α-Ketoheterocycles Able to Inhibit the Generation of Prostaglandin E2 (PGE2) in Rat Mesangial Cells

Prostaglandin E2 (PGE2) is a key mediator of inflammation, and consequently huge efforts have been devoted to the development of novel agents able to regulate its formation. In this work, we present the synthesis of various α-ketoheterocycles and a study of their ability to inhibit the formation of PGE2 at a cellular level. A series of α-ketobenzothiazoles, α-ketobenzoxazoles, α-ketobenzimidazoles, and α-keto-1,2,4-oxadiazoles were synthesized and chemically characterized. Evaluation of their ability to suppress the generation of PGE2 in interleukin-1β plus forskolin-stimulated mesangial cells led to the identification of one α-ketobenzothiazole (GK181) and one α-ketobenzoxazole (GK491), which are able to suppress the PGE2 generation at a nanomolar level.


Introduction
Prostaglandins are a class of highly bioactive eicosanoids, which are generated from arachidonic acid by the subsequent action of various enzymes [1,2]. Among them, prostaglandin E 2 (PGE 2 ) is the most abundant in humans, playing physiological and pathological roles [3]. The biosynthesis of PGE 2 begins when phospholipase A 2 (PLA 2 ) hydrolyzes membrane glycerophospholipids to release free fatty acids, including arachidonic acid ( Figure 1) [4]. Then, arachidonic acid is converted to prostaglandin H 2 (PGH 2 ) by the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) [1]. Finally, prostaglandin synthases, such as microsomal prostaglandin E synthase-1 (mPGES-1), catalyze the generation of PGE 2 [5,6], which exerts its actions interacting with PGE 2 receptors ( Figure 1). PGE 2 is a key mediator of inflammation [7,8], and consequently huge efforts have been devoted to the discovery of agents able to inhibit its production [9]. The involvement of PGE 2 in tumorigenesis and cancer is well described in recent review articles [10][11][12]. A wide variety of inhibitors targeting the various enzymes involved in the PGE 2 biosynthesis have been developed both in pharmaceutical industry and in academia. Inhibitors of PLA 2 targeting the release of arachidonic acid have been described, but none of them reached the market [13]. Numerous clinically validated COX-1 and COX-2 inhibitors are known. Non-steroidal anti-inflammatory drugs (NSAIDs) are non-selective COX inhibitors, while selective COX-2 inhibitors, such as celecoxib, overcome the gastrointestinal side effects of COX-1 inhibitors, however exhibiting potential cardiovascular toxicity [14]. Although mPGES-1 inhibitors have been proposed as safer alternatives to COX-2 inhibitors, lackingcardiovascular toxicity, further research is needed so that such inhibitors enter clinical practice [5,6]. Sometimes, although an inhibitor for one particular enzyme invo ation presents high potency in vitro, tremendous discrepancies can b is studied in cells. Thus, we have focused our attention on evaluatin flammatory compounds in a cellular system consisting of renal mesa previously shown that inhibitors of secreted PLA2 exhibit interesting production of PGE2 in mesangial cells [15], while small peptides were the generation of PGE2 [16]. Inspired by the anti-inflammatory prope thiazoles 1 and 2 ( Figure 2) and related compounds exhibit [17][18][19][20], w ous α-ketoheterocycles and studied their ability to inhibit the generat lular level. We present herein the synthesis of a number of α-ketoben lated heterocycles, and we demonstrate that two of them exhibit poten generation of PGE2 in rat mesangial cells. Sometimes, although an inhibitor for one particular enzyme involved in PGE 2 generation presents high potency in vitro, tremendous discrepancies can be observed when it is studied in cells. Thus, we have focused our attention on evaluating potential antiinflammatory compounds in a cellular system consisting of renal mesangial cells. We have previously shown that inhibitors of secreted PLA 2 exhibit interesting suppression of the production of PGE 2 in mesangial cells [15], while small peptides were also found to inhibit the generation of PGE 2 [16]. Inspired by the anti-inflammatory properties which α-keto-thiazoles 1 and 2 ( Figure 2) and related compounds exhibit [17][18][19][20], we synthesized various α-ketoheterocycles and studied their ability to inhibit the generation of PGE 2 at a cellular level. We present herein the synthesis of a number of α-ketobenzothiazoles and related heterocycles, and we demonstrate that two of them exhibit potent suppression of the generation of PGE 2 in rat mesangial cells.
Biomolecules 2020, 10, x 2 of 18 inhibitors, lackingcardiovascular toxicity, further research is needed so that such inhibitors enter clinical practice [5,6]. Sometimes, although an inhibitor for one particular enzyme involved in PGE2 generation presents high potency in vitro, tremendous discrepancies can be observed when it is studied in cells. Thus, we have focused our attention on evaluating potential anti-inflammatory compounds in a cellular system consisting of renal mesangial cells. We have previously shown that inhibitors of secreted PLA2 exhibit interesting suppression of the production of PGE2 in mesangial cells [15], while small peptides were also found to inhibit the generation of PGE2 [16]. Inspired by the anti-inflammatory properties which α-ketothiazoles 1 and 2 ( Figure 2) and related compounds exhibit [17][18][19][20], we synthesized various α-ketoheterocycles and studied their ability to inhibit the generation of PGE2 at a cellular level. We present herein the synthesis of a number of α-ketobenzothiazoles and related heterocycles, and we demonstrate that two of them exhibit potent suppression of the generation of PGE2 in rat mesangial cells.

General Chemistry Methods
Chromatographic purification of products was accomplished using forced-flow chromatography on Merck ® (Merck, Darmstadt, Germany) Kieselgel 60 F 254 230-400 mesh. Thin-layer chromatography (TLC) was performed on aluminum-backed silica plates (0.2 mm, 60 F 254 ). Visualization of the developed chromatogram was performed by fluorescence quenching using phosphomolybdic acid, ninhydrin, or potassium permagnate stains. Melting points were determined on a Buchi ® 530 (Buchi, Flawil, Switzerland) spectrometer and were uncorrected. 1 H and 13 C NMR spectra were recorded on a Varian ® Mercury (Varian, Palo Alto, CA, USA) (200 MHz and 50 MHz, respectively), a Bruker Avance Neo (400 MHz and 100 MHz, respectively) (Bruker, Faellanden, Switzerland), or a Bruker Avance (500 MHz and 125 MHz, respectively) (Bruker, Santa Barbara, CA, USA), and are internally referenced to residual solvent signals. Data for 1 H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br s = broad signal), coupling constant, integration, and peak assignment. Data for 13 C NMR are reported in terms of chemical shift (δ ppm). IR spectra were recorded with an OSTEC, IROS-05, FTIR spectrophotometer equipped with ATR diamond crystal (Simex Co., Ltd., Nizhny Novgorod, Russia). Mass spectra (ESI) were recorded on a Finnigan ® Surveyor MSQ LC-MS spectrometer (Thermo, Darmstadt, Germany). Highresolution mass spectrometry (HRMS) spectra were recorded on a Bruker ® Maxis Impact QTOF (Bruker Daltonics, Bremen, Germany) spectrometer. A microwave synthesizer, Discover (CEM, Charlotte, NC, USA), was used for the microwave synthesis. 1 H NMR and 13 C NMR spectra of the final products are shown in the Supplementary Materials.
Compounds 18, 19, 20a, 21a, and 22a were synthesized as previously described [21], and their analytical data were in accordance with literature.
General procedure for the synthesis of Weinreb amides 4a-f from carboxylic acids.
To a stirred solution of ester 5 or 6 (1 mmol) in dry tetrahydrofuran (2 mL) at −20 • C, N,O-dimethyl hydroxylamine hydrochloride (1.5 mmol) was added. Isopropyl magnesium chloride was then added dropwise over 15 min and the reaction mixture was left stirring for 35 min at −20 • C. The reaction mixture was quenched with a saturated solution of NH 4 Cl (5 mL) and the reaction mixture was extracted with diethyl ether (2 × 10 mL). The combined extracts were dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by flash chromatography eluting with the appropriate mixture of EtOAc:petroleum ether (40-60 • C) afforded the desired product.
N-Methoxy-N-methyl-2-(naphthalen-2-yloxy)acetamide (4g General procedure for the synthesis of α-ketobenzothiazoles 8a-h. To a stirred solution of benzothiazole (3 mmol) in dry Et 2 O (20 mL) at −78 • C, under a dry argon atmosphere, a solution of n-BuLi (1.6 M in hexane, 3 mmol) was added dropwise over a period of 10 min. The resulting orange solution was stirred for 45 min. Then, a solution of the Weinreb amide (1 mmol) in dry Et 2 O (2 mL) was slowly added giving the mixture a dark brown color. After stirring for 30 min at −78 • C, the mixture was allowed to warm up to room temperature over a period of 2 h. Then, saturated aqueous ammonium chloride solution was added, and the reaction mixture was extracted with diethyl ether (2 × 10 mL). The combined extracts were washed with brine (10 mL) and then dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by flash chromatography eluting with the appropriate mixture of EtOAc:petroleum ether (40-60 • C) afforded the desired product.
To a stirred solution of aldehydes 13a,b, 11 (1 mmol) in CH 2 Cl 2 (1.3 mL), a solution of NaHSO 3 (1.5 mmol, 156 mg) in water (0.3 mL) was added at room temperature. After stirring for 30 min, the organic solvent was concentrated under reduced pressure, water (0.3 mL) was added, and the reaction mixture was cooled to 0 • C. Then, a solution of KCN (1.5 mmol, 98 mg) in water (0.3 mL) was added dropwise over 1 h, and the reaction mixture was left stirring for 16 h. After the completion of the reaction, CH 2 Cl 2 (5 mL) was added to extract the product and the organic layer was washed with brine (10 mL), dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by flash chromatography eluting with the appropriate mixture of EtOAc:petroleum ether (40-60 • C) afforded the desired product.
To  General procedure for the synthesis of O-acyl-amidoximes (20b,c).
To a stirred solution of α-hydroxy-heterocyclic compounds 16a-e and 21b,c (1 mmol) in dry CH 2 Cl 2 (0.2 M), under an inert argon atmosphere, Dess-Martin periodinane was added (1.3 mmol, 551 mg). The reaction mixture was stirred for 1 h and after completion of the reaction the solvent was evaporated under reduced pressure and Et 2 O (30 mL) was added. The organic phase was washed with saturated aqueous NaHCO 3 (20 mL) containing Na 2 S 2 O 3 (1.  Rat renal mesangial cells (clone MZ B1) were isolated and characterized as previously described [22] and cultivated in medium consisting of RPMI 1640 supplemented with 10% fetal bovine serum, 10 mM Hepes, pH 7.4, 6 µg/mL bovine insulin, 5 mg/mL transferrin, 5 nM sodium selenite, 100 units/mL penicillin, and 100 µg/mL streptomycin. Prior to stimulation, cells were incubated for 4 h in DMEM containing 10 mM Hepes, pH 7.4, and 0.1 mg/mL fatty acid-free bovine serum albumin (BSA).

Quantification of Prostagladin E 2
Confluent mesangial cells in 24-well plates were stimulated for 24 h in a total volume of 400 µL DMEM containing 0.1 mg/mL BSA with the stimuli and inhibitors as indicated in the figure legends. Thereafter, supernatants were removed and centrifuged for 5 min at 1000 x g. The supernatant was taken for PGE 2 quantification using an enzyme-linked immunoassay (Enzo Life Sciences, Lörrach, Germany) following exactly the manufacturer's recommendations.

Statistical Analysis
Statistical analysis of data was performed using one-way analysis of variance (ANOVA) followed by a Bonferroni's post hoc test for multiple comparisons (GraphPad Prism version 5.00, San Diego, CA, USA). Half-maximal effective concentrations (EC 50 ) of the compounds were calculated using the same software.
Biomolecules 2020, 10, x 11 of 18 Carboxylic acid 3d, required for the synthesis of 8d, was prepared in two steps via etherification of alcohol 9 with tert-butyl 2-bromoacetate, followed by deprotection using trifluoroacetic acid (TFA) (Figure 4). α-Hydroxy-derivative 12 was synthesized through a reaction between aldehyde 11 and benzothiazolyl lithium ( Figure 5) [25]. For the benzoxazoles and benzimidazoles 17a-e, the synthesis started from the corresponding aldehydes 13a,b and 11, which were converted to the corresponding cyanohydrins 14a,b and 15. The formation of the heterocyclic rings was accomplished by the treatment of these compounds with 2-aminophenol (for the benzoxazole derivatives 16a,c,d) or 2-phenylenediamine (for the benzimidazole derivatives 16b,e) in the presence of acetyl chloride [26]. Hydroxy compounds 16a-e were finally oxidized to the α-ketoheterocycles 17a-e using Dess-Martin periodinane ( Figure 6). For the benzoxazoles and benzimidazoles 17a-e, the synthesis started from the corresponding aldehydes 13a,b and 11, which were converted to the corresponding cyanohydrins 14a,b and 15. The formation of the heterocyclic rings was accomplished by the treatment of these compounds with 2-aminophenol (for the benzoxazole derivatives 16a,c,d) or 2-phenylenediamine (for the benzimidazole derivatives 16b,e) in the presence of acetyl chloride [26]. Hydroxy compounds 16a-e were finally oxidized to the α-ketoheterocycles 17a-e using Dess-Martin periodinane ( Figure 6). hydrins 14a,b and 15. The formation of the heterocyclic rings was acc treatment of these compounds with 2-aminophenol (for the benzox 16a,c,d) or 2-phenylenediamine (for the benzimidazole derivatives 16b, of acetyl chloride [26]. Hydroxy compounds 16a-e were finally oxidized erocycles 17a-e using Dess-Martin periodinane ( Figure 6). The synthesis of keto-1,2,4-oxadiazoles was accomplished following a previously published procedure, as depicted in Figure 7. Amidoxime 19 was synthesized from aldehyde 13a through the corresponding O-tert-bytyldimethylsilyl cyanide 18 [21]. It was then coupled with either pivalic acid [21], benzoic acid, or isobutyric anhydride, using N,N -dicyclohexylcarbodiimide (DCC) as the coupling reagent to afford compounds 20a-c. The cyclization of these O-acyl-amidoximes took place in the presence of tetrabutylammonium fluoride (TBAF) under microwave irradiation, giving the desired hydroxy-oxadiazole derivatives 21a-c, which were then subjected to oxidation with Dess-Martin periodinane, providing the final α-keto-oxadiazoles 22a-c (Figure 7). coupled with either pivalic acid [21], benzoic acid, or isobutyric anhydride, using N,N′dicyclohexylcarbodiimide (DCC) as the coupling reagent to afford compounds 20a-c. The cyclization of these O-acyl-amidoximes took place in the presence of tetrabutylammonium fluoride (TBAF) under microwave irradiation, giving the desired hydroxy-oxadiazole derivatives 21a-c, which were then subjected to oxidation with Dess-Martin periodinane, providing the final α-keto-oxadiazoles 22a-c (Figure 7). In the 1 H-NMR spectra of the final heterocyclic compounds, the most characteristic peaks are those corresponding to the protons of the aromatic fused ring, which are located closest to the heteroatoms. These protons are the most downfield shifted aromatic ones, appearing at 8.24−7.92 ppm in the case of α-ketobenzothiazoles 8a-h, and at 7.90−7.31 in the case of α-ketobenzoxazoles 17a,c,d and α-ketobenzimidazoles 17b,e. In addition, the 1 H-NMR spectra of α-ketobenzimidazoles 17b,e show a characteristic chemical shift of N-H above 10 ppm. In the 13 C-NMR spectra of α-ketobenzothiazoles 8a,b,e,f, the carbon atom of the carbonyl group resonates at 195. 5-194.4 ppm, while the presence of an oxygen or a sulfur atom at the β-position of the alkyl chain (compounds 8c,d,g,h) causes an upfield shift to 191.2−188.7 ppm. A characteristic chemical shift for the carbonyl carbon atom at 190. 2−186.4 ppm, 194.7−194.4 ppm, and 191.9-191.8 ppm, is observed in the 13 C-NMR spectra of α-ketobenzoxazoles 17a,c,d, α-ketobenzimidazoles 17b,e, and 22b,c, α-keto-1,2,4-oxadiazoles, respectively.

Study of the Suppression of PGE2 Generation in Mesangial Cells
Renal mesangial cells were chosen as a model to evaluate the ability of our synthetic compounds to suppress the production of PGE2, based on our previous studies on PLA2 inhibitors [15,16]. Mesangial cells located in the renal glomerulus are involved in various pathological processes, including inflammation, of the renal glomerulus. As shown by Huwiler et al. [27,28], different PLA2s operate in mesangial cells to initiate the generation of PGE2. Stimulation of rat renal mesangial cells by interleukin-1β (IL-1β) plus forskolin (Fκ) results in huge increase of PGE2 synthesis, as previously described [15,16,29]. All the synthetic compounds were tested at a concentration of 3 µM and the results are summarized in Table 1.
The α-ketobenzothiazolyl derivative GK181, where the naphthalene group was placed at a distance of four carbon atoms from the carbonyl group, exhibited 85% inhibition of PGE2 release at a concentration of 3 µM (entry 1). When the distance between the In the 1 H-NMR spectra of the final heterocyclic compounds, the most characteristic peaks are those corresponding to the protons of the aromatic fused ring, which are located closest to the heteroatoms. These protons are the most downfield shifted aromatic ones, appearing at 8.24−7.92 ppm in the case of α-ketobenzothiazoles 8a-h, and at 7.90−7.31 in the case of α-ketobenzoxazoles 17a,c,d and α-ketobenzimidazoles 17b,e. In addition, the 1 H-NMR spectra of α-ketobenzimidazoles 17b,e show a characteristic chemical shift of N-H above 10 ppm. In the 13 C-NMR spectra of α-ketobenzothiazoles 8a,b,e,f, the carbon atom of the carbonyl group resonates at 195. 5-194.4 ppm, while the presence of an oxygen or a sulfur atom at the β-position of the alkyl chain (compounds 8c,d,g,h) causes an upfield shift to 191.2−188.7 ppm. A characteristic chemical shift for the carbonyl carbon atom at 190.2−186. 4 ppm, 194.7−194.4 ppm, and 191.9-191.8 ppm, is observed in the 13 C-NMR spectra of α-ketobenzoxazoles 17a,c,d, α-ketobenzimidazoles 17b,e, and 22b,c, α-keto-1,2,4-oxadiazoles, respectively.

Study of the Suppression of PGE 2 Generation in Mesangial Cells
Renal mesangial cells were chosen as a model to evaluate the ability of our synthetic compounds to suppress the production of PGE 2 , based on our previous studies on PLA 2 inhibitors [15,16]. Mesangial cells located in the renal glomerulus are involved in various pathological processes, including inflammation, of the renal glomerulus. As shown by Huwiler et al. [27,28], different PLA 2 s operate in mesangial cells to initiate the generation of PGE 2 . Stimulation of rat renal mesangial cells by interleukin-1β (IL-1β) plus forskolin (Fκ) results in huge increase of PGE 2 synthesis, as previously described [15,16,29]. All the synthetic compounds were tested at a concentration of 3 µM and the results are summarized in Table 1. as the benzothiazole derivatives GK455 (entry 14), GK516 (entry 15), GK518 (entry 1 and GK519 (entry 17) were proven unable to cause any inhibition. as the benzothiazole derivatives GK455 (entry 14), GK516 (entry 15), GK518 (entry 16) and GK519 (entry 17) were proven unable to cause any inhibition. and GK519 (entry 17) were proven unable to cause any inhibition. and GK519 (entry 17) were proven unable to cause any inhibition.   The activity of compounds GK181, GK299, and GK491, which exhibited the highes potency at 3 µM, was further explored at various concentrations and the results are shown in Figure 8. GK181, GK299, and GK491 compounds presented potent inhibition of PGE generation with EC50 values of 0.71 µM, 1.42 µM, and 0.79 µM, respectively. In conclusion we have identified one α-ketobenzothiazolyl derivative (GK181) and one α-ketobenzoxa zolyl derivative (GK491), being able to inhibit the generation of PGE2 in renal mesangia cells at a nanomolar level. The activity of compounds GK181, GK299, and GK491, which exhibited the highes potency at 3 µM, was further explored at various concentrations and the results are shown in Figure 8. GK181, GK299, and GK491 compounds presented potent inhibition of PGE generation with EC50 values of 0.71 µM, 1.42 µM, and 0.79 µM, respectively. In conclusion we have identified one α-ketobenzothiazolyl derivative (GK181) and one α-ketobenzoxa zolyl derivative (GK491), being able to inhibit the generation of PGE2 in renal mesangia cells at a nanomolar level.

Docking Studies
We have previously shown that inhibition of secreted PLA2 is a possible path via no inhibition The α-ketobenzothiazolyl derivative GK181, where the naphthalene group was placed at a distance of four carbon atoms from the carbonyl group, exhibited 85% inhibition of PGE 2 release at a concentration of 3 µM (entry 1). When the distance between the naphthalene and the heterocyclic group was reduced to two carbon atoms (GK517, entry 2), the activity was abolished. Similarly, GK489 (entry 3), where an oxygen atom was introduced at the β-carbon atom to the carbonyl did not present any inhibitory activity. The reduction of the carbonyl group of GK181 to the corresponding alcohol (GK490, entry 4) also destroyed the inhibitory activity, highlighting the importance of the carbonyl group. Replacement of the benzothiazolyl group of GK181 by a benzoxazolyl one (GK491, entry 5) led led to a slight decrease of the activity (77%) in comparison to GK181, while the corresponding benzimidazolyl derivative (GK492, entry 6) exhibited even lower activity (57%).
Then, the naphthalene group was replaced by a p-methoxy-phenyl group. Compound GK299 (entry 7) exhibited a slightly decreased inhibitory activity (79%) in comparison to GK181 (entry 1). Keeping constant the p-methoxy-phenyl group and its distance from the carbonyl (four carbon atoms), the effect of various heterocyclic rings was examined. In all cases (GK355, GK358, GK367 GK368, and GK369, entries 8-12, respectively), the inhibitory potency was either reduced or diminished. Only compounds GK355 and GK368 (entries 8 and 11, respectively) containing a benzoxazolyl or a phenyl substituted oxadiazolyl ring, respectively, presented inhibitory activity (25% and 68%, respectively). Compound GK358 (entry 9), containing a benzimidazolyl group; GK367 (entry 10); and GK369 (entry 12), containing either a tert-butyl or an isopropyl substituted oxadiazolyl ring, did not present any inhibitory activity. Taking into account the results obtained for the various heterocyclic systems, either for the naphthalene-containing compounds or for the p-methoxy-phenylcontaining compounds, it seems that the benzothiazolyl group is the optimum heterocyclic system. The benzoxazole derivative GK453 (entry 13), as well as the benzothiazole derivatives GK455 (entry 14), GK516 (entry 15), GK518 (entry 16), and GK519 (entry 17) were proven unable to cause any inhibition.
The activity of compounds GK181, GK299, and GK491, which exhibited the highest potency at 3 µM, was further explored at various concentrations and the results are shown in Figure 8. GK181, GK299, and GK491 compounds presented potent inhibition of PGE 2 generation with EC 50 values of 0.71 µM, 1.42 µM, and 0.79 µM, respectively. In conclusion, we have identified one α-ketobenzothiazolyl derivative (GK181) and one αketobenzoxazolyl derivative (GK491), being able to inhibit the generation of PGE 2 in renal mesangial cells at a nanomolar level. The activity of compounds GK181, GK299, and GK491, which exhibited the highest potency at 3 µM, was further explored at various concentrations and the results are shown in Figure 8. GK181, GK299, and GK491 compounds presented potent inhibition of PGE2 generation with EC50 values of 0.71 µM, 1.42 µM, and 0.79 µM, respectively. In conclusion, we have identified one α-ketobenzothiazolyl derivative (GK181) and one α-ketobenzoxazolyl derivative (GK491), being able to inhibit the generation of PGE2 in renal mesangial cells at a nanomolar level.

Docking Studies
We have previously shown that inhibition of secreted PLA2 is a possible path via which synthetic compounds may evoke the suppression of PGE2 release [15,16]. Thus, we performed docking calculations to understand how these compounds may interact with the active site of secreted GIIA sPLA2. AutoDock Vina [30] was used for docking the most potent compounds GK181 and GK491 in GIIA sPLA2. The crystal structure of the enzyme was retrieved from the Brookhaven Protein Databank (PDB: 1KQU).
As shown in Figure 9 for both GK181 and GK491, the naphthyl group is accommodated at the lipophilic pocket of the site and is involved in a T-shape interaction with His6. The carbonyl group points towards Ca 2+ ion and is close enough to Gly29 to form a hydrogen bond. The extended heterocyclic aromatic system is involved in π-π interactions with the catalytic His47. These two models reproduce the key interactions, as known by co-crystallized ligands [31], and therefore may suggest the mode of interactions between either GK181 or GK491 and secreted sPLA2. . Supernatants were taken for PGE 2 quantification using an enzyme-linked immunoassay as described in the Methods section. Data are presented as % of maximal IL-1/Fk stimulation and are means S.D. (n = 3). *** p < 0.001 considered statistically significant when compared to the unstimulated samples; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the IL-1/Fk-stimulated samples.

Docking Studies
We have previously shown that inhibition of secreted PLA 2 is a possible path via which synthetic compounds may evoke the suppression of PGE 2 release [15,16]. Thus, we performed docking calculations to understand how these compounds may interact with the active site of secreted GIIA sPLA 2 . AutoDock Vina [30] was used for docking the most potent compounds GK181 and GK491 in GIIA sPLA 2 . The crystal structure of the enzyme was retrieved from the Brookhaven Protein Databank (PDB: 1KQU).
As shown in Figure 9 for both GK181 and GK491, the naphthyl group is accommodated at the lipophilic pocket of the site and is involved in a T-shape interaction with His6. The carbonyl group points towards Ca 2+ ion and is close enough to Gly29 to form a hydrogen bond. The extended heterocyclic aromatic system is involved in π-π interactions with the catalytic His47. These two models reproduce the key interactions, as known by cocrystallized ligands [31], and therefore may suggest the mode of interactions between either GK181 or GK491 and secreted sPLA 2 .

Conclusions
We present herein the synthesis of eight α-ketobenzothiazoles, using the reaction between benzothiazolyl lithium and the appropriate Weinreb amide as the key step. A series of α-ketobenzoxazoles, α-ketobenzimidazoles, and α-keto-1,2,4-oxadiazoles were also synthesized. All the synthetic heterocycles were evaluated for their ability to suppress the generation of PGE2 in renal mesangial cells after stimulation with IL-1 plus forskolin. Interestingly, two heterocycles were identified, which were found able to inhibit PGE2 formation at a nanomolar level. These structures may serve as leads for the development of novel potent inhibitors of PGE2 formation with potential anti-inflammatory and/or anticancer properties.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Copies of 1 H NMR and 13 C NMR spectra of the final products.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Conclusions
We present herein the synthesis of eight α-ketobenzothiazoles, using the reaction between benzothiazolyl lithium and the appropriate Weinreb amide as the key step. A series of α-ketobenzoxazoles, α-ketobenzimidazoles, and α-keto-1,2,4-oxadiazoles were also synthesized. All the synthetic heterocycles were evaluated for their ability to suppress the generation of PGE 2 in renal mesangial cells after stimulation with IL-1 plus forskolin. Interestingly, two heterocycles were identified, which were found able to inhibit PGE 2 formation at a nanomolar level. These structures may serve as leads for the development of novel potent inhibitors of PGE 2 formation with potential anti-inflammatory and/or anticancer properties.