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Scientia Pharmaceutica

Article 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic Acid. Peculiarities of Preparation, Structure, and Biological Properties

Igor V. Ukrainets 1,*, Ganna M. Hamza 1, Anna A. Burian 1, Svitlana V. Shishkina 2,3, Natali I. Voloshchuk 4 ID and Oxana V. Malchenko 4

1 Department of Pharmaceutical Chemistry, National University of Pharmacy, 53 Pushkinska st., 61002 Kharkiv, Ukraine; [email protected] (G.M.H.); [email protected] (A.A.B.) 2 STC “Institute for Single Crystals”, National Academy of Sciences of Ukraine, 60 Nauki ave., 61001 Kharkiv, Ukraine; [email protected] 3 Department of Inorganic Chemistry, V. N. Karazin Kharkiv National University, 4 Svobody sq., 61077 Kharkiv, Ukraine 4 Department of Pharmacology, N. I. Pirogov Vinnitsa National Medical University, 56 Pirogov st., 21018 Vinnitsa, Ukraine; [email protected] (N.I.V.); [email protected] (O.V.M.) * Correspondence: [email protected]; Tel.: +38-0572-679-185

Received: 27 February 2018; Accepted: 6 March 2018; Published: 8 March 2018

Abstract: In order to determine the regularities of the structure– activity relationship, the peculiarities of obtaining, the spatial structure, and biological properties of 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid and some of its derivatives have been studied. Using nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction analysis, it has been proven that varying the reaction conditions using alkaline hydrolysis of methyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate makes it possible to successfully synthesize a monohydrate of the target acid, its sodium salt, or 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine. The derivatographic study of the thermal stability of 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid monohydrate has been carried out; based on this study, the optimal conditions completely eliminating the possibility of unwanted decomposition have been proposed for obtaining its anhydrous form. It has been shown that 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine is easily formed during the decarboxylation of not only 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid, but also its sodium salt, which is capable of losing CO2 both in rather soft conditions of boiling in an aqueous solution, and in more rigid conditions of dry heating. The NMR spectra of the compounds synthesized are given; their spatial structure is discussed. To study the biological properties of 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid and its sodium salt, the experimental model of inflammation caused by subplantar introduction of the carrageenan solution in one of the hind limbs of white rats was used. The anti-inflammatory activity and analgesic effect were assessed by the degree of edema reduction and the ability to affect the pain response compared to the animals of control groups. According to the results of the tests performed, it has been found that after intraperitoneal injection, the substances synthesized demonstrate a moderate anti-inflammatory action and simultaneously increase the pain threshold of the experimental animals very effectively, exceeding and in a similar dose by their analgesic activity.

Keywords: esters; hydrolysis; 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid; 2,1-benzothiazine; crystal structure; anti-inflammatory action; analgesic activity

Sci. Pharm. 2018, 86, 9; doi:10.3390/scipharm86010009 www.mdpi.com/journal/scipharm Sci. Pharm. 2018, 86, 9 2 of 14 Sci. Pharm. 2018, 86, 9 2 of 14

1. Introduction Even a cursory review of reference works on drugs reveals the fact that in the arsenal of modern non-narcoticnon-narcotic products products against against pain pain,, the the important important role role belongs belongs to tovarious various carboxylic carboxylic acids acids [1,2]. [1 ,In2]. Inaccordance accordance with with the the chemical chemical structure structure,, analg analgesicesic acids acids can can be be divided divided into into several several groups. groups. First First of all, it is, undoubtedly, derivatives of longlong-known-known I (Figure 1 1).). AA separateseparate group group isis derivatives of of anthranilic anthranilic and and 2-aminonicotinic 2-aminonicotinic acids acidsII (X =II C (X and = N, C respectively) and N, respectively) that are structurally that are closestructurally to salicylic close acid.to salicylic Especially acid. importantEspecially important and popular and popular analgesics were created were on created the basis on the of phenylaceticbasis of phenylacetic acid III. Furthermore,acid III. Furthermore, the most representativethe most representative group of analgesics group of analgesics officially permitted officially forpermitted use is comprisedfor use is comprised of 2-phenylpropionic of 2-phenylpropionic acids IV .acids Derivatives IV. Derivatives of succinic of succinic acid ( acid (FenbufenV and ),V and Suxibuzone), hetarylcarboxylic hetarylcarboxylic acid (Cinchophen, acid (Cinchophen, and Etodolac clinically and important clinically drug important drugVI), andKetorolac hetarylacetic VI), and acids hetarylaceticVII (, acids VII ,(Indometacin, etc.) shouldSulindac, also etc. be) should mentioned. also be mentioned.

Me R' R R COOH COOH COOH COOH

O X NH R R I II III IV Acetylsalicylic acid Enfenamic acid Actarit Almiprofen 5-Bromosalicylic acid acetate Bermoprofen Fosfosal Diclofenac Gentisic acid Narceine Salicylsulfuric acid Ibufenac

COOH Clometacin X Indometacin R4 1 COOH COOH Y R Isofezolac N z O O Sulindac 3 2 V VI R R Fenbufen Ketorolac VII

Figure 1. AnalgesicAnalgesic-acids-acids that are officially officially registered drugsdrugs [[11,,2].2].

These substances substances remain remain important important despite despite the the fact fact that that the thenegative negative impact impact of substances of substances with witha free acarboxyl free carboxyl group, group, first of first all, on of all,the gastrointestinal on the gastrointestinal tract, is tract,well known is well from known medical from practice. medical practice.However, However,the presence the of presence the acid offragment the acid (or fragment easily converted (or easily into converted it in vivo into) is often it in reflected vivo) is often very reflectedpositively very on thepositively biological on the properties biological exhibited properties by exhibited one or another by one molecule or another [3]. molecule Therefore, [3]. Therefore,carboxylic carboxylicacids are always acids areof particular always of interest particular for interestthe searc forh thefor new search promising for new promisingpain killers pain [4], killersand their [4], possibleand their side possible effects side are effects eliminated are eliminated by subsequent by subsequent chemical chemical transformation transformation to labile to labilenon-acidic non-acidic groups groups or by the or by special the special conditions conditions of their of administration their administration [5]. [5]. Taking into account the the above, above, it it is is of of interes interestt to to study study the the structure structure and and biological biological properties properties of 6 of4-methyl 4-methyl-2,2-dioxo-1-2,2-dioxo-1H-2λH-2,1λ-benzothiazine6,1-benzothiazine-3-carboxylic-3-carboxylic acids acids IX. TheIX. The main main prerequisites prerequisites for this for thisresearch research were were the expressedthe expressed analgesic analgesic activity activity demonstrated demonstrated by theirby their synthetic synthetic precursors precursors — 6 —methyl methyl-4-methyl-2,2-dioxo-1-4-methyl-2,2-dioxo-1H-2λH-2,1λ-benzothiazine6,1-benzothiazine-3-carboxylates-3-carboxylates VIIIVIII [6][ 6as] as well well as as excellent results obtained during the transition from quinolone esters X, which are similar in terms of their structure, to acids XI [7] (Figure 2). Another incentive to study the behavior of 4-methyl-substituted benzothiazine esters VIII in alkaline hydrolysis conditions was a simple scientific curiosity: as is

Sci. Pharm. 2018, 86, 9 3 of 14 results obtained during the transition from quinolone esters X, which are similar in terms XI Sci.of Pharm. their 2018 structure,, 86, 9 to acids [7] (Figure2). Another incentive to study the behavior3 of of 14 4-methyl-substituted benzothiazine esters VIII in alkaline hydrolysis conditions was a simple knownscientific [8], curiosity: the corresponding as is known acids [8], thecannot corresponding be obtained acids from cannottheir 4- behydroxy obtained analogs from theirXII since 4-hydroxy in the processanalogs XII ofsince the inir the process formation of, theirthey formation, are they immediately are immediately decarboxylated decarboxylated to 6 44-oxo-3,4-dihydro-1-oxo-3,4-dihydro-1H--22λλ6,1,1-benzothiazin-2,2-diones-benzothiazin-2,2-diones XIII..

Me O OH O OH O R' OMe OEt OMe S O S O N N O N O O R R R VIII X XII

Me O OH O O R' OH OH S O S O N N O N O O R R R IX XI XIII

Figure 2. ProbableProbable (IX (IX) ) and and known known (XI (XI andand XIIIXIII) ) hydrolysis hydrolysis products products of of esters esters of of 44-hydroxy-2-oxoquinoline--hydroxy-2-oxoquinoline- [7] [7] and 4 4-hydroxy-2,2-dioxo-2,1-benzothiazine--hydroxy-2,2-dioxo-2,1-benzothiazine- [[8]8] 3-carboxylic3-carboxylic acids.acids.

2. Materials Materials and and Methods Methods

2.1. Chemistry Chemistry 11НH-- and and 1313СC-NMR-NMR (proton (proton and and carbon carbon nuclear nuclear magnetic magnetic resonance resonance)) spectra spectra were were acquired acquired on on a Varian Mercury Mercury-400-400 ( (VarianVarian Inc., Inc., Palo Palo Alto, Alto, CA, CA, USA USA)) instrument instrument (400 (400 and and 100 100 MHz, MHz, respectively) respectively) in hexadeuterodimethylin hexadeuterodimethyl sulfoxide sulfoxide (DMSO (DMSO--d6) withd6) with tetramethylsilane tetramethylsilane as the as internal the internal standard. standard. The chemicalThe chemical shift shift values values were were recorded recorded on a onδ scale a δ scale and the and coupling the coupling constants constants (J) in (J hertz.) in hertz. The followingThe following abbreviatio abbreviationsns were were used used in reporting in reporting spectra: spectra: s = singlet, s = singlet, d = doublet, d = doublet, t = triplet, t = triplet, m = multiplet.m = multiplet. Melting Melting points pointswere determined were determined in a capillary in a capillary using a using Electrothermal a Electrothermal IA9100X1 IA9100X1 (Bibby Scientific(Bibby Scientific Limited, Limited, Stone, Stone, UK) UK)digital digital melting melting point point apparatus. apparatus. The The derivatographi derivatographicc study study of 44-methyl-2,2-dioxo-1-methyl-2,2-dioxo-1H-2-2λλ66,1-benzothiazine-3-carboxylic,1-benzothiazine-3-carboxylic acidacid monohydratemonohydrate ( 4(4)) was was conducted conducted using using a aMettler Mettler TA TA 3000 3000 (Mettler-Toledo (Mettler-Toledo GmbH, GmbH, Bussigny, Bussigny VD,,Switzerland) VD, Switzerland) thermoanalytical thermoanalytical unit. The unit. sample The samplewas heated was inheated the inert in the gas inert atmosphere gas atmosphere (argon, 20(argon, mL/min), 20 mL/min), and the and rate the of heating rate of washeating 5 ◦C/min. was 5 °C/minThe synthesis. of the The starting methyl synthesis 4-methyl-2,2-dioxo-1 of H-2λ6 the,1-benzothiazine-3-carboxyate starting methyl (1) was 4carried-methyl out-2,2 by-dioxo the method-1H-2λ6,1 described-benzothiazine in the- study3-carboxyate [6]. (1) was carried out by the method described in the study [6]. 2.2. Sodium 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Monohydrate (3) 2.2. Sodium 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Monohydrate (3) Add sodium hydroxide (1.20 g, 0.03 mol) to the suspension of ester 1 (2.53 g, 0.01 mol) in 15 mL of H2AddO, then sodium boil underhydroxide reflux (1.20 for 4g, h. 0.03 Cool, mol) and to acidify the suspension with HCl of to ester pH 3.5. 1 (2.53 Allow g, 0.01 to stand mol) for in 1015 hmL at ◦ ofthe H temperature2O, then boil of under 5–7 refluxC. Filter for the 4 h. crystalline Cool, and sodium acidify saltwith monohydrate HCl to рН 3.5.3 precipitated, Allow to stand and for dry 10 in h air.at theYield: temperature 2.06 g (74%); of after5–7 °C. recrystallization Filter the crystalline from methanol, sodium salt colorless monohydrate crystals are3 precipitated, obtained; melting and dry point in ◦ 1 air.(mp) Yield: 190–192 2.06 gC (74%); (decomposition after recrystallization without melting); from methanolH-NMR, (400 colorless MHz, crystals DMSO- ared6): obtained;δ 11.45 (br. melting s, 1H, 1 pointSO2NH), (mp) 7.50 190 (d,–192 1H, °CJ =(decomposition 8.2 Hz, H-5), 7.23 without (t, 1H, melting);J = 7.6 Hz, H H-7),-NMR 6.96–6.89 (400 MHz, (m, DMSO 2H, H-6,8),-d6): δ 2.33 11.45 (s, (br. 3H, s, 1H, SO2NH), 7.50 (d, 1Н, J = 8.2 Hz, Н-5), 7.23 (t, 1Н, J = 7.6 Hz, Н-7), 6.96–6.89 (m, 2Н, Н-6,8), 2.33 (s, 3Н, 4-CH3). Analytically calculated (Anal. Calcd.) for C10H8NNaO4S • H2O: C, 43.01; H, 3.61; N, 5.02; S, 11.48. Found: C, 42.94; H, 3.53; N, 5.11; S, 11.40.

2.3. 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic Acid Monohydrate (4)

Sci. Pharm. 2018, 86, 9 4 of 14

4-CH3). Analytically calculated (Anal. Calcd.) for C10H8NNaO4S • H2O: C, 43.01; H, 3.61; N, 5.02; S, 11.48. Found: C, 42.94; H, 3.53; N, 5.11; S, 11.40.

2.3. 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic Acid Monohydrate (4) Obtained by a similar method—see the previous example. Dilute the reaction mixture to the volume of 10 mL, then acidify with HCl to pH 2.5–3.0. Yield: 2.26 g (88%); colorless crystals; ◦ 1 mp 191–193 C (decomposition, a double-end sealed capillary); H-NMR (400 MHz, DMSO-d6): δ 11.82 (br. s, 2H, COOH + SO2NH), 7.76 (d, 1H, J = 8.1 Hz, H-5), 7.50 (t, 1H, J = 7.6 Hz, H-7), 7.23 (t, 1H, J = 7.6 Hz, H-6), 7.14 (d, 1H, J = 7.9 Hz, H-8), 2.47 (s, 3H, 4-CH3, coincides with the signal of residual 13 protons DMSO-d6). C-NMR (100 MHz, DMSO-d6): δ 162.9 (C=O), 145.0, 138.5, 132.7, 129.5, 128.4, 123.7, 121.6, 118.9, 17.8 (4-CH3). Anal. Calcd. for C10H9NO4S • H2O: C, 46.69; H, 4.31; N, 5.44; S 12.46%. Found: C, 46.75; H, 4.38; N, 5.39; S 12.54%.

2.4. 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic Acid (5) Place 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid monohydrate (4) (2.57 g, 0.01 mol) in a drying cabinet at the temperature of 100–110 ◦C for 10 h. Yield: 2.39 g (quantitative); ◦ colorless crystals; mp 210–212 C (decomposition); Anal. Calcd. for C10H9NO4S: C, 50.20; H, 3.79; N, 5.85; S 13.40%. Found: C, 50.26; H, 3.83; N, 5.94; S 13.34%.

2.5. 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine (7) • Add sodium hydroxide (1.20 g, 0.03 mol) to the suspension of ester 1 (2.53 g, 0.01 mol) in 15 mL of H2O, then boil under reflux for 20 h. Cool, and acidify the resulting aqueous solution of salt 6 with HCl to pH 3.5. Filter the precipitate of benzothiazine 7 obtained, wash with cold water, and dry in air. Yield: 1.66 g (85%); colorless crystals; mp 179–181 ◦C (ethanol); 1H-NMR (400 MHz, DMSO-d6): δ 11.31 (br. s, 1H, SO2NH), 7.22 (d, 1H, J = 7.9 Hz, H-5), 6.98 (td, 1H, J = 7.6 and 1.4 Hz, H-7), 6.61 (d, 1H, J = 7.6 Hz, H-8), 6.47 (t, 1H, J = 7.4 Hz, H-6), 6.07 (s, 1H, H-3), 2.12 (s, 3H, 4-CH3). Anal. Calcd. for C9H9NO2S: C, 55.37; H, 4.65; N, 7.17; S 16.42%. Found: C, 55.46; H, 4.73; N, 7.10; S 16.36%. • Keep 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid (5) (2.39 g, 0.01 mol) at the ◦ temperature of 220-225 C for 10 min. After CO2 evolution is stopped, cool the reaction mixture. Triturate the residue with 10 mL of cold ethanol. Filter the precipitate of benzothiazine 7, and dry in air. Yield: 1.77 g (91%). • Keep sodium 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate monohydrate (3) (2.79 g, 0.01 mol) at the temperature of 200–210 ◦C for 15 min. The initially colorless salt 3 turns into a yellow powder without melting. Dissolve the resulting sodium salt 6 in 15 mL of H2O and acidify with HCl to pH 3.5. Filter the precipitate of 7, wash with cold water, and dry in air. Yield: 1.81 g (93%).

The mixed samples of benzothiazine 7 obtained by different methods did not result in a depression of the melting point, and the 1H-NMR spectra of the compounds were identical.

2.6. X-ray Structural Analysis of Sodium 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Monohydrate (3) ◦ The crystals of sodium salt 3 (C10H8NNaO4S • H2O) were monoclinic, colorless. At 23 C: ◦ 3 a 5.241(2), b 30.040(8), c 7.119(1) Å; β 95.86(2) ; V 1114.8(5) Å , Z 4, space group P21/c, 3 −1 dcalc 1.664 g/cm , µ(MoKα) 0.341 mm , F(000) 576. The unit cell parameters and intensities of 7403 reflections (1962 independent reflections, Rint = 0.103) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited, Oxford, UK) using MoKα radiation, a Charge Coupled ◦ Device (CCD) detector, a graphite monochromator, and ω-scanning to 2θmax 50 . The structure was solved by the direct method using the SHELXTL program package (Institute of Inorganic Chemistry, Sci. Pharm. 2018, 86, 9 5 of 14

Göttingen, Germany) [9]. The positions of the hydrogen atoms were found from the electron density difference map and refined using the “rider” model with Uiso = nUeq for the non-hydrogen atom bonded to a given hydrogen atom (n = 1.5 for methyl, and n = 1.2 for the other hydrogen atoms). The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.204 for 1909 reflections (R1 0.086 for 1280 reflections with F > 4σ (F), S 1.088). CCDC 1826303 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Center [10].

2.7. X-ray Structural Analysis of 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic Acid Monohydrate (4)

◦ The crystals of acid monohydrate 4 (C10H9NO4S • H2O) were monoclinic, colorless. At 23 C: ◦ 3 a 7.7755(7), b 10.1006(9), c 14.856(1) Å; β 100.533(3) ; V 1147.1(2) Å , Z 4, space group P21/c, 3 −1 dcalc 1.391 g/cm , µ(MoKα) 0.280 mm , F(000) 500. The unit cell parameters and intensities of 7106 reflections (1971 independent reflections, Rint = 0.036) were measured on a Bruker-Apex2 diffractometer (Bruker Corporation, Billerica, MA, USA) using MoKα radiation, a CCD detector, ◦ a graphite monochromator, and ω-scanning to 2θmax 50 . The structure was solved by the direct method using the SHELXTL program package (Institute of Inorganic Chemistry) [9]. The positions of the hydrogen atoms were found from the electron density difference map and refined in the isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.116 for 1913 reflections (R1 0.045 for 1549 reflections with F > 4σ (F), S 1.127). CCDC 1826302 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Center [11].

2.8. X-ray Structural Analysis of 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine (7) ◦ The crystals of benzothiazine 7 (C9H9NO2S) were monoclinic, colorless. At 23 C: a 12.793(2), ◦ 3 3 b 4.9346(5), c 14.834(2) Å; β 105.06(1) ; V 904.3(2) Å , Z 4, space group P21/c, dcalc 1.434 g/cm , µ(MoKα) 0.321 mm−1, F(000) 408. The unit cell parameters and intensities of 8114 reflections (2624 independent reflections, Rint = 0.073) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) ◦ using MoKα radiation, a CCD detector, a graphite monochromator, and ω-scanning to 2θmax 60 . The structure was solved by the direct method using the SHELXTL program package (Institute of Inorganic Chemistry) [9]. The positions of the hydrogen atoms were found from the electron density difference map and refined using the “rider” model with Uiso = nUeq for the nonhydrogen atom bonded to a given hydrogen atom (n = 1.5 for methyl and hydroxyl groups, and n = 1.2 for the other hydrogen atoms). The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.129 for 2547 reflections (R1 0.050 for 1482 reflections with F > 4σ (F), S 0.903). CCDC 1826304 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Center [12].

2.9. Pharmacology

Anti-Inflammatory and Analgesic Test All biological experiments were carried out in full accord with the European Convention on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and the Ukrainian Law No. 3447-IV “On protection of animals from severe treatment” [13] (project ID 3410U14, approved 15 October 2015). The biological studies were conducted on white Wistar male rats weighing 220–250 g. All experimental animals were under quarantine for 10 days. One day before the experiments, the animals were transferred to the research laboratory for adaptation. During the experiments, the animals Sci. Pharm. 2018, 86, 9 6 of 14 were kept in standard conditions of the 12 h light-and-dark regimen with access to water and food ad libitum. The anti-inflammatory activity of the compounds synthesized was studied on the model of the experimental inflammation process caused by subplantar introduction of 0.1 mL of 1% carrageenan solution [14,15] in one of the hind limbs of rats. The test compounds and the reference drugs were injected intraperitoneally into the animals of the experimental groups in 2 h after the introduction of carrageenan. In 3 h after the carrageenan injection (at the peak of inflammation), the volume of healthy and swollen limbs (mm3) was measured using a plethysmometer (Ugo Basile Biological Research Apparatus Company, Varese, Italy). The anti-inflammatory effect (in %) was assessed by the degree of edema reduction in the experimental animals compared to the animals of the control group. The analgesic activity was detected on the same model by determining the pain threshold—the smallest pressure force (g/mm2) on the rat’s foot, which caused a painful reaction of the animal expressed by localization of pain and/or paw withdrawal—the healthy and the injured limb. The measurements were performed using a baseline dolorimeter (Fabrication Enterprises, White Plains, NY, USA). The analgesic effect (in %) of the compounds studied was assessed by the ability to increase the pain threshold in the experimental groups compared to the animals of the control group. All substances under research, Lornoxicam (Nycomed Austria GmbH, Linz, Austria) and Diclofenac (Hemofarm A.D., Vrsac, Serbia), were introduced intraperitoneally in the form of aqueous solutions or fine aqueous suspensions stabilized with Tween-80 at a screening dose of 20 mg/kg. The animals of the control group received an equivalent amount of water with Tween-80. Seven experimental animals were involved to obtain statistically reliable results in testing each of the compounds 3 and 4, the reference drugs, and the control. Processing of the results obtained was performed using STATISTICA 6.1 software package (StatSoft Inc., Tulsa, OK, USA). Descriptive statistics included calculations of the arithmetic means (M) and standard errors of the mean (±m). The significance of differences within one group was performed using Wilcoxon’s non-parametric test. The reliability of intergroup differences was determined using non-parametric Mann-Whitney U-criteria. Differences were considered to be statistically significant at p ≤ 0.05.

3. Results and Discussion

3.1. Chemistry A number of simple experiments conducted showed that methyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (1) in a boiling aqueous solution of sodium hydroxide was hydrolyzed quite easily and rapidly (Scheme1). According to 1H-NMR monitoring in 4 h after the beginning of hydrolysis, the initial ester in the reaction mixture was no longer detected. Unlike 4-hydroxy analogs XII [8], in this case, highly undesirable decarboxylation of hetaryl-3-carboxylic acid initially formed in the conditions of synthesis was not observed during this time. However, this does not mean that 4-methylsubstituted 2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acids are not subjected to decarboxylation at all. As it turned out, this side process is typical for them too, and in relatively mild conditions. It is only necessary to increase the reaction time by several hours and decarboxylation becomes inevitable. For example, after the 20 h alkaline hydrolysis of ester 1, exclusively 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine (7) was isolated instead of the expected acid 5. Sci. Pharm. 2018, 86, 9 7 of 14 Sci. Pharm. 2018, 86, 9 7 of 14 Sci. Pharm. 2018, 86, 9 7 of 14

NaOH, H2O o Reflux, 20 h HCl 225 C NaOH, H2O o Reflux, 20 h pHHCl 3.5 225 C - CO2 pH 3.5 - CO2 1 6 7 5

1 NaOH, H2O 6 7 5 210 oC o - H O Reflux, 4 h - CO2 100-110 C, 10 h 2 NaOH, H2O 210 oC o - H O Reflux, 4 h - CO2 100-110 C, 10 h 2 x H O x H2O 2 HCl HCl x H O x H2O 2 pHHCl 3.5 pH HCl2.5-3.0

pH 3.5 pH 2.5-3.0 2 3 4 Scheme2 1. The peculiarities of 4-methyl3 -2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic4 acid (5)

preparation. 6 SchemeScheme 1. 1. TheThe peculi peculiaritiesarities of of4-methyl 4-methyl-2,2-dioxo-1-2,2-dioxo-1H-2λH-2,1-λbenzothiazine6,1-benzothiazine-3-carboxylic-3-carboxylic acid acid (5) preparation. It(5 ) is preparation. clear that the initial product of alkaline hydrolysis of ester 1, regardless of the reaction duration, is always disodium salt 2. But the subsequent attempt of the seemingly quite trivial ItIt is clear clear that that the the initial initial product product of alkaline of alkaline hydrolysis hydrolysis of ester of 1 ester, regardless 1, regardless of the reaction of the duration,reaction transformation in acid revealed a number of interesting features. In particular, after acidifying the durationis always, disodiumis always salt disodium2. But the salt subsequent 2. But the attempt subsequent of the attempt seemingly of quitethe seemingly trivial transformation quite trivial in reaction mixture with hydrochloric acid to the usual pH for such cases (approximately 3.5), there transformationacid revealed a in number acid revealed of interesting a number features. of interesting In particular, features after acidifying. In particular, the reaction after acidifying mixture withthe was an abundant white precipitate of the presumable target acid 5. As is known, the spatial crystal reactionhydrochloric mixture acid with to the hydrochloric usual pH for acid such to casesthe usual (approximately pH for such 3.5), cases there (approximately was an abundant 3.5), whitethere structure of derivatives of 2,1-benzothiazine-3-carboxylic acids largely determines their analgesic wasprecipitate an abundant of the presumablewhite precip targetitate of acid the5 presumable. As is known, target the spatialacid 5. crystalAs is known, structure the of spatial derivatives crystal of activity [16,17]. Hydrolysis of ester 1 into the corresponding acid from the very beginning was structure2,1-benzothiazine-3-carboxylic of derivatives of 2,1-benzothiazine acids largely determines-3-carboxylic their acids analgesic largely activity determines [16,17 ]. their Hydrolysis analgesic of considered as a possible variant of strengthening analgesic properties. Therefore, it is quite natural activityester 1 into[16,17] the. Hydrolysiscorresponding of ester acid from1 into the the very corresponding beginning was acid considered from the very as a possible beginning variant was that the resulting product was immediately subjected to X-ray structural analysis. In addition, it was consideredof strengthening as a possible analgesic variant properties. of strengthening Therefore, analgesic it is quite properties. natural that Therefore, the resulting it is quite product natural was found, quite unexpectedly, that this was not thatimmediately the resulting subjected product to was X-ray immediately structural subjected analysis. to In X addition,-ray structural it was analysis. found, quite In addition, unexpectedly, it was 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid, but only its sodium salt, monohydrate foundthat this, was notquite 4-methyl-2,2-dioxo-1 unexpectedlyH-2λ6,,1-benzothiazine-3-carboxylic that this acid, but was only its sodium not 3 (Figure 3). 6 4salt,-methyl monohydrate-2,2-dioxo-31H(Figure-2λ ,1-3benzothiazine). -3-carboxylic acid, but only its sodium salt, monohydrate The anion negative charge of this salt is localized at the deprotonated carboxyl group. It is 3 (FigureThe 3). anion negative charge of this salt is localized at the deprotonated carboxyl group. It is confirmed by the С(9)–О(1) 1.261(7) Å and С(9)–О(2) 1.248(7) Å bond lengths which are characteristic for confirmedThe anion by the negative C(9)–O (1) charge1.261(7) of thisÅ and salt C (9) is–O localized(2) 1.248(7) at the Å bond deprotonated lengths which carboxyl are characteristic group. It is the carboxylate anion (the mean value of such a bond length is [18] 1.255 Å). confirmedfor the carboxylate by the С(9) anion–О(1) 1.261(7) (the mean Å and value С(9) of–О such(2) 1.248(7) a bond Å length bond islengths [18] 1.255 which Å). are characteristic for the carboxylate anion (the mean value of such a bond length is [18] 1.255 Å).

Figure 3. The standard representation of different atoms in different colors. The molecular structure of Figure 3. The standard representation of different atoms in different colors. The molecular structure sodium salt monohydrate 3 with atoms represented by thermal vibration ellipsoids of 50% probability. Figureof sodium 3. The salt standard monohydrate representation 3 with of atoms different represented atoms in different by thermal colors. vibration The molecular ellipsoids structure of 50% ofprobability. sodium salt monohydrate 3 with atoms represented by thermal vibration ellipsoids of 50% probability.

Sci. Pharm. 2018, 86, 9 8 of 14 Sci. Pharm. 2018, 86, 9 8 of 14

The dihydrothiazine cycle of the studied anion adopts a conformation which is intermediate between twist-boat and sofa (the puckering parameters [19] are: S = 0.51, Θ = 44.8◦, Ψ = 32.5◦). The S(1) between twist-boat and sofa (the puckering parameters [19] are: S = 0.51, Θ = 44.8 , Ψ = 32.5 ). The S(1) and С(8) atoms deviate from the mean plane of the remaining atoms of this cycle on 0.66 Å and and C(8) atoms deviate from the mean plane of the remaining atoms of this cycle on 0.66 Å and 0.23 Å, respectively.0.23 Å, respectively. The nitrogen The nitrogen atom has atom a planar has configuration,a planar configuration, and the sum and of the the sum bond of angles the bond centered angles at centered◦ at it is 360°. The carboxyl substituent is turned significantly relative to the С(7)–С(8) it is 360 . The carboxyl substituent is turned significantly relative to the C(7)–C(8) endocyclic double endocyclic double bond (the С(7)–С(8)–С(9)–О(1) torsion angle◦ is −42.2(8)°), in spite of the formation of bond (the C(7)–C(8)–C(9)–O(1) torsion angle is −42.2(8) ), in spite of the formation of the C(10)–H ... the С(10)–Н…О(1) intramolecular hydrogen bond (Н…О 2.18 Å, О◦–Н…О 136°). The steric repulsion O(1) intramolecular hydrogen bond (H ... O 2.18 Å, O–H ... O 136 ). The steric repulsion between the methylbetween group the methyl and atoms group of and the aromaticatoms of cyclethe aromatic is also obtained cycle is inalso the obtained anion of in the the salt anion3 (the of shortenedthe salt 3 (the shortened intramolecular contacts are Н(5)…С(10) 2.53 Å and Н(10а)…С(5) 2.71 Å compared to the intramolecular contacts are H(5) ... C(10) 2.53 Å and H(10a) ... C(5) 2.71 Å compared to the van der Waalsvan der radii Waals sum radii [20] sum 2.87 [ Å).20] 2.87 Å). In the crystal phase,phase, the anions of the salt 3 formform columnscolumns along the [1 00 0]0] crystallographiccrystallographic direction. These columns are bound through sodium cations (Figure 44).). TheThe coordinationcoordination numbernumber ofof the sodium cation is six. Each cation is is bound wi withth four anions anions,, belongs to to three different columns columns,, and has has additional additional coordination coordination with with a water a water molecule. molecule. The position The position of the water of the molecule water molecule is stabilized is stabilized by the intermolecular hydrogen bonds (N(1)–H…O(1w)’ (−x, 1 − y, 2 − z) H…O 2.10 Å, by the intermolecular hydrogen bonds (N(1)–H ... O(1w)’ (−x, 1 − y, 2 − z)H ... O 2.10 Å, N–H ... O N-H…O◦ 132°; O(1w)–H(1wa)…O(1)′ (1 − x, 1 – y, 1 − z) H…O 1.83 Å O–H…O 173°◦ and O(1w)–H(1wb)…O(2)′ 132 ;O(1w)–H(1wa) ... O(1)0 (1 − x, 1 – y, 1 − z)H ... O 1.83 Å O–H ... O 173 and O(1w)–H(1wb) ... (−x, 1 − y, 1 − z) H…O 2.00 Å, O–H…O 174°). ◦ O(2)0 (−x, 1 − y, 1 − z) H . . . O 2.00 Å, O–H . . . O 174 ).

Figure 4. PackingPacking of of Sodium salt monohydrate 3 in the the crystal crystal phase. phase. Projection Projection along along [1 [1 0 0]0] crystallographic direction.

Sodium 4-methyl-2,2-dioxo-14-methyl-2,2-dioxo-1H--22λλ6,1,1-benzothiazine-3-carboxylate-benzothiazine-3-carboxylate monohydrate monohydrate ( (33)) is is readily soluble in water, and its unexpected easy release from the reaction mixture is most likely due to the salting out effect caused by the simultaneous presence of a rather large amount of sodium chloride and hydrochloric acid acid in in addition addition to to salt salt 3 3inin the the solution solution.. If necessary, If necessary, this this effect effect can can be beremoved removed by bysimply simply adding adding a solvent, a solvent, i.e. i.e.,, water water.. However, However, sodium sodium salt salt monohydrate monohydrate 3 3isis only only dissolved. dissolved. Additionally, forfor itsits transitiontransition into the acidic form,form, additional acidificationacidification of the reaction mixture to lower pH values of 2.5–3.02.5–3.0 isis requiredrequired (see(see MaterialsMaterials andand Methods).Methods). The X X-ray-ray diffraction study confirms confirms the obtaining of the benzothiazine-3-carboxylicbenzothiazine-3-carboxylic acid 4 and indicatesindicates that that this this acid acid exists exists as a as monohydrate a monohydrate in the in crystal the crystal phase (Figure phase 5 (Figure). The molecular 5). The molecular structure ofstructure the acid of is the very acid close is very to the close structure to the ofstructure its anion of inits salt anion3. in salt 3. The dihydrothiazine cycle cycle of of4 4adopts adopts a conformationa conformation which which is intermediateis intermediate between between twist-boat twist-boat and ◦ ◦ sofaand (thesofa puckering(the puckering parameters parameters [19] are: [19 S] =are: 0.59, S =Θ 0.59,= 55.9 Θ ,=Ψ 55.9= 22.4, Ψ). = The22.4 S(1)). Theand S C(1)(8) andatoms С(8) deviate atoms deviate from the mean plane of the remaining atoms of this cycle on 0.82 Å and 0.25 Å, respectively. The nitrogen atom has a planar configuration, and the sum of the bond angles centered at it is 360°.

Sci. Pharm. 2018, 86, 9 9 of 14 Sci. Pharm. 2018, 86, 9 9 of 14

TheSci. Pharm. carboxyl 2018, substituent86, 9 is turned significantly relative to the С(7)–С(8) endocyclic double bond9 (theof 14 fromС(7)–С the(8)– meanС(9)–О plane(1) torsion of the angle remaining is 44.8(4)°), atoms in of spite this cycleof the on formation 0.82 Å and of 0.25the С Å,(10) respectively.–Н…О(1) intr Theamolecular nitrogen atomhydrogenThe carboxyl has abond planar substituent (Н…О configuration, 2.30 is Å,turned О– andН…О significantly the 131°). sum ofThe relative the steric bond repulsionto angles the С(7) centered between–С(8) endocyclic at methyl it is 360 doublegroup◦. The and bond carboxyl atoms (the С(7)–С(8)–С(9)–О(1) torsion angle is 44.8(4)°), in spite of the formation of the С(10)–Н…О(1) intramolecular substituentof the aromatic is turned cycle significantly is also obtained relative to in the the C (7) acid–C(8) 4 endocyclic(the shortened double intramolecular bond (the C(7)–C contacts(8)–C(9)–O are(1) hydrogen bond (Н…О◦ 2.30 Å, О–Н…О 131°). The steric repulsion between methyl group and atoms torsionН(5)…С angle(10) 2.57 is Å 44.8(4) and Н),(10а) in…С spite(5) of2.79 the Å formation compared of to the the C (10)van–H der... WaalsO(1) intramolecular radii sum [20] hydrogen2.87 Å). bond (Hof ... theO aromatic 2.30 Å, O–H cycle... isO also 131◦ ). obtained The steric in repulsion the acid between 4 (the shortenedmethyl group intramolecular and atoms of thecontacts aromatic are Н(5)…С(10) 2.57 Å and Н(10а)…С(5) 2.79 Å compared to the van der Waals radii sum [20] 2.87 Å). cycle is also obtained in the acid 4 (the shortened intramolecular contacts are H(5) ... C(10) 2.57 Å and H(10a) ...C(5) 2.79 Å compared to the van der Waals radii sum [20] 2.87 Å).

Figure 5. The molecular structure of acid monohydrate 4 with atoms represented by thermal Figurevibration 5. Theellipsoids molecular of 50% structure probability. of acid monohydrate 4 with atoms represented by thermal vibration ellipsoidsFigure 5. ofThe 50% molecular probability. structure of acid monohydrate 4 with atoms represented by thermal Invibration the crystal ellipsoids phase of 50%, 4- methylprobability.-2,2- dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid and water 6 moleculesIn the are crystal bound phase, by the 4-methyl-2,2-dioxo-1 set of the intermolecularH-2λ hydrogen,1-benzothiazine-3-carboxylic bonds forming the three acid-dimensional and water molecules are bound by the set of the intermolecular hydrogen6 bonds forming the three-dimensional net (FigureIn the 6): crystal N(1)– H…O phase(1w)′, 4 (-xmethyl, 0.5 − -y2,2, −0.5-dioxo + z)- H…O1H-2λ 2.11,1-benzothiazine Å, N–H…O -175°;3-carboxylic O(2)–H…O acid(1w) H…O and water 1.82 ◦ net (Figure6): N –H ... O 0 (x, 0.5 − y, −0.5 + z)H ... O 2.11 Å, N–H ... O 175 ;O –H ... O Å,molecules O–H…O are 172°; bound(1) O(1w) by–H the(1wa)(1w) set…O of(1)′ the (−x intermolecular, 1 − y, 1 − z) H…O hydrogen 1.92 Å, bonds O–H…O forming 168°; theand three O(1w)(2)-–dimensionalH(1wb)…O(1w)(3)′ ◦ 0 − − − H(1net ...− (Figurex, O1 − 1.82 y, 6):1 Å, − N z O–H)(1) H…O–H…O... 1.93O(1w)′ 172 Å,(x, O;O0.5–H…O (1w)− y,–H −0.5 167°.(1wa) + z )... H…OO(1) 2.11( x Å,, 1 N–yH…O, 1 z175°;)H ... O(2)O–H…O 1.92 Å,(1w) O–H H…O... 1.82O ◦ ◦ 168Å, O;– andH…O O(1w) 172°;–H O(1wb)(1w)–...OH(1wa)(3)…O0 (1(1)′− (−xx,, 1 1− − y, 1 − zz) )H…O H . . . 1.92 O 1.93 Å, Å,O– O–HH…O . .168°; . O 167and. O(1w)–H(1wb)…O(3)′ (1 − x, 1 − y, 1 − z) H…O 1.93 Å, O–H…O 167°.

Figure 6. Packing of acid monohydrate 4 in the crystal phase. Projection along [0 1 0] crystallographicFigure 6. Packing direction. of acid monohydrate 4 in the crystal phase. Projection along [0 1 0] crystallographic direction. 6 TheFigure existence 6. Packing of of 4-methyl-2,2-dioxo-1 acid monohydrate 4 inH the-2 λcrystal,1-benzothiazine-3-carboxylic phase. Projection along [0 1 0] acid crystallographic in the form of monohydrateThedirection. existence4 does of not 4- mattermethyl fundamentally-2,2-dioxo-1H- at2λ this6,1-benzothiazine stage of our research.-3-carboxylic However, acid if in this the compound form of ismonohydrate used as a basis4 does for not further matter chemical fundamentally transformations, at this stage the presence of our research. of crystallization However, water if this in itscompound moleculeThe existence is can used become asof a 4 - basismethyl extremely for-2,2 further-dioxo undesirable - chemical1H-2λ6,1 or - transformations,benzothiazine even unacceptable.-3- carboxylic the Aspresence a rule, acid of the in crystallization the easiest form and of mostwatermonohydrate availablein its mol 4 wayecule does to can removenot become matter water extremely fundamentally from crystallohydrates undesirable at this or stageeven is to unacceptable. of dry our them research. in As different a However, rule, conditions,the easiest if this moreandcompound most often whenavailable is used heated. as way a Therefore,basis to remove for further in order water chemical to from determine crystallohydratestransformations, the thermal stability the is presenceto dry of acid them of monohydrate crystallization in different4, conditions,water in its moremolecule often can when become heated. extremely Therefore, undesirable in order or to even determine unacceptable. the thermal As a srule,tability the ofeasiest acid and most available way to remove water from crystallohydrates is to dry them in different conditions, more often when heated. Therefore, in order to determine the thermal stability of acid

Sci. Pharm. 2018, 86, 9 10 of 14 Sci. Pharm. 2018, 86, 9 10 of 14 monohydrate 4, the derivatographic study was conducted under conditions of dry heating. The experimental data obtained (Figure 7) have convincingly shown that the smooth loss in mass begins immediatelythe derivatographic after the studystart of was heating conducted (see therm underogravimetric conditions of curve); dry heating. it obviously The experimental indicates the data gradualobtained removal (Figure of 7water.) have Otherwise, convincingly the showntest sample that theis sufficiently smooth loss stable in mass to a beginstemperature immediately of 200 °C; after the start of heating (see thermogravimetric curve); it obviously indicates the gradual removal of on the differential thermogravimetric curve at this temperature, there is a break◦ that goes into the peawater.k at 240 Otherwise, °C. The rapid thetest loss sample in mass is stops sufficiently at 255 °C stable and tothen a temperature there is only of uniform 200 C; volatilization on the differential of thermogravimetric curve at this temperature, there is a break that goes into the peak at 240 ◦C. the product. Within the temperature range◦ from 200 to 255 °C, approximately 18% of the mass is lost, whichThe rapid uniquely loss in masscorresponds stops at 255 C to and then decarboxylation there is only uniform and volatilization transformation of the product. into ◦ 4-methylWithin-2,2 the-dioxo temperature-1H-2λ6,1 range-benzothiazine from 200 to (7 255). C, approximately 18% of the mass is lost, which uniquely corresponds to decarboxylation and transformation into 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine (7).

Temperature (°C)

Figure 7. 4 Figure 7. DerivatogramsDerivatograms of acid ofmonohydrate acid monohydrate 4: thermogravimetric: thermogravimetric curve (black); curve differential (black); differential thermogravimetric curve (blue). Sample weight 7.57 mg. thermogravimetric curve (blue). Sample weight 7.57 mg.

Therefore,Therefore, the derivatographic the derivatographic study conducted study conductedsuggests that suggests for the removal that of for crystallization the removal waterof crystallization from acid water 4 frommonohydrate acid 4 monohydrate and obtaining and obtaining of of anhydrous anhydrous λ6 4-methyl4-methyl-2,2-dioxo-1-2,2-dioxo-1H-2λH-26,1-benzothiazine,1-benzothiazine-3-carboxylic-3-carboxylic acid acid (5), a ( 5 conventional), a conventional drying drying oven at oven a ◦ temperatureat a temperature of 100– of150 100–150 °C can beC safely can be used safely. used. λ6 Interestingly,Interestingly, 4-methyl 4-methyl-2,2-dioxo-1-2,2-dioxo-1H-2λH-26,1-benzothiazine,1-benzothiazine (7) ( can7) can be beobtained obtained not not only only by by the the longlong-term-term alkaline alkaline hydrolysis hydrolysis of ester of ester 1 and1 and thermal thermal decarboxylation decarboxylation of acid of acid 5 or5 itsor monohydrate its monohydrate 4. 4. λ6 Thus,Thus, initially initially colorless colorless sodium sodium 4- 4-methyl-2,2-dioxo-1methyl-2,2-dioxo-1H-H2λ-26,1-,1-benzothiazine-3-carboxylatebenzothiazine-3-carboxylate monohydratemonohydrate (3) (when3) when heated heated without without melting melting rapidly rapidly turns into turns a powder into a powderof an intense of an yellow intense λ6 color,yellow which color, appeared which appeared to be the to be sodium the sodium salt of salt 4- ofmethyl 4-methyl-2,2-dioxo-1-2,2-dioxo-1H-2λH6-2,1-benzothiazine,1-benzothiazine (6), (6), which,which, in turn, in turn, can can be beeasily easily conver convertedted into into the the acidic acidic form form 7. 7. ΑccordingAccording to to the the X-ray X-ray diffraction diffraction data data,, thethe dihydrothiazine dihydrothiazine cycle cycle of of λ6 4-methyl4-methyl-2,2-dioxo-1-2,2-dioxo-1H-H2λ-26,1-benzothiazine,1-benzothiazine ( (77)) (Figure(Figure8 ) adopts 8) adopts a conformation a conformation which is intermediate which is ◦ intermediatebetween twist-boat between andtwist sofa-boat similar and sofa to 3 similarand 4 (the to 3 puckering and 4 (the parameters puckering [parameters19] are: S = 0.50,[19] are:Θ = S 52.0 = , Ψ = 17.7◦). The S and C atoms deviate from the mean plane of the remaining atoms of this cycle on 0.50, Θ = 52.0, Ψ = 17.7(1) ). The(8) S(1) and С(8) atoms deviate from the mean plane of the remaining atoms of this0.67 cycle Å and on 0.17 0.67 Å, Å respectively. and 0.17 Å The, respectively. nitrogen atom The has nitrogen a planar atom configuration, has a planar and configuration, the sum of the and bond angles centered at it is 360◦. The absence of the substituent at the C atom causes the significantly the sum of the bond angles centered at it is 360°. The absence of the(8) substituent at the С(8) atom lower length of the C –C bond (1.337(4) Å) compared to the substituted dihydrothiazines 3 and causes the significantly lower(7) (8) length of the С(7)–С(8) bond (1.337(4) Å) compared to the substituted dihydrothiazines4. The steric repulsion 3 and 4. The between steric the repulsion methyl between group and the atoms methyl of group the aromatic and atoms cycle of isthe obtained aromatic in 7 (the shortened intramolecular contacts are H ... C 2.59 Å and H ... C 2.85 Å compared to cycle is obtained in 7 (the shortened intramolecular(5) contacts(9) are Н(5)…С(9c)(9) 2.59 Å(5) and Н(9c)…С(5) 2.85 Å comparedthe van der to Waals the van radii der sum Waals [20 radii] 2.87 sum Å). [20] 2.87 Å).

Sci. Pharm. 2018, 86, 9 11 of 14

Sci. Pharm. 2018, 86, 9 11 of 14 Sci. Pharm. 2018, 86, 9 11 of 14

Figure 8. The molecular structure of benzothiazine 7 with atoms represented by thermal vibration Figureellipsoids 8. Theof 50% molecular probability. structure of benzothiazine 7 with atoms represented by thermal vibration ellipsoids of 50% probability. Figure 8. The molecular structure of benzothiazine 7 with atoms represented by thermal vibration In the crystal phase, the 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine (7) molecules form chains ellipsoidsIn the crystal of 50% phase, probability. the 4-methyl-2,2-dioxo-1 H-2λ6,1-benzothiazine (7) molecules form chains along the [0 1 0] crystallographic direction (Figure 9) due to formation of the N(1)–H…O(2)′ (x, 1 + y, z) along the [0 1 0] crystallographic direction (Figure9) due to formation of the N (1)–H ... O 0 (x, 1 + y, z) intermolecularIn the crystal hydrogen phase, thebond 4- methyl(H…O- 2.092,2-dioxo Å, N–-1H…OH-2λ6 169°).,1-benzothiazine (7) molecules (2)form chains intermolecular hydrogen bond (H . . . O 2.09 Å, N–H . . . O 169◦). along the [0 1 0] crystallographic direction (Figure 9) due to formation of the N(1)–H…O(2)′ (x, 1 + y, z) intermolecular hydrogen bond (H…O 2.09 Å, N–H…O 169°).

FigureFigure 9.9. The chain of benzothiazine 77 moleculesmolecules boundbound byby hydrogenhydrogen bonds.bonds.

3.2.3.2. Evaluation ofFigure the Anti-InflammatoryAnti 9. The-Inflammatory chain of benzothiazine andand AnalgesicAnalgesic 7 molecules ActivityActivity bound by hydrogen bonds. TheThe pharmacological pharmacological tests conducted tests have shown conducted that 4-methyl-2,2-dioxo-1 have H-2λ shown6,1-benzothiazine that 3.2.-3-carboxylic4-methyl Evaluation-2,2 acid- dioxoof the monohydrate -Anti1H--2λInflammatory6,1-benzothiazine (4) and and its sodiumAnalgesic-3-carboxylic salt Activity3 demonstrate acid monohydrate moderate (4 and) and statistically its sodium reliable salt 3 anti-inflammatorydemonstrateThe moderate pharmacological properties and statistically on the carrageenan tests reliable edema anti conducted-inflammatory model in rats haveproperties (Table 1). on shown the carrageenan that 4edema-methyl model-2,2-dioxo in rats-1H (Table-2λ6,1- 1).benzothiazine -3-carboxylic acid monohydrate (4) and its sodium salt 3 demonstrate moderate and statistically reliable anti-inflammatory properties on the carrageenan edema model in rats (Table 1).

Sci. Pharm. 2018, 86, 9 12 of 14 Sci. Pharm. 2018, 86, 9 12 of 14

Table 1. The Anti-Inflammatory Activity of Sodium Salt 3, Acid 4, and Reference Drugs. Table 1. The Anti-Inflammatory Activity of Sodium Salt 3, Acid 4, and Reference Drugs.

R

x H2O

Volume of Anti- Inflammatory Volume of Damaged Entry Product R Volume of VolumeNon-Damaged of ∆Volume (Volume Increase) Activity, Compared Extremity (mm3) ΔVolume Anti- Inflammatory Damaged NonExtremity-Damage (mm3) to Control (%) Entry Product R (Volume Activity, Compared to 1 3 NaExtremity 516.3 ± 15.8 d Extremity 304.9 ± 14.0 211.4 ± 16.5 1,2,3 48.9 1,2,3 2 4 H 568.6 ±3 16.5 268.73± 7.59 Increase)299.9 ± 20.1 Control (%)27.5 3 Lornoxicam –(mm 360.5 ±)26.4 (mm 263.9 )± 19.8 96.58 ± 7.62 1 76.7 1 4 Diclofenac3 Na– 516.3 397.6 ± ±15.811.9 304.9 306.6 ± 14.0± 9.36 211.4 ± 16.591.05 1,2,3± 5.52 1 48.9 78.0 ± ± ± 2 5 Control4 –H 568.6 768.7 ± 16.527.3 268.7 354.9 ± 7.5911.6 299.9 ± 20.1 413.7 1,2,332.2 27.5 0 3 1 DifferencesLornoxicam statistically – significant360.5 for± 26.4p ≤ 0.05 vs.263.9 control. ± 19.82 Differences96.58 statistically ± 7.62 1 significant for76.7p ≤ 0.05 vs. 4 Diclofenac.Diclofenac3 Differences – statistically397.6 significant± 11.9 for306.6p ≤ 0.05 ± 9.36 vs. Lornoxicam.91.05 ± 5.52 1 78.0 5 Control – 768.7 ± 27.3 354.9 ± 11.6 413.7 ± 32.2 0 Furthermore, the compounds synthesized deserve closer attention as potential analgesics 1 Differences statistically significant for p ≤ 0.05 vs. control. 2 Differences statistically significant for and further study with the involvement of other experimental models and possible chemical p ≤ 0.05 vs. Diclofenac. 3 Differences statistically significant for p ≤ 0.05 vs. Lornoxicam. optimization since they exceed Diclofenac and structurally similar Lornoxicam by the intensity of the analgesicFurthermore, effect the (Table compounds2). The causesynthesized for the deserve noticeable closer increase attention in itsas activitypotential in analgesics sodium salt and3 furthercompared study to acid with4 canthe beinvolvement its increased of bioavailability. other experimental However, models transition and from possible derivatives chemical of 6 optimization2,2-dioxo-1H since-2λ ,1-benzothiazine-3-carboxylic they exceed Diclofenac and structurally acids that are similar practically Lornoxicam insoluble by inthe water intensity to their of thereadily analgesic soluble effect forms (Table due 2). to saltThe formationcause for the is not noticeable always accompaniedincrease in its by activity an increase in sodium in analgesic salt 3 comparedproperties to [16 acid]. 4 can be its increased bioavailability. However, transition from derivatives of 2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acids that are practically insoluble in water to their Table 2. The Analgesic Activity of Sodium Salt 3, Acid 4, and Reference Drugs. readily soluble forms due to salt formation is not always accompanied by an increase in analgesic properties [16]. Pain Threshold on Pain Threshold on Analgesic Activity, Entry Product R Damaged Extremity Non-Damaged ∆Pain Threshold Compared to Control (g/mm2) Extremity (g/mm2) (%) Table 2. The Analgesic Activity of Sodium Salt 3, Acid 4, and Reference Drugs. 1 3 Na 350.0 ± 15.8 306.0 ± 19.6 44.0 ± 8.12 1,3 86.2 2 4 H 294.0 ± 32.3 208.0 ± 17.7 86.0 ± 18.6 1 73.0 Pain Pain Threshold 1,2 3 Lornoxicam – 441.0 ± 25.6 346.0 ± 23.4 95.0 ± 4.47 Analgesic70.1 4 Diclofenac –Threshold 738.0 ± 18.3 on 679.0 ± on25.4 59.0 ± 9.27 1 81.4 5 Control – 593.0 ± 56.3 275.0 ± 32.1 318.0 ±ΔPain34.9 Activity, 0 Entry Product R Damaged Non-Damaged 1 ≤ 2 Threshold Compared≤ to Differences statistically significantExtremity for p 0.05 vs. control.ExtremityDifferences statistically significant for p 0.05 vs. Diclofenac. 3 Differences statistically significant for p ≤ 0.05 vs. Lornoxicam. Control (%) (g/mm2) (g/mm2) 1,3 4. Conclusions1 3 Na 350.0 ± 15.8 306.0 ± 19.6 44.0 ± 8.12 86.2 2 4 H 294.0 ± 32.3 208.0 ± 17.7 86.0 ± 18.6 1 73.0 3 TheLornoxicam preparative method– for441.0 obtaining ± 25.6 4-methyl-2,2-dioxo-1346.0 ± 23.4 H-295.0λ6,1-benzothiazine-3-carboxylic ± 4.47 1,2 70.1 acid4 hasDiclofenac been proposed, – which738.0 allows ± 18.3 researchers 679.0 to± 25.4 synthesize 59.0 the ± 9.27 target 1 compound81.4 with a high5 yieldControl and purity. – It has been593.0 ± shown56.3 that275.0 as products± 32.1 of318.0 alkaline ± 34.9 hydrolysis0 of methyl 4-methyl-2,2-dioxo-11 Differences statisticallyH-2λ6 significant,1-benzothiazine-3-carboxylate, for p ≤ 0.05 vs. control. 2 Differences both statistically benzothiazine-3-carboxylic significant for acidp and≤ 0.05 its vs. monohydrate,Diclofenac. 3 Differences sodium statistically salt, as wellsignificant as 4-methyl-2,2-dioxo-1 for p ≤ 0.05 vs. Lornoxicam.H-2λ 6,1-benzothiazine, can be isolated. Using X-ray structural analysis, the peculiarities of the spatial 4.structure Conclusions of all compounds synthesized have been studied. The biological properties of 4-methyl-2,2-dioxo-1The preparative methodH-2λ6,1-benzothiazine-3-carboxylic for obtaining 4-methyl-2,2-dioxo acid-1H and-2λ6,1 its-benzothiazine sodium salt-3- havecarboxylic been acidtested. has been In addition, proposed, a moderatewhich allows anti-inflammatory researchers to synthesize activity of the the target substances compound studied with has a high been yielddetermined and purity. and their It high has analgesic been shown effect has that been as observed. products of alkaline hydrolysis of methyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate, both benzothiazine-3-carboxylic acid and Acknowledgments: We are grateful to Candidate of Chemistry Magda D.6 Tsapko (Taras Shevchenko National itsUniversity, monohydrate, Kiev, Ukraine) sodium for salt, her as help well in registrationas 4-methyl of-2,2 NMR-dioxo spectra-1H- of2λ the,1- compoundsbenzothiazine synthesized., can be isolated. Using X-ray structural analysis, the peculiarities of the spatial structure of all compounds Author Contributions: The synthesis of the compounds presented in this work and analysis of their characteristics synthesizedwere performed by have I.V.U. and G.M.H. been Derivatographic studied. and TheX-ray structural biological studies were properties performed by A.A.B. of 4and-methyl S.V.S.,-2,2 respectively.-dioxo-1H The-2λ6 pharmacological,1-benzothiazine studies-3-carboxylic were conducted acid and by N.I.V.its sodium and O.V.M. salt have The manuscript been tested. was Inwritten addition, by I.V.U., a moderate G.M.H., andanti O.V.M.-inflammatory activity of the substances studied has been determined andConflicts their ofhigh Interest: analgesicThe authorseffect has declare been no observed. conflict of interest.

Sci. Pharm. 2018, 86, 9 13 of 14

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