Scientia Pharmaceutica

Article Synthesis, Crystal Structure, and Biological Activity of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine- 3-carboxylate Polymorphic Forms

Igor V. Ukrainets 1,* ID , Anna A. Burian 1, Vyacheslav N. Baumer 2, Svitlana V. Shishkina 2,3, Lyudmila V. Sidorenko 1 ID , Igor A. Tugaibei 4, Natali I. Voloshchuk 5 ID and Pavlo S. Bondarenko 5

1 Department of Pharmaceutical Chemistry, National University of Pharmacy, 53 Pushkinska st., 61002 Kharkiv, Ukraine; [email protected] (A.A.B.); [email protected] (L.V.S.) 2 SSI “Institute for Single Crystals”, National Academy of Sciences of Ukraine, 60 Nauki ave., 61001 Kharkiv, Ukraine; [email protected] (V.N.B.); [email protected] (S.V.S.) 3 Department of Inorganic Chemistry, V. N. Karazin Kharkiv National University, 4 Svobody sq., 61077 Kharkiv, Ukraine 4 Department of Clinical Biochemistry, Forensic Toxicology, and Pharmacy, Kharkiv Medical Academy of Postgraduate Education, 58 Amosov st., 61176 Kharkiv, Ukraine; [email protected] 5 Department of Pharmacology, N. I. Pirogov Vinnitsa National Medical University, 56 Pirogov st., 21018 Vinnitsa, Ukraine; [email protected] (N.I.V.); [email protected] (P.S.B.) * Correspondence: [email protected]; Tel.: +380-572-679-185



Received: 11 April 2018; Accepted: 21 May 2018; Published: 30 May 2018


Abstract: Continuing the search for new potential analgesics among the derivatives of 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid, the possibility of obtaining its esters by the alkylation of the corresponding sodium salt with iodoethane in dimethyl sulfoxide (DMSO) at room temperature was studied. It was found that under such conditions, together with the oxygen atom of the carboxyl group, a heteroatom of nitrogen is also alkylated. Therefore, the product of the reaction studied is a mixture of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (major) and its 1-ethyl-substituted analog (minor). A simple but very effective method of preparative separation of these compounds was proposed. Moreover, the heterogeneous crystallization from ethanol was revealed to result in a monoclinic polymorphic form of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate, while the homogeneous crystallization results in its orthorhombic form. The molecular and crystal structures of both forms were confirmed by X-ray diffraction analysis, and the phase purity by powder diffraction study. The pharmacological tests carried out on the model of a carrageenan edema showed that the screening dose of 20 mg/kg of 1-ethyl-substituted ester and the orthorhombic form of its analog unsubstituted in position 1 exhibited weak anti-inflammatory and moderate analgesic effects. At the same time, the monoclinic form of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate appeared to be both a powerful analgesic and an anti-inflammatory agent that exceeded Piroxicam and Meloxicam in the same doses by these indicators. A detailed comparative analysis of the molecular and crystal structures of two polymorphic forms of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate was carried out using quantum chemical calculations of the energies of pairwise interactions between molecules. An explanation of the essential differences of their biological properties based on this was offered.

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

Sci. Pharm. 2018, 86, 21; doi:10.3390/scipharm86020021 www.mdpi.com/journal/scipharm Sci. Pharm. 2018, 86, 21x 2 2of of 17

1. Introduction 1. Introduction Ester compounds as officially permitted drugs are widely represented in practically all pharmacologicalEster compounds groups. as Agents officially for the permitted control of drugs pain areand widelypain syndromes represented of different in practically etiologies all pharmacologicalare no exception. groups. More Agents than for50 thesynthetic control ofanalgesics pain and painwith syndromesa central ofand different peripheral etiologies action, are nonon-steroidal exception. anti-inflammatory More than 50 synthetic drugs, analgesics as well as with local a anesthetics, central and peripheralbelong to ester action, compounds non-steroidal due anti-inflammatoryto their chemical structure drugs, as [1,2]. well as local anesthetics, belong to ester compounds due to their chemical structureEster [ 1compounds,2]. attract the attention of scientists engaged in a targeted search for new biologicallyEster compounds active substances attract because the attention of their of high scientists efficiency engaged and relatively in a targeted small searchnumber for of newside biologicallyeffects [3]. They active are substances also very becauseconvenient of theirfor this high kind efficiency of research and relativelybecause of small the fact number that ofany side of effectstheir components [3]. They are (both also veryacid convenientand alcoholic) for thiscan kind be easily of research modified because if necessary. of the fact Moreover, that any of these their componentscompounds (bothare almost acid and unlimited alcoholic) and, can last be easilybut not modified least, ifthey necessary. are well-studied Moreover, theseusing compoundsrepeatedly aretested almost methods. unlimited As another and, last positive but not point, least, it they shou areld be well-studied noted that usingesterases repeatedly that are testedresponsible methods. for Asthe anotherbiodegradation positive point,of such it drugs should are be present noted that in all esterases human that organs are responsibleand tissues forin large the biodegradation quantities [4]. ofTherefore, such drugs the arerisk present of an “overload” in all human of organsthe enzyme and tissues systems in largeof a patient quantities with [4 a]. drug Therefore, ester is the reduced risk of anto a “overload” minimum of[3]. the enzyme systems of a patient with a drug ester is reduced to a minimum [3]. Often, the transformation of a group with acid pr propertiesoperties in the ester group leads to changes in the pharmacokinetic,pharmacokinetic, pharmaceutical, and/or and/or pharma pharmacologicalcological properties properties of of drug drug acids acids in in a desired direction. Therefore, it is not surprising that this property is widely used to transform pharmaceutical acids, alcohols, or phenols of differentdifferent pharmacologicalpharmacological orientations (including analgesics, which are well-proven in medical practice (Figure 11)))) intointo moremore suitablesuitable andand safersafer esterester prodrugsprodrugs [[1–3].1–3]. At the same time, enol ( I) or phenolic ( (II)) hydroxyl, hydroxyl, and and the the carboxyl carboxyl group group ( (IIIIII andand IVIV),), as as well well as phenolic hydroxyl and the COOH-group ((V),), cancan bebe successfullysuccessfully subjectedsubjected toto modification.modification.

Me O Me O O COOH N Me Me O O O O O O Cl H N N N H N S Me Cl OO HN Me

O I: Ampiroxicam II: Propacetamol III: Aceclofenac

O Me Me Me COOH O Me MeO O Me O OH N

O Cl

IV: Indometacin farnesil V: Salsalate

FigureFigure 1. TheThe ester-prodrugs ester-prodrugs of analgesic acids and phenol phenolss that are officiallyofficially registered drugsdrugs [[1,2].1,2].

To date, organic chemistry has an extremely wide range of methods and reagents that provides To date, organic chemistry has an extremely wide range of methods and reagents that provides for the gain of ester compounds virtually without any restrictions [5–21]. In principle, modern for the gain of ester compounds virtually without any restrictions [5–21]. In principle, modern pharmaceutical chemistry is capable of applying all the advanced scientific achievements in this pharmaceutical chemistry is capable of applying all the advanced scientific achievements in this field. However, in real industrial-scale syntheses, as a rule, manufacturers are guided not only by field. However, in real industrial-scale syntheses, as a rule, manufacturers are guided not only by the the novelty of technologies, but also by their safety (primarily environmental), reliability, and novelty of technologies, but also by their safety (primarily environmental), reliability, and economic economic feasibility. Therefore, in the schemes for obtaining drug esters, the time-tested methods for feasibility. Therefore, in the schemes for obtaining drug esters, the time-tested methods for the the formation of ester fragments are the most common. These are Fisher esterification—a generally formation of ester fragments are the most common. These are Fisher esterification—a generally recognized method—and the acylation of alcohols (phenols) with anhydrides or halides of carboxylic recognized method—and the acylation of alcohols (phenols) with anhydrides or halides of carboxylic acids [1]. In some cases, another traditional version of the synthesis of ester compounds (the

Sci. Pharm. 2018, 86, 21 3 of 17

Sci. Pharm. 2018, 86, x 3 of 17 acids [1]. In some cases, another traditional version of the synthesis of ester compounds (the alkylation of carboxylic acid salts with alkyl halides) worked well. This is the method that we used to transform alkylation of carboxylic acid salts with alkyl halides) worked well. This is the method that we used 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid into an ester. Naturally, the lower to transform 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid into an ester. Naturally, the alkyl esters of this acid are easier to obtain according to the usual linear scheme [22,23] when the lower alkyl esters of this acid are easier to obtain according to the usual linear scheme [22,23] when required substituent is introduced at the preliminary stage of the synthesis of the corresponding alkyl the required substituent is introduced at the preliminary stage of the synthesis of the corresponding (chlorosulfonyl) acetate VII (Scheme1). It must be remembered that the use of bases at the final stage alkyl (chlorosulfonyl) acetate VII (Scheme 1). It must be remembered that the use of bases at the final of the hetericyclization provides an opportunity to avoid easy transesterification [24,25]. stage of the hetericyclization provides an opportunity to avoid easy transesterification [24,25].

Me Me Me O ClSO CH COO Alk (VII) O 2 2 O NaOAlk O Alk S O NH N SO -CH -COO Alk N 2 2 O R R R VI VIII IX

NaO Alk'

Me O Me O

O Alk O Alk' + S O S O N N O O R R IX X

Scheme 1. 1. TheThe conventional conventional method method for forthe thesynthesis synthesis of alkyl of alkyl 1-R-4-methyl-2,2-dioxo-1 1-R-4-methyl-2,2-dioxo-1H-2λ6H,1--2 λ6,1- benzothiazine-3-carboxylates IX [22–25]. [22–25].

However, we deliberately focused on the alkylation of the benzothiazine-3-carboxylic acid salt However, we deliberately focused on the alkylation of the benzothiazine-3-carboxylic with the alkyl halide in order to involve the most diverse esters in the alcoholic part of the acid salt with the alkyl halide in order to involve the most diverse esters in the alcoholic molecule, in addition to the already studied methyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3- part of the molecule, in addition to the already studied methyl 4-methyl-2,2-dioxo-1H-2λ6,1- carboxylates [23]. It is the alkylation of the salts of the carboxylic acids with the alkyl halides benzothiazine-3-carboxylates [23]. It is the alkylation of the salts of the carboxylic acids with the alkyl characterized by a very wide synthetic potential (unlike the method presented in Scheme 1) that halides characterized by a very wide synthetic potential (unlike the method presented in Scheme1) allows for the solution of similar problems. In addition, the reactions of the alkylation of ambident that allows for the solution of similar problems. In addition, the reactions of the alkylation of ambident nucleophiles are always interesting from a theoretical point of view since, in most cases, they are nucleophiles are always interesting from a theoretical point of view since, in most cases, they are ambiguous and not always predictable. ambiguous and not always predictable.

2. Materials Materials and and Methods Methods

2.1. Chemistry Chemistry 11НH-NMR-NMR (proton (proton nuclear nuclear magnetic magnetic resonance) resonance) spec spectratra were were acquired acquired on on a a Varian Mercury-400 (Varian Inc.,Inc., Palo Palo Alto, Alto, CA, CA, USA) USA) instrument instrument (400 MHz) (400 in MHz) hexadeutero-dimethyl in hexadeutero-dimethyl sulfoxide (DMSO-dsulfoxide6) (DMSO-dwith tetramethylsilane6) with tetramethylsilane as the internal as the standard. internal The standard. chemical The shift chemical values shift were values recorded were on recorded a δ scale onand a the δ scale coupling and constants the coupling (J) in hertz.constants The following(J) in hertz. abbreviations The following were abbreviations used in reporting were the used spectra: in reportings = singlet, the d = spectra: doublet, s t == triplet,singlet, q =d quartet.= doublet, The t melting = triplet, points q = werequartet. determined The melting in a capillary points usingwere determineda electrothermal in a capillary IA9100X1 using (Bibby a electrothermal Scientific Limited, IA9100X1 Stone, (Bibby UK) digital Scientific melting Limited, point Stone, apparatus. UK) digitalThe synthesis melting point of the apparatus. starting The sodium synthesis 4-methyl-2,2-dioxo-1 of the starting sodiumH-2λ6 ,1-benzothiazine-3-carboxylate4-methyl-2,2-dioxo-1H-2λ6,1- benzothiazine-3-carboxylatemonohydrate (1) was carried monohydrate out by the method (1) was described carried out in [ 26by]. the method described in [26].

2.2. Monoclinic Form of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (2M) Iodoethane (1.56 g, 0.01 mol) was added to the solution of sodium salt 1 (2.79 g, 0.01 mol) in 10 mL dimethyl sulfoxide (DMSO), and the mixture was stirred for 5 h at 25°С. The mixture was diluted with cold water and acidified with dilute HCl to рН 3. The precipitate formed was filtered, washed with cold water, and dried in the air. A total of 2.00 g of the product composed of ester 2

Sci. Pharm. 2018, 86, 21 4 of 17

2.2. Monoclinic Form of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (2M) Iodoethane (1.56 g, 0.01 mol) was added to the solution of sodium salt 1 (2.79 g, 0.01 mol) in 10 mL dimethyl sulfoxide (DMSO), and the mixture was stirred for 5 h at 25◦C. The mixture was diluted with cold water and acidified with dilute HCl to pH3. The precipitate formed was filtered, washed with cold water, and dried in the air. A total of 2.00 g of the product composed of ester 2 and its N-ethyl-substituted analog 3 (according to the data of the 1H-NMR spectrum—68 and 32%, respectively) was isolated. The mixture of the crystals of esters 2M and 3 obtained by the crystallization from ethanol in 10 mL of hexane was boiled for 5 min, then the hexane was carefully decanted, avoiding the sediment. The procedure was repeated five times. Finally, a pure monoclinic form of 2M was obtained. The yield was: 1.26 g (47%); colorless crystals; melting point (mp) 171–173 ◦C; 1H-NMR (400 MHz, DMSO-d6): δ 11.83 (br. s, 1H, SO2NH), 7.78 (d, 1H, J = 8.0 Hz, H-5), 7.50 (t, 1H, J = 7.7 Hz, H-7), 7.22 (t, 1H, J = 7.7 Hz, H-6), 7.12 (d, 1H, J = 8.0 Hz, H-8), 4.31 (q, 2H, J = 7.1 Hz, OCH2), 2.43 (s, 3H, 4-CH3), 1.27 (t, 3H, J = 7.1 Hz, OCH2CH3). This was analytically calculated (Anal.Calcd.) for C12H13NO4S: C, 53.92; H, 4.90; N, 5.24; S, 12.00. We found: C, 53.98; H, 4.99; N, 5.17; S, 11.91. The filtrate obtained after the isolation of esters 2 and 3 was kept for several days at room temperature. Gradually, crystals of the product identified by the 1H-NMR spectrum and the absence of the depression of the melting point of the sample mixed with the known reference 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid monohydrate [26] formed in the solution.

2.3. Orthorhombic Form of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (2O) The mixture of esters 2 and 3 obtained was treated after the alkylation of salt 1 (see the previous example) with 15 mL of 10% aqueous solution of Na2CO3, and filtered. The filtrate was acidified with dilute HCl to pH3. The precipitate of the pure ester 2 formed was filtered, washed with cold water, and dried in the air. After the crystallization from ethanol, an orthorhombic form 2O was obtained. ◦ 1 The yield was: 1.31 g (49%); colorless crystals; m.p. 165–167 C; H-NMR (400 MHz, DMSO-d6): δ 7.84 (d, 1H, J = 8.0 Hz, H-5), 7.61 (t, 1H, J = 7.8 Hz, H-7), 7.51 (d, 1H, J = 7.9 Hz, H-8), 7.33 (t, 1H, J = 7.8 Hz, H-6), 4.32 (q, 2H, J = 7.0 Hz, OCH2), 3.96 (q, 2H, J = 7.1 Hz, NCH2), 2.43 (s, 3H, 4-CH3), 1.27 (t, 3H, J = 7.0 Hz, OCH2CH3), 1.14 (t, 3H, J = 7.1 Hz, NCH2CH3). The Anal.Calcd. was C12H13NO4S: C, 53.92; H, 4.90; N, 5.24; S, 12.00. We found: C, 54.00; H, 4.96; N, 5.31; S 11.93%.

2.4. Ethyl 1-Ethyl-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (3) The residue was washed on the filter (see the previous example) undissolved in an aqueous solution of Na2CO3 with cold water, and dried in the air. The yield was: 0.67 g (23%); colorless ◦ 1 crystals; m.p. 96–98 C (ethanol); H-NMR (400 MHz, DMSO-d6): δ 7.84 (d, 1H, J = 8.0 Hz, H-5), 7.61 (t, 1H, J = 7.8 Hz, H-7), 7.51 (d, 1H, J = 7.9 Hz, H-8), 7.33 (t, 1H, J = 7.8 Hz, H-6), 4.32 (q, 2H, J = 7.0 Hz, OCH2), 3.96 (q, 2H, J = 7.1 Hz, NCH2), 2.43 (s, 3H, 4-CH3), 1.27 (t, 3H, J = 7.0 Hz, OCH2CH3), 1.14 (t, 3H, J = 7.1 Hz, NCH2CH3). The Anal.Calcd. was C14H17NO4S: C, 56.93; H, 5.80; N, 4.74; S 10.86%. We found: C, 56.85; H, 5.74; N, 4.82; S 10.95%.

2.5. X-ray Structural Analysis of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Monoclinic Form (2M)

The crystals of ester 2M (C12H13NO4S) were monoclinic and colorless at 293 K: a 8.0177(6), ◦ 3 3 b 10.259(1), c 7.4995(9) Å; β 90.130(2) ; V 616.9(1) Å , Z 2, space group Pc, dcalc 1.439 g/cm , −1 µ(MoKα) 0.268 mm , F(000) 294. The unit cell parameters and intensities of 6074 reflections (3187 independent reflections, Rint = 0.073) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited, Oxford, UK) 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, Göttingen, Germany) [27]. The positions of the hydrogen atoms were found from the electron density difference map and refined Sci. Pharm. 2018, 86, 21 5 of 17

using the riding model with Uiso = nUeq for the non-hydrogen atom bonded to a given hydrogen atom (n = 1.5 for methyl groups, and n = 1.2 for the other hydrogen atoms). The hydrogen atom 2 at the N(1) atom was refined using isotropic approximation. The structure was refined using F full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR2 0.136 for 3,118 reflections (R1 0.054 for 2,350 reflections with F > 4σ(F), S 0.989). The final atomic coordinates, and the crystallographic data for the molecule of ester 2M 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 1835802 [28].

2.6. X-ray Structural Analysis of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Orthorhombic Form (2O)

The crystals of ester 2O (C12H13NO4S) were orthorhombic and colorless at 293 K: a 8.957(1), 3 3 −1 b 15.866(3), c 17.955(3) Å; V 2551.7(8) Å , Z 8, space group Pbca, dcalc 1.392 g/cm , µ(MoKα) 0.260 mm , F(000) 1120. The unit cell parameters and intensities of 23,700 reflections (3717 independent reflections, Rint = 0.159) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) 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) [27]. 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 groups, and n = 1.2 for the other hydrogen atoms). The hydrogen atom at the N(1) atom was refined using isotropic approximation. The structure was refined using F2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to 0.190 for 3,675 reflections (R1 0.070 for 1880 reflections with F > 4σ(F), S 0.889). The final atomic coordinates, and the crystallographic data for the molecule of ester 2R 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 1835803 [29].

2.7. X-ray Structural Analysis of Ethyl 1-Ethyl-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (3)

The crystals of the N-ethyl substituted ester 3 (C14H17NO4S) were triclinic and colorless at 293 K: a 10.2546(7), b 11.3609(9), c 13.1329(9) Å; β 105.068(6)◦; γ 100.765(6)◦; V 1433.3(2) Å3, Z 4, 3 −1 space group P1, dcalc 1.369 g/cm , µ(MoKα) 0.238 mm , F(000) 624. The unit cell parameters and intensities of 14,670 reflections (8344 independent reflections, Rint = 0.030) were measured on an Xcalibur-3 diffractometer (Oxford Diffraction Limited) 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) [27]. 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 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 0.145 for 8,264 reflections (R1 0.051 for 5404 reflections with F > 4σ(F), S 0.967). The final atomic coordinates, and the crystallographic data for the molecule of ester 3 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 1835804 [30]. Sci. Pharm. 2018, 86, 21 6 of 17

2.8. Powder Diffraction Study of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Monoclinic and Orthorhombic Form (2M and 2O) The powder diffraction study of the monoclinic and orthorhombic forms of ethyl 4-methyl-2,2- dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (2M and 2O) was performed using a Siemens D500 diffractometer (Siemens Analytical X-ray Instruments Inc., Madison, WI, USA) with Bragg–Brentano geometry, a curved graphite monochromator on the counter arm, scanned with ∆2θ = 0.02◦, with an accumulation time at each point of 30 s. The Rietveld refinement was carried out with the WinPLOTR and FullProf programs (Institute Laue-Langevin, Grenoble, France) [31–33] using the model structures obtained by the monocrystal diffraction method.

2.9. Pharmacology

Anti-Inflammatory and Analgesic 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 Ukrainian Law No. 3447-IV “On the protection of animals from severe treatment” [34] (project ID 3410U14, approved 15 October 2015). In this study, male Wistar rats (200–250 g) were obtained from vivarium of the Institute of Pharmacology and Toxicology of the Academy of Medical Sciences of Ukraine (Kiev, Ukraine). All animals received standard food for rodents and water. They were acclimatized within 10 days. One day before the experiments, the animals were transferred to the scientific laboratory for adaptation. All the time they were maintained at 20–22 ◦C, 40–60% relative humidity and a 12 h/12 h (light/dark) cycle. The anti-inflammatory action of the ester 2 two polymorphic modifications and ester 3 was studied on the model of the experimental inflammation process caused by subplantar introduction of 0.1 mL of 1% carrageenan solution [35,36] in one of the hind limbs of the rats. The test compounds and the reference drugs were injected intraperitoneally into the animals of the experimental groups 2 h after the introduction of carrageenan. Three hours 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 rats in the control group. The analgesic action was detected on the same model by determining the pain threshold—the smallest pressure force (g/mm2) on the rat’s foot that 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 rats in the control group. All test substances, Piroxicam (Jenapharm, Jena, Germany) and Meloxicam (Boehringer Ingelheim, Ingelheim am Rhein, Germany), were introduced intraperitoneally in the form of 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 esters 2M, 2O and 3, the reference drugs and the control. The processing of the results obtained was performed using the STATISTICA 6.1 software package (StatSoft Inc., Tulsa, OK, USA). Descriptive statistics included calculations of the arithmetic means (M) and the standard errors of the mean (±m). The significance of the differences within one group was found using Wilcoxon’s non-parametric test. The reliability of intergroup differences was determined using non-parametric Mann–Whitney U-criteria. Effects were regarded as statistically significant at p ≤ 0.05. Sci. Pharm. 2018, 86, 21 7 of 17

2.10. Quantum-Chemical Calculations The packing analysis of the 2M and 2O polymorphic forms was performed within an energetic approach that has been suggested earlier [37]. The first coordination sphere of the crystal building unit (molecule or dimer of molecules) was determined using the “Molecular Shell calculation” option within the Mercury program, version 3.8 (Cambridge Crystallographic Data Centre, Cambridge, UK) [38]. The molecular geometries of the building unit dimers were taken from X-ray diffraction studies. Taking into account the well-known effect of shortening the lengths of the X-H bonds in the X-ray diffraction data [39], the positions of the hydrogen atoms were normalized according to the results of the geometry optimization of the isolated molecule. Other bonds and torsion angles were not changed. The pairwise interaction energies were calculated using the B97-D3/Def2-TZVP density functional method [40–42] and corrected for the basis set superposition error with the counterpoise method [43]. All calculationsSci. Pharm. 2018 were, 86, x performed using ORCA 3.0 software (Max Planck Institute, Mülheim7 of 17 an der Ruhr, Germany) [44]. The results of the calculations were visualized with energy-vector diagrams [37], The pairwise interaction energies were calculated using the B97-D3/Def2-TZVP density where the vector lengths were calculated using the following equation: L = (R E )/2E , where R is a functional method [40–42] and corrected for the basis set superposition errori withi thei counterpoisestr i distancemethod between [43]. the All geometrical calculations were centers performed of the interactingusing ORCA molecules, 3.0 software E i (Maxis the Planck energy Institute, of interaction betweenMülheim these two an moleculesder Ruhr, andGermany) Estr is [44]. the energyThe results of the of strongestthe calculations pairwise were interaction visualized inwith the crystal. Such a diagramenergy-vector is a moleculediagrams [37], image where and maythe vector be multiplied lengths were with calculated all symmetry using operationsthe following similar to a molecule.equation: Li = (RiEi)/2Estr, where Ri is a distance between the geometrical centers of the interacting molecules, Ei is the energy of interaction between these two molecules and Estr is the energy of the 3. Resultsstrongest and Discussionpairwise interaction in the crystal. Such a diagram is a molecule image and may be multiplied with all symmetry operations similar to a molecule. 3.1. Chemistry 3. Results and Discussion The reaction of the sodium 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate monohydrate3.1. Chemistry (1) and the equivalent amount of iodoethane in the DMSO solution proceeds rather easily at roomThe temperature.reaction of the sodi Theum1 H-NMR4-methyl-2,2-dioxo-1 spectrumH-2 ofλ6 the,1-benzothiazine-3-car crude productboxylate showed monohydrate that, as expected, alkylation(1) and did the not equivalent clearly proceed.amount of iodoethane Along with in th thee DMSO expected solution ethyl proceeds ester rather2, its Neasily-ethyl-substituted at room temperature. The 1Н-NMR spectrum of the crude product showed that, as expected, alkylation did analog 3 was also formed, and in a fairly large amount—approximately 36% (Scheme2). It is of interest not clearly proceed. Along with the expected ethyl ester 2, its N-ethyl-substituted analog 3 was also that theformed, original and product in a fairly was large naturally amount—approximatel present (not iny 36% the form(Scheme of salt2). It1 is, butof interest in the correspondingthat the 6 4-methyl-2,2-dioxo-1original productH was-2λ ,1-benzothiazine-3-carboxylicnaturally present (not in the form acid) of salt in the1, but reaction in the corresponding mixture. However, we failed4-methyl-2,2-dioxo-1 to detect the productH-2λ6,1-benzothiazine-3-carboxylic of monoalkylation solely acid) by the in the nitrogen reaction heteroatom. mixture. However, Hence, we the ethyl iodide initiallyfailed to detect attacks the the product oxygen of monoalkylation atom of the carboxylate solely by the anion.nitrogen However, heteroatom. while Hence, ethyl the ethyl ester 2 was formingiodide as a initially new potential attacks the target oxygen for atom electrophilic of the carboxylate attack, anion. it began However, to seriously while ethyl compete ester 2 was with salt 1. forming as a new potential target for electrophilic attack, it began to seriously compete with salt 1. O,N 3 As a result,As a result, there wasthere awas relatively a relatively high high content content ofof O,N-dialkylation-dialkylation product product 3. .

Me O Me O Me O

EtI, DMSO ONa O Et OEt + S O x H O o S O S O N 2 25 C, 5 h N N O O O H H Et 1 2 3 Scheme 2. Alkylation of sodium 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate monohydrate Scheme 2. Alkylation of sodium 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (1) (Et = C2H5). monohydrate (1) (Et = C2H5). With regard to their solubility in ethanol, esters 2 and 3 differ insignificantly, therefore, it is not Withpossible regard to separate to their them solubility by conventional in ethanol, crys esterstallization2 andfrom 3thisdiffer solvent. insignificantly, However, quite therefore, unexpectedly, much more interesting results were obtained than the trivial separation of the it is not possible to separate them by conventional crystallization from this solvent. However, reaction mixture into its individual components. Thus, the crystallization of a crude product from quite unexpectedly,ethanol produced much two moreoutwardly interesting different resultstypes of were crystals: obtained parallelepipeds than the and trivial thick separationplates. of the reactionAccording mixture to the intoX-ray its structural individual analysis, components. the first of them—parallelepipeds—appeared Thus, the crystallization of to a crudebe ethyl product from ethanol1-ethyl-4-methyl-2,2-dioxo-1 produced two outwardlyH-2λ6,1-benzothiazine-3-carboxylate different types of crystals: (3) (Figure parallelepipeds 2). and thick plates. According toThere the were X-ray two structural molecules analysis, (A and B the) of first this ofcompound them—parallelepipeds—appeared that differed in some geometrical to be ethyl parameters in the asymmetric part of the unit cell. The benzothiazine cycle adopted a distorted sofa 1-ethyl-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (3) (Figure2). conformation in molecules A and B (the puckering parameters [45] are: S = 0.65, Θ = 56.3, Ψ = 33.1 in molecule A and S = 0.65, Θ = 56.6, Ψ = 34.9 in molecule B). The C(8) and S(1) atoms deviated from the mean plane of the remaining atoms of this cycle by 0.36 Å and 0.95 Å in molecule A and 0.34 Å and 0.95 Å in molecule B. The nitrogen atom had a slightly pyramidal configuration in both molecules (the sum of the bond angles centered at the N(1) atom is 352° in molecule A or 353° in molecule B). The C(1)–N(1) bond was elongated (1.422(3) Å in A and 1.426(2) Å in B) as compared to the mean value [46] 1.371 Å. The carboxylic fragment was turned in relation to the endocyclic double bond (the C(7)–C(8)–C(9)–O(1) torsion angle was −52.9(3)° in A and 51.5(3)° in B) and the C(7)–C(8) bond (1.344(3) Å in A and 1.335(2) Å in B) was elongated as compared to the mean value 1.326 Å) due to the

Sci. Pharm. 2018, 86, 21 8 of 17

There were two molecules (A and B) of this compound that differed in some geometrical parameters in the asymmetric part of the unit cell. The benzothiazine cycle adopted a distorted sofa conformation in molecules A and B (the puckering parameters [45] are: S = 0.65, Θ = 56.3, Ψ = 33.1 in molecule A and S = 0.65, Θ = 56.6, Ψ = 34.9 in molecule B). The C(8) and S(1) atoms deviated from the mean plane of the remaining atoms of this cycle by 0.36 Å and 0.95 Å in molecule A and 0.34 Å and 0.95 Å in molecule B. The nitrogen atom had a slightly pyramidal configuration in both molecules ◦ ◦ (the sum of the bond angles centered at the N(1) atom is 352 in molecule A or 353 in molecule B). The C(1)–N(1) bond was elongated (1.422(3) Å in A and 1.426(2) Å in B) as compared to the mean value [46] 1.371 Å. The carboxylic fragment was turned in relation to the endocyclic double bond ◦ ◦ (the C(7)–C(8)–C(9)–O(1) torsion angle was −52.9(3) in A and 51.5(3) in B) and the C(7)–C(8) bond (1.344(3) Å in A and 1.335(2) Å in B) was elongated as compared to the mean value 1.326 Å) Sci. Pharm. 2018, 86, x 8 of 17 due to the repulsion between the vicinal methyl and ester substituents. The ethyl group of the ester substituentSci.repulsion Pharm. 2018 between was, 86, x localized the vicinal in methylsp-conformation and ester substituents. relatively The toethyl the group C(9)–O of (1)the bondester substituent and8 wasof 17 almost was localized in sp-conformation relatively to the C(9)–O(1) bond and was almost orthogonal to the orthogonal to the carboxylic fragment (the O(1)–C(9)–O(2)–C(10) and C(9)–O(2)–C(10)–C(11) torsion angles are −3.3(3)repulsioncarboxylic◦ and between 82.0(2)fragment ◦the in(the vicinal molecule O(1) –Cmethyl(9)–OA (2) andand–C(10) 0.5(3)ester and subs ◦Cand(9)tituents.–O−(2)–C84.5(2)(10) The–C (11)ethyl◦ intorsion moleculegroup angles of theB ). areester The − 3.3(3)°substituent ethyl substituentand was82.0(2)° localized in molecule in sp-conformation A and 0.5(3)° relatively and −84.5(2)° to the inC (9)molecule–O(1) bond B ).and The was ethyl almost substituent orthogonal at the to Nthe(1) at the N(1) atom was turned relatively to the C(1)–N(1) endocyclic bond (the C(1)–N(1)–C(12)–C(13) carboxylicatom was turnedfragment relatively (the O to(1)–C the(9) –OC(1)(2)–N–C(1)(10) endocyclic and C(9)–O bond(2)–C (10)(the–C C(11)(1) –Ntorsion(1)–C(12) angles–C(13) torsionare −3.3(3)° angle wasand torsion angle was 63.7(2)◦ in molecule A and −69.8(2)◦ in molecule B). 82.0(2)°63.7(2)° inin moleculemolecule A A and and − 69.8(2)°0.5(3)° andin molecule −84.5(2)° B ).in molecule B). The ethyl substituent at the N(1) atom was turned relatively to the C(1)–N(1) endocyclic bond (the C(1)–N(1)–C(12)–C(13) torsion angle was 63.7(2)° in molecule A and −69.8(2)° in molecule B).

Figure 2. The spatial structure of 1-ethyl substituted ester 3 according to the X-ray diffraction data. Figure 2. The spatial structure of 1-ethyl substituted ester 3 according to the X-ray diffraction data. The The atoms are represented by thermal vibration ellipsoids of 50% probability. atoms are represented by thermal vibration ellipsoids of 50% probability. Figure 2. The spatial structure of 1-ethyl substituted ester 3 according to the X-ray diffraction data. TheAccording atoms are to represented the X-ray by structural thermal vi brationanalysis, ellipsoids the second of 50% probability.type of crystals from the mixture 6 Accordingobtained after to the alkylation X-ray structural of ester 1—thick analysis, plates—were the second ethyl 4-methyl-2,2-dioxo-1 type of crystalsH from-2λ ,1- the mixture benzothiazine-3-carboxylate (2) (Figure 3). obtained afterAccording the alkylationto the X-ray of structural ester 1—thick analysis, plates—werethe second type ethyl of crystals 4-methyl-2,2-dioxo-1 from the mixtureH -2λ6,1- obtained after the alkylation of ester 1—thick plates—were ethyl 4-methyl-2,2-dioxo-1H-2λ6,1- benzothiazine-3-carboxylate (2) (Figure3). benzothiazine-3-carboxylate (2) (Figure 3).

Figure 3. The spatial structure of ethyl ester 2 according to the X-ray diffraction data. The atoms are represented by thermal vibration ellipsoids of 50% probability. Figure 3. The spatial structure of ethyl ester 2 according to the X-ray diffraction data. The atoms are Figure 3. The spatial structure of ethyl ester 2 according to the X-ray diffraction data. The atoms are representedUnlike its Nby-ethyl-substituted thermal vibration ellipsoids analog 3of, 50%ester probability. 2 is readily soluble in the aqueous solution of representedsodium carbonate by thermal due vibration to salt form ellipsoidsation by of the 50% cyclic probability. sulfamide group. This property was used for simpleUnlike and itseffective N-ethyl-substituted separation of analog the mixture 3, ester of2 isesters readily 2 andsoluble 3. Esterin the 2 ,aqueous obtained solution after the of sodiumacidification carbonate of the due alkaline to salt solution,formation was by theagain cyclic recrystallized sulfamide group. in its Thispure property form from was the used same for simplesolvent, andi.e., effectiveethanol. Surprisingly,separation of this the timemixture the crystalof esters habit 2 andchanged—the 3. Ester 2crystals, obtained became after thinthe acidificationslices. Moreover, of the X-ray alkaline diffraction solution, analysis was again showed recrystallized that the inchanges its pure affected form from not theonly same the solvent,appearance, i.e., butethanol. also Surprisingly,the crystal st ructure.this time In th othere crystal words, habit ester changed—the 2 was able crystalsto crystallize became in thintwo slices.polymorphic Moreover, modifications: X-ray diffraction in the presence analysis of compoundshowed that 3 it theformed changes a monoclinic affected 2M not form only with the a appearance,spatial Рс group, but also and the in crystalits pure st formructure. it formedIn other an words, orthorhombic ester 2 was 2O ableform towith crystallize a spatial in Pbcatwo polymorphicgroup. Comparison modifications: of the geometri in the presencecal characteristics of compound of the 3 it molecule formed ain monoclinic different crystalline 2M form withforms a spatialof ester Рс2 showedgroup, andthat intheir its molecularpure form (but it formed not crystal an line)orthorhombic structure 2Owas form almost with the asame. spatial In Pbcaboth group.cases, theComparison benzothiazine of the cyclegeometri wacals in characteristics conformation of of the a moleculedistorted in sofa, different as evidenced crystalline by forms the ofparameters ester 2 showed of puckering that their and molecular deviation (but of Snot(1)and crystal С(8) line)atoms structure from the was mean almost square the planesame. ofIn otherboth cases, the benzothiazine cycle was in conformation of a distorted sofa, as evidenced by the parameters of puckering and deviation of S(1)and С(8) atoms from the mean square plane of other

Sci. Pharm. 2018, 86, 21 9 of 17

Unlike its N-ethyl-substituted analog 3, ester 2 is readily soluble in the aqueous solution of sodium carbonate due to salt formation by the cyclic sulfamide group. This property was used for simple and effective separation of the mixture of esters 2 and 3. Ester 2, obtained after the acidification of the alkaline solution, was again recrystallized in its pure form from the same solvent, i.e., ethanol. Surprisingly, this time the crystal habit changed—the crystals became thin slices. Moreover, X-ray diffraction analysis showed that the changes affected not only the appearance, but also the crystal structure. In other words, ester 2 was able to crystallize in two polymorphic modifications: in the presence of compound 3 it formed a monoclinic 2M form with a spatial Pc group, and in its pure form it formed an orthorhombic 2O form with a spatial Pbca group. Comparison of the geometrical characteristics of the molecule in different crystalline forms of ester 2 showed that their molecular (but not crystalline) structure was almost the same. In both cases, the benzothiazine cycle was in conformation of a distorted sofa, as evidenced by the parameters of puckering and deviation of S(1)and C(8) atoms from the mean square plane of other atoms of the cycle (see Table1). The steric repulsion between the methyl group and the ester substituent (shortened intramolecular contact C(12) ... O(1) compared to the sum of the van der Waals radii [47] 3.00 Å) lead to the elongation of the C(7)–C(8) bond and the turn of the ethoxycarbonyl fragment with respect to the endocyclic double bond (C(7)–C(8)–C(9)–O(1) torsion angle). The ethyl group is in an ap- conformation with respect to the C(8)–C(9) bond and somewhat turned in relation to C(9)–O(2) bond (C(8)–C(9)–O(2)–C(10) and C(9)–O(2)–C(10)–C(11) torsion angles, Table1).

Table 1. Some geometric characteristics of the polymorphic form ester 2.

Entry Parameter Monoclinic (2M) Orthorhombic (2O) 1 S 0.58 0.63 2 Θ, deg 49.8 54.2 3 Ψ, deg 37.5 31.4 4 S(1),Å 0.78 0.89 5 C(8),Å 0.22 0.33 6 C(7)–C(8),Å 1.361(4) 1.362(4) 7 N(1)–C(1),Å 1.412(4) 1.398(3) 8 C(7)–C(8)–C(9)–O(1), deg −36.1(5) −37.8(5) 9 C(8)–C(9)–O(2)–C(10), deg 177.9(3) −179.8(2) 10 C(9)–O(2)–C(10)–C(11), deg −160.4(4) 173.3(3) 11 C(12) ...O(1),Å 2.94 2.96

Interestingly, all our attempts to obtain a monoclinic polymorph modification 2M by the homogeneous crystallization of pure ester 2 from different solvents (ethanol, ethyl acetate, aqueous N,N-dimethylformamide or methylene chloride) failed; furthermore, an exclusively orthorhombic form 2O was always isolated. For some yet unknown reasons, the formation of the monoclinic form 2M appeared to be possible only under conditions of heterogeneous crystallization, i.e., with the obligatory joint presence of foreign particles of N-ethyl-substituted ester 3 in the solution. It is quite difficult (if possible at all) to provide a theoretical explanation for this fact, which was accidentally discovered. Therefore, such findings often remain either a carefully guarded secret or a daily practice, but, unfortunately, they do not have any scientific background [48]. However, currently we were presented with the solution to the practical problem of the isolation of the monoclinic form 2M in its pure form, which is of interest for pharmacological tests. It is clear that the method of separation of the mixture of esters 2M and 3 described above is unsuitable for this, since it involves dissolving the target product. Thus, it was found that the impurity of N-ethyl-substituted ester 3 can be transferred into a solution, but the monoclinic form 2M is left unchanged in a crystalline form using hot hexane (see Section2). The phase purity of crystal forms is known to be very important for pharmaceutical study. Any impurity of by-product or other crystal form (polymorphic modification, hydrate, solvate, etc.) Sci. Pharm. 2018, 86, 21 10 of 17 leads to unclear or wrong data regarding the biological activity. The phase purity of the monoclinic and orthorhombic modifications of ester 2 was confirmed by powder diffraction study (Figure4). The data obtained from the powder diffraction experiments were compared to the data calculated from the single crystal structures and showed clearly that both polymorphic modifications were pure and did not contain any impurities. Thus, it may be expected that the biological experiment was informative andSci. Pharm. reasonably 2018, 86, carriedx out. 10 of 17

Figure 4. X-ray powder patterns of the monoclinic 2M (top) and orthorhombic 2M (down) forms of Figure 4. X-ray powder patterns of the monoclinic 2M (top) and orthorhombic 2M (down) forms of ester 2 and their Rietveld refinement: red—experimental X-ray diffraction patterns, black—calculated ester 2 and their Rietveld refinement: red—experimental X-ray diffraction patterns, black—calculated X-ray diffraction patterns, green—Bregg position of diffraction maximum, blue—difference between X-ray diffraction patterns, green—Bregg position of diffraction maximum, blue—difference between experimental and calculated intensity values in each point. experimental and calculated intensity values in each point.

3.2. Evaluation of the Anti-Inflammatory and Analgesic Activity 3.2. Evaluation of the Anti-Inflammatory and Analgesic Activity The analysis of the results of our tests (Tables 2 and 3) suggests that the dialkylation product of The analysis of the results of our tests (Tables2 and3) suggests that the dialkylation product of salt 1, i.e.,ethyl 1-ethyl-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (3), is of little salt 1, i.e.,ethyl 1-ethyl-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (3), is of little interest interest as an object for further pharmacological study. The basis for this conclusion was a very as an object for further pharmacological study. The basis for this conclusion was a very moderate moderate analgesic and an extremely weakly expressed anti-inflammatory effect demonstrated by analgesic and an extremely weakly expressed anti-inflammatory effect demonstrated by this compound this compound in the animal experiments. However, when revealing the regularities of the in the animal experiments. However, when revealing the regularities of the “structure–activity” “structure–activity” relationship, which are important for further purposeful searches for new relationship, which are important for further purposeful searches for new biologically active substances, biologically active substances, such information is also valuable. such information is also valuable. Much more attention should be paid to the study and comparison of the biological properties Much more attention should be paid to the study and comparison of the biological of the different crystal modifications of ester 2. These studies have recently aroused keen interest properties of the different crystal modifications of ester 2. These studies have recently aroused among a wide range of researchers engaged in the creation of drugs, since they involve the keen interest among a wide range of researchers engaged in the creation of drugs, since they intellectual field called polymorphism of crystals, which is insufficiently studied, still poorly involve the intellectual field called polymorphism of crystals, which is insufficiently studied, understood and not always predictable. Indeed, the crystal modifications of ethyl still poorly understood and not always predictable. Indeed, the crystal modifications of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (2) obtained were surprisingly different in their biological properties (Tables 2 and 3), although, in fact, they are the same substance. Thus, if the orthorhombic form 2O, with a sufficiently pronounced analgesic action, had a very weak anti-inflammatory effect, then the monoclinic form 2M under the same conditions and in the same dose was both a powerful analgesic and anti-exudative agent significantly exceeding not only Piroxicam, but its more active analog Meloxicam by these indicators. All this gives grounds for a detailed study of the features of the crystal structure of esters 2M and 2O, as an obvious factor that largely contributes to the observed therapeutic effects. In comparison, in the case of N-benzyl-4-hydroxy-1-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide and the solvates of its 4-O-sodium salt, this factor was the spatial structure [49].

Sci. Pharm. 2018, 86, 21 11 of 17

4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (2) obtained were surprisingly different in their biological properties (Tables2 and3), although, in fact, they are the same substance. Thus, if the orthorhombic form 2O, with a sufficiently pronounced analgesic action, had a very weak anti-inflammatory effect, then the monoclinic form 2M under the same conditions and in the same dose was both a powerful analgesic and anti-exudative agent significantly exceeding not only Piroxicam, but its more active analog Meloxicam by these indicators. All this gives grounds for a detailed study of the features of the crystal structure of esters 2M and 2O, as an obvious factor that largely contributes to the observed therapeutic effects. In comparison, in the case of N-benzyl-4-hydroxy-1-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamide and the solvates of its Sci. Pharm. 2018, 86, x 11 of 17 4-O-sodium salt, this factor was the spatial structure [49].

Table 2. The anti-inflammatory activity of esters 2M, 2O, 3, and reference drugs. Table 2. The anti-inflammatory activity of esters 2M, 2O, 3, and reference drugs.

Me O

OEt S O N O R Volume of Volume of Volume Volumeof of Δ Volume∆ Volume Anti-Anti-InflammatoryInflammatory Entry EntryProduct Product R Damaged R ExtremityDamaged Non-DamagedNon-Damaged Extremity (Volume(Volume Activity,Activity, Compared Compared to 3 3 (mm3) Extremity (mm ) (mmExtremity3) (mm ) Increase)Increase) toControl Control (%) (%) 1 1 2M 2MH 377.1H ± 29.9 377.1 ± 29.9 318.3 ± 20.4 318.3 ± 20.4 58.858.8 ± 5.8± 1,2,35.8 1,2,3 85.885.8 2 2 2O 2O H 736.2H ± 23.3 736.2 ± 23.3 449.5 ± 23.9 449.5 ± 23.9 286.7286.7 ± 8.6± 1,2,38.6 1,2,3 30.730.7 1,2,3 3 33 3Et 743.5 Et ± 32.0 743.5 ± 32.0 455.2 ± 21.2 455.2 ± 21.2 288.2288.2 ± 8.0± 1,2,38.0 30.330.3 1 4 4Piroxicam Piroxicam − 566.7− ± 160.4 566.7 ± 160.4 342.8 ± 19.8 342.8 ± 19.8 223.8223.8 ± 6.1± 16.1 45.945.9 − ± ± ± 1 5 5Meloxicam Meloxicam − 492.6 ± 39.3 492.6 39.3 361.2 ± 26.1 361.2 26.1 131.3131.3 ± 8.0 18.0 68.368.3 6 Control − 768.7 ± 27.3 354.9 ± 11.6 413.7 ± 32.2 0 6 Control − 768.7 ± 27.3 354.9 ± 11.6 413.7 ± 32.2 0 1 p ≤ 2 p ≤ 1 Differences statistically statistically significant significant for for 0.05p ≤ 0.05 vs.control. vs. control.Differences 2 Differences statistically statistically significant significant for 0.05 for vs. p Piroxicam. 3 Differences statistically significant for p ≤ 0.05 vs. Meloxicam. ≤ 0.05 vs. Piroxicam. 3 Differences statistically significant for p ≤ 0.05 vs. Meloxicam. Table 3. The Analgesic Activity of Esters 2M, 2O, 3, and Reference Drugs. Table 3. The Analgesic Activity of Esters 2M, 2O, 3, and Reference Drugs. Pain Threshold on Pain Threshold on Analgesic Activity, Pain Threshold on Pain Threshold on ∆ Pain Analgesic Activity, Entry Product R Damaged Non-Damaged Δ Pain Compared to Entry Product R Damaged Extremity Non-Damaged2 Extremity2 Threshold Compared to Control Extremity (g/mm ) Extremity (g/mm ) Threshold Control (%) (g/mm2) (g/mm2) (%) 1 2M H 370.0 ± 15.2 340.0 ± 17.0 30.0 ± 4.5 1,2,3 90.6 1 2M H 370.0 ± 15.2 340.0 ± 17.0 30.0 ± 4.5 1,2,3 90.6 2 2O H 344.0 ± 28.8 236.0 ± 11.4 108.0 ± 8.6 1,2,3 66.0 1,2,3 2 3 2O 3 H Et 344.0 ± 28.8 380.0 ± 10.5 236.0 220.0 ± 11.4± 13.4 160.0 108.0± ±10.5 8.61,2,3 49.766.0 3 43 PiroxicamEt − 380.0 ± 10.5504.0 ± 18.1 220.0 340.0 ± 13.4± 15.2 160.0164.0 ± 10.58.1 11,2,3 48.449.7 4 5Piroxicam Meloxicam − −504.0 ± 18.1414.0 ± 19.6 340.0 326.0 ± 15.2± 26.4 88.0 164.0± 11.6± 8.11 1 72.348.4 5 6Meloxicam Control − −414.0 ± 19.6593.0 ± 56.3 326.0 275.0 ± 26.4± 32.1 318.0 88.0 ± 11.634.9 1 0 72.3 6 1 DifferencesControl statistically− significant593.0 ± 56.3 for p ≤ 0.05 vs. control. 275.0 2± Differences32.1 statistically 318.0 ± significant34.9 for p ≤ 0.05 0 vs. 3 1Piroxicam. DifferencesDifferences statistically statistically significant significant for p ≤ for 0.05p ≤ vs.0.05 control. vs. Meloxicam. 2 Differences statistically significant for p ≤ 0.05 vs. Piroxicam. 3 Differences statistically significant for p ≤ 0.05 vs. Meloxicam. 3.3. The Crystal Structure Study 3.3. TheAs Crystal mentioned Structure above, Study the spatial structure of ester 2 was almost same in the two polymorphic formsAs2M mentionedand 2O .above, In contrast, the spatial the structure crystal structure of ester 2 of was the almost two polymorphic same in the two forms polymorphic was very 6 formsdifferent. 2M and In the 2O monoclinic. In contrast, crystals, the crystal the 2Mstructuremolecules of the of two ethyl polymorphic 4-methyl-2,2-dioxo-1 forms wasH-2 veryλ ,1- different.benzothiazine-3-carboxylate In the monoclinic crystals, formed the the 2M infinite molecules chains of ethyl (Figure 4-methyl-2,2-dioxo-15, left) along the crystallographicH-2λ6,1- benzothiazine-3-carboxylatedirection [001]. The molecules formed within the the infinite chain ch areains bound (Figure by the5, left) N(1) –Halong... theO(4 0)crystallographicintermolecular ◦ directionhydrogen [001]. bonds The (x, 1molecules−y, −0.5 within + z;H the... Ochain 2.14 are Å, N–Hbound... byO the 165 N)(1) and–H…O stacking(4′) intermolecular interactions hydrogen(the distance bonds between (x, 1 aromatic−y, −0.5 + rings z; H…O was 3.472.14 Å),Å, N–H…O simultaneously. 165°) and There stacking were interactions not any specific (the distanceinteractions between between aromatic the molecules rings was belonging 3.47 Å), to simultaneously. the neighboring There chains. were In not the any case specific of the interactionsorthorhombic between modification the molecules2O (Figure belonging5, right), to the the molecules neighboring of the chains. ester formedIn the thecase infinite of the orthorhombicchains in the modification crystallographic 2O (Figure direction 5, right), [100] the and molecules were bound of the ester only formed by the the N(1) infinite–H ... chainsO(40) ◦ inintermolecular the crystallographic hydrogen direction bonds ([100]−0.5 and + x, were 0.5 − boundy, 1 − onlyz) (Hby ...theO N 2.10(1)–H…O Å, N–H(4′) intermolecular... O 161 ) hydrogenwithin the bonds chain. (− Stacking0.5 + x, 0.5 interactions − y, 1 − z) were (H…O observed 2.10 Å, betweenN–H…O the 161°) molecules within the of thechain. neighboring Stacking interactions were observed between the molecules of the neighboring chains (the distance between the aromatic rings was 3.36 Å). The geometric characteristics of the hydrogen bonds and stacking interactions allowed us to presume that the molecules of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1- benzothiazine-3-carboxylate were bound slightly stronger in the form 2O. It should be noted that the analysis of the geometric characteristics of intermolecular interactions does not allow discussion of the interaction energies between molecules in the crystal in full measure. At the same time, the difference in the biological activity of the two polymorphic modifications of one compound with the same spatial structure can be caused by the rate of the crystal structure destruction and therefore depends on the interactions between molecules.

Sci. Pharm. 2018, 86, 21 12 of 17 chains (the distance between the aromatic rings was 3.36 Å). The geometric characteristics of the hydrogen bonds and stacking interactions allowed us to presume that the molecules of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate were bound slightly stronger in the form 2O. It should be noted that the analysis of the geometric characteristics of intermolecular interactions does not allow discussion of the interaction energies between molecules in the crystal in full measure. At the same time, the difference in the biological activity of the two polymorphic modifications of one compound with the same spatial structure can be caused by the rate of the crystal structure destruction and therefore depends on the interactions between molecules. Sci. Pharm. 2018, 86, x 12 of 17

2M 2O

Figure 5. The chains of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate molecules in 6 Figure 5. Thethe monoclinic chains of 2M ethyl (left) 4-methyl-2,2-dioxo-1 and orthorhombic 2O (rightH-2) polymorphicλ ,1-benzothiazine-3-carboxylate forms. molecules in the monoclinic 2M (left) and orthorhombic 2O (right) polymorphic forms. The earlier suggested method of crystal structure analysis based of the comparison of the pairwise interaction energies between molecules in the crystal phase [37,50], allows one to turn The earlierfrom the suggested qualitative method analysis of of crystal the obtained structure intermolecular analysis based interactions of the comparisonto the quantitative of the pairwise interactionestimation energies of betweenthe full interaction molecules energies in the between crystal molecules. phase [37According,50], allows to this onemethod, to turn any from the qualitativemolecule analysis in ofa crystal the obtained may be chosen intermolecular as the basic interactionsmolecule M0, which to the is quantitativea building unit estimation of a crystal. of the full interactionThe energies first coordination between sphere molecules. of this molecule According is determined to this method,using a standard any molecule option and in contains a crystal a may be quantity of neighboring molecules М1,2…n, interacting with the basic M0. The interaction energies chosen as thewithin basic the moleculedimers М M0–М0,1, whichМ0–М2, is М a0– buildingМ3 … М0– unitМn are of aestimated crystal. Theusing first quantum-chemical coordination sphere of this moleculecalculations. is determined The normalization using a standard of the obtained option andvalues contains of interaction a quantity energies of neighboringrelatively the molecules strongest for this basic molecule interaction energy causes the replacement of the absolute energies M1,2 . . . n, interacting with the basic M0. The interaction energies within the dimers M0–M1,M0–M2, M –M ...valuesM –M by theirare relative estimated values using which quantum-chemicalare used for the construction calculations. of the energy-vector The normalization diagram of the 0 3 (EVD).0 Thisn diagram is an image of the basic molecules reflecting its interaction energies with all obtained valuesneighboring of interaction molecules. energies relatively the strongest for this basic molecule interaction energy causes the replacementThe application of the of absolutethe suggested energies method values to the bymonoclinic their relative polymorphic values modification which are 2M used for the constructionrevealed of the that energy-vector the first coordination diagram sphere (EVD). of the This basic diagram molecule is ancontained image 14 of neighboring the basic molecules molecules bound to it by some energy and symmetry operations. The summary interaction energy reflecting its interaction energies with all neighboring molecules. of the basic molecule with all those belonging to its first coordination sphere was −78.1 kcal/mol. The applicationThe strongest of interactions the suggested were observed method with to the two monoclinic neighboring molecules polymorphic causing modification the formation2M of revealed that the firsttwo coordination identical dimers sphere 2M_1 ofand the 2M_2 basic with molecule equal interaction contained energies 14 neighboring (Table 4). Thismolecules caused the bound to it by someformation energy andof a zigzag symmetry chain (Figure operations. 6) in the Thecrystallographic summary direction interaction [001]. energyThe interaction of the energy basic molecule of the basic molecule with the two neighboring molecules within this chain was −35.9 kcal/mol or with all those belonging to its first coordination sphere was −78.1 kcal/mol. The strongest interactions 46.0% of the full interaction energy with all molecules belonging to its first coordination sphere. The were observedmolecules with within two the neighboring chain were moleculesbound by the causing N–H…O theintermolecular formation hydrogen of two identicalbonds and dimersthe 2M_1 and 2M_2stackingwithequal interaction, interaction as it was energies detected (Table using 4the). Thistraditional caused analysis the formationof the intermolecular of a zigzag chain (Figure6) ininteractions the crystallographic in the crystal phase. direction The separated [001]. The chain interaction can be considered energy the ofprimary the basic basic moleculestructural with the motif of the monoclinic polymorphic modification of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1- two neighboring molecules within this chain was −35.9 kcal/mol or 46.0% of the full interaction energy benzothiazine-3-carboxylate. with all moleculesIt should belonging be noted tothat its the first traditional coordination analysis sphere.of the crystal The packing molecules for this within structure the is chain were bound by theconfined N–H by... theO separation intermolecular out of hydrogenthe chain. It bonds is caused and by the the stacking absence of interaction, specific interactions as it was detected using the traditionalbetween molecules analysis belonging of the to intermolecularthe neighboring chains. interactions The application in the of crystal the quantum-chemical phase. The separated calculations allowed us to estimate the interaction energies between the molecules bound by chain can be considered the primary basic structural motif of the monoclinic polymorphic modification non-specific interactions. As with the 2M crystal, it was shown that the interaction energies 6 of ethyl 4-methyl-2,2-dioxo-1between molecules of theH -2neighboringλ ,1-benzothiazine-3-carboxylate. chains were not equal. The chains arranged within the plane It shouldparallel be to noted the crystallographic that the traditional plane (011) analysis interacted of more the crystalstrongly packing(the interaction for this energy structure between is confined by the separationthe molecules outof belonging the chain. neighboring It is caused chains by was the −17.6 absence kcal/mol of specificor 22.5% interactionsfrom the total interaction between molecules energy within the first coordination sphere). As a result, the layer was formed as the second basic belonging to the neighboring chains. The application of the quantum-chemical calculations allowed us structural motif. The interaction energy of the basic molecule with all the neighboring molecules

Sci. Pharm. 2018, 86, 21 13 of 17 to estimate the interaction energies between the molecules bound by non-specific interactions. As with the 2M crystal, it was shown that the interaction energies between molecules of the neighboring chains were not equal. The chains arranged within the plane parallel to the crystallographic plane (011) interacted more strongly (the interaction energy between the molecules belonging neighboring chains was −17.6 kcal/mol or 22.5% from the total interaction energy within the first coordination sphere). As a result, the layer was formed as the second basic structural motif. The interaction energy of the basic molecule with all the neighboring molecules within the layer was −53.5 kcal/mol (68.5%), while the interaction energy with molecules of the neighboring layer was only −9.9 kcal/mol (12.7%). Thus, the monoclinic crystal structure of ester 2 was built by the molecules as building units and may be divided on two levels of organization from the viewpoint of interaction energies between molecules: chains as the primary level of organization and layers of strongly interacting chains as the secondary level of organization. Sci. Pharm. 2018, 86, x 13 of 17

Table 4. withinSymmetry the layer codes, was bonding−53.5 kcal/mol type, (68.5%), interaction while the energy interaction of the energy basic with molecule molecules with of neighboring the −1 ones (Eintneighboring, kcal mol layer), withwas only the highest−9.9 kcal/mol values (12.7%). (more Thus, than the 5% monoclinic of the total crystal interaction structure of energy) ester 2 and the contributionwas built of this by the energy molecules to the as totalbuilding interaction units and may energy be divided (%) in on the two crystals levels of2M organizationand 2O. from the viewpoint of interaction energies between molecules: chains as the primary level of organization and layers of strongly interacting chains as the secondary level of organization. Contribution to the Total Dimer Symmetry Operation E (kcal · mol−1) Interaction Type int Interaction Energy (%) Table 4. Symmetry codes, bonding type, interaction energy of the basic molecule with neighboring ones (Eint, kcal mol−1), with the highest valuesCrystals (moreof than2M 5% of the total interaction energy) and the 2M_1 contributionx, 1 − ofy this, 0.5 energy + z to the total interaction−17.95 energy (%) in the 22.98crystals 2M and 2O. N–H . . . O, stacking 2M_2 x, 1 − y, −0.5 + z −17.95 22.98 N–H . . . O, stacking Symmetry Contribution to the Total Interaction Dimer − Eint (kcal · mol−−1) Interaction Type 2M_3 x, 2 Operationy, 0.5 + z 8.78Energy (%) 11.24 non-specific − − − 2M_4 x, 2 y, 0.5 + z 8.78Crystals of 2M 11.24 non-specific 2M_5 2M_1 x1, 1 + −x y,, y0.5, z + z −17.95 −5.8622.98 7.50N–H…O, non-specific stacking 2M_6 2M_2 x−, 1 − + yx, −,0.5y, z+ z −17.95 −5.8622.98 7.50N–H…O, non-specific stacking 2M_3 x, 2 − y, 0.5 + z Crystals−8.78 of 2O, building unit is a11.24 molecule non-specific 2M_4 x, 2 − y, −0.5 + z −8.78 11.24 non-specific 2O_1 2M_5 1 − x,1 1 +− x, y, z 1 − z −5.86 −16.567.50 22.41 C–Hnon-specific . . . O, C–H . . . π 2O_2 2M_6 0.5 + x,− 0.51 + x−, yy, z, 1 − z −5.86 −11.517.50 15.58non-specific N–H . . . O 2O_3 −0.5 + x, 0.5 − y, 1 − z Crystals− of11.51 2O, building unit is a molecule 15.58 N–H . . . O 2O_4 2O_1 −x1 ,− 1 x,− 1 −y ,y, 1 1− − zz −16.56 −7.9822.41 10.81С–Н…О, stackingС–Н…π 2O_5 2O_2 0.50.5 ++ x,, 0.5y, 0.5− y, −1 −z z −11.51 −5.0315.58 6.81 N–H…O C–H . . . π 2O_6 2O_3 −−0.50.5 + xx, ,0.5y, − 0.5 y, 1− − z −11.51 −5.0315.58 6.81 N–H…O C–H . . . π 2O_4 −x, 1 − y, 1 − z −7.98 10.81 stacking 2O_5 0.5 + x, y, 0.5 − z Crystals−5.03 of 2O, building unit is6.81 a dimer С–Н…π 2O_d_12O_6 1/2 +−0.5x, 1/2+ x, y−, 0.5y, − 1 z − z −5.03 −16.116.81 13.25С–Н N–H…π . . . O 2O_d_2 1/2 + x, 3/2 − y, 1 − z Crystals− 16.11of 2O, building unit is a dimer 13.25 N–H . . . O 2O_d_32O_d_1− 1/21/2 + +x x,, 1/2 1/2 −− y, y1, − 1 z− z −16.11 −16.1113.25 13.25 N–H…O N–H . . . O 2O_d_2 1/2 + x, 3/2 − y, 1 − z −16.11 13.25 N–H…O 2O_d_4 −1/2 + x, 3/2 − y, 1 − z −16.11 13.25 N–H . . . O 2O_d_3 −1/2 + x, 1/2 − y, 1 − z −16.11 13.25 N–H…O − 2O_d_52O_d_4 −1/21 + +x, x3/2, y −, zy, 1 − z −16.11 10.4713.25 8.61 N–H…O stacking 2O_d_62O_d_5 −11 ++ x,, yy, ,z z −10.47 −10.478.61 8.61 stacking stacking 2O_d_72O_d_6 0.5 − x−,1 1 +− x, yy,, z 0.5 + z −10.47 −7.528.61 6.18 C–H stacking . . . O, C–H . . . π 2O_d_82O_d_71/2 −0.5x −, 1x, −1 −y y,, −0.51/2 + z + z −7.52 −7.526.18 6.18С C–H–Н…О ., . С .– O,Н… C–Hπ . . . π 2O_d_92O_d_83/2 1/2− −x x,, 1 1 −− yy, ,−1/2 + +z z −7.52 −7.526.18 6.18С C–H–Н…О ., . С .– O,Н… C–Hπ . . . π 2O_d_102O_d_93/2 −3/2x −, 1x, −1 −y y,, −1/21/2 + z + z −7.52 −7.526.18 6.18С C–H–Н…О ., . С .– O,Н… C–Hπ . . . π 2O_d_10 3/2 − x, 1 − y, −1/2 + z −7.52 6.18 С–Н…О, С–Н…π

Figure 6. The packing of ester 2 in the monoclinic polymorphic form 2M presented as the packing of Figure 6. Themolecules packing (left of) and ester packing2 in theof energy-vector monoclinic diagrams polymorphic (right). The form primary2M basicpresented structural asmotif the packing of molecules (left(chain)) and is highlighted packing in of blue. energy-vector The secondary basic diagrams structural ( rightmotif (layer)). The is highlighted primary in basic pink. structural motif (chain) is highlighted in blue. The secondary basic structural motif (layer) is highlighted in pink. The analysis of the orthorhombic polymorphic modification 2O showed that the first coordination sphere of the ester 2 contained 14 neighboring molecules, similar the monoclinic modification. However, the total interaction energy of the basic molecule with all those in its first coordination sphere was slightly weaker compared to the monoclinic modification (−73.9 kcal/mol). In contrast to the monoclinic crystal form, the basic molecules formed the strongest interaction with

Sci. Pharm. 2018, 86, 21 14 of 17

The analysis of the orthorhombic polymorphic modification 2O showed that the first coordination sphere of the ester 2 contained 14 neighboring molecules, similar the monoclinic modification. However, the total interaction energy of the basic molecule with all those in its first coordination sphere was slightly weaker compared to the monoclinic modification (−73.9 kcal/mol). In contrast to the monoclinic crystal form, the basic molecules formed the strongest interaction with only one neighboring moleculeSci. Pharm. 2018,in 86, thex orthorhombic crystal (Table4). Taking into account that14 theof 17 interaction energy in the dimer 2O_1 is significantly stronger compared to the next dimer, we may assert that the building unitonly in crystal one neighboring2O was molecule the dimer in the (Figure orthorhombic7) rather crystal than (Table the 4). molecule.Taking into account The molecules that the in such a interaction energy in the dimer 2O_1 is significantly stronger compared to the next dimer, we may complex buildingassert unitthat the are building bound unit by in very crystal weak 2O was C–H the ...dimerO (Figure and C–H 7) rather... πthanhydrogen the molecule. bonds The and mainly by a lot of differentSci.molecules Pharm. 2018 types in, 86 ,such x of non-specifica complex building interactions unit are bound (total by electrostatic, very weak C–H…O dispersion, and C–H…14 polarization, of 17π etc.). It should be notedhydrogen that bonds this stronglyand mainly bound by a lot dimer of different was not types separated of non-specific out by interactions the traditional (total analysis of onlyelectrostatic, one neighboring dispersion, molecule polarization, in the orthorhombicetc.). It should crystal be noted (Table that 4). this Taking strongly into bound account dimer that thewas the intermolecularinteractionnot separated interactions energy out byin thethe in dimertraditional the 2O_1 crystal. analysis is significantly of the intermolecular stronger compared interactions to the innext the dimer, crystal. we may assert that the building unit in crystal 2O was the dimer (Figure 7) rather than the molecule. The molecules in such a complex building unit are bound by very weak C–H…O and C–H… π hydrogen bonds and mainly by a lot of different types of non-specific interactions (total electrostatic, dispersion, polarization, etc.). It should be noted that this strongly bound dimer was not separated out by the traditional analysis of the intermolecular interactions in the crystal.

Figure 7. DimerFigure of the7. Dimer molecules of the ofmolecules ester 2 of, which ester 2 is, which the building is the building unit ofunit the of orthorhombic the orthorhombic polymorphic polymorphic form 2O. form 2O. In the next stage of the orthorhombic crystal structure analysis, the first coordination sphere of In the nextthe stagedimer of2O_1 the was orthorhombic constructed. The crystal interaction structure energies analysis, with all 14 the neighboring first coordination dimers were sphere of the dimer 2O_1 wascalculated.Figure constructed. 7.The Dimer analysis of The the of molecules interactionthe interaction of ester energies energi 2, whiches showed withis the allbuildingthat 14 the neighboring unitbasic of dimer the orthorhombic formed dimers the werefour calculated. strongestpolymorphic interactions form 2O with. the neighboring dimers. It caused the formation of the layer parallel to the The analysis of the interaction energies showed that the basic dimer formed the four strongest crystallographic plane (110) as the primary basic structural motif (Figure 8). The neighboring dimers interactions withwereIn bound the the next neighboringby stage the N–H…Oof the orthorhombic intermolecular dimers. crystal It hydrog caused struencture bonds the analysis, within formation the the first layer coordination of and the the layer interactionsphere parallel of to the crystallographictheenergy dimer plane of the2O_1(110) basic was dimer as constructed. the within primary the The layer interaction basic was − structural85.4 energies kcal/mol with motif(70.2% all of (Figure14 the neighboring total8 ).interaction The dimers neighboring energy). were dimers calculated.The interaction The analysisenergy with of the the interactionneighboring energi layerses was showed much thatweaker: the basic−15.0 kcal/moldimer formed or 12.4%. the Thus,four were bound by the N–H ... O intermolecular hydrogen bonds within the layer and the interaction strongestthe orthorhombic interactions crystal with the structure neighboring of the dimers. ester It2 causedwas built the formationby the dimers of the layerof strongly parallel boundto the energy of thecrystallographicmolecules basic dimer as building plane within (110) units the as and the layer only primary one was level basic− of85.4 st organizationructural kcal/mol motif was (Figure (70.2% able to8). beThe of separated theneighboring total out. interaction dimers energy). The interactionwere energy bound by with the theN–H…O neighboring intermolecular layers hydrog wasen muchbonds within weaker: the layer−15.0 and kcal/mol the interaction or 12.4%. Thus, energy of the basic dimer within the layer was −85.4 kcal/mol (70.2% of the total interaction energy). the orthorhombicThe interaction crystal energy structure with the of neig thehboring ester layers2 was was built much by weaker: thedimers −15.0 kcal/mol of strongly or 12.4%. boundThus, molecules as building unitsthe orthorhombic and only onecrystal level structure of organization of the ester 2 waswas built able by to the be separateddimers of strongly out. bound molecules as building units and only one level of organization was able to be separated out.

Figure 8. The packing of the dimers 2O_1 in the orthorhombic polymorphic form presented as the packing of energy-vector diagrams. The layer as the primary basis structural motif is highlighted in blue.

Figure 8. The packing of the dimers 2O_1 in the orthorhombic polymorphic form presented as the Figure 8. Thepacking packing of energy-vector of the dimers diagrams.2O_1 The layerin as the the orthorhombicprimary basis structural polymorphic motif is highlighted form in blue. presented as the packing of energy-vector diagrams. The layer as the primary basis structural motif is highlighted

in blue. Sci. Pharm. 2018, 86, 21 15 of 17

Thus, the analysis of interaction energies between the molecules showed that two polymorphic modifications of the ester 2 had different building units (the molecule in monoclinic 2M and the dimer of the molecules in orthorhombic 2O), but a similar layered type of crystal packing. Moreover, the relations between the interaction energies within the layer and between the neighboring layers were very close in two polymorphic modifications. This allows us to presume that the difference between the two modifications might be minimal in the first stage of crystal destruction. However, the layer was homogeneous and was made of strongly bound dimers in the orthorhombic form, while the layer of the monoclinic form was not homogeneous and was made of chains. We may presume that this difference was the factor causing the best bioavailability, dissolution rate and/or solubility of the monoclinic form 2M, due to the easier destruction of the layers.

4. Conclusions The reaction of sodium4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate monohydrate and an equivalent amount of iodoethane in DMSO solution was studied. It was found that not only the oxygen atom of the carboxyl group is subjected to alkylation, but to a significant extent a heteroatom of nitrogen. As a result, two esters—ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate and its 1-ethyl-substituted analog—were isolated as products of the reaction studied. In all cases, the true direction of the alkylation was determined by X-ray diffraction analysis. Moreover, the ability to exist in two polymorphic forms—monoclinic and orthorhombic—was found in ethyl 4-methyl-2,2- dioxo-1H-2λ6,1-benzothiazine-3-carboxylate. According to the data from the pharmacological tests, the monoclinic form showed strong analgesic and anti-inflammatory effects, whereas the orthorhombic form appeared to be relatively less active. Based on a detailed analysis of the molecular and crystal structure of two polymorphic forms of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate, the possible causes of the much higher activity of the monoclinic crystal form were suggested.

Author Contributions: The synthesis of the compounds presented in this work and analysis of their characteristics were performed by I.V.U. and A.A.B. Single crystal and powder X-ray diffraction structural studies and quantum-chemical calculations were performed by S.V.S., V.N.B., L.V.S. and I.A.T. The pharmacological studies were conducted by N.I.V. and P.S.B. The manuscript was written by I.V.U., S.V.S. and P.S.B. Acknowledgments: We are grateful to Candidate of Chemistry Magda D. Tsapko (Taras Shevchenko National University, Kiev, Ukraine) for her help in the registration of the NMR spectra of the compounds synthesized. Conflicts of Interest: The authors declare no conflicts of interest.

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