Scientia Pharmaceutica

Article The Crystal Structure of N-(1-Arylethyl)-4-methyl- 2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides as the Factor Determining Biological Activity Thereof

Igor V. Ukrainets 1,* , Ganna M. Hamza 1, Anna A. Burian 1 , Natali I. Voloshchuk 2 , Oxana V. Malchenko 2, Svitlana V. Shishkina 3,4, Irina A. Danylova 1 and Galina Sim 5

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.); [email protected] (I.A.D.) 2 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.) 3 STC “Institute for Single Crystals”, National Academy of Sciences of Ukraine, 60 Nauki ave., 61001 Kharkiv, Ukraine; [email protected] 4 Department of Inorganic Chemistry, V.N. Karazin Kharkiv National University, 4 Svobody sq., 61077 Kharkiv, Ukraine 5 Department of Pharmaceutical Chemistry, Far Eastern State Medical University, 35 Murav’eva-Amurskogo st., 680000 Khabarovsk, Russia; [email protected] * Correspondence: [email protected]; Tel.: +380-572-679-185



Received: 27 February 2019; Accepted: 4 April 2019; Published: 19 April 2019


Abstract: In order to detect new structural and biological patterns in a series of hetaryl-3-carboxylic acid derivatives, the optically pure (S)- and (R)-enantiomers of N-(1-arylethyl)-4-methyl- 2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides, their true racemates, and mechanical racemic mixtures have been synthesized in independent ways. The particular features of the 1Н-and 13C-NMR spectra of all synthesized substances, liquid chromato-mass spectrometric behavior thereof under electrospray ionization conditions, and also the results of polarimetric and X-ray diffraction studies have been discussed. Pharmacological screening on a model of carrageenan inflammation has found a clear relationship between the spatial structure of the studied objects and biological activity thereof. Enantiomers with chiral centers having (S)-configuration showed weak inhibition of pain and inflammatory reactions, while their mirror (R)-isomers exhibited very powerful analgesic and antiphlogistic properties under the same conditions, with the level of specific activity exceeding that of Lornoxicam and Diclofenac. Taking obtained data into account, a noticeable decrease in the activity of mechanical racemic mixtures, consisting of one-half of the “wrong” (S)-enantiomers, is quite natural. The true racemate of N-(1-phenylethyl)-amide proved itself in a similar way, while 4-methoxy-substituted analog thereof stood out against this background with unexpectedly high analgesic and anti-inflammatory activities. A comparative analysis of X-ray diffraction data has found that crystalline and molecular structure of racemic N-[1-(4-methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide is completely different from that of the original enantiomers and, moreover, very unusual for racemates. Obviously, it is the factor determining the unique character of the biological effects of the said substance.

Keywords: 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylamide; 2,1-benzothiazine; chiral switches, crystal structure; molecular conformation; analgesic activity; anti-inflammatory action

Sci. Pharm. 2019, 87, 10; doi:10.3390/scipharm87020010 www.mdpi.com/journal/scipharm Sci. Pharm. 2019, 87, 10 2 of 20

1. Introduction

ChiralInt. J. Mol. substances Sci. 2019, 20, x always FOR PEER attract REVIEW increased attention from researchers of various 2 of specialties. 21 The appearance of at least one asymmetric carbon atom in the molecule provides the substance with a number of uniqueChiral substances properties, always the study attract ofincreased which constitutesattention from the researchers range of of scientific various specialties. interests of The synthetic chemists,appearance analysts, of spectroscopists, at least one asymmetric optics, carbon crystallographers, atom in the molecule andvarious providesother the substance scientists. with However, a number of unique properties, the study of which constitutes the range of scientific interests of the practical benefits of studying chiral substances are, perhaps, to the greatest extent pharmacists and synthetic chemists, analysts, spectroscopists, optics, crystallographers, and various other scientists. medicalHowever, chemists. the It practical is true thatbenefits the of awareness studying chiral of the substances need for are, research perhaps, in to this the areagreatest did extent not come to them immediately.pharmacists and It was medical only chemists. after racemic It is true drugs that th weree awareness criticized of the for need containing for research 50% in of this impurities area [1] (although,did not in ourcome opinion, to them immediately. this is not alwaysIt was only true) after did racemic the pharmacydrugs were criticized take up for this containing problem 50% seriously. Even a wholeof impurities direction [1] (although, known inin theour literatureopinion, this as is “chiral not always switches” true) did appeared the pharmacy [2–5 ],take thanks up this to which problem seriously. Even a whole direction known in the literature as "chiral switches" appeared [2– the “lifetime” of a number of the originally racemic drugs was prolonged in the form of their more 5], thanks to which the "lifetime" of a number of the originally racemic drugs was prolonged in the active enantiomersform of their more [2,4] active (Figure enantiomers1). [2,4] (Figure 1).

Me OH H N Me Me COOH HO Me Me Me HO

Dexibuprofene Levalbuterol [(S)-(+)-ibuprofen] [(R)-(-)-salbutamol]

O F COOH Me Me O N N N N N O H Me Me Me

Levofloxacin Levobupivacaine [(S)-(-)-ofloxacin] [(S)-(-)-bupivacaine]

F

H Me N O N S N Me N O CF3 Me O N

Escitalopram Dexlansoprazole

[(S)-(+)-citalopram] [(R)-(+)-lansoprazole] Figure 1.FigureImproved 1. Improved enantiomerically enantiomerically pure pure “copies” "copies" of theof the known known drug-racemates drug-racemates createdcreated by thethe “chiral switches”"chiral technology switches" [technology2–4]. [2–4]. However, it should be kept in mind that obtaining enantiomerically pure substances in general, However, it should be kept in mind that obtaining enantiomerically pure substances in general, and drugs in particular, as a rule, is a technologically complicated and costly practice. In addition, all and drugsthe ineffort particular, expended as does a rule, notis inevitably a technologically lead to positive complicated results. When and costlycarrying practice. out pharmacological In addition, all the effort expendedtests of substances does not containing inevitably asymmetric lead to positive carbon results. atoms, researchers When carrying often outencounter pharmacological the fact that tests of substancesonly containing one of the enantiomers asymmetric causes carbon the desired atoms, biol researchersogical effect, often while encounter the mirror theisomer fact has that low only or one of the enantiomersno activity causes [6–9]. theIt is desiredwithin the biological realm of e ffpoect,ssibility while that the enantiomers mirror isomer would has exhibit low or completely no activity [6–9]. It is withindifferent, the realm and sometimes of possibility directly that opposite, enantiomers physiolo wouldgical properties exhibit completely [2], or one of di thefferent, isomers and would sometimes prove itself as unambiguously harmful or even dangerous [10]. Enantiomers sometimes demonstrate directly opposite, physiological properties [2], or one of the isomers would prove itself as unambiguously harmful or even dangerous [10]. Enantiomers sometimes demonstrate a completely identical clinical picture [11] that makes the “chiral switch” attempt irrelevant. One must also be prepared for the fact that Sci. Pharm. 2019, 87, 10 3 of 20 the separation of the racemate may not lead to products with individual activity because enantiomers in a living organism are easily metabolized into one another [2]. It is also possible that the racemate would be much more active than each of the enantiomers [12]. Usually, such cases are interpreted as manifestations of synergism of effects, which are peculiar to each of the mirror isomers, separately [13]. However, in the case of observable pharmacological effects caused by enantiomers and racemates formed thereof, this explanation may be quite insufficient. This is exactly the situation we encountered when studying N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides, which are the object of this message.

2. Materials and Methods

2.1. Chemistry 1Н-and 13C-NMR (proton and carbon nuclear magnetic resonance) spectra were obtained on a Varian Mercury-400 (Varian Inc., Palo Alto, CA, USA) instrument (400 and 100 MHz, respectively) in hexadeuterodimethyl sulfoxide (DMSO-d6) with tetramethylsilane as the internal standard. The chemical shift values were recorded on a δ scale and the coupling constants (J) in Hertz. The following abbreviations were used in reporting spectra: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet. The electrospray ionization liquid chromato-mass spectra (ESI-LC/MS) were recorded on a modular Agilent Technologies 1260 Infinity system with 6530 Accurate-Mass Q-TOF LC/MS (G6530B#200 ESI) mass-spectrometric detector (Agilent Technologies, Inc., Santa Clara, CA, USA). The chromatography conditions were: Agilent Extend-C18 column of 2.1 50 mm; the sorbent particle size—1.8 µm; the mobile phase flow rate—0.25 mL/min; the column × temperature—30 ◦C; the injection volume—1.0 µL; the mobile phase composition—0.1% formic acid in methanol. The elemental analysis was performed on a Euro Vector EA-3000 (Eurovector SPA, Redavalle, Italy) microanalyzer. The melting points were determined in a capillary using an electrothermal IA9100X1 (Bibby Scientific Limited, Stone, UK) digital melting point apparatus. The specific rotations of optically active amides 3 and 4 were measured on a WWZ-2S automatic polarimeter (Zhejiang Nade Scientific instrument Co., Ltd., Yuhang, Hangzhou, Zhejiang, China). Synthesis of these compound used commercial S(–)- and R(+)-1-phenyl- and 1-(4-methoxyphenyl)- ethylamines from Fluka company (Fluka Chemie AG, Buchs, Switzerland), with optical purities of at least 99.5 and 99.0% respectively. In the synthesis of all N-(1-arylethyl)-4-methyl-2,2-dioxo-1H- 2λ6,1-benzothiazine-3-carboxamides, 3–5 described in this article the commercial N,N0-carbonyldiimidazole (CDI) and the anhydrous N,N-dimethylformamide (DMF) for peptide synthesis of Aldrich company (St. Louis, MO, USA) were used. The synthesis of the starting anhydrous 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid (1) was carried out by the method described in [14].

2.2. General Procedure for the Synthesis of N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3- carboxamides 3, 4 and 5a

6 N,N0-Carbonyldiimidazole (1.78 g, 0.011 mol) was added to a solution of 4-methyl-2,2-dioxo-1H-2λ , 1-benzothiazine-3-carboxylic acid 1 (2.39 g, 0.01 mol) in anhydrous DMF (5 mL) and protected from atmospheric moisture using a CaCl2 tube. The mixture was then heated for about 2 h at 90 ◦C until the evolution of CO2 ceased. The reaction mixture was purged with dry argon through a thin capillary for 5 min to remove residual CO2, and then 0.01 mol of the corresponding 1-arylethylamine was added, and the mixture was kept for 5 h at the temperature of 90 ◦C in a tightly closed vial of thick dark glass (it is convenient to use vials of suitable volume designed for chemicals). The reaction mixture was cooled, diluted with cold water, and adjusted to pH ~3 with hydrochloric acid. The precipitate was formed, which was filtered off, washed with cold water, dried, and recrystallized from ethanol. 1-Arylethylamides 3, 4 and 5a were colorless crystals. Sci. Pharm. 2019, 87, 10 4 of 20

N-[(1S)-1-Phenylethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide (3a). The yield was: α 20 3.21 g (94%); colorless crystals; melting point (mp) 216–218 ◦C; [ ] D = +14.5◦, c = 5, dimethyl sulfoxide 1 (DMSO); H-NMR (400 MHz, DMSO-d6): δ 11.70 (br. s, 1H, SO2NH), 9.15 (d, 1H, J = 7.6, CONH), 7.73 (d, 1Н, J = 7.7, Н-5), 7.50 (t, 1H, J = 7.4, H-7), 7.42 (d, 2H, J = 7.1, H-20,60), 7.36 (t, 2H, J = 7.2, H-30,50), 7.27 (t, 1H, J = 6.3, H-40), 7.23 (t, 1H, J = 7.6, H-6), 7.15 (d, 1H, J = 7.8, H-8), 5.18-5.05 (m, 1H, NCН), 2.26 13 (s, 3H, 4-CН3), 1.45 (d, 3H, J = 6.7, CН-CН3). C-NMR (100 MHzd6ript6): δ 159.5 (C=O), 144.5, 139.3, 138.1, 131.8, 131.5, 128.8 (20,60-C), 127.6, 127.4, 126.8 (30,50-C), 123.2, 121.1, 118.5 (3-C), 49.1 (CН), 22.7 + (CHCH3), 17.7 (4-CH3). ESI-LC/MS (m/z, %): 343 (48) [M + H] , 239 (26), 222 (28), 105 (13). This was analytically calculated (Anal. Calcd.) for C18H18N2O3S: C, 63.14; H, 5.30; N, 8.18; S 9.36%. We found: C, 63.17; H, 5.39; N, 8.11; S 9.25%. N-[(1R)-1-Phenylethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide (4a). The yield was: 3.07 g (90%); colorless crystals; m.p. 216–218 C; [α] 20 = 14.5 , c = 5, DMSO; 1H-NMR (400 MHz, ◦ D − ◦ DMSO-d6): δ 11.70 (br. s, 1H, SO2NH), 9.15 (d, 1H, J = 7.6, CONH), 7.73 (d, 1Н, J = 7.7, Н-5), 7.50 (t, 1H, J = 7.4, H-7), 7.42 (d, 2H, J = 7.1, H-20,60), 7.36 (t, 2H, J = 7.2, H-30,50), 7.27 (t, 1H, J = 6.3, H-40), 7.23 (t, 1H, J = 7.6, H-6), 7.15 (d, 1H, J = 7.8, H-8), 5.18-5.05 (m, 1H, NCН), 2.26 (s, 3H, 4-CН3), 1.45 13 (d, 3H, J = 6.7, CН-CН3). C-NMR (100 MHz, DMSO-d6): δ 159.5 (C=O), 144.5, 139.3, 138.1, 131.8, 131.5, 128.8 (20,60-C), 127.6, 127.4, 126.8 (30,50-C), 123.2, 121.1, 118.5 (3-C), 49.1 (CН), 22.7 (CHCH3), 17.7 + (4-CH3). ESI-LC/MS (m/z, %): 343 (44) [M + H] , 239 (23), 222 (33), 105 (15). The Anal. Calcd. was for C18H18N2O3S: C, 63.14; H, 5.30; N, 8.18; S 9.36%. We found: C, 63.20; H, 5.37; N, 8.27; S 9.28%. N-(1-Phenylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide (5a). The yield was: 3.18 g 1 (93%); colorless crystals; m.p. 212–214 ◦C; H-NMR (400 MHz, DMSO-d6): δ 11.71 (br. s, 1H, SO2NH), 9.15 (d, 1H, J = 7.6, CONH), 7.72 (d, 1Н, J = 7.7, Н-5), 7.50 (t, 1H, J = 7.4, H-7), 7.42 (d, 2H, J = 7.1, H-20,60), 7.37 (t, 2H, J = 7.2, H-30,50), 7.27 (t, 1H, J = 6.3, H-40), 7.23 (t, 1H, J = 7.6, H-6), 7.16 (d, 1H, 13 J = 7.8, H-8), 5.18-5.04 (m, 1H, NCН), 2.26 (s, 3H, 4-CН3), 1.45 (d, 3H, J = 6.7, CН-CН3). C-NMR (100 MHz, DMSO-d6): δ 159.5 (C=O), 144.5, 139.3, 138.1, 131.8, 131.5, 128.8 (20,60-C), 127.6, 127.4, 126.8 (30,50-C), 123.2, 121.1, 118.5 (3-C), 49.1 (CН), 22.7 (CHCH3), 17.7 (4-CH3). ESI-LC/MS (m/z, %): 343 (40) + [M + H] , 239 (22), 222 (25), 105 (10). The Anal. Calcd. was for C18H18N2O3S: C, 63.14; H, 5.30; N, 8.18; S 9.36%. We found: C, 63.22; H, 5.41; N, 8.25; S 9.30%. N-[(1S)-1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide (3b). The yield α 20 1 was: 3.34 g (90%); colorless crystals; m.p. 183–185.◦C; [ ] D = +15.2◦, c = 5, DMSO; H-NMR (400 MHz, DMSO-d6): δ 11.69 (br. s, 1H, SO2NH), 9.07 (d, 1H, J = 7.8, CONH), 7.70 (d, 1Н, J = 7.9, Н-5), 7.45 (t, 1H, J = 7.6, H-7), 7.32 (d, 2H, J = 8.1, H-20,60), 7.21 (t, 1H, J = 7.7, H-6), 7.12 (d, 1H, J = 8.0, H-8), 6.90 (d, 2H, J = 8.0, H-30,50), 5.14-5.04 (m, 1H, NCН), 3.74 (s, 3H, 40-OMe), 2.23 (s, 3H, 4-CН3), 1.41 (d, 3H, J = 6.8, 13 CН-CН3). C-NMR (100 MHz, DMSO-d6): δ 159.5 (C=O), 158.6 (40-C-О), 139.3, 137.9, 136.3, 131.8, 131.6, 127.8 (20,60-C), 127.5, 123.2, 121.2, 118.5 (3-C), 114.1 (30,50-C), 55.5 (OCH3), 48.7 (CH), 22.7 (CHCH3), 17.6 + (4-CH3). ESI-LC/MS (m/z, %): 373 (39) [M + H] , 265 (27), 222 (14), 108 (2). The Anal. Calcd. was for C19H20N2O4S: C, 61.27; H, 5.41; N, 7.52; S 8.61. We found: C, 61.34; H, 5.49; N, 7.58; S 8.54%. N-[(1R)-1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide (4b). The yield was: 3.27 g (88%); colorless crystals; m.p. 183–185 C; [α] 20 = 15.2 , c = 5, DMSO; 1H-NMR (400 MHz, ◦ D − ◦ DMSO-d6): δ 11.68 (br. s, 1H, SO2NH), 9.07 (d, 1H, J = 7.8, CONH), 7.71 (d, 1Н, J = 7.9, Н-5), 7.45 (t, 1H, J = 7.6, H-7), 7.33 (d, 2H, J = 8.1, H-20,60), 7.21 (t, 1H, J = 7.7, H-6), 7.13 (d, 1H, J = 8.0, H-8), 6.90 (d, 2H, J = 8.0, H-30,50), 5.15-5.04 (m, 1H, NCН), 3.74 (s, 3H, 40-OMe), 2.23 (s, 3H, 4-CН3), 1.41 (d, 3H, J = 6.8, 13 CН-CН3). C-NMR (100 MHz, DMSO-d6): δ 159.5 (C=O), 158.6 (40-C-О), 139.3, 137.9, 136.3, 131.8, 131.6, 127.8 (20,60-C), 127.5, 123.2, 121.2, 118.5 (3-C), 114.1 (30,50-C), 55.5 (OCH3), 48.7 (CH), 22.7 (CHCH3), 17.6 + (4-CH3). ESI-LC/MS (m/z, %): 373 (43) [M + H] , 265 (25), 222 (19), 108 (1). The Anal. Calcd. was for C19H20N2O4S: C, 61.27; H, 5.41; N, 7.52; S 8.61. We found: C, 61.21; H, 5.35; N, 7.45; S 8.53%. Sci. Pharm. 2019, 87, 10 5 of 20

2.3. N-[1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide (5b) 1.86 g (0.005 mol) of N-[(1S)-1-(4-methoxyphenyl)ethyl]-amide 3b and 1.86 g (0.005 mol) of N-[(1R)-1-(4-methoxyphenyl)ethyl]-amide 4b were dissolved in 35 ml of boiling ethanol and filtered. The filtrate was cooled and left for 12 h at a temperature of about 10 ◦C. The precipitated crystals of racemate 5b were filtered off and air dried. The yield was: 3.57 g (96%); colorless crystals; m.p. 177–179 ◦C.

2.4. Mechanical Racemic Mixture of Optically Active N-(1-Phenylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1- benzothiazine-3-carboxamides (6a) 1.71 g (0.005 mol) of N-[(1S)-1-phenylethyl]-amide 3a and 1.71 g (0.005 mol) of N-[(1R)-1- phenylethyl]-amide 4a were thoroughly mixed until a homogeneous mass was formed. The yield was quantitative; colorless crystals; m.p. 207–209 ◦C.

2.5. Mechanical Racemic Mixture of Optically Active N-[1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo- 1H-2λ6,1-benzothiazine-3-carboxamides (6b) 1.86 g (0.005 mol) of N-[(1S)-1-(4-methoxyphenyl)ethyl]- amide 3b and 1.86 g (0.005 mol) of N-[(1R)-1-(4-methoxyphenyl)ethyl]-amide 4b were mixed thoroughly until a homogeneous mass was formed. The yield was quantitative; colorless crystals; m.p. 168–170 ◦C.

2.6. X-ray Structural Analysis of N-[(1S)-1-Phenylethyl]-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3- carboxamide (3a)

The crystals of (1S)-1-phenylethylamide 3a (C18H18N2O3S) were orthorhombic and colorless. 3 3 At 20 ◦C: a 7.8554(5), b 8.8432(6), c 24.288(2) Å; V 1687.2(2) Å , Z 4, space group P212121, dcalc 1.348 g/cm , 1 µ(MoKα) 0.210 mm− , F(000) 720. The unit cell parameters and intensities of 10,394 reflections (4805 independent reflections, Rint = 0.077) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited, Oxford, UK) using MoKα radiation, a CCD detector, 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, Göttingen, Germany) [15]. The positions of the hydrogen atoms were found from the electron density difference maps and refined using the “riding” 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 hydrogen atoms of amino groups were refined within isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.119 for 4794 reflections (R1 0.060 for 2317 reflections with F > 4σ (F), S = 0.910). The final atomic coordinates, and the crystallographic data for the molecule of (1S)-1-phenylethylamide 3a have been deposited to with the Cambridge Crystallographic Data Centre, 12 Union Road, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: [email protected]) and are available on request quoting the deposition number CCDC 1899013 [16].

2.7. X-ray Structural Analysis of N-(1-Phenylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3- carboxamide (5a)

The crystals of racemic 1-phenylethylamide 5a (C18H18N2O3S) were monoclinic and colorless. 3 At 20 ◦C: a 7.7803(9), b 8.8579(6), c 24.674(3) Å; β 98.37(1)◦; V 1682.3(3) Å , Z 4, space group P21/с, 3 1 dcalc 1.352 g/cm , µ(MoKα) 0.211 mm− , F(000) 720. The unit cell parameters and intensities of 12,542 reflections (2959 independent reflections, Rint = 0.074) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) using MoKα radiation, a CCD detector, 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) [15]. The positions of the hydrogen atoms were found from the electron density difference maps and refined using the “riding” 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 Sci. Pharm. 2019, 87, 10 6 of 20 hydrogen atoms). The hydrogen atoms of amino groups were refined within isotropic approximation. The hydrogen atoms of amino groups were refined within isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.257 for 2801 reflections (R1 0.090 for 2075 reflections with F > 4σ (F), S = 1.054). The final atomic coordinates and the crystallographic data for the molecule of racemic 1-phenylethylamide 5a have been deposited to with the Cambridge Crystallographic Data Centre, 12 Union Road, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: [email protected]) and are available on request quoting the deposition number CCDC 1899015 [17].

2.8. X-ray Structural Analysis of N-[(1S)-1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6, 1-benzothiazine-3-carboxamide (3b)

The crystals of N-[(1S)-1-(4-methoxyphenyl)ethyl]-amide 3b (C19H20N2O4S) were monoclinic 3 and colorless. At 20 ◦C: a 13.490(2), b 9.2913(9), c 15.033(2) Å; β 103.46(1)◦; V 1832.4(4) Å , Z 4, space 3 1 group Р21, dcalc 1.350 g/cm , µ(MoKα) 0.203 mm− , F(000) 784. The unit cell parameters and intensities of 18,921 reflections (10,140 independent reflections, Rint = 0.082) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) using MoKα radiation, a Charge Coupled Device (CCD) detector, 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) [15]. The positions of the hydrogen atoms were found from the electron density difference map and refined using the “riding” 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 hydrogen atoms participating in the formation of intermolecular hydrogen bonds were refined in an isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.215 for 9948 reflections (R1 0.093 for 4991 reflections with F > 4σ (F), S 0.954). The final atomic coordinates, and the crystallographic data for the molecule of N-[(1S)-1-(4-methoxyphenyl)ethyl]-amide 3b have been deposited with the Cambridge Crystallographic Data Centre, 12 Union Road, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: [email protected]) and are available on request quoting the deposition number CCDC 1899014 [18].

2.9. X-ray Structural Analysis of N-[(1R)-1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6, 1-benzothiazine-3-carboxamide (4b)

The crystals of N-[(1R)-1-(4-methoxyphenyl)ethyl]-amide 4b (C19H20N2O4S) were monoclinic 3 and colorless. At 20 ◦C: a 13.491(1), b 9.2746(6), c 15.050(2) Å; β 103.644(8)◦; V 1829.8(3) Å , Z 4, space 3 1 group Р21, dcalc 1.352 g/cm , µ(MoKα) 0.204 mm− , F(000) 784. The unit cell parameters and intensities of 18,889 reflections (10,263 independent reflections, Rint = 0.077) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) using MoKα radiation, a Charge Coupled Device (CCD) detector, 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) [15]. The positions of the hydrogen atoms were found from the electron density difference map and refined using the “riding” 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 hydrogen atoms participating in the formation of intermolecular hydrogen bonds were refined in an isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.119 for 10,185 reflections (R1 0.065 for 4502 reflections with F > 4σ (F), S 0.887). The final atomic coordinates, and the crystallographic data for the molecule of N-[(1R)-1-(4-methoxyphenyl)ethyl]-amide 4b have been deposited to with the Cambridge Crystallographic Data Centre, 12 Union Road, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: [email protected]) and are available on request quoting the deposition number CCDC 1899016 [19]. Sci. Pharm. 2019, 87, 10 7 of 20

2.10. X-ray Structural Analysis of N-[1-(4-Methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ6, 1-benzothiazine-3-carboxamide (5b)

The crystals of racemic N-[1-(4-methoxyphenyl)ethyl]-amide 5b (C19H20N2O4S) were 3 orthorhombic and colorless. At 20 ◦C: a 16.644(2), b 24.533(3), c 9.0188(7) Å; V 3682.5(6) Å , Z 8, 3 1 space group Pca21, dcalc 1.344 g/cm , µ(MoKα) 0.203 mm− , F(000) 1568. The unit cell parameters and intensities of 22,382 reflections (6429 independent reflections, Rint = 0.143) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) using MoKα radiation, a Charge Coupled Device (CCD) detector, 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) [15]. The positions of the hydrogen atoms were found from the electron density difference map and refined using the “riding” 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 hydrogen atoms participating in the formation of intermolecular hydrogen bonds were refined in an isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.091 for 6405 reflections (R1 0.063 for 3090 reflections with F > 4σ (F), S 0.865). The final atomic coordinates and the crystallographic data for the molecule of racemic N-[1-(4-methoxyphenyl)ethyl]-amide 5b have been deposited to with the Cambridge Crystallographic Data Centre, 12 Union Road, CB2 1EZ, UK (fax: +44-1223-336033; e-mail: [email protected]) and are available on request quoting the deposition number CCDC 1899017 [20].

2.11. Pharmacology

Analgesic and Anti-Inflammatory Tests 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” [21] (project ID 3410U14, approved 15 October 2015). The pharmacological research was carried out with the permission and under the supervision of the Commission on Bioethics (N.I. Pirogov Vinnitsa National Medical University, Vinnitsa, Ukraine). The analgesic effect with the simultaneous assessment of the anti-inflammatory activity of all N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides 3–6a,b synthesized was studied on the standard model of carrageenan edema [22,23]. The studies were conducted on white Wistar male rats weighing 200–250 g. The test substances, Lornoxicam (Wasserburger Arzneimittelwerk GmbH, Wasserburger, Germany) and Diclofenac (KRKA, Novo Mesto, Slovenia) were introduced intraperitoneally in the form of fine aqueous suspensions stabilized with Tween-80 in the screening dose of 20 mg/kg. The animals of the control group received an equivalent amount of water with Tween-80. Other details of pharmacological experiments, as well as the statistical processing of the results, were described in detail earlier [14].

3. Results and Discussion

3.1. Chemistry Chiral pharmaceutical substances are prepared by various methods. First of all, it is an obvious, although far from easily accomplished, separation of racemic mixtures which were originally synthesized or isolated from natural raw materials. The options for the practical implementation of this approach are constantly being improved upon and at present they allow obtaining the desired enantiomer having a high degree of optical purity, on a commercial scale [24–27]. Unfortunately, despite all the positive aspects, the separation of racemates always has one major drawback residing in the fact that it can be considered effective only if all the isolated components of the mixture find a practical use. Otherwise, at least half of the finished product will just become fruitless ballast. Therefore, more Sci. Pharm. 2019, 87, 10 8 of 20 attention has been paid to the development of modern technologies of asymmetric synthesis, which would allow to immediately obtain biologically active substances with the known-good configuration of chiral centers [28–31]. The assembling of compounds of complex structure based on commercially available enantiomerically pure building blocks of natural or synthetic origin (chiral pool synthesis) has gained particular popularity [32]. In this case, the chiral fragment, as a rule, is introduced into the molecule at the final stage of the synthetic scheme, which allows for minimizing the risk of possible racemization. This strategy was implemented in the synthesis of the target objects in the λ6 presentInt. J. study: Mol. Sci. the2019, previously 20, x FOR PEER formed REVIEWbicyclic 4-methyl-2,2-dioxo-1H-2 ,1-benzothiazine-3-carboxylic 8 of 21 acid (1) was transformed into imidazolide 2, which was further reacted with optical high-purity S(–)-benzothiazine-3-carboxylicand R(+)-1-phenyl- and acid 1-(4-methoxyphenyl)-ethylamines (1) was transformed into imidazolide 2 forming, which was corresponding further reactedchiral N-(1-arylethyl)-4-methyl-2,2-dioxo-1with optical high-purity S(–)- and H-R(+)-2λ1-phenyl-6,1-benzothiazine-3-carboxamides and 1-(4-methoxyphenyl)-ethylamines3–4 (Scheme forming1). By the samecorresponding principle, except chiral N- using(1-arylethyl)-4-methyl-2,2-dioxo-1 racemic 1-phenylethylamine,H-2λ racemate6,1-benzothiazine-3-carboxamides5a (R = H) was synthesized, 3-4 which(Scheme is of 1). interest By the forsame structural principle, andexcept biological using racemic comparative 1-phenylethylamine, studies. Dueracemate to lack5а (R of = H) racemic was synthesized, which is of interest for structural and biological comparative studies. Due to lack of 1-(4-methoxyphenyl)ethylamine, 4-methoxy-substituted analog thereof, 5b (R = OMe), was obtained racemic 1-(4-methoxyphenyl)ethylamine, 4-methoxy-substituted analog thereof, 5b (R = OMe), was by crystallizationobtained by crystallization of a mixture of of a enantiomersmixture of enantiomers3b and 4b 3bin and a 1:1 4b ratioin a 1:1 from ratio ethanol. from ethanol. As will As bewill shown below,be theshown samples below, obtained the samples in thisobtained way in were this way true were single true crystal single racemates, crystal racemates, i.e., each i.e., crystal each crystal contained bothcontained enantiomers both in enantiomers a 1:1 ratio. Thisin a 1:1 is their ratio. fundamental This is their difundamentalfference from difference mechanical fromracemic mechanical mixtures 6a,b,racemic obtained mixtures by simple 6a,b mixing, obtained of equimolar by simple amounts mixing of of eq theuimolar corresponding amounts opticallyof the corresponding pure enantiomers 3 andoptically4, also pure in a 1:1enantiomers ratio, but 3 withoutand 4, also subsequent in a 1:1 ratio, crystallization. but without subsequent crystallization.

CDI, DMF, 80 oC

1 2

(S)-1-arylethylamine (R)-1-arylethylamine Racemic 1-phenylethylamine

3b Me Me Crystallisation

3a,b 4a,b 5a,b

Mixing in a 1:1 ratio

Me Me

6a,b SchemeScheme 1. Synthesis 1. Synthesis of chiral ofN -(arylethyl)chiral N-(arylethyl)-4-methyl-2,2-dioxo-1-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-H-2λ6,1-benzothiazine- 3-carboxamides 3- 3 and 4carboxamides, their true racemates 3 and 5 4and, their mechanical true racemates racemic mixtures 5 and6 . mechanical3–6: a R = H; racemicb R = OMe. mixtures 6. 3-6: а R = H; b R = OMe.

All obtained N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides are colorless crystalline substances, which at room temperature are moderately soluble in DMSO and DMF, slightly soluble in alcohols, and insoluble in water. Taking into account the structure of the studied compounds (the presence of benzothiazine bicycle, aryl ethylamide fragments, several methyl groups in different chemical environments, optical activity, etc.), we have used a standard set of analytical methods to confirm their structure: elemental analysis, 1Н- and 13С-NMR spectroscopy,

Sci. Pharm. 2019, 87, 10 9 of 20

All obtained N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides are colorless crystalline substances, which at room temperature are moderately soluble in DMSO and DMF, slightly soluble in alcohols, and insoluble in water. Taking into account the structure of the studied compounds (the presence of benzothiazine bicycle, aryl ethylamide fragments, several methyl groups inInt. diJ. Mol.fferent Sci. 2019 chemical, 20, x FOR environments, PEER REVIEW optical activity, etc.), we have used a standard set of analytical 9 of 21 methods to confirm their structure: elemental analysis, 1Н-and 13C-NMR spectroscopy, electrospray ionizationelectrospray liquid ionization chromato-mass liquid chromato-mass spectrometry, sp andectrometry, polarimetry. and However, polarimetry. the mostHowever, important the most and interestingimportant and features interesting of the features spatial structure of the spatial of this struct groupure of this substances group of in subs thetances crystalline in the phase crystalline have beenphase detected have been using detected X-ray using diffraction. X-ray diffraction. As expected, 11HH and and 1313C CNMR NMR spectra spectra of ofall allsamples samples in each in each of the of thetriads: triads: (S)-enantiomer(S)-enantiomer – (R) –- (R)enantiomer-enantiomer – racemate – racemate (for (for example, example, 3a −3a 4a–4a − –5a5a)) were were the the same. same. All All the the signals signals of of protons protons and most of thethe signals signals of theof carbonthe carbon atoms atoms composing composing these spectra these werespectra easily were interpreted easily interpreted by the characteristic by the chemicalcharacteristic shifts, chemical intensity, shifts, and multiplicity intensity, and (see Sectionmultiplicity 2.2). In(see other Section words, 2.2). NMR In spectroscopyother words, allowsNMR reliablespectroscopy confirmation allows ofreliable the chemical confirmation structure of ofthe the chemical studied structure substances, of andthe evenstudied their substances, purity to some and extenteven their (on thepurity absence to some of “irrelevant”extent (on the peaks absence in the of experimental "irrelevant" peaks spectra). in the experimental spectra). Similar information is provided by electrospray ionization liquid liquid chromato-mass chromato-mass spectrometry, spectrometry, which, in addition to chemical identity, makes it possiblepossible toto establishestablish anan analyticallyanalytically important molecular weight weight of of a substance. a substance. Additional Additional information information on the onstructure the structure of the sample of the is sample provided is providedby the analysis by the of the analysis initial molecule of the initial fragments molecule formed fragments upon registration formed uponof the mass registration spectrum. of For the massexample, spectrum. it has been For observed example, that, it under has been the influence observed of that,electrospray under theionization, influence N-(1-phenylethyl)- of electrospray ionization,amides 3a, 4a,N-(1-phenylethyl)-amides and 5a behave in the same3a, 4a,mannerand 5aandbehave firstly inform the protonated same manner molecular and firstly ions form[M + protonatedH]+ (Scheme molecular 2). Primary ions fragmentation [M + H]+ (Scheme of said 2substances). Primary occurs fragmentation similarly ofto saidN-benzyl-4-methyl-2,2- substances occurs similarlydioxo-1H-2 toλ6N,1-benzothiazine-3-carboxamides-benzyl-4-methyl-2,2-dioxo-1H-2 asλ6 ,1-benzothiazine-3-carboxamidesdescribed in our earlier works [33], as i.e., described with the in ourbenzyl earlier bond works -CONH [33‒],CH(Me)Ph i.e., with the breaking benzyl ( bondβ-decay –CONH–CH(Me)Ph [34]). This produces breaking phenethyl (β-decay cation [ 347 and]). This the producesunstable protonated phenethyl cation of7 and4-methyl-2,2-dioxo-1 the unstable protonatedH-2λ6,1- cationbenzothiazine-3-carboxamide of 4-methyl-2,2-dioxo-1 8H, -2whichλ6,1- benzothiazine-3-carboxamiderapidly loses NH3 molecule and8, becomes which rapidly more losesstable, NH and3 moleculetherefore andtypical becomes for all morederivatives stable, of and 4- thereforemethyl-2,2-dioxo-1 typical forH all-2λ derivatives6,1-benzothiazine-3-carboxylic of 4-methyl-2,2-dioxo-1 acid acylium-cationH-2λ6,1-benzothiazine-3-carboxylic 9 with m/z 222 (Scheme acid 2). acylium-cationHowever, the mass9 with spectrometricm/z 222 (Scheme behavior2). of the However, phenethylamides the mass 3a spectrometric, 4a and 5a were behavior different of from the phenethylamidesthat of the benzylamides3a, 4a mentionedand 5a were above. diff erentIn particular, from that one of of the the benzylamidesdirections of primary mentioned destruction above. Inof protonated particular, molecular one of the ions directions in their ofspectra primary impl destructionying the breaking of protonated of the terminal molecular amide ions bond in -CO their‒ spectraNHCH(Me)Ph, implying which the breaking was usual of the for terminal 4-methyl-2,2-dioxo-1 amide bond –CO–NHCH(Me)Ph,H-2λ6,1-benzothiazine-3-carboxamides, which was usual for 4-methyl-2,2-dioxo-1was not observable. H-2λ6,1-benzothiazine-3-carboxamides, was not observable.

Me

3a: [M + H]+: 343 (48%)

HC+

- NH3

9: 222 (28%) 8: 239 (26%) 7: 105 (13%) Scheme 2.2. The primaryprimary fragmentation fragmentation of of the theN -[N(1S)-[(1S)-1-phenylethyl]-amide-1-phenylethyl]-amide3a protonated3a protonated molecular molecular ion. ion.

When going to 4-methoxy-substituted analogs (enantiomers 3,4b and racemate 5b), a part of the regularities mentioned above remains. At the same time, the 4-methoxy group of the phenethyl fragment implies its peculiarity on the mass spectrometric behavior of the studied compounds. We have found that it promotes the appearance of a new and very unusual path of primary fragmentation of protonated molecular ions for the studied class of compounds. During its implementation, the bond between the aryl substituent and the ethylcarbamide fragment breaks first (α-decay [34]),

Sci. Pharm. 2019, 87, 10 10 of 20

When going to 4-methoxy-substituted analogs (enantiomers 3,4b and racemate 5b), a part of the regularities mentioned above remains. At the same time, the 4-methoxy group of the phenethyl fragment implies its peculiarity on the mass spectrometric behavior of the studied compounds. We Int.have J. Mol. found Sci. 2019 that, it20, promotes x FOR PEER the REVIEW appearance of a new and very unusual path of primary fragmentation 10 of 21 of protonated molecular ions for the studied class of compounds. During its implementation, the bond resultingbetween thein the aryl peaks substituent of 4-methoxyphenyl and the ethylcarbamide 10 and benzothiazine-3- fragment breaks carbonylaminoethyl first (α-decay [34]), resulting 11 cations in withthe peaks m/z 108 of 4-methoxyphenyland 265, respectively10 and(Scheme benzothiazine-3- 3). carbonylaminoethyl 11 cations with m/z 108 and 265, respectively (Scheme3).

Me

3b: [M + H]+: 373 (39%)

10: 108 (3%) 11: 265 (27%) Scheme 3. TheThe primary primary fragmentation fragmentation of of the the NN-[-[(1S)(1S)-1-(4-methoxyphenyl)ethyl]-amide-1-(4-methoxyphenyl)ethyl]-amide 3b protonatedprotonated molecular ion.

Finally, another another important important analytical analytical characteristic characteristic of the studied of the N-(1-arylethyl)- studied N- 4-methyl-2,2-(1-arylethyl)- dioxo-14-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamidesH-2λ6,1-benzothiazine-3-carboxamides 3 and 4, which3 requiresand 4, which a mandatory requires definition, a mandatory are theirdefinition, specificare rotation their values. specific Our rotation polarimetric values. studies Ourconfirmed polarimetric that N-(1-arylethyl)-amides studies confirmed 3 thatand 4N- are(1-arylethyl)-amides true optically active3 and substances.4 are true opticallyMoreover, active the substances.fact that enantiomer Moreover, pairs the fact 3a–4a that and enantiomer 3b–4b, obtainedpairs 3a– 4ain independentand 3b–4b, obtainedways using in independentdifferent asymmetric ways using reagents different had asymmetricthe same specific reagents rotation had valuesthe same (see specific Section rotation 2.2), which values were (see different Section only2.2), in which sign, werewas a di fairlyfferent reliable only in hallmark sign, was of aoptical fairly high-purityreliable hallmark [35]. Meanwhile, of optical high-puritythe issue of [the35]. true Meanwhile, configuration the issueof the ofobtained the true chiral configuration amides 3a,b of andthe obtained4a,b may arise chiral from amides the fact3a,b thatand they4a,b rotatedmay the arise plane from of thepolarization fact that in they the opposite rotated the direction plane thanof polarization the original in 1-arylethylamines. the opposite direction However, than thethis originalfact, though 1-arylethylamines. curious, is not principal, However, since this fact,the configurationthough curious, of isthe not substance principal, and since the thedirection configuration of the polarization of the substance plane androtation the directionare completely of the differentpolarization characteristics plane rotation unrelated are completely to each other. different It is characteristics well known unrelatedthat specific to eachrotation other. values It is welland theirknown sign that (+ specificor –) can rotation vary greatly values andin the their same sign substance (+ or ) depending can vary greatly on concentration, in the same substancenature of − solvent,depending temperature, on concentration, etc. [35,36]. nature In addition, of solvent, as will temperature, be shown etc.below, [35 ,neit36].her In racemization addition, as nor will the be conversionshown below, of configuration neither racemization occurred nor in the the synthesis conversion of N- of(1-arylethyl)-4-methyl-2,2-dioxo-1 configuration occurred in the synthesisH-2λ6,1- of benzothiazine-3-carboxamidesN-(1-arylethyl)-4-methyl-2,2-dioxo-1 3 andH- 42,λ 6i.e.,,1-benzothiazine-3-carboxamides asymmetric carbon atoms in3 theand finished4, i.e., asymmetric products retainedcarbon atoms the configuration in the finished of productsthe original retained chiral the building configuration blocks. of the original chiral building blocks.

3.2. Evaluation of the Analgesic and Anti-Inflammatory Activity A comparative analysis of the data obtained from the pharmacological tests of N-(1-arylethyl)- 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides (Tables 1 and 2) revealed a close relationship between their spatial structure and biological activity. Therefore, enantiomers 3a,b with chiral centers having (S)-configuration showed extremely weak inhibition of the pain reaction, whereas their mirror isomers having (R)-configuration 4a,b were very powerful analgesics, superior to lornoxicam and diclofenac, under the same conditions. Such a significant difference in the activity

Sci. Pharm. 2019, 87, 10 11 of 20

3.2. Evaluation of the Analgesic and Anti-Inflammatory Activity A comparative analysis of the data obtained from the pharmacological tests of N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides (Tables1 and2) revealed a close relationship between their spatial structure and biological activity. Therefore, enantiomers 3a,b with chiral centers having (S)-configuration showed extremely weak inhibition of the pain reaction, whereas their mirror isomers having (R)-configuration 4a,b were very powerful analgesics, superior to lornoxicam and diclofenac, under the same conditions. Such a significant difference in the activity of optical antipodes indicates that their interaction with pain receptors includes at least three sites. With regards to the anti-inflammatory properties of the enantiomeric pairs 3a–4a and 3b–4b, the similar structural and biological patterns have been detected, although in a slightly less pronounced form. Stereospecificity of action was demonstrated here only by 4-methoxy derivatives 3b and 4b; meanwhile their unsubstituted analogs 3a and 4a exhibit an anti-exudative effect of similar strength regardless of the configuration of the chiral center (Table2). However, the appearance of a direct relationship between the spatial structure of N-R-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine- 3-carboxamides and analgesic activity thereof in the absence of any anti-inflammatory activity has been noted in our earlier works [33]. The significant difference between the biological properties of enantiomers of N-(1-arylethyl)- 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides 3 and 4 have been, of course, unknown without experimental testing, but it was quite predictable. Meanwhile, the pharmacological study of their true racemates 5 and racemic mixtures 6 presented some very interesting surprises. At the same time, nothing unusual was observed in the behavior of racemic N-(1-phenylethyl)-amide 5a; as one would expect, it showed extremely weak analgesic activity because it consists of one half of the “wrong” (S)-enantiomer 3a. It was not surprising that the racemic mixture 6a showed approximately the same result (Table1). For the same reason, a weak analgesic e ffect of the racemic mixture 6b was quite predictable. Moreover, only racemic N-[1-(4-methoxyphenyl)ethyl]-amide 5b did not fit into this almost perfect picture and clearly stood out against the background of other samples due to surprisingly high activity. We tried to find an explanation for this phenomenon in a detailed study of the spatial structure of both the racemate 5b itself and analogs thereof presented in this work. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 21 Sci. Pharm. 2019, 87, 10 12 of 20

Table 1. The analgesic activity of N-(1-arylethyl)-amides 3-6, and reference drugs. Table 1. The analgesic activity of N-(1-arylethyl)-amides 3–6, and reference drugs.

*

ConfigurationConfiguration of PainPain Thresholdon Threshold Damaged PainPain Threshold Threshold onon Non-DamagedNon-Damaged Δ Pain AnalgesicAnalgesic Activity, Activity, EntryEntry Product Product R R ∆ Pain Threshold Chiralof Chiral Centre Centre on DamagedExtremity Extremity (g/mm (g/mm2) 2) ExtremityExtremity (g (g/mm/mm22)) Threshold ComparedCompared to to Control Control (%) (%) 1,2,3 1 1 3a 3a (S)(S) H H 514.0 514.0 ± 33.233.2 265.0 265.0 ±29.0 29.0 249.0 249.022.1 ± 22.11,2,3 +21.7+21.7 ± ± ± 1,2,3 2 2 3b 3b (S)(S) OMeOMe 450.0 450.0 ± 29.329.3 236.0 236.0 ±15.6 15.6 214.0 214.019.0 ± 19.01,2,3 +32.7+32.7 ± ± ± 1,2,3 3 3 4a 4a (R)(R) H H 369.0 369.0 ± 15.015.0 335.2 335.2 ±24.1 24.1 33.8 33.85.2 ± 5.21,2,3 +89.4+89.4 ± ± ± 4 4 4b 4b (R)(R) OMeOMe 416.0 416.0 ± 37.937.9 382.0 382.0 ±32.2 32.2 34.0 34.05.5 ± 5.51,2 1,2 +89.3+89.3 ± ± ± 5 5 5a 5a (±)( )H H 469.0 469.0 ± 20.120.1 228.0 228.0 ±10.1 10.1 241.0 241.042.2 ± 42.21,2,3 1,2,3 +24.2+24.2 ± ± ± ± 6 6 5b 5b (±)( )OMe OMe 408.0 408.0 ± 10.310.3 333.0 333.0 ±18.5 18.5 75.0 75.07.3 ± 7.31,2,3 1,2,3 +76.4+76.4 ± ± ± ± 7 7 6a 6a (S)(S) & &(R)(R) H H 501.0 501.0 ± 54.454.4 282.0 282.0 ±15.4 15.4 219.0 219.029.8 ± 29.81,2 1,2 +31.1+31.1 ± ± ± 8 8 6b 6b (S)(S) & &(R)(R) OMeOMe 356.0 356.0 ± 15.215.2 186.0 186.0 ±12.6 12.6 170.0 170.033.4 ± 33.41,2 1,2 +46.5+46.5 ± ± ± 9 9Lornoxicam Lornoxicam − − 441.0441.0 ± 33.133.1 346.0 346.0 ±30.2 30.2 95.0 95.012.7 ± 12.71 1 +70.1+70.1 − − ± ± ± 10 10Diclofenac Diclofenac − − 538.0538.0 ± 23.623.6 479.0 479.0 ±32.8 32.8 59.0 59.015.3 ± 15.31 1 +81.4+81.4 − − ± ± ± 1 11 Control − − 593.0 62.7 257.0 41.4 318.0 58.2 1 0 11 Control − − 593.0 ± 62.7± 257.0± ± 41.4 318.0± ± 58.2 0 1 DifferencesDifferences statistically statistically significant significant for p for0.05 p ≤ vs. 0.05 non-damaged vs. non-damaged extremity; extremity;2 Differences 2 Differences statistically significantstatistically for significantp 0.05 vs. Diclofenac;for p ≤ 0.053 Divs.ff erencesDiclofenac; statistically 3 Differences significant statistically for p 0.05 vs. Lornoxicam. ≤ ≤ ≤ significant for p ≤ 0.05 vs. Lornoxicam.

Table 2. The anti-inflammatory activity of N-(1-arylethyl)-amides 3–6, and reference drugs. Table 2. The anti-inflammatory activity of N-(1-arylethyl)-amides 3-6, and reference drugs. Configuration Volume of Damaged Volume of Non-Damaged ∆ Volume Anti- Inflammatory Activity, Entry Product Configuration R Volume of Damaged Volume of Non-Damaged Δ Volume Anti- Inflammatory Activity, Entry Product of Chiral Centre R Extremity (mm3) Extremity (mm3) (Volume Increase) Compared to Control (%) of Chiral Centre Extremity (mm3) Extremity (mm3) (Volume Increase) Compared to Control (%) 1 1 3a3a (S)(S) H H 607.8 607.8 ± 15.515.5 500.9 500.9 ± 17.617.6 106.9106.9 ± 7.77.7 1,2,31,2,3 ++74.274.2 ± ± ± 2 2 3b3b (S)(S) OMeOMe 660.4 660.4 ± 47.447.4 311.6 311.6 ± 25.125.1 348.7348.7 ± 22.822.8 1,2,31,2,3 ++15.715.7 ± ± ± 1,2,3 3 3 4a4a (R)(R) H H 520.9 520.9 ± 87.787.7 478.9 478.9 ± 87.387.3 41.941.9 ± 3.63.6 1,2,3 ++89.989.9 ± ± ± 1,2,3 4 4 4b4b (R)(R) OMeOMe 636.2 636.2 ± 74.974.9 552.0 552.0 ± 53.853.8 84.284.2 ± 6.96.9 1,2,3 ++79.779.7 ± ± ± 1,2 5 5 5a5a (±)( )H H 648.6 648.6 ± 42.142.1 464.2 464.2 ± 13.913.9 184.4184.4 ± 26.326.3 1,2 ++55.455.4 ± ± ± ± 1,2,3 6 5b ( ) OMe 535.7 64.3 374.5 31.8 161.1 19.31,2,3 +61.1 6 5b (±)± OMe 535.7 ± 64.3± 374.5 ±± 31.8 161.1 ±± 19.3 +61.1 7 6a (S) &(R) H 687.3 94.0 360.5 32.7 326.8 59.4 1,2,3 +21.0 7 6a (S) & (R) H 687.3 ± 94.0± 360.5 ±± 32.7 326.8 ±± 59.4 1,2,3 +21.0 8 6b (S) &(R) OMe 743.8 98.6 379.1 91.6 364.6 87.5 1,2,3 +11.9 8 6b (S) & (R) OMe 743.8 ± 98.6± 379.1 ±± 91.6 364.6 ±± 87.5 1,2,3 +11.9 9 Lornoxicam 360.5 82.5 263.9 60.9 96.6 22.7 1 +76.7 9 Lornoxicam − −− − 360.5 ± 82.5± 263.9 ±± 60.9 96.6 ±± 22.7 1 +76.7 10 Diclofenac 397.6 22.4 306.6 17.6 91.0 16.0 1 +78.0 10 Diclofenac − −− − 397.6 ± 22.4± 306.6 ±± 17.6 91.0 ±± 16.0 1 +78.0 11 Control 768.7 61.0 354.9 22.9 413.7 72.1 1 0 11 Control − −− − 768.7 ± 61.0± 354.9 ±± 22.9 413.7 ±± 72.1 1 0 1 Differences statistically significant for p 0.05 vs. non-damaged extremity; 2 Differences statistically significant for p 0.05 vs. diclofenac; 3 Differences statistically significant for p 0.05 1 Differences statistically significant for p ≤ 0.05 vs. non-damaged extremity; 2 Differences statistically significant for p ≤ 0.05 vs. diclofenac; 3 Differences statistically vs. lornoxicam. ≤ ≤ ≤ significant for p ≤ 0.05 vs. lornoxicam.

Sci. Pharm. 2019, 87, 10 13 of 20

3.3. The Molecular and Crystal Structure Study The basis for studying the molecular and crystalline structure of the synthesized N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides was the desire to solve several problems at one stroke, both analytical, structural, and biological. Although all the methods described above confirm the structure of the studied substances, they do it only indirectly. Additionally, the character of this work required accurate information on the conformation of chiral centers in optically active amides 3 and 4, as also on the actual structure of racemates 5a,b. Otherwise, all the revealed patterns of the “structure-activity” relationships, as well as the final conclusions, would be inaccurate. The only “absolute” method that allows unambiguous solving such problems is X-ray diffraction. Here it must be remembered that the basis for determining the absolute configuration of one or another chiral center of the molecule from X-ray diffraction data is the phenomenon of X-ray scattering [37]. Noticeably, this effect appears only on atoms of the third and subsequent periods. Therefore, a direct determination of the absolute configuration of substances that do not contain atoms “heavier” than oxygen is usually impossible (see, for example, the case of 2-carbonyl analogs of amides 3 and 4 [38]). For this purpose, the molecule must be subjected to the appropriate modification: salt formation, halogenation, etc. [39]. The appearance of the fairly “heavy” sulfur atom in the molecules of N-(1-arylethyl)-4-methyl- 2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides 3–4 allows solving all the issues of the absolute conformation of chiral centers without any additional chemical transformations. Therefore, in particular, it was found that N-[(1S)-1-phenylethyl]-amide 3a crystallizes in the chiral space group P212121, which indicates the presence of only one enantiomer in the crystal. The (S)-configuration of the chiral center at the C atom is unambiguously determined from experimental data using the Flack parameter ( 0.06(12)). (10) − The dihydrothiazine heterocycle in this compound (Figure2) has conformation, which is intermediate between the twist-boat and chair conformations (folding parameters [40]: S = 1.71, Θ = 88.7◦, Ψ = 8.9◦). The deviations of the S(1) and C(8) atoms from the mean plane of the remaining atoms of this cycle were 0.67 Å and 0.09 Å, respectively. The cyclic nitrogen atom has a planar − − configuration, wherein the sum of the valence angles centered on it equals to 360◦. Steric repulsion has been found between the atoms of the methyl substituent at C(7) and the aromatic cycle of C(1) ... C(6), as evidenced by the shortened intramolecular contacts Н(5) ... C(18) 2.55 Å, H(18a) ... C(5) 2.83 Å, H(18b) ... C(5) 2.80 Å with the van der Waals radii sum 2.87 Å [41]. The carbamide group in the substituent at the C(8) atom is noticeably rotated relative to the endocyclic double C(7)–C(8) bond (torsion angle C –C –C О 71.9(4) ). The methyl group at the C atom is situated in the position (7) (8) (9)− (3) − ◦ (10) intermediate between aс- and aр- relatively the C(9)–N(2) bond (torsion angle C(9)–N(2)–C(10)–C(17) 157.6(3)◦), and the phenyl substituent at the same atom is in the –sc- conformation relatively the C(9)–N(2) bond and is rotated relatively the bond N(2)–C(10) (torsion angles C(9)–N(2)–C(10)–C(11) 78.1(4) Int.,N J. Mol.–C Sci. 2019–C, 20, x–C FOR PEER 47.1(4)REVIEW ). 14 of 21 − ◦ (2) (10) (11) (12) − ◦

Figure 2. The molecular structure of N-[(1S)-1-phenylethyl]-amide 3a with atoms represented by Figure 2. The molecular structure of N-[(1S)-1-phenylethyl]-amide 3a with atoms represented by thermal vibrationthermal vibration ellipsoids ellipsoi ofds 50% of 50% probability. probability.

In a crystal, the molecules of N-[(1S)-1-phenylethyl]-amide 3a are linked via intermolecular hydrogen bonds N(1)−H…O(3’) (1 − x, 0.5 + y, 1.5 − z) H…O 1.99 Å, N−H…O 161°; N(2)−H…O(1’)(1 − x, 0.5 + y, 1.5 − z) H…O 2.06 Å, O−H…O 154° forming chains along the crystallographic direction [010] (Figure 3). The formation of hydrogen bonds leads to the elongation of the С(9)−О(3) bond to 1.234(4) Ǻ (average value [42] 1.210 Å) and to the non-equivalence of S(1)−O(1) and S(1)−O(2) bonds (1.441(2) Å and 1.430(2) Å, respectively).

Figure 3. Packing of N-[(1S)-1-phenylethyl]-amide 3a in the crystal phase. Projection along [0 1 0] crystallographic direction.

Based on X-ray diffraction data, N-(1-phenylethyl)-amide 5a is a true racemate consisting of two mirror isomers 3а and 4a in a 1:1 ratio (Figure 4). In this case, the conformations of one of the enantiomers thereof and above-described N-[(1S)-1-phenylethyl]-amide 3a were almost the same. The nature of the packing of molecules in a crystal was also not changed. Unfortunately, it was not possible to study the structure of the second enantiomer, N-[(1R)-1-phenylethyl]-amide 4a on an individual basis. However, its structure is identical to that of one of the components of racemate 5а. Therefore, in the case of the triad 3a – 4a – 5a we have a classic example with two optically pure enantiomers and racemate formed thereof, respectively. Hence, the results shown by enantiomers 3а

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 14 of 21

Sci. Pharm. 2019, 87, 10 14 of 20 Figure 2. The molecular structure of N-[(1S)-1-phenylethyl]-amide 3a with atoms represented by thermal vibration ellipsoids of 50% probability. In a crystal, the molecules of N-[(1S)-1-phenylethyl]-amide 3a are linked via intermolecular In a crystal, the molecules of N-[(1S)-1-phenylethyl]-amide 3a are linked via intermolecular hydrogen bonds N(1)–H ... O(30) (1 x, 0.5 + y, 1.5 z)H ... O 1.99 Å, N–H ... O 161◦;N(2)–H ... hydrogen bonds N(1)−H…O(3’) (1 −− x, 0.5 + y, 1.5 − z) H…O− 1.99 Å, N−H…O 161°; N(2)−H…O(1’)(1 − x, O (1 x, 0.5 + y, 1.5 z)H ... O 2.06 Å, O–H ... O 154 forming chains along the crystallographic (10) −0.5 + y, 1.5 − z) H…O− 2.06 Å, O−H…O 154° forming chains◦ along the crystallographic direction [010] direction [010] (Figure3). The formation of hydrogen bonds leads to the elongation of the C –O (Figure 3). The formation of hydrogen bonds leads to the elongation of the С(9)−О(3) bond to 1.234(4) (9) (3) bond toǺ 1.234(4) (average Åvalue (average [42] 1.210 value Å) and [42 ]to 1.210 the non-equivalence Å) and to the of non-equivalence S(1)−O(1) and S(1)−O(2) of bonds S(1)–O (1.441(2)(1) and Å S (1)–O(2) bonds (1.441(2)and 1.430(2) Å andÅ, respectively). 1.430(2) Å, respectively).

Figure 3. Packing of N-[(1S)-1-phenylethyl]-amide 3a in the crystal phase. Projection along [0 1 0] Figure 3. Packing of N-[(1S)-1-phenylethyl]-amide 3a in the crystal phase. crystallographic direction. Projection along [0 1 0] crystallographic direction.

BasedBased on X-ray on X-ray diff diffractionraction data, data, N-(1-phenylethyl)-amide-(1-phenylethyl)-amide 5a is a5a trueis racemate a true racemate consisting of consisting two of two mirrormirror isomers isomers 3a3а andand 4a in a 1:1 1:1 ratio ratio (Figure (Figure 4).4 ).In Inthis this case, case, the theconformations conformations of one ofof onethe of the enantiomersenantiomers thereof thereof and above-describedand above-describedN-[ N(1S)-[(1S)-1-phenylethyl]-amide-1-phenylethyl]-amide 3a3a werewere almost almost the the same. same. The nature ofThe the nature packing of the of packing molecules of molecules in a crystal in a crystal was also was not also changed. not changed. Unfortunately, Unfortunately, it it was was not not possible possible to study the structure of the second enantiomer, N-[(1R)-1-phenylethyl]-amide 4a on an to study the structure of the second enantiomer, N-[(1R)-1-phenylethyl]-amide 4a on an individual individual basis. However, its structure is identical to that of one of the components of racemate 5а. basis. However,Therefore, in its the structure case of the is identicaltriad 3a – to4a that– 5a ofwe onehave of a theclassic components example with of two racemate optically5a pure. Therefore, in the caseenantiomers of the triad and racemate3a–4a–5a formedwe have thereof, a classic respectively. example Hence, with the tworesults optically shown by pure enantiomers enantiomers 3а and racemate formed thereof, respectively. Hence, the results shown by enantiomers 3a and 4a, as well as the biological effects (especially analgesic) of the true racemate 5a and the mechanical racemic mixture 6a very similar in strength are quite expectable (see Tables1 and2). When studying the structural features of the triad 3b–4b–5b, a slightly different picture was noted. Optically pure enantiomers 3b and 4b do not raise questions. As expected, they crystallize in the non-centrosymmetric space group Р21. The difference from the unsubstituted analogs 3a and 4a described above, perhaps, is only in the fact that in the independent part of the unit cell of each of these products, two molecules (A and B) were founded, which differ from each other in both the orientation of the methoxy group and the angle of rotation of the aryl substituent around the NHCH(Me)–C bond (Figure5). However, these di fferences are quite insignificant, for example, the torsion angles N(2)–C(10)–C(11)–C(12) determining the nature of rotation in molecules A and B are 150.7(6)◦ and 134.0(5)◦, respectively. The structural similarity of enantiomers 3a/4a and 3b/4b, respectively, attracts attention. The spatial arrangement of the methyl substituent in the methoxy group, as well as the appearance of this methoxy group in the molecule, are not principal for the manifestation of analgesic and anti-inflammatory action. As a result, we have a high total activity of the 4b-A/4b-B pair having the (R)-configuration of the chiral centers and low activity of their optical antipodes 3b-A/3b-B. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 15 of 21 Sci. Pharm. 2019, 87, 10 15 of 20 and 4a, as well as the biological effects (especially analgesic) of the true racemate 5а and the mechanical racemic mixture 6а very similar in strength are quite expectable (see Tables 1 and 2).

Figure 4. CrystalFigure 4. conformers Crystal conformers of N-[(1S) of N-1-phenylethyl]-amide-[(1S)-1-phenylethyl]-amide3a 3aand and racemateracemate 5a5a (according(according to the to the data of data of X-ray diffraction analysis), and N-[(1R)-1-phenylethyl]-amide 4a (presumably). The X-ray diffraction analysis), and N-[(1R)-1-phenylethyl]-amide 4a (presumably). The benzothiazine fragment Int.benzothiazine J. Mol. Sci. 2019 ,fragment 20, x FOR PEER is located REVIEW vertically and is turned to the viewer by the C-atom of the 4-methyl 16 of 21 is located verticallygroup (colored and isblue). turned to the viewer by the C-atom of the 4-methyl group (colored blue).

When studying the structural features of the triad 3b – 4b – 5b, a slightly different picture was noted. Optically pure enantiomers 3b and 4b do not raise questions. As expected, they crystallize in the non-centrosymmetric space group Р21. The difference from the unsubstituted analogs 3a and 4a described above, perhaps, is only in the fact that in the independent part of the unit cell of each of these products, two molecules (А and В) were founded, which differ from each other in both the orientation of the methoxy group and the angle of rotation of the aryl substituent around the NHCH(Me)–C bond (Figure 5). However, these differences are quite insignificant, for example, the torsion angles N(2)−C(10)−C(11)−C(12) determining the nature of rotation in molecules А and В are 150.7(6)° and 134.0(5)°, respectively. The structural similarity of enantiomers 3a/4a and 3b/4b, respectively, attracts attention. The spatial arrangement of the methyl substituent in the methoxy group, as well as the appearance of this methoxy group in the molecule, are not principal for the manifestation of analgesic and anti-inflammatory action. As a result, we have a high total activity of the 4b-A/4b-B pair having the (R)-configuration of the chiral centers and low activity of their optical antipodes 3b-A/3b-B. 3b-A 3b-B4b-B 4b-A

Relative analgesic activity Relative anti-inflammatory activity

5b-A 5b-B 5b-C 5b-D

Figure 5. Crystal conformers of N-[(1S)-1-(4-methoxyphenyl)ethyl]-amide 3b, its mirror isomer 4b, and Figure 5. Crystal conformers of N-[(1S)-1-(4-methoxyphenyl)ethyl]-amide 3b, its mirror isomer 4b, racemate 5b (experimentaland racemate 5b (experimental data). The data). benzothiazine The benzothiazine fragment fragment is is located vertically vertically and is andturned is turned to the viewer by theto C-atom the viewer of by the the 4-methylC-atom of the group 4-methyl (colored group (colored blue). blue).

The crystal structures of the mirror isomers 3b and 4b have the same unit cell parameters (see Section 2.8 and 2.9), which is usual for classical enantiomers. The appearance of the "heavy" sulfur atom in the studied molecules and the resulting violation of the Fridel law allows unambiguous determining the true configuration of their chiral centers using the calculated Flack parameter [37]. Therefore, in the amides 3b-A/3b-B, С(10) atoms have (S)-configuration (the Flack parameter is

Sci. Pharm. 2019, 87, 10 16 of 20

The crystal structures of the mirror isomers 3b and 4b have the same unit cell parameters (see Sections 2.8 and 2.9), which is usual for classical enantiomers. The appearance of the “heavy” sulfur atom in the studied molecules and the resulting violation of the Fridel law allows unambiguous Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 17 of 21 determining the true configuration of their chiral centers using the calculated Flack parameter [37]. Therefore,0.05(10)), inwhereas the amides in the3b-A amides/3b-B,C 4b-A(10) atoms/4b-B havethey (S)have-configuration (R)-configuration (the Flack (the parameter Flack parameter is 0.05(10)), is whereas0.05(11)). in the amides 4b-A/4b-B they have (R)-configuration (the Flack parameter is 0.05(11)). InIn crystals, crystals, moleculesmolecules AА and ВB composingcomposing both both enantiomers enantiomers 3b3b andand 4b4b formform chains chains of ofboth both А– AА–A–А–A–А–A andand ВB–В–B–В–B–В–B typestypes (Figure (Figure 66)) alongalong thethe crystallographiccrystallographic directiondirection [010]. [010]. These These chains chains are are simultaneouslysimultaneously interconnected interconnected by by two two intermolecular intermolecular hydrogen hydrogen bonds bonds N (1)N(1)–H–H…O... O(1’)(10 )andand N N(2)(2)–H…O–H ...(3’) O (symmetry operations 1 – x, 0.5 + y, 1 z in chain A–A and x, 0.5 + y,–z in chain B–B). (symmetry(30) operations 1 – x, 0.5 + y, 1 –z in− chain А–А and –x, –0.5− +− y, –z in chain В–В)

Figure 6. The main fragment of the package in enantiomers 3b and 4b. Figure 6. The main fragment of the package in enantiomers 3b and 4b. Similar to the triad of derivatives 3a 4a 5a with the unsubstituted aromatic core, it was logical to Similar to the triad of derivatives 3a− – 4a− – 5a with the unsubstituted aromatic core, it was logical assume that the racemate 5b is the sum of the conformers composing the optically active amides 3b to assume that the racemate 5b is the sum of the conformers composing the optically active amides and 4b. However, in reality, everything was completely different. First of all, the non-centrosymmetric 3b and 4b. However, in reality, everything was completely different. First of all, the non- space group of racemate 5b (Рсa21) was surprising. In theory, this should indicate the appearance of centrosymmetric space group of racemate 5b (Рса21) was surprising. In theory, this should indicate only one enantiomer in a crystal. However, it was found that two molecules (A and B) are present the appearance of only one enantiomer in a crystal. However, it was found that two molecules (A in the independent part of the unit cell, which molecules have different configurations of their chiral and B) are present in the independent part of the unit cell, which molecules have different centers: (S)-configuration for A and (R)-configuration for B, which was confirmed by the calculated configurations of their chiral centers: (S)-configuration for А and (R)-configuration for В, which was value of the Flack parameter 0.03(12). confirmed by the calculated value of the Flack parameter 0.03(12). Additionally, unlike amide 5a, the methoxy-substituted analog 5b could not be considered a usual Additionally, unlike amide 5а, the methoxy-substituted analog 5b could not be considered a racemate, since constituent molecules thereof, 5b-A and 5b-B (Figure5), did not mirror reflections of usual racemate, since constituent molecules thereof, 5b-А and 5b-В (Figure 5), did not mirror each other and were not linked by a center of symmetry. In addition, a symmetry plane appeared reflections of each other and were not linked by a center of symmetry. In addition, a symmetry plane in the amide 5b crystal. Consequently, each of the molecules 5b-A and 5b-B had a pair of 5b-C and appeared in the amide 5b crystal. Consequently, each of the molecules 5b-А and 5b-В had a pair of 5b-D symmetric to them in the crystal. In other words, the structure of amide 5b contained four 5b-C and 5b-D symmetric to them in the crystal. In other words, the structure of amide 5b contained conformers of the studied compound (Figure5), which could be divided into two pairs, connected by a four conformers of the studied compound (Figure 5), which could be divided into two pairs, symmetry transformation relatively the plane. The unique character of amide 5b as a racemate also lies connected by a symmetry transformation relatively the plane. The unique character of amide 5b as a in the fact that structure of all the conformers 5b-A, 5b-B, 5b-C, and 5b-D composing said racemate racemate also lies in the fact that structure of all the conformers 5b-А, 5b-В, 5b-C, and 5b-D was significantly different from that of the original enantiomers 3b and 4b. For this reason, it was composing said racemate was significantly different from that of the original enantiomers 3b and 4b. not possible to determine, which of these conformers provided an unusually high level of analgesic For this reason, it was not possible to determine, which of these conformers provided an unusually and anti-inflammatory activities of amide 5b compared to the mechanical racemic mixture 6b, which high level of analgesic and anti-inflammatory activities of amide 5b compared to the mechanical mixture consisted by one half of the enantiomer 3b with obviously low activity. racemic mixture 6b, which mixture consisted by one half of the enantiomer 3b with obviously low Another factor that could additionally contribute to the biological properties of amide 5b was its activity. crystalline package. As in the case of enantiomers 3b and 4b, molecules A and B also form chains of Another factor that could additionally contribute to the biological properties of amide 5b both A–A–A–A and B–B–B–B types in the crystal of racemate 5b. However, these chains are different. was its crystalline package. As in the case of enantiomers 3b and 4b, molecules А and В also form More specifically, in the zigzag A–A chains, molecules were linked only by one intermolecular hydrogen chains of both А–А–А–А and В–В–В–В types in the crystal of racemate 5b. However, these chains bond N –H ... O , while the neighboring chains were not connected at all (Figure7). The chains of are different.(1) More(1) specifically, in the zigzag А–А chains, molecules were linked only by one intermolecular hydrogen bond N(1)–H…O(1), while the neighboring chains were not connected at all (Figure 7). The chains of molecules В were formed via the intermolecular hydrogen bonds N(2)– H…O(1) and were linked by the hydrogen bond N(1)–H…O(3). In the crystal, the chains А–А and В–В formed alternating layers (Figure 8).

Sci. Pharm. 2019, 87, 10 17 of 20

molecules B were formed via the intermolecular hydrogen bonds N(2)–H ... O(1) and were linked by theInt. hydrogen J. Mol. Sci. bond2019, 20 N, x(1) FOR–H PEER... O REVIEW(3). In the crystal, the chains A–A and B–B formed alternating layers 18 of 21 (FigureInt. J. Mol.8 ).Sci. 2019, 20, x FOR PEER REVIEW 18 of 21

Figure 7. The main fragment of the package of molecules А (left) and B (right) in the structure of Figure 7. The main fragment of the package of molecules A (left) and B (right) in the structure of Figureracemate 7. The 5b main. Projection fragment along of [0the 0 1]package crystallographic of molecules direction. А (left) and B (right) in the structure of racemate 5b. Projection Projection along along [0 [0 0 1] crystallographic direction.

FigureFigure 8. Packing8. thePacking chains in layersthe inchains a racemate in5b crystal.layers Moleculesin aA formracemate a layer highlighted5b crystal. in Figureyellow,Molecules molecules8. А formPackingB form a layer green. highlightedthe chains in yellow,in moleculeslayers Вin form a green. racemate 5b crystal. Molecules А form a layer highlighted in yellow, molecules В form green. Therefore,Therefore, our our study study clearly clearly showed showed that the that anti-inflammatory, the anti-inflammatory, and especially and analgesicespecially properties analgesic of N-(1-arylethyl)-4-methyl-2,2- dioxo-1H-2λ6,1-benzothiazine-3-carboxamides are largely determined propertiesTherefore, of N-our(1-arylethyl)-4-methyl-2,2- study clearly showed thatdioxo-1 theH- anti-inflammatory,2λ6,1-benzothiazine-3-carboxamides and especially analgesic are largely 6 propertiesdetermined of N- by(1-arylethyl)-4-methyl-2,2- molecular conformations dioxo-1 thereofH- initially2λ ,1-benzothiazine-3-carboxamides fixed in crystals, and also by arethe largelynature of determinedthe molecular by molecular package in conformations the crystalline thereof phase. initially fixed in crystals, and also by the nature of the molecular package in the crystalline phase. 4. Conclusions 4. Conclusions

Sci. Pharm. 2019, 87, 10 18 of 20 by molecular conformations thereof initially fixed in crystals, and also by the nature of the molecular package in the crystalline phase.

4. Conclusions This article is devoted to obtaining new optically pure enantiomers of N-(1-arylethyl)-4- methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides, their true racemates, and mechanical racemic mixtures, and also to a detailed study of the spatial structure and biological properties thereof. Elemental analysis, 1Нand 13C NMR spectroscopy, electrospray ionization liquid chromato-mass spectrometry, polarimetry, and X-ray diffraction have been used to confirm the structure of the synthesized compounds. Based on the data obtained from the pharmacological tests, chiral N-(1-arylethyl)-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides have shown apparent stereospecificity of biological properties. Therefore, against the background of low active (S)-enantiomers, mirror isomers thereof having (R)-configuration of chiral centers have proven to be more powerful analgesics and antiphlogistics than lornoxicam and diclofenac. At the same time, an unusually high level of activity has been noted in racemic N-[1-(4-methoxyphenyl)ethyl]- 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide. It was shown that the unexpected pharmacological effects of this compound had been caused by a unique crystalline and molecular structure, which is quite unusual for racemates.

Author Contributions: The synthesis of the compounds presented in this work and analysis of their characteristics were performed by I.V.U. and G.M.H. The liquid chromato-mass-spectrometric and X-ray structural studies were performed by I.A.D., G.S., and S.V.S., A.A.B., respectively. The pharmacological studies were conducted by N.I.V. and O.V.M. The manuscript was written by I.V.U., G.M.H., and O.V.M. Funding: This research received no external funding. Acknowledgments: We are grateful to Candidate of Chemistry Evgene S. Gladkov (SSI “Institute for Single Crystal” National Academy of Sciences of Ukraine, Kharkiv, Ukraine) for his help in registration of NMR spectra of the compounds synthesized. Conflicts of Interest: The authors declare no conflict of interest.

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