The synthesis, thermal behaviour, spectral and structural characterization, and in silico prediction of pharmacokinetic parameters of tetraalkylammonium salts of non-steroidal anti-inflammatory drug nimesulide

The synthesis, spectral properties, thermal analysis, structural characterization and in silico prediction of pharmacokinetic parameters of tetramethylammonium (compound 1) and tetraethylammonium (compound 2) salt of nimesulide were described in this article. Both compounds crystallize in the monoclinic P21/n space group, with one tetraalkylammonium cation and one nimesulide anion in the asymmetric unit and their crystal structures are stabilized by C–H···O hydrogen bonds between ions. Additionally, structures of title compounds are stabilized by π–π interactions (compound 1), or C–H···π interactions (compound 2) between nimesulide anions. The TG and DSC measurements show that compound 1 melts at a temperature higher than nimesulide, whereas the compound 2 melts at a temperature lower than nimesulide. The MALDI-TOF, 1H NMR, 13C NMR and ATR-FTIR analyses confirm the SCXRD study, that in compounds 1 and 2 nimesulide exists in an ionized form. Studies performed by SWISS ADME and ProTOX II tools, predict to be oral bioavailability of both salts obtained, and one of them (compound 1) is predicted to be well-absorbed by digestive system, while both compounds obtained are classified into toxicity class 4.


Experimental Materials
Tetramethylammonium hydroxide pentahydrate and tetraethylammonium hydroxide solution were purchased from Sigma-Aldrich, whereas nimesulide was received from Ambeed and they were used without further purification.The purity of nimesulide and compounds 1 and 2 and were controlled by TG/DSC methods.

Synthesis of compound 1
Nimesulide (0.05 g, 0.162 mmol) and tetramethylammonium hydroxide pentahydrate (0.026 g, 0.162 mmol) were dissolved in the mixture of solvents: 5 cm 3 of ethanol and 5 cm 3 of a methanol.After this, the mixture was stirred.The solution was allowed to evaporate in place without sunlight for a few days to give yellow crystals (m.p. = 179 °C).

Synthesis of compound 2
Nimesulide (0.05 g, 0.162 mmol) was dissolved in 0.12 ml tetraethylammonium hydroxide solution (20 wt.% in H 2 O, d = 1.01 g/cm 3 in 20 °C, 0.162 mmol).After this, the mixture was stirred.The solution was allowed to evaporate in place without sunlight for a few days to give yellow crystals (m.p. = 113 °C).

Nuclear magnetic resonance (NMR)
The 1 H NMR and 13 C NMR spectra were recorded on a Bruker III Avance 500 MHz spectrometer ( 1 H frequency 500.13Hz) operated at magnetic fields of 11.7 T, using standard experimental conditions in DMSO-d 6 solution.

Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR)
The ATR-FTIR spectra were acquired using a Perkin Elmer Spectrum 2™ instrument (Perkin Elmer, Waltham, USA) equipped with attenuated total reflectance (ATR) accessory.The spectra were recorded at room temperature in the spectral range from 4000 to 500/cm at a resolution of 4/cm averaging 16 scans for each measurement.

Thermogravimetry (TG) and differential scanning calorimetry (DSC)
Thermogravimetric measurements (TG) were performed with a Netzsch 209 thermobalance: samples weighing ~ 4.0 mg were placed in a platinum crucible and heated at 10.0 K/min in a dynamic Ar atmosphere.Differential scanning calorimetry (DSC) measurements were performed with a Perkin Elmer TGA 8000 instrument: samples weighing ~ 4.0 mg were placed in an aluminium crucible and heated at 10.0 K/min in a dynamic N atmosphere.1).The lattice parameters were obtained using CrysAlis CCD, while data were reduced using CrysAlis RED software (multi-scan absorption corrections were applied) 39 .Crystal structures were solved by direct methods using SHELXS-97 and refined by full-matrix least-squares on F 2 with anisotropic displacement parameters for non-H atoms using SHELXL-2017/1 40 .All H-atoms were placed geometrically and refined using a riding model with C-H = 0.93 ÷ 0.97 Å and U iso (H) = 1.2U eq (C) (C-H = 0.96 Å and U iso (H) = 1.5U eq (C) for the methyl groups).The phenoxy group in compound 1 has disordered orientations with refined site-occupancy factors of the disordered parts of 0.533(6) and 0.467(6) (the disordered benzene rings were refined as rigid ideal hexagons with C-C = 1.38 Å and constrained with isotropic displacement parameters).In all figures, the disordered phenoxy group is omitted for clarity.All interactions were found using the PLATON program 41 .The molecular graphics were prepared using ORTEPII 42 , PLUTO-78 43 , and Mercury 44 software.

ADME and Protox II analyses
The web-service SWISS-ADME tool by the Swiss Institute of Bioinformatics (http:// www.swiss adme.ch/) was used to calculate physicochemical descriptors, important for drug discovery 45 .Compounds were analyzed to predict ADME (absorption, distribution, metabolism, and excretion) parameters, pharmacokinetic properties, or drug-like nature in comparison to pure nimesulide.The web-service ProTOX II was used for the prediction of the toxicity of the title compounds 46 .

Spectral characterization
Negative-ion and positive-ion mode MALDI-TOF mass spectra for compounds  The 1 H and 13 C NMR spectra of pure nimesulide, and compounds 1 and 2 are shown in Fig. 4. The 1 H and 13 C NMR data are presented in the "Experimental" section.
Chemical shifts, multiplicities and the observed proton-proton coupling constants are in accordance with the structure of the studied compounds.The number of carbon signals corresponds to the expected number of carbon atoms of the studied compounds.A singlet at 10.2 ppm in the 1 H NMR spectra of pure nimesulide indicates N-H proton of sulfonamide group 47 .This resonance disappeared in the 1 H NMR spectra of compounds 1 and 2 due to the amide proton detachment (Fig. 4a).Resonances between 6.5 and 8.5 ppm correspond to aromatic protons.NMR resonances with chemical shifts in the range of 1-3.5 ppm correspond to aliphatic protons of the methylene and methyl groups.The signals in the range of 110-160 ppm in the 13 C NMR spectra indicate aromatic carbons, whereas those in the range of 0-55 ppm correspond to saturated functional groups (Fig. 4b).
Formation of the salts results in a disappearance of N-H band, which is related to proton detachment from N-amine atom.The changes are also observed within the stretching vibrations of SO 2 and NO 2 groups.The asymmetric and symmetric stretching vibrations of SO 2 group are observed at slightly lower wavenumbers as shoulder low-intensity bands for both salts, whereas those of nitro group remain strong but are shifted to lower wavenumbers by 25-35/cm.

TG/DSC studies
The thermal behaviour of pure nimesulide and compounds 1 and 2 are shown in Fig. 6.The TG-DSC diagrams for the nimesulide show a sharp endothermic peak at 149 °C, which represent the melting point, whereas the first decomposition step occurs between 220 and 390 °C with a mass loss of 71.9% associated with an exothermic peak at 330 °C attributed to the decomposition of the compound.These temperatures are in agreement with literature results 26,34,[48][49][50] .The TG-DSC curves for compounds 1 and 2 exhibit sharp endothermic peaks at 179 www.nature.com/scientificreports/and 113 °C, which represent the melting points of both compounds, respectively.For the compound 1, the first decomposition step occurs between 219 °C and 377 °C with a mass loss of 64.7% associated with exothermic peaks at 269 and 297 °C.For the compound 2, the first decomposition step is observed in range of 205-360 °C with a mass loss of 75.4% associated with exothermic peaks at 258 and 297 °C.

Single-Crystal X-Ray Diffraction (SCXRD) studies
Compounds 1 and 2 crystallize in the monoclinic P2 1 /n space group with one tetraalkylammonium cation and one nimesulide anion in the asymmetric unit (Fig. 7, Table 1).
In both crystal structures, the proton detachment from N7-atom of nimesulide is observed which is confirmed by d(C-N) and d(N-S) bond length and ∠(C-N-S) angle within the sulfonamide group.In the crystals of title  35 .For the structures of cocrystals of nimesulide with pyridine analogues, the values of these bond length are 1.42 and 1.64 Å, respectively 37 .In the crystals of title compounds, the values of ∠(C-N-S) angle are 122.2°and 119.2°, for compounds 1 and 2, respectively, which indicates the sp 2 hybridization of the nitrogen atom.For comparison, in the crystal structures of nimesulide the ∠(C-N-S) angle is 124.6 and 124.1°, for form I and II, respectively 26,[34][35][36] .In turn, in the crystals of complexes of silver with nimesulide the ∠(C-N-S) angle is in the range 118.7 ÷ 122.3°3 5 , whereas for the structures of cocrystals of nimesulide with pyridine analogues, this angle is in the range 119.9 ÷ 124.0°3 7 .

ADME analysis
Bioavailability Radar is one of results, which the Swiss ADME web tool is giving.It is prediction on six physicochemical properties, such as lipophilicity, size, polarity, insolubility, insaturation, flexibility 51 .Descriptors used for it, in comparison to pure nimesulide, are presented in Table 2.
The pink area illustrates optimal range for six parameters.Nimesulide represents proper values for 5 properties.Lipophilicity, calculated by XLOGP3 is between − 7 and + 5 and size (MW) is between 150 and 500 g/ mol.Other properties, such as polarity (TPSA) and solubility (logS), are in range 20-130 Å 2 and not higher than 6, respectively.One of properties of nimesulide, saturation, which is described as a number of carbons in the sp 3 hybridization-is out of range.The value of the parameter should be more than 0.25.All parameters for compounds 1 and 2 are in the range of the pink area, which means that substances are predicted to be orally bioavailable.Another parameter, referring to drug-likeness, is described by Lipinski's rule of five 55 from Pfizer and Bioavailability Score 56 by Abbott.Both criteria, for all structures are in the acceptable range-Lipinski's filter causing 0 violation and Bioavailability score is 0.55.Amount PAINS #alarm 57 for all compounds is 0 alarm and the value of Synthetic accessibility 58 for all structures is in an optimal range.
Method to predict at the same time two properties: the passive gastrointestinal absorption (HIA), brain access (BBB) is called BOILED-Egg.This model is based on two physicochemical parameters-WLOGP and TPSA.The BOILED-Egg plot (Fig. 10) contains 3 areas: the yellow one is assigned for most likely BBB permeation, the white one is a space for most likely HIA absorption molecules and the grey one stands for structures predicted to have weak absorption and low brain penetration.Yellow and white compartments are not mutually exclusive 59 .Occurred points are colored.Molecules, which are not effluated from the central nervous system by P-glycoprotein substrates (PGP-), are marked as red dots.Blue dots are for substances predicted to dispose from the central nervous system by P-glycoprotein substrates (PGP+) 60 .Nimesulide and compound 1 are predicted to be wellabsorbed and not crossing the blood-brain barrier, which is confirmed in the literature for the nimesulide 61,62 , whereas compound 2 is predicted not to be absorbed and not accessing the brain.Compounds 1 and 2 are rid of the nervous system (PGP+), while nimesulide is not pumped-out by P-glycoprotein (PGP-).The Servis ProTox II has classified all molecules (nimesulide, compound 1 and 2) into toxicity class 4 (predicted LD 50 : 997 mg/kg).

Conclusion
The synthesis, spectral properties, thermal analysis, and structural characterization of tetramethylammonium (compound 1) and tetraethylammonium (compound 2) salts of nimesulide were described in this article.The MALDI-TOF, 1 H NMR, 13 C NMR and ATR-FTIR analyses confirm SCXRD study, that nimesulide exists in an ionized form in both studied compounds.MALDI-TOF mass spectra of the compounds 1 and 2 showed the peaks of nimesulide anion (found at m/z 307.045, and 307.008, respectively), and tetramethylammonium and tetraethylammonium cations (found at m/z 74.218 and m/z 130.198, respectively).A singlet at 10.2 ppm in the 1 H NMR spectra of pure nimesulide corresponding to H-N proton of sulfonamide group, disappeared in the 1 H NMR spectra of both title compounds due to the amide proton detachment.Likewise, formation of the salts resulted in a disappearance of N-H stretching band at 3278/cm in the ATR-FTIR spectra.The TG and DSC measurements confirmed that compound 1 melts at a temperature higher than nimesulide (179 °C), whereas the compound 2 melts at a temperature lower than nimesulide (113 °C).Compounds 1 and 2 crystallize in the monoclinic P2 1 /n space group, with one tetraalkylammonium cation and one nimesulide anion in the asymmetric unit, and their crystal structures are stabilized by C-H•••O hydrogen bonds between ions.Additionally, structure of compound 1 is stabilized through π-π interactions, whereas in the crystal packing of compound 2 the C-H•••π interactions between nimesulide anions playing an important role.Studies performed by SWISS ADME and ProTOX II tools, predict to be oral bioavailability of both salts obtained, and one of them (compound 1) was predicted to be well-absorbed by digestive system.Both compounds were predicted to be classified into toxicity class 4 (predicted LD50: < 1000 mg/kg).

Figure 4 .
Figure 4. 1 H NMR (a) and 13 C NMR spectra (b) of nimesulide and compounds 1 and 2 in DMSO-d 6 at 298 K.

Figure 5 .
Figure 5. ATR-FTIR spectra of studied compounds in the range of 4000-500/cm (left panel) with spectra enlargement in the range of 1800-1000/cm (right panel).

Figure 7 .
Figure 7. Crystal structures of compounds 1 and 2 with the atom-labelling scheme (hydrogen bonds are represented by dashed lines).

Figure 8 .
Figure 8. Crystal packing of compound 1 viewed along b-axis (interactions between nimesulide anions are highlighted by green, whereas hydrogen bonds between nimesulide anion and tetramethylammonium cation are highlighted by orange).

Figure 9 .
Figure 9. Crystal packing of compound 2 viewed along b-axis (interactions between nimesulide anions are highlighted by green, whereas hydrogen bonds between nimesulide anion and tetraethylammonium cation are highlighted by orange).

Table 1 .
Crystal data and structure refinement for title compounds.

Table 2 .
ADME analysis for nimesulide and compounds 1 and 2. a Parameter for lipophilicity calculations 52 .b Molecular weight [g/mol].c Topological polar surface area [Å 2 ] 53 .d Estimated solubility 54 .e Ratio of sp 3 hybridized carbons over the total amount of carbons in molecule.f Number of rotatable bonds.