Antinociceptive properties of new coumarin derivatives bearing substituted 3,4-dihydro-2H-benzothiazines

Background Coumarins are an important class of widely distributed heterocyclic natural products exhibiting a broad pharmacological profile. In this work, a new series of coumarins bearing substituted 3,4-dihydro-2H-benzothiazines were described as potential analgesic agents. The clinical use of NSAIDs as traditional analgesics is associated with side effects such as gastrointestinal lesions and nephrotoxicity. Therefore, the discovery of new safer drugs represents a challenging goal for such a research area. Results The target compounds 3-(3-methyl-3,4-dihydro-2H-benzo[b][1,4]thiazin-3-yl)-2H-chromen-2-ones 2a-u were synthesized and characterized by spectral data. The antinociceptive properties of target compounds were determined by formalin-induced test and acetic acid-induced writhing test in mice. Among the tested compounds, compound 2u bearing 2-(4-(methylsulfonyl)benzoyl)- moiety on benzothiazine ring and 4-(methylsulfonyl)phenacyloxy- group on the 7 position of coumarin nucleus showed better profile of antinocecieption in both models. It was more effective than mefenamic acid during the late phase of formalin-induced test as well as in the acetic acid-induced writhing test. Conclusion Considering the significant antinoceciptive action of phenacyloxycoumarin derivatives, compound 2u prototype might be further used as model to obtain new more potent analgesic drugs.


Introduction
Pain is an uncomfortable sensation that alerts the human organs about a current or potential damage to tissues [1]. It has been accepted that pain can widely affect human life quality, and its management is considered as a main challenge in pharmacotherapy [2]. NSAIDs are one of major classes of traditional analgesics for treatment of pain. The clinical use of NSAIDs is associated with side effects such as gastrointestinal lesions and nephrotoxicity [3]. Therefore, the discovery of new safer drugs represents a challenging goal for such a research area.
Coumarins are an important class of widely distributed heterocyclic natural products exhibiting a broad pharmacological profile [4]. Several coumarin derivatives have been synthesized with diverse biological activities [5][6][7][8][9] especially analgesic/anti-inflammatory activity [10][11][12][13]. Recently, the synthesis and anti-inflammatory/analgesic activities of several coumarin derivatives with various substitutions on 3position of coumarin nucleus have been reported [14][15][16]. On the other hand, benzothiazine derivatives are also important heterocyclic compounds with wide spectrum of biological activities [17,18]. In view of the above facts and in continuation of our research program on the synthesis of biologically active heterocyclic compounds [19,20], we introduced herein the new coumarin derivatives bearing substituted 3,4-dihydro-2H-benzothiazines as analgesic agents. The antinociceptive properties of target compounds were determined by formalin-induced paw licking test and acetic acid-induced writhing test in mice. Indeed, the formalin-induced paw licking method is used to investigate both peripheral and central mechanisms whereas the acetic acid test is believed to demonstrate the involvement of peripheral mechanisms in the control of pain [21,22].

Chemistry
The target compounds 3-(3-methyl-3,4-dihydro-2H-benzo [b] [1,4]thiazin-3-yl)-2H-chromen-2-ones 2a-r (Additional file 1: Table S1) were synthesized according to the pathway outlined in Scheme 1 [23]. All reagents and chemicals were commercially available and used as received. Aluminasupported potassium fluoride (KF/Al 2 O 3 ) was prepared by literature method [24]. The dihydrobenzothiazole derivatives 1 were prepared as reported method by us [19,20]. The synthesis of compounds 2a-d, 2f-i and 2p-r was described in our previous paper [23]. Column chromatography was carried out on silica gel (70-230 mesh). TLC was conducted on silica gel 250 micron, F254 plates. Melting points were measured on a Kofler hot stage apparatus and are uncorrected. The IR spectra were taken using Nicolet FT-IR Magna 550 spectrographs (KBr disks). 1 H NMR spectra were recorded on a Bruker 400 or 500 MHz NMR instruments. The chemical shifts (δ) and coupling constants (J) are expressed in parts per million and Hertz, respectively. Mass spectra of the products were obtained with an HP (Agilent technologies) 5937 Mass Selective Detector. Elemental analyses were carried out by a CHN-Rapid Heraeus elemental analyzer. The results of elemental analyses (C, H, N) were within ± 0.4% of the calculated values.
General procedure for the synthesis of compounds 2 A suspension of dihydrobenzothiazole derivatives 1 (1.0 mmol), KF/Al 2 O 3 (0.7 g), and quinine hydrochloride (10 mol%) in ethanol (3.0 mL) was stirred at room temperature for 5 min. Then, appropriate phenacyl halide (1.2 mmol) was added to the mixture and stirring was continued. After completion of the reaction (3-5 h), the solvent was removed under reduced pressure. The residue was mixed with ethyl acetate (5 mL) and the catalyst was filtered and washed with ethyl acetate (3 × 5 mL). After evaporation of the solvent at reduced pressure, the crude product was purified by column chromatography (nhexane/ethyl acetate, 9:1) and crystallized from ethanol for further purification.

Pharmacology Animals
Male NMRI mice weighing 20-30 g were used for studying in vivo antinociceptive activities of target compounds. Animals were maintained under standard conditions (24 ± 2°C, 60-70% humidity) and allowed food and water ad libitum. They were housed in appropriate cages with 12 h light/dark cycle. Before each experiment animals randomly selected and allocated into groups. The whole protocol was approved by the Ethics Committee of the Faculty of Pharmacy at Tehran University of Medical Sciences.

Formalin-induced pain test
All target compounds 2a-u were subjected for testing their analgesic activity using formalin paw test [25]. The compounds or standard drug mefenamic acid were administered i.p. (30 mg/kg, 0.2 mL/20 g body weight) as a suspension in saline and tween 80 (4% w/v). Each group of mice (n = 6 animals per group) were pretreated by test compounds, mefenamic acid or vehicle, 30 minutes before injection of formalin (20 μL, 0.5%, s.c.) into the planar surface of the right hind paw. The amount of time that the animal spent licking injected paw was measured during the first 10 minutes (phase 1, neurogenic) and 10-30 minutes (phase 2, inflammatory) after formalin injection. The percentage inhibition was determined by using the following formula: Inhibition % = 100 × (controlexperiment)/control. The asterisks denote the levels of significance in comparison with control groups (*P <0.05, **P <0.01 and ***P <0.001). c Activity relative to mefenamic acid was determined by using the following formula: Relative Activity = Inhibition % of compound/Inhibition % of mefenamic acid.

Acetic acid-induced writhing test
The analgesic activity was also determined in vivo by the abdominal constriction test induced by acetic acid (0.6%; 0.1 mL/10 g) in mice [21]. An acetic acid solution was administered i.p. 30 minutes after administration of compounds or mefenamic acid. After the treatment, pairs of mice were placed in separate boxes and the numbers of constrictions of the abdominal muscles, together with stretching, were counted cumulatively over a period of 60 minutes. Antinociceptive activity was expressed as the percentage of inhibition of constrictions when compared with the vehicle control group.

Statistical analysis
The nociception data are expressed as means ± SEM. Variance analysis (ANOVA) followed by Bonferroni's test was used to compare means. P-values less than 0.05 were considered to be statistically significant.

Chemistry
The dihydrobenzothiazole derivatives 1 were quantitatively obtained by reaction of 3-acetylcoumarins with 2aminothiophenol derivatives in the presence of acetic acid under reflux condition or microwave irradiation [19,20]. The intramolecular Mannich-type reaction of compounds 1 with different phenacyl halides in the present of KF/ Al 2 O 3 and catalyzing by quinine hydrochloride in ethanol afforded 3,4-dihydro-2H-benzothiazine derivatives 2a-r via a ring expansion. When 7-hydroxy-3-(benzothiazol-2-yl) coumarin derivative 1e was treated with 2.5 equivalents of phenacyl halides, without protection of hydroxyl group, O-phenacyl derivatives 2s-u was obtained in excellent yields (Scheme 1). The physicochemical and spectral data of new compounds 2e, 2j-o, and 2s-u are described in experimental section.

Biological activity Formalin-induced nociception test
All target compounds 2a-u were tested using formalininduced pain test in mice [25]. The obtained results were reported as mean ± SEM of licking time and as percent of inhibition in Table 1. In general, the results showed that most of compounds were significantly able to reduce the licking time with percent of inhibition in the range of 25% to 60% at the first phase. The standard drug mefenamic acid showed 89% reduction of the licking time during the first phase. Amongst the tested compounds, 2a, 2c, 2f, 2h, 2i, 2l-n and 2r-u significantly reduced the formalin induced licking time in the range of 39-98% as compared to mefenamic acid with 85% of inhibition during the second phase. Compounds 2m and 2r-u showed more effective antinociceptive activity in the second phase rather than first phase, indicating their ability to inhibit nociception associated with inflammatory response. Indeed, 7-hydroxy-and 7-phenacyloxycoumarin derivatives (2r and 2s-u, respectively) were more effective than mefenamic acid. Compounds 2s and 2t were the most effective compounds at the dose of 30 mg/kg. The percentage inhibition was determined by using the following formula: Inhibition% = 100 × (controlexperiment)/control. b Activity relative to mefenamic acid was determined by using the following formula: Relative Activity = Inhibition % of compound/Inhibition % of mefenamic acid. c Tween 80 in saline (4% w/v). ***P <0.001 vs. control.