New Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Inhibitors, Nalidixic Acid Linked to Isatin Schiff Bases via Certain l-Amino Acid Bridges

A series of new Schiff bases were synthesized by condensation of isatins with the nalidixic acid-l-amino acid hydrazides. Prior to hydrazide formation, a peptide linkage has been prepared via coupling of nalidixic acid with appropriate l-amino acid methyl esters to yield 3a–c. The chemical structures of the new Schiff bases (5b and 5d–h) were confirmed by means of IR, NMR, mass spectroscopic, and elemental analyses. The anti-inflammatory activity of these Schiff bases was evaluated via measurement of the expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells model. The Schiff bases exhibited significant dual inhibitory effect against the induction of the pro-inflammatory iNOS and COX-2 proteins with variable potencies. However, they strongly down-regulated the iNOS expression to the level of 16.5% ± 7.4%–42.2% ± 19.6% compared to the effect on COX-2 expression (<56.4% ± 3.1% inhibition) at the same concentration (10 μM). The higher iNOS inhibition activity of the tested Schiff bases, relative to that of COX-2, seems to be a reflection of the combined suppressive effects exerted by their nalidixic acid, isatins (4a–c), and l-amino acid moieties against iNOS expression. These synthesized nalidixic acid-l-amino acid-isatin conjugates can be regarded as a novel class of anti-inflammatory antibacterial agents.


Introduction
Since the introduction of the first quinolone, nalidixic acid (1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid) for clinical use in 1967, structural modifications have continued and resulted in five generations of quinolones with an improved spectrum of antimicrobial coverage and pharmacokinetics [1,2]. Moreover, chemical modifications of nalidixic acid are still working to develop new macromolecules with enhanced therapeutic properties to combat the emergence of multidrug-resistant bacteria [3]. Microbial infections cause inflammation that attract and activate phagocytes (e.g., neutrophils) and induce immunity against the infectious agents [4]. However, the consequent release of inflammatory mediators together with the increased production of oxygen species contributes to the killing of bacteria but also damages the surrounding tissue. Therefore, resolution of the acute inflammatory response is crucial to avoid excessive damage to tissues [5]. Sulphasalazine represents the best example of an antibiotic that became an anti-inflammatory drug. Mounting evidence has shown inhibitory effects of antibiotics, belonging to macrolides, tetracyclines and quinolones, on the generation of inflammatory mediators and on immune responses [6,7]. The antibacterial 4-quinolone derivatives such as ciprofloxacin, tosufloxacin, levofloxacin and moxifloxacin, and other quinolone substituted thiazolidin-4-ones, were recently proposed as anti-inflammatory agents [8,9], and some could significantly protect mice injected with a lethal dose of LPS against death. It is also known that isatin has exhibited wide antimicrobial activity [10,11] and has been reported, along with its 5-, 6-, and 7-substituted analogs, to inhibit the production of inflammatory mediators [12]. Moreover, certain branched amino acids (BAAs) such as Isoleucine, Leucine, and Valine have been found to exhibit in vitro and in vivo anti-inflammatory activity [13,14]. An improvement for the anti-inflammatory effect of some compounds, including nonsteroidal antiinflammatory drugs (NSAIDs), was also manifested when N-linked with certain amino acids [15][16][17]. Besides that, a selective COX-2 inhibitory activity [18] was a beneficial effect.
In this work, a series of nalidixic acid-based conjugates were constructed from L-amino acid and an isatin analog through carboxamide, hydrazide, and Schiff base formation, successively, with the aim of modification and potentiation of the biological activity of naldixic acid nucleus. The carboxamide was done using one of three naturally occurring amino acids (L-Val, L-Leu, or L-Phe) through the amidation of the carboxylic group at the 3-position of the nalidixic acid molecule by mixed anhydride coupling methods [19]. Our preliminary antibacterial screening of these nalidixic acid-based conjugates has shown a wide spectrum antimicrobial activity relative to nalidixic acid itself [20]. However, on the basis of the previously reported anti-inflammatory activity of certain amino acids [13,21] and isatin analogs [12], the anti-inflammatory activity of the new nalidixic acid-based conjugates containing these moieties were thus evaluated in vitro. The anti-inflammatory activity of the conjugates was determined in comparison to their individual moieties via measurement of the expressed pro-inflammatory iNOS and COX-2 proteins, using the LPS-stimulated RAW264.7 macrophage cells model [22].

Chemistry
The strategy for this work is to construct a linear nalidixic acid-based conjugate that possesses a carboxamide with amino acid and a Schiff base of indoline-2,3-dione (isatin) moieties that may add new biological activity for nalidixic acid. The selection of isatin and appropriate L-amino acid moieties has been considered on the basis of their previously reported anti-inflammatory effects and the antimicrobial activity of istains. By the mixed anhydride method [23], the methyl esters of L-Valine (Val), L-Leucine (Leu) and L-Phenyl alanine (Phe) have been coupled smoothly and quickly, by mixed anhydride method, with nalidixic acid to yield the corresponding nalidixic acid amides (2a-c) in high yield. Hydrazinolysis of 2a-c with hydrazine hydrate afforded the corresponding hydrazides (3a-c), which were then condensed with isatin analogs (4a-c), in acidified ethanol under microwave irradiation (420 W/110˝C/5-10 min), to give the corresponding Schiff bases (5a-h) in a high yield and high purity (Scheme 1). Microwave-assisted Schiff bases synthesis has been accomplished in a small amount of solvent and very short reaction time (5-10 min) with a high yield of pure products. Microwave irradiation had been reported to trigger heating to carry out the chemical reaction by dipolar polarization and ionic conduction [24]. Finally, the chemical structures of the hydrazides (3a-c) and final conjugates (5a-h) were established on the basis of their mass spectroscopy (MS) and NMR spectral data.

The Anti-Inflammatory Activity
LPS is a major component of Gram-negative bacteria cell walls, which have been implicated in septic shock. This is accompanied by an overproduction of inflammatory mediators such as NO and prostaglandins [25,26], which resulted from the expression of iNOS and COX-2 proteins, respectively. Therefore, compounds capable of reducing/blocking LPS-induced expression of iNOS and/or COX-2 proteins might be beneficial in management of the inflammatory responses. In this study, an in vitro assay of LPS-treated RAW264.7 macrophage cells linked with immunoblot analysis was carried out to identify the iNOS and/or COX-2 inhibitory compound. Dexa-methasone (10 μM), a well-known anti-inflammatory drug and an inhibitor of iNOS and COX-2, [27][28][29] was used as a positive control. Prior to carrying out the anti-inflammatory assay, the cytotoxicity of isatins (4a-c), nalidixic acid (1) and conjugates (3b, 3c, 5b, 5d-h) was measured at the same concentration (10 μM) against RAW264.7 macrophage cells, via Alamar Blue assay. The compounds showed no cytotoxicity (cell viability >90%).

The Anti-Inflammatory Activity
LPS is a major component of Gram-negative bacteria cell walls, which have been implicated in septic shock. This is accompanied by an overproduction of inflammatory mediators such as NO and prostaglandins [25,26], which resulted from the expression of iNOS and COX-2 proteins, respectively. Therefore, compounds capable of reducing/blocking LPS-induced expression of iNOS and/or COX-2 proteins might be beneficial in management of the inflammatory responses. In this study, an in vitro assay of LPS-treated RAW264.7 macrophage cells linked with immunoblot analysis was carried out to identify the iNOS and/or COX-2 inhibitory compound. Dexa-methasone (10 µM), a well-known anti-inflammatory drug and an inhibitor of iNOS and COX-2, [27][28][29] was used as a positive control. Prior to carrying out the anti-inflammatory assay, the cytotoxicity of isatins (4a-c), nalidixic acid (1) and conjugates (3b, 3c, 5b, 5d-h) was measured at the same concentration (10 µM) against RAW264.7 macrophage cells, via Alamar Blue assay. The compounds showed no cytotoxicity (cell viability >90%).

The Effect of Nalidixic Acid and Its Conjugates on iNOS and COX-2 Expression
In another experiment, nalidixic acid (1) exerted a moderate but significant (p < 0.05) downregulation of both iNOS and COX-2 expression to 75.9%˘9.0% and 67.4%˘7.6%, respectively, at 10 µM relative to that of the control LPS-stimulated cells ( Figure 1). Although the Schiff bases synthesized from isatins with amino acid conjugates of nalidixic acid (5b and 5d-h) inhibited both iNOS and COX-2 expression, their anti-iNOS-based anti-inflammatory activity was found to be very pronounced (cf. the proximate equal potency of nalidixic acid alone). Therefore, 5b and 5d-h exhibited powerful reduction in iNOS expression to the level of 16.5%˘7.4%-42.2%˘19.6% compared to their inhibitory effect on COX-2 expression (<56.4%˘3.1% inhibition) at the same concentration (10 µM) ( Figure 2). Although L-Leu and L-Phe did strongly reduce the iNOS expression ( Figure 1), their conjugates with nalidixic acid (3b and 3c) either maintain or partially reduce the anti-iNOS expression activity of nalidixic acid, respectively ( Figure 2) compared to their Schiff bases with isatins 5d-h, which exhibited strong iNOS inhibitory activity. Although neither isatins nor amino acids exhibited significant anti-COX-2-based anti-inflammatory effect (Figure 1), it seems that they maintained the basic COX-2 expression inhibitory activity of nalidixic acid (1) in the molecules of 3b, 3c, and 5d-f (81.5%˘9.2%-69.1%˘4.5% inhibition) ( Figure 2). However, a probable synergism in the anti-COX-2-based anti-inflammatory effect may be found in the case of conjugates synthesized from halogenated isatins with L-Phe conjugates of nalidixic acid as observed in 5g and 5h (61.8%˘7.3% and 56.4%˘3.1% inhibition, respectively). It appears that 5g and 5h structures, containing halogenated isatin and L-Phe with nalidixic acid, represent the most active biphasic iNOS and COX-2-based anti-inflammatory agents that may be regarded as a new class of nalidixic acid-based antiinflammtory agents. The authors would like to apologize for any inconvenience caused to the readers by these changes.

Discussion
In this work, we have tried to analyze the anti-inflammatory activity of isatins and certain L-amino acids with the aim to add an anti-inflammatory potential to the known antibacterial nalidixic acid. This was achieved through the synthesis of new isatin Schiff bases linked to nalidixic acid through an amino acid bridge. The design of this study was based on the following facts.
It is known that isatin (1H-indole-2,3-dione) is a plant-derived natural compound, and, in humans, it is a metabolic derivative of adrenaline [30]. It has shown antibacterial, anti-tubercular, and antifungal activities [10,11]. Moreover, it has been reported to inhibit the production of nitric oxide in LPS-interferon-γ-stimulated RAW 264.7 macrophage cells and inhibit the production of prostaglandin E2 (PGE2) and tumor necrosis factor (TNF-α) through its effect on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively [12]. The same anti-inflammatory-related activities were also observed with isatin analogs e.g., 5-, 6-, or 7-chloroisatin and 5-methylisatin [12]. The synthetic versatility of isatin has also led to its extensive use as a precursor in organic synthesis of interesting molecules with anticancer [31], antiviral [32], anti-HIV [33], and anticonvulsant [34] activities. The incorporation of amino acids was also supposed to improve the possible anti-inflammatory activity of the whole molecule containing nalidixic acid and isatin moieties on the basis of the following facts. BAAs such as Isoleucine and Leucine have been reported to exhibit anti-inflammatory activity, which were thought to be related to the interference with the action and/or synthesis of prostaglandins [13]. A supplementation of BAA mixture (Leucine, Isoleucine, and Valine) effectively reduces the muscle soreness and fatigue sensation, which could be attributed to the attenuation of muscle damage and inflammation [14]. Other amino acids, e.g., Glycine, Histidine and Cysteine, exhibited anti-inflammatory effects in human coronary arterial endothelial cells though inhibition of NF-κB activation and IL-6 production [21]. Some amino acid conjugates with other compounds have been found to create, maintain, or improve the anti-inflammatory effect. Therefore, some N-linked amino acid conjugates with linoleic acid, 2,5-diaryl substituted furans, and substituted 1,3-dioxanes showed potential anti-inflammatory effect [15][16][17]. Moreover, amino acid conjugates (e.g., with the NSAID pirprofen) kept the anti-inflammatory activity with the advantage of a significant elimination of ulcerogenicity as compared to the parent drug [35]. A similar phenomenon was also observed for the NSAID celecoxib bound with amino acids, which showed selective COX-2 inhibitory activity [18].
On the basis of the above findings, the anti-inflammatory activity of the selected L-amino acids, isatin moieties, and nalidixic acid in this study was primarily evidenced. These starting materials exhibited in vitro high selective capacity to down-regulate iNOS protein expression in the LPS-stimulated RAW264.7 macrophage cells. Nalidixic acid alone also showed a moderate down-regulation effect but against both iNOS and COX-2 expressions with a balanced potency. As a result, the integrated anti-inflammatory activities of amino acid, isatin, and nalidixic acid moieties were thus disclosed in the newly synthesized Schiff bases (5b and 5d-h). The Schiff bases have been shown to inhibit both iNOS and COX-2 expression but with much more powerful anti-iNOS expression effect at the same concentration. The compounds also still retained the antibacterial properties of nalidixic acid [20]. However, it seems that amino acids might have no significant role or merely maintain the anti-iNOS and anti-COX expression activity of nalidixic acid as indicated by comparison of the effect of nalidixic acid alone with that of the tested nalidixic acid-amino acid hydrazide (3b and 3c). Nevertheless, in a molecular docking study, it was found that 5-chloro-and 5-bromoisatin-based hydrazide derivatives possessed good anti-inflammatory activity because of good binding scores into the active site of COX-2 enzyme through hydrogen bond and aromatic interaction with Gln-275 and His-193, respectively [36]. This fact may add another mechanism for the anti-COX-2 activity of the new 5-haloisatin Schiff bases with nalidixic acid-amino acid hydrazide (e.g., 5g and 5h). However, these kind of Schiff bases exhibited significant biphasic iNOS and COX-2-based anti-inflammatory activity. Thus, it was concluded that the new synthesized nalidixic acid-based conjugates reported herein could provide suitable templates in a drug discovery program of novel anti-inflammatory antibiotics for promoting the resolution of chronic microbial and/or non-microbial inflammation. However, the effect of these compounds on the cellular mechanisms of iNOS and COX-2 protein expression, e.g., by inhibiting the activation of the nuclear factor (NF)-κB, and on the activity of the two enzymes is worth being investigated in our future studies.

General
The organic solvents and chemicals, including L-configured amino acids, were obtained from Sigma-Aldrich (

Synthesis of Nalidixic Acid Carboxamides of Amino Acids Esters (2a-c)
To a cold and stirred dry dichloromethane solution (25 mL,´20˝C) of nalidixic acid (1 mmol), ethyl chloroformate (1 mmol) and triethylamine (1 mmol) were successively added. After 10 min, a cold methylene chloride solution (10 mL,´20˝C) of an amino acid methyl ester, namely L-Valine methyl ester, L-Leucine methyl ester, or L-Phenylalanine methyl ester (1 mmol) was added, with continuous stirring for 3 h and at RT overnight. The solution was then washed with water, 1 N HCl, 1 N NaHCO 3 , and finally with water (250 mL). The solution was dried on anhydrous CaCl 2 and evaporated. The oily residue was solidified by trituration with dry ether, filtered off, dried under vacuum, and recrystallized to afford the esters 2a-c, respectively, as previously described [37].

Cell Viability Assay
The RAW264.7 macrophage cells obtained from the American Type Culture Collection (ATCC, #TIB-71) were cultured on 96-well plates. The testing compound at 10 mM was incorporated into the cell culture in DMEM medium in humidified incubator within a 5% CO 2 atmosphere at 37˝C for 24 h. AlamarBlue ® (Invitrogen, Carlsbad, CA, USA) was added according to the manufacturer instruction, the absorbance (A) was measured at 570 nm, and the cell viability was then analyzed [38].

In Vitro Anti-inflammatory Assay
The method described by Ho et al. [39] was modified by our group [22] and employed to measure the in vitro anti-inflammatory activity of amino acids, isatins (4a-c ), and the synthesized compounds (3b, 3c, 5b and 5d-h) in comparison with dexamethasone, a positive control. Briefly, cultured RAW264.7 cells were incubated with LPS (0.01 mg/mL) in the presence (10 µM) or absence of the compounds. The intracellular protein containing-supernatant fraction was then used for measuring COX-2 and iNOS protein expression using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. β-actin was used as an internal loading control.

Conclusions
A series of nalidixic acid, amino acid, and isatin-based Schiff bases have been synthesized and evaluated for their anti-inflammatory activity. The compounds showed an anti-inflammatory effect via inhibition of the proinflammatory iNOS and COX-2 proteins expression with variable potencies. This is the first report to point out that the newly synthesized linear macromolecules of nalidixic acid linked to isatin via certain L-amino acid bridges can be regarded as a novel class of anti-inflammatory antibacterial agents.