N-Acylated and N-Alkylated 2-Aminobenzothiazoles Are Novel Agents That Suppress the Generation of Prostaglandin E2

The quest for novel agents to regulate the generation of prostaglandin E2 (PGE2) is of high importance because this eicosanoid is a key player in inflammatory diseases. We synthesized a series of N-acylated and N-alkylated 2-aminobenzothiazoles and related heterocycles (benzoxazoles and benzimidazoles) and evaluated their ability to suppress the cytokine-stimulated generation of PGE2 in rat mesangial cells. 2-Aminobenzothiazoles, either acylated by the 3-(naphthalen-2-yl)propanoyl moiety (GK510) or N-alkylated by a chain carrying a naphthalene (GK543) or a phenyl moiety (GK562) at a distance of three carbon atoms, stand out in inhibiting PGE2 generation, with EC50 values ranging from 118 nM to 177 nM. Both GK510 and GK543 exhibit in vivo anti-inflammatory activity greater than that of indomethacin. Thus, N-acylated or N-alkylated 2-aminobenzothiazoles are novel leads for the regulation of PGE2 formation.


Introduction
The release of arachidonic acid (AA) from membrane glycerophospholipids, catalyzed by the action of phospholipases A 2 (PLA 2 s), initiates the formation of a variety of bioactive lipids, which act as potent signaling mediators and are collectively referred to as eicosanoids [1,2]. Prostaglandins constitute a major and important class of such lipid signaling molecules [3]. In particular, prostaglandin E 2 (PGE 2 ) has attracted great interest because of its participation in both physiological and pathological processes. PGE 2 plays a key role in inflammatory diseases [4]; however, it is also involved in tumorigenesis and cancer [5][6][7] as well as in the pathogenesis of Alzheimer's disease (AD) [8]. As a consequence, the quest for agents able to inhibit and regulate the formation of PGE 2 has been a highly active field during recent decades [9].
In addition to PLA 2 , which is the rate-limiting enzyme for the release of free arachidonic acid [10], a number of enzymes are involved in the biosynthesis of PGE 2 , and all of these enzymes have been targeted in the effort to produce anti-inflammatory agents.
Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) catalyze the oxidation of AA to prostaglandin H 2 (PGH 2 ) [1]. Then, prostaglandin synthases, such as microsomal prostaglandin E synthase-1 (mPGES-1), catalyze the formation of PGE 2 [11]. Once this lipid is formed, it may interact with four distinct receptors (EP1 to EP4) to exert its action [12]. The widely used non-steroidal anti-inflammatory drugs (NSAIDs) are non-selective COX inhibitors, which usually exhibit gastrointestinal side effects, while selective COX-2 inhibitors, such as celecoxib, overcome the side effects of NSAIDs; however, they present potential cardiovascular toxicity [13]. PLA 2 inhibitors have been developed and studied for decades [10,14]; however, none of them have reached the market. Currently, there is a great interest in mPGES-1 inhibitors [11,15,16] because they are considered a safer alternative to COX-2 inhibitors, as they lack cardiovascular toxicity, although further research is required to prove their clinical efficiency and safety.
As part of our effort to develop PLA 2 inhibitors as novel anti-inflammatory agents [17][18][19], we have shown that inhibitors of secreted PLA 2 are able to suppress the production of PGE 2 in mesangial cells [20]. Inspired by thiazolyl ketones exhibiting an interesting ability to inhibit PGE 2 formation and in vivo anti-inflammatory properties [19], we have most recently presented a series of α-ketoheterocycles and we have demonstrated that the α-ketobenzothiazole derivative GK181 (1, Figure 1a) and α-ketobenzoxazole derivative GK491 (2, Figure 1a) inhibit PGE 2 formation in rat mesangial cells with half maximal effective concentrations (EC 50 ) values of 0.71 µM and 0.79 µM, respectively [21]. Benzothiazole is indeed an important heterocyclic skeleton that plays an important role in medicinal chemistry as a key template for the development of various therapeutic agents [22][23][24][25]. Among the various derivatives based on the privileged benzothiazole molecular scaffold, 2-aminobenzothiazoles exhibit diverse biological properties; for example, riluzole is a marketed drug (Figure 1b) for treating amyotrophic lateral sclerosis [26]. In 2012, a series of 2-aminothiazoles were reported and their evaluation concluded that disubstituted 2-N-arylaminothiazoles may inhibit PGE 2 production in cells [27]. Recently, Chini et al., employing a combinatorial virtual screening, have identified substituted 2benzoylaminobenzothiazoles able to suppress PGE 2 levels [28]. The promising properties of α-ketobenzothiazole 1 and α-ketobenzoxazole 2 in inhibiting PGE 2 generation at a cellular level, observed by our group [21], prompted us to further explore compounds based on the privileged benzothiazole scaffold. We present, herein, routes for the synthesis of a variety of N-acylated and N-alkylated 2-aminobenzothiazoles and the corresponding benzoxazoles and benzimidazoles. A structure-activity relationship study for the ability of such compounds to inhibit the cytokine-stimulated generation of PGE 2 in rat mesangial cells resulted in the discovery of novel naphthalene-containing N-acylated or N-alkylated 2-aminobenzothiazoles (Figure 1c), which inhibited PGE 2 generation at a nanomolar level and presented anti-inflammatory in vivo activity in a rat-paw carrageenan-edema assay.

General Chemistry Methods
The chromatographic purification of products was accomplished using forced-flow chromatography on a 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 ). The visualization of the developed chromatograms was performed by fluorescence quenching using phosphomolybdic acid, ninhydrin or potassium permanganate stains. The melting points were determined on a Buchi ® 530 apparatus (Buchi, Flawil, Switzerland) 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) or a Bruker Avance Neo (Bruker, Faellanden, Switzerland) (400 MHz and 100 MHz, respectively) and were internally referenced to residual solvent signals. The data for 1 H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, quint = quintet, m = multiplet, and br = broad signal), coupling constant, integration and peak assignment. The data for 13 C NMR are reported in terms of the chemical shift (δ ppm). High-resolution mass spectrometry (HRMS) spectra were recorded on a Bruker ® Maxis Impact QTOF (Bruker Daltonics, Bremen, Germany) spectrometer. The purity of all the compounds subjected to biological tests was determined using analytical HPLC and was found to be ≥95%. The HPLC analyses were carried out on a Shimadzu LC-2010AHT system and a Phenomenex, Luna C18(2) 100A (150 × 2 mm, 5 µm) analytical column, using H 2 O/acetonitrile 65/35 v/v, with a gradient to 40:60 v/v, at a flow rate of 1.0 mL/min.

General Procedure for the Synthesis of Carbamates 16a,b
To a stirred solution of alcohol 15a,b (1.7 mmol) in dry CH 2 Cl 2 (17 mL), cooled to 0 • C and under argon, triphosgene (297 mg, 1.0 mmol) and Et 3 N (0.25 mL, 1.7 mmol) were added and the mixture was stirred for 15 min at 0 • C and for 15 min at room temperature. Then, a cold saturated aqueous solution of NaHCO 3 (20 mL) was added dropwise, the mixture was extracted with CH 2 Cl 2 (20 mL), and the organic layer was washed with brine (15 mL). The organic layer was dried over Na 2 SO 4 , and the solvent was evaporated under reduced pressure. The residue was dissolved in dry tetrahydrofuran (THF, 2.8 mL) and Et 3 N (0.55 mL) and placed in a pressure vessel. 3a (255 mg, 1.7 mmol) and a catalytic amount (12 mg, 0.1 mmol) of 4-(dimethylamino)pyridine (4-DMAP) were added, and the reaction mixture was left stirring for 48 h. Then, the mixture was concentrated under reduced pressure, and purification by flash chromatography, eluting with the appropriate mixture of solvents, provided the desired product.

General Procedure for the Synthesis of Hemiaminal Ethers 18a-h
To a stirred solution of alcohol 17a-d (1.0 mmol) in dry CH 2 Cl 2 (2 mL), (2,2,6,6τetramethylpiperidin-1-yl)oxyl (TEMPO, 16 mg, 0.1 mmol) and iodobenzene diacetate (360 mg, 1.2 mmol) were added consecutively, and the reaction mixture was left stirring at room temperature for 1 h. The mixture was transferred to a separatory funnel and washed with an aqueous solution of 10% Na 2 S 2 O 3 (5 mL), an aqueous solution of 5% NaHCO 3 (5 mL), and brine (5 mL), consecutively. The organic layer was dried over Na 2 SO 4 , and the solvent was evaporated under reduced pressure. The crude aldehyde was used directly in the next step.

General Procedure for the Reduction of Hemiaminal Ethers to 2-Aminobenzothiazoles 19a-h
To a stirred solution of hemiaminal ethers 18a-h (1.0 mmol) in absolute MeOH (1 mL), placed in a pressure vessel, NaBH 4 (76 mg, 2.0 mmol) was added. The vessel was sealed, and the reaction mixture was left stirring at 80 • C for 1 h. The solvent was then evaporated under reduced pressure; the residue was dissolved in H 2 O (5 mL) and ethyl acetate (10 mL), transferred to a separating funnel and extracted with ethyl acetate (3 × 5 mL). Purification by flash chromatography, eluting with an appropriate mixture of EtOAc:petroleum ether (40-60 • C), afforded the desired product.

Quantification of PGE 2
Confluent mesangial cells in 24-well plates were pretreated for 20 min with varying concentrations of inhibitors and then stimulated for 24 h in a total volume of 400 µL of DMEM containing 0.1 mg/mL of BSA, in the absence or presence of 1 nM interleukin 1β (IL-1) plus 5 µM forskolin (Fk). Thereafter, the supernatants were collected and centrifuged for 5 min at 1000× g. The PGE 2 in the supernatant was quantified using an enzymelinked immunoassay (Enzo Life Sciences, Lörrach, Germany) following the manufacturer's recommendations.

Statistical Analysis
Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by a Bonferroni's post hoc test for multiple comparisons (GraphPad Prism 8.4.3., San Diego, CA, USA). The half maximal effective concentrations (EC 50 ) of the inhibitors were calculated using the same software.

Rat-Paw Carrageenan-Induced-Edema Assay
The anti-inflammatory activities of selected aminobenzothiazole derivatives were determined by employing the rat-paw carrageenan-induced-edema assay, as previously described [18]. Only male animals (180-220 g body weight) were used. Each group was composed of five animals. The animals were divided into three five-membered groups, and all the tested compounds were suspended in water (0.01 mmol/mL/kg body weight), with a few drops of Tween 80, and ground in a mortar before being administered intraperitoneally simultaneously. After treatment with the tested compounds in group 1, a 2nd group was used as a positive control (indomethacin at 0.01 mmol/mL/kg body weight, i.p.), and another group (3rd) served as the control in which water was administered (negative control). The compounds were injected intraperitoneally at the same time as carrageenan was given by intradermal injection. The rats were euthanized 3.5 h post-injection. The weight of the uninjected with carrageenan paw was subtracted from the weight of the injected paw for each animal. The change in paw weight for the treated animals was compared to that for the control animals and expressed as the percent inhibition of edema. The values of CPE % are the means from three different experiments with standard errors of the mean less than 10%. The statistical significance of the results was established with Student's t-test, for * p < 0.01 and ** p < 0.05. All the animal experiments performed in the manuscript were conducted in compliance with institutional guidelines. Our studies were in accordance with recognized guidelines on animal experimentation (guidelines for the care and use of laboratory animals published by the Greek Government 160/1991, based on EU regulations 86/609). The rats were kept in the Centre of the School of Veterinary Medicine (EL54 BIO42), Aristotle 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).
Carbamates 16a,b were prepared in a two-step reaction from benzylic alcohols 15a,b. The first step included conversion into chloroformates by treatment with triphosgene, which then reacted with 2-aminobenzothiazole (3a) in the presence of triethylamine and 4-DMAP (Scheme 3).  N-Alkylated 2-aminobenzoxazoles 23a-d were synthesized through a metal-free oxidative amination of benzoxazole with amines 22a-d, using TBAI, TBHP and acetic acid [34], as depicted in Scheme 5.

Study of the Suppression of PGE 2 Generation in Mesangial Cells
All the compounds synthesized were evaluated for their ability to suppress the production of PGE 2 , using renal mesangial cells as a model, as described in our previous studies [20,21]. As demonstrated in the past, mesangial cells are involved in various pathological processes, including inflammation of the renal glomerulus [35]. Upon the stimulation of rat renal mesangial cells with interleukin-1β (IL-1β) plus forskolin (Fk), a huge increase in PGE 2 formation is observed, permitting the evaluation of synthetic compounds as inhibitors of this generation [20,21,35,36]. The results obtained for the effect of the newly synthesized compounds at a concentration of 3 µM are summarized in Tables 1 and 2.  Initially, an analog of compound 1 (compound 14, entry 1, Table 1), where the carbonyl group of 1 was incorporated into an amide bond, and two N-acylated derivatives of 2aminobenzothiazole (5a and 5b (GK510), entries 2 and 3, Table 1), where the naphthalene ring is situated at a distance corresponding to two or three carbon atoms away from the carbonyl group, were evaluated. Compound 14 exhibited weaker inhibitory activity (64%) in comparison to 1 (85%) [21]. Both compounds 5a and 5b inhibited the generation of PGE 2 (50% and 96%, respectively); however, 5b exhibited potent inhibition, indicating that the optimum distance between the naphthalene ring and the amide bond corresponds to two carbon atoms. The insertion of a double bond at the α,β-position (9a, entry 4, Table 1) or replacement of the amide functionality by a carbamate one (16b, entry 5, Table 1) destroyed the inhibitory activity (28% and 39%, respectively). The replacement of the benzothiazole group of 5a and 5b by a benzoxazole group led to active derivatives. The benzoxazole derivative 5d (entry 6, Table 1), carrying the naphthalene group at a distance of three carbon atoms from the carbonyl group, exhibited higher inhibitory activity (73%) than the benzoxazole derivative 5c (49%, entry 7, Table 1), with a shorter linker. When a benzimidazole group replaced the benzothiazole group of 5a and 5b, a remarkable decrease in activity was observed. Compound 5f (entry 8, Table 1) inhibited it by 66%, while 5e (entry 9, Table 1) inhibited it by 29%. The replacement of the naphthalene ring of 5b by a p-methoxyphenyl ring (compound 11, entry 10, Table 1) resulted in a decrease in inhibitory potency (80%). Similarly, derivatives carrying a p-methoxyphenyl ring, either 9b (entry 11, Table 1) bearing an α,β-double bond or 16a (entry 12, Table 1) bearing a carbamate group, presented very weak activity (25% and 15%, respectively). Finally, the insertion of an oxygen atom, replacing the methylene attached to the naphthalene ring (compound 7, entry 13, Table 1), led, again, to a decrease in the inhibitory activity (41%).
Next, we explored the replacement of the amide bond of the previous compounds by either a hemiaminal ether or a methyleneamino functionality. Compound 18a (entry 1, Table 2), where the carbonyl group of 5b was replaced by a methoxy group (hemiaminal ether), was found to present 46% inhibitory activity. Interestingly, the N-alkylated 2aminobenzothiazole derivative 19a (GK543) (entry 2, Table 2) proved to be a potent inhibitor, causing 98% inhibition at 3 µM. Similar potent inhibitory activity (95%) was observed for the N-alkylated 2-aminobenzoxazole derivative 23a (entry 3, Table 2). The insertion of an oxygen atom (compound 25a, entry 4, Table 2), replacing the methylene group attached to the naphthalene ring, resulted in a considerable decrease in the inhibitory potency (63%), in comparison to 19a. However, in the case of the benzoxazole derivatives, similar replacement (compound 23c, entry 5, Table 2) led to a slight decrease (87%). Changing the position of the substituent on the naphthalene group from 2-to 1-(compound 19d, entry 6, Table 2) caused a decrease in the activity (82%), in comparison to 19a. An additional decrease was observed for compound 25b (entry 7, Table 2), where an oxygen atom replaced a methylene of 19d. The benzoxazole derivative 23d (entry 8, Table 2), bearing an oxygen atom, caused an even weaker effect (45%).
Taken together, eight novel compounds-N-acylated 2-aminobenzothiazoles 5b and 11; N-alkylated 2-aminobenzothiazoles 19a, 19b and 19d; N-alkylated 2-aminobenzoxazoles 23a and 23c; and N-alkylated 2-aminothiazole 19e-were identified to inhibit the generation of PGE 2 at levels higher than 80% at 3 µM in rat renal mesangial cells. The effect of these compounds was further studied at various concentrations ranging from 0.1 to 3 µM, and the results are shown in Figure 2. The EC 50 values are summarized in Tables 1 and 2. N-Acylated 2-aminobenzothiazole 5b and N-alkylated 2-aminobenzothiazoles 19a and 19b were found to exhibit the most potent inhibitory activity, with EC 50 values of 173 nM, 118 nM and 177 nM, respectively. As concluded, either N-acylatedor N-alkylated 2aminobenzothiazole derivatives were demonstrated to be better inhibitors of PGE 2 generation than the corresponding benzoxazole derivatives. As shown in Figure 2, 19e presented an unusual biphasic effect, as low concentrations enhanced PGE 2 , while at higher concentrations of 1 µM and 3 µM, PGE 2 was efficiently reduced. The reason for this biphasic effect is presently unclear; however, NSAIDs have been reported to exhibit such biphasic effects [37]. , 19e (f), 23a (g) and 23c (h) on IL-1/Fk-stimulated PGE 2 generation in rat mesangial cells. Cells were pretreated for 20 min with the indicated concentrations of inhibitors and then stimulated for 24 h in the absence (−) or presence (+) of 1 nM interleukin 1β (IL-1) plus 5 µM forskolin (Fk). PGE 2 was quantified in supernatants using an enzyme-linked immunoassay, as described in the Experimental section. Data are presented as % of IL-1/Fk stimulation and are means ± S.D.s (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered statistically significant when comparing with the IL-1/Fk-stimulated samples.

Study of the In Vivo Anti-Inflammatory Activity
The rat-paw carrageenan-induced edema assay was employed as a model for acute inflammation to evaluate the anti-inflammatory activity of selected benzothiazole derivatives, as we have previously described for the evaluation of PLA 2 inhibitors [18]. Two of the benzothiazole derivatives, presenting the most potent inhibitory activity in vitro (5b, EC 50 173 nM, and 19a, EC 50 118 nM), as well as 19c, presenting weaker activity (62% at 3 µM), were evaluated at a dose of 0.01 mmol/kg. Indomethacin was used in these experiments as a reference drug and led to 37.3% inhibition of inflammation at the same dose. The results for the in vivo anti-inflammatory activity of 5b, 19a and 19c are presented in Table 3 and are in accordance with their in vitro inhibitory activity of PGE 2 generation. Two novel benzothiazole derivatives synthesized in the present study, 5b and 19a, exhibited anti-inflammatory activity higher than that of indomethacin.

Inhibition of Phospholipases A 2 by 19a
To obtain insight as to the mechanism of action of compound 19a, which was identified in the present study to exert the most potent in vitro and in vivo activity, its effect on NO formation in cytokine-stimulated rat mesangial cells and on the ability to inhibit PLA 2 activity in vitro was studied. No effect on NO production was observed (Supplementary Materials, Figure S1), suggesting that 19a is not involved in the NF-kB pathway and gene transcription [38]. We determined the ability of 19a to inhibit three different human PLA 2 s, namely, cytosolic calcium-dependent PLA 2 (GIVA cPLA 2 ), secreted PLA 2 (GV sPLA 2 ) and calcium-independent PLA 2 (GVIA iPLA 2 ), utilizing a lipidomics assay, as previously described [32]. Compound 19a was found to inhibit GVIA iPLA 2 with an X I (50) value of 0.03, but did not inhibit GIVA cPLA 2 or GV sPLA 2 significantly (Figure 3). The X I (50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme activity by 50%. Although this inhibitory activity is not particularly potent, this finding suggests that the anti-inflammatory activity of 19a may be attributed, at least in part, to the inhibition of GVIA iPLA 2 and its specific interference with the prostaglandin pathway. We have previously shown that compound AX048 (ethyl 4-(2oxohexadecanamido)butanoate) [17], which, in vivo, presents anti-hyperalgesic activity and inhibits spinal PGE 2 release, inhibits GVIA iPLA 2 in vitro with an X I (50) value of 0.027 (a value comparable to that measured for 19a) and GIVA cPLA 2 with an X I (50) value of 0.022 [17]. We have also shown that arachidonoyl trifluoromethyl ketone, a widely used inhibitor of GVIA iPLA 2 , inhibits the cytokine-stimulated generation of PGE 2 in rat mesangial cells [36] and modulates allodynia after facial carrageenan injection in mice [39]. Furthermore, the selective GVIA iPLA 2 inhibitor FKGK18 (1,1,1-trifluoro-6-(2naphthalenyl)-2-hexanone) has been demonstrated to inhibit the generation of PGE 2 in CD4 + T cells [40]. Taken together, the inhibition of PGE 2 release and the anti-inflammatory activity of 19a may be attributed, at least in part, to the inhibition of GVIA iPLA 2 . However, in addition, 19a may exert its anti-inflammatory activity by acting on additional target(s). The curve was generated using GraphPad Prism with a nonlinear regression targeted to a symmetrical sigmoidal curve based on plots of % inhibition vs. log(inhibitor concentration). The reported X I (50) value was calculated from the resultant plot.

Conclusions
We have developed various routes for the synthesis of N-acylated and N-alkylated 2-aminobenzothiazoles, and we have synthesized a series of such compounds and related benzoxazoles and benzimidazoles. All the compounds synthesized were evaluated for their ability to inhibit the cytokine-stimulated PGE 2 release at a cellular level, employing a rat mesangial cell model. We have identified three novel compounds exhibiting potent inhibition of PGE 2 generation. 2-Aminobenzothiazole acylated by a 3-(naphthalen-2yl)propanoyl moiety (5b), as well as 2-aminobenzothiazole N-alkylated by a three carbonatom chain carrying either a naphthalene (19a) or a phenyl (19b) ring, was found to suppress PGE 2 formation with EC 50 values of 173 nM, 118 nM and 177 nM, respectively. The inhibitors 5b and 19a were also found to exhibit anti-inflammatory activity greater than that of indomethacin in a rat-paw carrageenan-induced edema assay. The inhibition of PGE 2 release and the anti-inflammatory activity of the most potent compound, 19a, identified in the present study may be attributed, at least in part, to the inhibition of GVIA iPLA 2 . All the above findings suggest that N-acylated and N-alkylated 2-aminobenzothiazoles are potential novel leads for the development of agents inhibiting PGE 2 generation.

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