LDL cholesterol with simvastatin. The tested compounds effectively reduced triglyceride levels, while simvastatin had no effect. These results may be related to the antioxidant and the NSAID moiety. Antioxidant and anti-inflammatory activity may contribute to this effect in parallel or separately. Thus, compound 4 had low activity in carrageenan edema inhibition, and compound 5 had a modest antioxidant capacity, but both produced a significant hypolipidemic effect. However, the dual antioxidant and anti-inflammatory activity may offer a more powerful combination as is the case with compound 9.

General
All commercially available chemicals were of the appropriate purity. IR spectra were recorded on a Perkin Elmer Spectrum BX FT-IR spectrometer. 1 H NMR and 13 C NMR spectra were recorded using a BRUKER Avance III-300 MHz or an AGILENT DD2-500 MHz spectrometer. All chemical shifts are reported in δ (ppm), and signals are given as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Melting points (mp) were determined with a MEL-TEMPII (Laboratory Devices, USA) instrument and are uncorrected. The microanalyses were performed on a Perkin-Elmer 2400 CHN elemental analyzer.
κ-Carrageenan and lipoxygenase type I-B from soybean were purchased from Sigma. For the in vivo experiments, 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). The statistical analysis of the results was performed using GraphPad Software (version 7).

General Methods for the Synthesis of Compounds 4-6
For compounds 5 and 6, in a suspension of serine ethyl ester hydrochloride (1.2 mmol) in tetrahydrofuran (10 mL), triethylamine (Et 3 N) (1.4 mmol) was added, and after 5 min, the corresponding acid (1 mmol) and N,N -dicyclohexylcarbodiimide (DCC, 1.3 mmol) were added. After stirring for 12 h at room temperature, the precipitated material was filtered off, and THF was evaporated. The resulting mixture was dissolved in CH 2 Cl 2 (10 mL), successively washed with water and 5% NaHCO 3 solution and dried (Na 2 SO 4 ), and the final compounds were isolated with flash chromatography using petroleum ether and ethyl acetate as eluents.
For compound 4, the acid (1 mmol) was dissolved in THF (10 mL), and a solution of carbonyldiimidazole (CDI, 1.05 mmol), in the same solvent, was added. After 45 min, the solution was poured into a suspension of the amine (1.2 mmol) in THF (5 mL), and the mixture was left for 12 h with stirring at room temperature. Then, THF was evaporated, the resulting mixture was dissolved in CH 2 Cl 2 (10 mL), successively washed with water and 5% NaHCO 3 solution and dried (Na 2 SO 4 ) and the final compounds were isolated with flash chromatography using petroleum ether and ethyl acetate as eluents.

General Method for the Synthesis of Compounds 1-3
The corresponding ethyl ester amides (3 mmol) were dissolved in CHCl 3 (10 mL), 5 mL of a 5% aqueous NaOH solution was added and the mixture was left for 1 h with stirring at room temperature. Then, CHCl 3 and the produced ethanol were evaporated, the resulting mixture was dissolved in CHCl 3 (10 mL) and the aqueous solution was acidified with aqueous solution of 5% HCl. The aqueous phase was extracted three times with 20mL CHCl 3 and dried (Na 2 SO 4 ), and the final compounds were purified with recrystallization from acetone and petroleum ether.

General Method for the Synthesis of Compounds 7-12
The acid (1 mmol) was dissolved in CH 2 Cl 2 (20 mL), the corresponding serine derivative (1.05 mmol), N,N -dicyclohexylcarbodiimide (DCC, 1.3 mmol) and 4-(dimethylamino)pyridine (DMAP, 0.1 mmol), in the same solvent, were added and the mixture was left for 4 h with stirring at room temperature. Then, the resulting mixture was filtered, and the final compounds were isolated with flash chromatography using petroleum ether and ethyl acetate as eluents.  at room temperature (22 ± 2 • C). Absorbance (517 nm) was recorded after 30 min [26]. For the calculation of percent interaction, the residual absorbance of DPPH Ai (at a fixed concentration) after its interaction with the examined compounds (at various concentrations) was compared with the control absorbance Ao, without the addition of compounds, following the equation:

Carrageenan-Induced Paw Edema
An amount of 0.1 mL of an aqueous carrageenan solution (1% w/v) was injected into the hind paw of adult Wistar rats (6 rats were used for each compound). The tested compounds (in water with a few drops of Tween 80) were administered i.p. (0.15 mmol/kg) 5 min before the carrageenan administration. The produced edema, after 3.5 h, was estimated as paw weight increase [32].

In Vitro Evaluation of Lipoxygenase Activity
The reaction mixture contained the test compounds, dissolved in absolute ethanol (0.1 mL) at various concentrations, or the solvent (control, 0.1 mL), soybean lipoxygenase dissolved in 0.9% NaCl solution (250 u/mL, 0.2 mL) and sodium linoleate (100 µM) in Tris-HCl buffer, pH 9.0 (2.7 mL). The reaction was followed for 7 min at 28 • C, recording the absorbance of a conjugated diene structure at 234 nm, due to the formation of 13-hydroperoxy-linoleic acid. The performance of the assay was checked using nordihydroguaiaretic acid as a reference. 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 [32].

Effect on Plasma Cholesterol and Triglyceride Levels
Hyperlipidemia was induced by the i.p. administration of Triton WR 1339 (200 mg/kg) to adult Wistar rats (6 rats were used for each compound). The examined compounds (0.15 mmol/kg) were administered i.p. one hour later. Blood was taken from the aorta after 24 h, for the determination of plasma total cholesterol, LDL cholesterol and triglyceride concentration, using commercial kits [48].

Conclusions
In the present study, the described derivatives of antioxidant acids with L-serine, with or without NSAID conjugation, were designed to attain a series of biological properties aiming at the prevention or restoration of various pathological implications of degenerative and inflammatory conditions. L-serine incorporation seems to offer at least equal or several times more improved antioxidant activity, especially in the case of NSAID incorporation on the parent antioxidant amino acid derivatives. However, the 3,5-di-tertbutyl-4-hydroxybenzoic acid moiety offers a less effective antioxidant capacity, potentially due to steric and physicochemical reasons. All the compounds may offer considerable anti-inflammatory activity that may, in part, especially in the case of compounds 8 and 11, be derived from the lipoxygenase inhibitory ability, since they bear a very weak antioxidant effect, whilst the effect of NSAID derivatives and especially of compound 9 may be related to both these actions and to other factors that remain to be clarified. Finally, as for the hypolipidemic activity, the tested compounds may offer an improvement in the lipidemic indices, in comparison to the parent acids, a fact that may also be related to the L-serine moiety incorporation. Compound 9 showed antioxidant and radical scavenging characteristics accompanied by anti-inflammatory, hypolipidemic and especially cholesterol-lowering abilities.
Keeping in mind the adverse effects that may derive from multiple-drug therapy for multicausal diseases, it could be concluded that multifunctional compounds such as compound 9 and some others described in this report may assist in achieving avoidance of drug interactions and better patient tolerance and compliance.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.