Saccharin salts of biologically active hydrazone derivatives

Transcription

1 See discussions, stats, and author profiles for this publication at: Saccharin salts of biologically active hydrazone derivatives Article in New Journal of Chemistry September 2015 DOI: /C5NJ01532D CITATIONS 12 READS 88 6 authors, including: Artem Surov Institute of Solution Chemistry of RAS 37 PUBLICATIONS 296 CITATIONS SEE PROFILE Alexander Voronin Institute of Solution Chemistry of RAS 20 PUBLICATIONS 160 CITATIONS SEE PROFILE Andrei V. Churakov Russian Academy of Sciences 305 PUBLICATIONS 2,046 CITATIONS SEE PROFILE German L Perlovich Institute of Solution Chemistry of RAS 183 PUBLICATIONS 2,153 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Monte Carlo Studies of Drug Nucleation View project Nitrosyl carboxylate palladium complexes as catalysts for aerobic oxidation of alcohols View project All content following this page was uploaded by Artem Surov on 31 October The user has requested enhancement of the downloaded file.

2 PAPER View Journal View Issue Cite this: New J. Chem., 2015, 39, 8614 Received (in Montpellier, France) 17th June 2015, Accepted 25th August 2015 DOI: /c5nj01532d 1. Introduction Hydrazones represent an important class of Schiff base compounds that exhibit different types of biological activity. 1 There are a lot of data in the literature suggesting that hydrazone derivatives have great potency against resistant forms of tuberculosis and can be used as an inexpensive substitution for isoniazid (an important first-line antituberculosis drug). 2 It has been found that the combination of isoniazid with some hydroxyaldehydes leads to the formation of stable hydrazones that display conserved activity and less toxicity due to the inactivation of the NH 2 group of isoniazid. 3 In addition, hydroxy- and methoxy-derivatives of hydrazones possess antioxidant activity due to their ability to capture free radicals. 4 The presence of hydroxyl groups on the benzene ring also plays an important role in the anticancer activity of hydrazones, especially when it is located in the ortho-position. 5 Although the biological activity is undoubtedly the key feature of potent active pharmaceutical ingredients (APIs), other factors may be equally important for application in vivo a Institution of Russian Academy of Sciences, G.A. Krestov Institute of Solution Chemistry RAS, , Ivanovo, Russia. glp@isc-ras.ru b Institute of General and Inorganic Chemistry RAS, Leninskii Prosp. 31, , Moscow, Russia c Russian Research Center for Safety of Bioactive Substances, , Staraya Kupavna, Russia Electronic supplementary information (ESI) available: Details of the DFT calculations, packing arrangements of the pure APIs, results of PXRD analysis. CCDC and For ESI and crystallographic data in CIF or other electronic format see DOI: /c5nj01532d Saccharin salts of biologically active hydrazone derivatives Artem O. Surov, a Alexander P. Voronin, a Anna A. Simagina, a Andrei V. Churakov, b Sophia Y. Skachilova c and German L. Perlovich* a The crystal structures of two saccharin salts with derivatives of an anti-tubercular drug isoniazid, namely vanillin isoniazid saccharinate (salt I) and salinazid saccharinate (salt II), were obtained in a X-ray diffraction experiment. The pattern of intermolecular interactions in the crystals was quantified by solidstate DFT followed by the Bader analysis of periodic electron density. It was established that ca. 42% of lattice energy is contributed by C HO contacts, while conventional hydrogen bonds have only ca. 28%. Salt I was found to show a 12-fold aqueous solubility improvement compared to pure API, whereas salt II is approximately 20 times more soluble than the starting salinazid. The standard thermodynamic functions of the salt formation were determined. The Gibbs energy change of the process was found to be negative, indicating that the formation of the salts from individual components is a spontaneous process. The most significant contribution to the Gibbs energy is provided by the enthalpy term, while the entropy change of the process has a negative value, introducing a positive contribution to DG f. such as solubility. Unfortunately, in many cases this important aspect is subject to later studies in drug discovery and drug development research. This is a serious drawback for a drug candidate on its way to become a useful pharmaceutical agent as it is hard to compensate for weak solubility properties. It has been reported that currently almost 40% of marketed drugs face the major problem of poor aqueous solubility. 6 One of the best approaches to overcoming the solubility challenge without modification of the pharmacophore structure of an API is developing new crystalline forms such as salts or co-crystals. In fact, salt formation is the most common method for improving solubility and today more than 50% of APIs are marketed as salts. 7 There are various pharmaceutically relevant organic counter ions for salt formation. In this work, well-known artificial sweeter called saccharin was employed as a salt former for the development of new crystalline forms of hydrazone derivatives. Saccharin is currently approved by FDA for use in food as a non-nutritive sweetener, which is a significant advantage in terms of further biopharmaceutical studies of API saccharinates. It has been reported in the literature that salt or co-crystal formation with saccharin often leads to a considerable enhancement of the aqueous solubility of APIs. 8 The presence of saccharin as a potent sweetener may improve organoleptic properties of a formulation, masking the bitter taste of a drug. 9 In addition, the ph of the saccharinate solutions is higher than that of, for example, the corresponding hydrochlorides, which makes them more suitable for injections and drops. Therefore, saccharin is one of the most frequently chosen co-formers/salt formers for preparation of new solid forms of APIs New J. Chem., 2015, 39, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015

3 Fig. 1 Molecular structures of the studied hydrazones and saccharin. In this work, we report crystal structures, thermal analysis, aqueous solubility and formation thermodynamics of the saccharin salts with two biologically active hydrazone compounds, namely vanillin isoniazid ((2-hydroxy-3-methoxybenzaldehyde)isonicotinoyl hydrazone) and salinazid ((2-hydroxybenzaldehyde)isonicotinoyl hydrazone) (Fig. 1). Additionally, the DFT computations complemented with the Bader analysis of the periodic electron density were performed to describe quantitatively the patterns of the intermolecular interactions in the saccharin salts as well as to estimate their lattice energies. 2. Materials and methods 2.1 Compounds and solvents Vanillin isoniazid ((2-hydroxy-3-methoxybenzaldehyde)isonicotinoyl hydrazone, C 14 H 13 N 3 O 3, MW , 99%) and salinazid ((2-hydroxybenzaldehyde)isonicotinoyl hydrazone, C 13 H 11 N 3 O 2, MW , 99%) were obtained from Interbioscreen Ltd. Saccharin (C 7 H 5 NO 3 S, MW , 99%) was purchased from Acros Organics. All the solvents were of analytical grade and used as received without further purification. 2.2 Salt synthesis Solvent-drop grinding experiments were performed using a Fritsch planetary micro mill, model Pulverisette 7, in 12 ml agate grinding jars with ten 5 mm agate balls at a rate of 600 rpm for 60 min. The experiments were carried out with stoichiometric amounts of vanillin isoniazid or salinazid and saccharin and a few drops of solvent (methanol) were added using a micropipette. For slurry experiments, 100 mg of the hydrazone and an equimolar amount of saccharin were stirred in methanol for 12 h. 2.3 Crystallization procedure For each salt, 30 mg of the hydrazone and an equimolar amount of saccharin were dissolved in methanol and stirred at 50 1C. The resulting clear solution was slowly cooled. The solution was kept in a fume hood at room temperature. Diffraction quality yellow crystals of saccharin salts were grown over a period of several days. Crystals obtained from the crystallisation batches were air dried before being subjected to further analysis. 2.4 X-ray diffraction experiments Single-crystal X-ray diffraction data were collected on a Bruker SMART APEX II diffractometer using graphite-monochromated MoKa radiation (l = Å). The structures were solved by direct methods and refined by full matrix least-squares on F 2 with anisotropic thermal parameters for all non-hydrogen atoms. 11 Absorption corrections based on measurements of equivalent reflections were applied. 12 All hydrogen atoms were found from the difference Fourier map and refined isotropically. X-ray powder diffraction (PXRD) data were recorded under ambient conditions in Bragg Brentano geometry by a Bruker D8 Advance diffractometer with CuKa 1 radiation (l = Å). 2.5 Differential scanning calorimetry (DSC) Thermal analysis was carried out using a Perkin Elmer DSC 4000 differential scanning calorimeter with a refrigerated cooling system (USA). The sample was heated in sealed aluminum sample holders at a rate of 10 K min 1 in a nitrogen atmosphere. The unit was calibrated with indium and zinc standards. The accuracy of the weighing procedure was 0.01 mg. 2.6 Solubility experiments and formation thermodynamics study Dissolution measurements with the solids were made using the shake-flask method at K. The samples were suspended in 10 ml of degassed water in pyrex glass tubes. The amount of the substance dissolved was measured by taking aliquots of 1 ml of the respective media. The solid phase was removed by filtration (Rotilabo s syringe filter, PTFE, 0.2 mm), and the concentration was determined by UV-vis spectroscopy (Varian Cary 50). The following reference wavelengths were used: 300 nm for vanillin isoniazid and salt I, 330 nm for salinazid and salt II. The solubility of the compounds was also measured at 293.2, 298.2, and K. An excess of the solid was placed in an Eppendorf tube and 2 ml of water was added. After 24 h the suspension was filtered through a Rotilabo s syringe filter (PTFE, 0.2 mm), and the concentration in the supernatant was determined by UV-vis spectroscopy as described above. The results are stated as the average of at least three replicated experiments. Concentrations were calculated according to an established calibration curve. In the case of 1 : 1 stoichiometry, the formation reaction of a multi-component compound from a pure API (A) and a pure co-former (B) may be described as A solid +B solid - AB solid (1) It has been established in the literature that the standard freeenergy change, DG f, for the above reaction may be expressed through consideration of the solubility data of each of the materials. 13 In the case of salt dissolution, however, ionised species are usually formed. Therefore, the dissociation constants of A (pk a,a ) and B (pk a,b ) have to be taken into account. Hence, all the solubility experiments should be conducted in water solutions to ensure proximity of the intrinsic pk a value of This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem., 2015, 39,

4 each compound. If A is a weak acid and B is a weak base, then the Gibbs energy of formation of a (1 : 1) salt may be given by the following equation: 14! a;b pk a;a S p A Sp B DG f ¼ RT ln 10pK S salt A Ssalt B where S p A and S p B are the solubility of pure A and B in a solvent, while S salt A and S salt B are the solubility of the salt components in a solution when in equilibrium with the pure salt. For the sake of simplicity, the activities of the components are approximated by molar concentrations. The product of S salt A and S salt B is generally known as K sp of a salt. Thus, eqn (2) may be written as: DG f ¼ RT ln 10pK a;b pk a;a S p A Sp B (3) K sp In addition, comparison of eqn (2) and/or (3) with the expression for the standard free-energy change for the isothermal reaction (2) DG1 = RTln K (4) shows that the ratio appearing in the logarithmic term of eqn (2) and/or (3) has the character of an equilibrium constant at the given temperature. Therefore, this quantity can be defined as K f. If the K f values are known at different temperatures, the van t Hoff relation may be used to derive the enthalpy of formation, DHf, of a multi-component compound: dlnk f dð1=tþ ¼ DH f (5) R Finally, the entropy change of the formation process can be estimated from the general relationship relating different thermodynamic functions: DG f ¼ DH f T DS f (6) In order to verify the salt stability in aqueous media, a composition of the saturated solutions of salts at each temperature was also analysed by HPLC. HPLC was performed on a Shimadzu Prominence model LC-20AD equipped with a PDA detector and a C-18 column (150 mm 4.6 mm ID, 5 mmparticlesizeand100å pore size). Elution was achieved by a mobile phase made of 0.1% trifluoroacetic acid in water (55%) and methanol (45%) by the isocratic method. An injection volume of 20 ml was used with an eluent flow rate of 1 ml min Solid-state DFT calculations and energy of intermolecular interactions The DFT computations with periodic boundary conditions (solid-state DFT calculations) were performed using the Crystal14 program. 15 Details of the DFT computation procedures are given in the ESI. All the calculations were carried out using the B3LYP/6-31G** approximation and a Grimme modified empirical dispersion correction (f (R)C 6 /R 6 ). 16 The quantum theory of atoms in molecule and crystal (QTAIMC) analysis 17 of the periodic electron density obtained from the crystalline wave function was performed with TOPOND The calculation methodology is presented elsewhere. 19 The following electron-density features at the (3; 1) bond critical point (BCP) are computed: (i) the values of the electron density, r b, (ii) the Laplacian of the electron density, r 2 r b, and (iii) the positively-defined local electronic kinetic energy density, G b. Within the QTAIMC, the particular noncovalent intermolecular interaction is associated with the existence of the bond path (i.e. the bond critical point) between the pair of atoms. The absence of the bond critical point implies that the two atoms do not interact. The network of the bond paths yields a comprehensive bond picture, the energy of each specific interaction (in our case it is the intermolecular hydrogen bonds, C HO contact, etc.) is considered to be totally independent of the others. The effects of the crystal environment, long-term electrostatic effects, etc. are taken into account implicitly, via the periodic electronic wavefunction, and are coded in the bond critical point features. The energy of the particular noncovalent interaction, E int,isevaluated according to Mata et al. 20 as E int = 0.429G b (in atomic units) (7) Eqn (7) yields reasonable E int values for molecular crystals with H-bonds, C HO and p-stacking contacts, etc. 21 In addition, this approach gives an opportunity to estimate the lattice energy of a crystal as a sum of the energies of noncovalent interactions between the considered molecule and its neighbors: 22 E latt ¼ X i X E int;j;i (8) where j and i denote the atoms belonging to different molecules. Eqn (8) is BSSE free. For the sake of simplicity, indexes j and i will be omitted below. It has to be pointed out that E int,j,i values are commonly determined using atom-atom potentials 23 or the PIXEL method based on semi-classical density sums. 24 In the case of crystals with proton transfer, however, the applicability of these schemes for E int and E latt evaluation is not straightforward, at least for the Gavezzotti model, as it was originally calibrated only for molecular crystals containing neutral atoms. Switching to ions thus requires preliminary parametrization of the force field. On the other hand, the QTAIMC concept is successfully used both for salts and neutral co-crystals, 21e,25 and eqn (7) was derived using the interaction data of both neutral and chargetransfer heterodimers. 20 Thus, Bader analysis of the calculated wavefunction was chosen in the present work as superior to the Gavezzotti model in estimating the energy of particular noncovalent interactions. j o i 3. Results and discussion 3.1 The salt formation and the pk a rule The saccharinate salts with two substituted hydrazones with potential antitubercular activity, vanillin isoniazid and salinazid, were obtained by solvent-drop grinding, slurry conversion and crystallization from solution. DSC and PXRD analysis of resulting solids revealed that different preparation procedures have led to identical phases New J. Chem., 2015, 39, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015

5 Vanillin isoniazid and salinazid are weak bases. Their pk a values based on pyridine nitrogen (3.57 and 3.60, respectively) 26 are quite close to that of isoniazid (3.50), which is the structural precursor of the studied molecules. Therefore, both compounds are able to form either co-crystals or salts with different organic co-formers/counterions. It has been established in the literature that the reaction of an acid with a base is expected to form a salt if the difference in DpK a =pk (base) a pk (acid) a is greater than 3, and it is most widely known as a rule of three. 27 This states that salt formation generally requires a difference of at least three pk a units between a conjugate base and a conjugate acid. Cruz-Cabeza has conducted a study on the relationship between the proton transfer probability and the DpK a value of 6465 acid base crystalline complexes taken from CSD. 28 It has been shown that components with DpK a o 1 form mostly co-crystals, while systems with DpK a 4 4 tend to form exclusively salts. This rule has been also adjusted to various drug/co-former pairs, 29 despite the fact that DpK a range between 0 and 3 remains hardly predictable. For the studied APIs and saccharin (pk a = 2.32) the DpK a value equals ca. 1.3, which is in the middle of the range of the salt-co-crystal continuum. Therefore, the formation of either a salt or a co-crystal between the components is almost equally probable. On the other hand, the analysis of the Cambridge Structural Database (CSD) 30 has revealed that saccharin has a greater propensity for salt formation. A search of the CSD (Version 5.36, 2014 release with Nov 2014 update) yields 68 twocomponent crystal structures of saccharinates and only 18 co-crystals (saccharin saccharinates were not considered). 3.2 Crystal structures and thermal analysis The crystal structures of pure vanillin isoniazid and salinazid have been described earlier. CSD 30 contains two records for vanillin isoniazid (ref. codes CANCOK and CANCOK01) and four records for salinazid (ref. codes WEHFEU, WEHFEU01-03). All of these structures were found to be identical, whereas different polymorphs were not observed. Crystal packing arrangement for the pure APIs is shown in Fig. S1 (see ESI ). Crystal structures of the compounds are quite similar and consist of distinct zigzag layers of the molecules packed in a head-to-head and tail-to-tail manner. Inside each layer, the molecules are linked by N HN hydrogen bonds to form infinite chains with graph set notation C(7). 31 Crystallographic data for the saccharin salts of vanillin isoniazid and salinazid are summarized in Table 1, and the packing arrangements of the salts are shown in Fig. 2. To simplify the discussion of the salts, the following nomenclature will be applied: salt I (saccharinate of vanillin isoniazid), salt II (saccharinate of salinazid). The single crystal X-ray diffraction data confirmed the protonation of the pyridine ring of the APIs, as evidenced by the proton location and bond length analysis. Both salts are found to be isostructural, and they crystallize in the monoclinic P2 1 /n space group with one API cation and one saccharinate anion in the asymmetric unit. The asymmetric unit contains API and saccharin molecules connected by N + 3 H3O6 hydrogen bonds (H-bonds) involving the pyridine ring of the API and the carbonyl oxygen atom attached to the thiazole fragment of the saccharin (Fig. 2a). In both structures, the pyridine ring of the API molecule lies approximately in the same plane as the saccharin molecule to which it is hydrogen-bonded (the angles between least-squares planes ca for salt I and ca for salt II). This spatial arrangement is probably stabilized by weak C1 H1N 4 interaction (green dash line in Fig. 2a), which completes a closedring dimeric unit. The second N1 H21O4 hydrogen bond (Fig. 2a) connects two neighboring [API + saccharin] units to form a hydrogen bonded chain consisting of alternating API and saccharinate ions along the c-axis, while the API molecules Table 1 Crystallographic data for the API saccharinates Compound reference Salt I Salt II Chemical formula C 14 H 14 N 3 O 3 C 7 H 4 NO 3 S C 13 H 12 N 3 O 2 C 7 H 4 NO 3 S Formula Mass Crystal system Monoclinic Monoclinic a/å (9) (11) b/å (4) (5) c/å (18) (2) b/ (2) (3) Unit cell volume/å (5) (6) Temperature/K 173(2) 173(2) Space group P21/n P21/n No. of formula units per unit cell, Z 4 4 Absorption coefficient, m/mm No. of reflections measured No. of independent reflections R int Final R 1 values (I 4 2s(I)) Final wr(f 2 ) values (I 4 2s(I)) Final R 1 values (all data) Final wr(f 2 ) values (all data) Goodness of fit on F Largest diff. peak & hole, e Å / / CCDC This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem., 2015, 39,

6 Table 2 Selected torsion angles and the dihedral angles between planes of aromatic rings, b, in the pure API molecules and the corresponding API saccharinates t 1 (C2 C3 C6 N1),1 t 2 (C6 N1 N2 C7),1 t 3 (N2 C7 C8 C9),1 b,1 Vanillin isoniazid Salt I Salinazid Salt II aromatic rings referenced below as b (the acute angle between the least-squares planes through the two rings). Table 2 shows that conformations of the API molecules in the salts with saccharin are similar to those in the crystals of the pure APIs. In each molecule, the t 2 torsion angle deviates by no more than 71 from In the case of t 3, all the values are generally located around 01. However, the orientation of the pyridine ring (t 1 ) is twisted from planarity relatively more significantly than the rest of the molecule, increasing the b values up to ca The DSC traces for the salts, APIs and saccharin are shown in Fig. 3, and the thermal data are presented in Table 3. The bulk materials were inspected by X-ray powder diffraction Fig. 2 (a) Schematic representation of the hydrogen bonds (red dash lines) occurring in the salt crystals. Flexible torsion angles in the hydrazone molecules are numbered and indicated by t 1, t 2 and t 3 ; (b) molecular packing projections for salt I along the a-axis and (c) salt II along thec-axis. are not directly connected with each other by hydrogen bonds (Fig. 2b). In addition, nearly planar [API + saccharin] in a chain forms an angle of ca. 681 with each other (Fig. 2c). This promotes a number of C HO and other weak interactions between the neighboring molecules in the crystal. The description and energies of these contacts will be discussed below. The conformations of each API molecule can be defined in terms of at least three torsion angles, one defining the conformation of the central spacer unit between the two rings (t 2 ) and two defining the orientation of the rings themselves (t 1 and t 3 ) (see Fig. 1). The values of the selected torsion angles for the pure APIs and the API saccharinates are represented in Table 2. In addition, we introduced an angle between the Fig. 3 DSC curves of the salts, saccharin, vanillin isoniazid and salinazid recorded at a 10 K min 1 heating rate. Table 3 Thermophysical data for the salts, compared to saccharin, vanillin isoniazid and salinazid T fus, K (onset) DH T fus a, kj mol 1 DS T fus a, J mol 1 K 1 Vanillin isoniazid Salinazid Saccharin Salt I Salt II a For the salts, the values correspond to a mole of molecules in the asymmetric units New J. Chem., 2015, 39, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015

7 before being subjected to thermal analysis. The experimental PXRD pattern was found to be in good agreement with those calculated from the single crystal analysis (Fig. S2, ESI ). As Fig. 3 indicates, DSC thermograms have only one endotherm for all the compounds which corresponds to the melting process. The melting temperature of vanillin isoniazid is found to be ca. 15 K lower than that of salinazid, however, the opposite trend is observed for the fusion enthalpies. Commonly, salt formation is applied to improve stability of APIs by increasing their melting point, but according to the DSC experiments (Fig. 3), the salt formation of the API compounds decreases their melting point. The melting temperature of salt I is ca. 30 K lower compared to that of the pure API (vanillin isoniazid), while for salt II, T fus decreases by ca. 53 K. It should be noted that the difference in melting points of the salts is less than that of the pure APIs. In addition, an inversion of thermal stability occurs, i.e. vanillin isoniazid saccharinate (salt I) melts at the temperature of 9 K higher than salinazid saccharinate (salt II) does. 3.3 Pattern of intermolecular interactions in the salts As mentioned above, there are a number of relatively short contacts between the neighboring molecules in the salt crystals. However, the existence of short intermolecular contact does not imply the existence of the intermolecular (noncovalent) interaction, e.g. see ref. 32. The Bader analysis of the computed periodical electron density enables us to detect and to quantify the noncovalent interactions (besides conventional N HO bonds) in the salts. TheobtainedresultsarecollectedinTableS1(seeESI ). According to the QTAIMC analysis, the charge assisted N + 3 H3O6 hydrogen bond is characterized by r b a.u. and belongs to the intermediate type of interactions. 33 Its energy reaches 57.1 kj mol 1 in salt I and 58.8 kj mol 1 in salt II (Fig. 4). This is the strongest intermolecular interaction in the crystals of both salts. It should be noted that the energy obtained for the N + 3 H3O6 H-bond is in good agreement with the value for the 3-hydroxypyridine benzoic acid complex, where a similar proton transfer forms an N + HO bond. 21e The QTAIMC analysis also confirms the existence of a weak C1 H1N 4 contact (E int = kj mol 1 ), which completes a closed-ring heterodimer between the molecules (Fig. 4). Therefore, the total energy of the supramolecular synthon formed by these two interactions is calculated to be 66.5 kj mol 1 for salt I and 68.3 kj mol 1 for salt II. Analysis of CSD showed that this type of synthon is quite robust, and it is readily formed in the saccharin salts with various pyridine derivatives. The second N1 H21O4 H-bond between API and saccharin molecules is characterized by small positive r 2 r b values and the electron density r b at the bond critical point lower than 0.02 a.u. According to Gatti, this intermolecular H-bond corresponds to the closed-shell interactions. 34 The energy of this H-bond is slightly larger in salt II (16.3kJmol 1 )comparedtothatinsalti (14.6 kj mol 1 ). In addition, the QTAIMC analysis reveals that the O4 and O5 atoms of saccharin act as acceptors of four C HO interactions from the neighboring API molecule (Fig. 4). The energies of these contacts vary from 6.1 to 13.5 kj mol 1 (Table S1, ESI ). It should be noted that the energy of the strongest contact Fig. 4 Intermolecular N HO hydrogen bonds (blue) and C HO interactions (green) in the crystals of (a) salt I and (b) salt II. The interaction energies are given in kj mol 1. (C7 H7O5) is found to be comparable to that of the conventional N1 H21O4 H-bond. It suggests an important role of C HO interactions in the lattice energy balance of the salts. In fact, the total energy of intermolecular interactions between API and saccharin molecules linked by the N1 H21O4 H-bond equals 53.2 kj mol 1 for salt I and 55.3 kj mol 1 for salt II, and the contribution of C HO contacts is approximately 72%. A number of the (3; 1) bond critical points corresponding to weak C HO contacts between the basic molecule of API and the neighboring saccharin molecules was located by the QTAIMC analysis (Fig. S3, ESI ). The E int values of these contacts vary from B4 to 11 kj mol 1 (Table S1, ESI ). The strongest interactions were found between the carbonyl O1-atom and H17-atom, which is attached to the phenyl ring of saccharin. The adjusted vanillin isoniazid molecules in salt I were also observed to interact with each other via the C14 H14BO3 and C14 H14BO2 contacts between the methoxy and hydroxy groups to form centrosymmetric dimers (Fig. S4 and Table S1, ESI ). In addition, a weak C14 H14CO2 interaction was found between the API molecules related by simple translation along the a-axis. Interestingly, such packing features are not seen in the crystal structure of the pure vanillin isoniazid compound. It might be reasonable to assume that the nearly planar orientation of the API molecules (b angle in Table 2 is close to zero) should promote a p-stacking in the salt crystals. Indeed, the fact that there were a set of BCPs in the structures can be attributed This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem., 2015, 39,

8 to p-stacking interactions between API API and API saccharin molecules. It was previously established that the energy of this type of intermolecular interaction may be confidently evaluated in the same manner as H-bonds or C HO contacts, e.g. using eqn (7). 21b Calculations for salt I resulted in the following values of the p-stacking interaction energy: ca. 38 kj mol 1 for API API stacks and ca. 15 kj mol 1 for API saccharin contacts (Fig. S5a, S6a and Table S1, ESI ). Similar values were obtained for salt II (Fig.S5b,S6bandTableS1,ESI ). The values of the lattice energies of the salts evaluated using eqn (8) were found to be kj mol 1 and kj mol 1 for salts I and II, respectively. Therefore, in both structures, conventional hydrogen bonds comprise ca. 28% of the lattice energy, while the most significant contribution is made by C HO(N) interactions, the value of which reaches ca. 42%.Therestofthepackingenergy corresponds to the p-stacking contacts (ca. 19%) and other weak intermolecular interactions (B11%). It would also be interesting to analyze the sums of the intermolecular interaction energies between different types of molecules in the crystal structure (Table S2, ESI ). It is evident that the API saccharin interactions provide the largest contribution to the lattice energy (more than 70%). The API API interactions comprise approximately a quarter of the total energy in salt I and ca. 16% in salt II,whilethereisalmostno interaction between the saccharin molecules. 3.4 Dissolution study It is known that solubility in aqueous media is a key parameter among other physicochemical properties for pharmaceutical solids. The dissolution profiles of vanillin isoniazid, salinazid and the corresponding salts in water at K are shown in Fig. 5. The solubility values are shown in Table 4. As Table 4 shows, the solubility of vanillin isoniazid at K is about 17% higher than that of salinazid. This may be caused by the influence of the hydrophilic methoxy group in the vanillin isoniazid molecule. In the case of the salts, an opposite tendency is observed: the solubility of salinazid saccharinate (salt II) at K is higher than the solubility of vanillin isoniazid saccharinate (salt I) by approximately a quarter. It should be noted that the solubility values of the pure APIs as well as the saccharinates qualitatively agree with the trend in their melting points, i.e. the compound with a lower solubility melts at a higher temperature. Therefore, salt I demonstrates a 12-fold solubility improvement compared to pure API (vanillin isoniazid), whereas salt II is approximately 20 times more soluble than the starting salinazid. 3.5 Thermodynamics of the salt formation In spite of great interest in the structure, preparation and properties of multi-component compounds such as co-crystals, salts, solvates, etc., there is relatively little data on their thermodynamic characteristics of formation, which are fundamental measures of their stability. 35 The solubility data of vanillin isoniazid, salinazid, saccharin and the corresponding salts in water from K to K are shown in Table 4. Congruent solubility of the salts was observed at each temperature, and the solid phase recovered after the experiment was identified by PXRD as the starting material (Fig. S7, ESI ). The1:1API saccharinmolarcompositioninthe saturated solutions was also confirmed by the HPLC analysis. As Table 5 shows, the Gibbs energy of formation of salt I calculated by eqn (3) is more negative than that of salt II, which indicates its greater thermodynamic stability. Negative values of DG f also suggest that the formation of the salts from individual components is a spontaneous process. The formation enthalpy of the salts was derived from eqn (5) (Fig. 6). It is evident that for both salts the enthalpy term provides the most significant contribution to the driving force of the Fig. 5 Dissolution profiles of vanillin isoniazid, salinazid and the salts in water at K. Table 5 Values of K f and standard thermodynamic functions of the salt formation K f DG f, kj mol 1 DH f, kj mol 1 DS f, J mol 1 K 1 Salt I Salt II Table 4 Temperature dependence of solubility, S 0 (mol L 1 ), of the APIs, saccharin and the corresponding salts Temperature Vanillin isoniazid Salinazid Saccharin Salt I Salt II S S S S a ln K sp S a ln K sp a The numbers represent concentration (in mol L 1 ) of each of API and saccharin in a stoichiometric solution in equilibrium with the salt New J. Chem., 2015, 39, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015

9 Fig. 6 The van t Hoff plots of ln K f against reciprocal temperature of salt I and salt II. formation process (Table 5). For salt II, the value of DHf is found to be ca. 5 kj mol 1 larger than that for salt I. It has to be pointed out that the enthalpy of formation is an integral parameter which can be considered as the difference between the crystal lattice energy of the salt and the individual components. However, the DFT calculations accompanied by the QTAIMC analysis showed that the lattice energies of salts I and II are closely comparable. Thus, the difference in the formation enthalpies of salts should be mostly caused by the difference in the crystal lattice energy of the pure APIs, since the lattice energy of saccharin is a constant. Small values of the formation enthalpies are in good agreement with the results of the DFT calculations, which indicates that the salt crystals are mainly stabilized by C HO(N), p-stacking and other weak intermolecular interactions, while the hydrogen bonds comprise only 30% of the lattice energy. Thenegativeentropychangeoftheprocessindicatesamore efficient packing of the molecules in the salt compared to their native crystal structures. For salt II, the absolute value of DSf is ca. 2.5 times greater than that for salt I, while the difference in the formation enthalpies is only ca. 25%. As a result of this competition between DHf and DSf, the Gibbs energy of salt II increases (i.e. becomes less negative) more appreciably compared to that of salt I. Since the thermodynamic parameters of the formation of a multi-component compound are not a function of the solvent, it would be interesting to analyze the thermodynamic data available for saccharin co-crystals. For example, it has been reported by Oliveira et al. 35a that the formation process of the carbamazepine saccharin (1 : 1) co-crystal is characterized by the following values at 306 K: DG f ¼ 4:4kJ mol 1, DHf ¼ 5:9 kj mol 1 and DSf ¼ 4:9Jmol 1 K 1. Similar to salts I and II, all the thermodynamic parameters of the co-crystal appeared with the negative sign. Their absolute values, however, are considerably lower than those of the salts. On the other hand, the co-crystal of saccharin with adefovir dipivoxil 36 shows that the Gibbs energy of formation equals ca. 12.8kJmol 1 at 303 K. 35d It indicates a greater affinity between adefovir dipivoxil and saccharin in the co-crystal compared to that of the APIs and saccharin in salts I and II, despite the fact that no charge transfer occurs in the adefovir dipivoxil saccharin system. The enthalpy and entropy terms of the formation of the adefovir dipivoxil saccharin co-crystal (DH f 40 kj mol 1, DS f 90 J mol 1 K 1 ) are also found to be significantly larger than those of salts I and II. Hence,a similar competition between DH f and DS f contributions is observed for the mentioned co-crystal. These examples demonstrate that thermodynamic parameters of the formation process may vary over a wide range. Nevertheless, a number of regularities have to be pointed out: (i) the largest contribution to the Gibbs energy of formation is provided by the enthalpy part, which characterizes the crystal packing energy gain via different intermolecular interactions between the components; (ii) the entropy change of the process has a negative value, introducing a positive contribution to DG f.asa result, the formation of a co-crystal/salt is seen to be a consequence of the competition between DH f and DS f terms. 4. Conclusions The saccharin salts with two biologically active hydrazone compounds were obtained and their crystal structures were determined. The Bader analysis of the periodic electron density computed by the solid-state DFT methods was performed to quantify the pattern of intermolecular interactions in the salts. It was found that conventional N HO hydrogen bonds comprise ca. 28% of the lattice energy, while the most significant contribution is made by C HO(N) interactions, the value of which reaches ca. 42%. The rest of the packing energy corresponds to p-stacking contacts (ca. 19%) and other weak intermolecular interactions (B11%). The dissolution study of the compounds in water has shown that salt formation of the drug molecules with saccharin substantially increases their solubility. The saccharine salt of vanillin isoniazid demonstrates a 12-fold solubility improvement compared to the pure base, whereas salinazid saccharinate is approximately 20 times more soluble than the starting drug. Both salts were found to be stable and to dissolve congruently in water. With the solubility of the salts and the corresponding solubility of the pure compounds in water determined at different temperatures, the thermodynamic functions of the formation of the salts were estimated. The Gibbs energy change of the process (DG f )was found to be 9.1 kj mol 1 for vanillin isoniazid saccharinate and 7.3kJmol 1 for salinazid saccharinate, which indicates that the formation of the salts from individual components is a spontaneous process. The most significant contribution to the Gibbs energy is provided by the enthalpy of formation, which characterizes the crystal packing energy gain via different intermolecular interactions between components. The entropy change of the process has a negative value, introducing a positive contribution to DG f.asa result, the formation of the salts is seen to be a consequence of the competition between enthalpy and entropy terms. Acknowledgements This work was supported by a grant from the President of the Russian Federation no. MK We thank The Upper Volga Region Centre of Physicochemical Research for the technical assistance of XRPD experiments. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem., 2015, 39,

10 Notes and references 1 (a) S. Rollas and S-.G.Küçükgüzel, Molecules, 2007, 12, 1910; (b) R. Narang, B. Narasimhan and S. Sharma, Curr. Med. Chem., 2012, 19, (a) E. Vavríková, S. Polanc, M. Kocevar, J. Kosmrlj, K. Horváti, S. B+osze, J. Stolaríková, A. Imramovský and J. Vinsová, Eur. J. Med. Chem., 2011, 46, 5902; (b) F. Martins, S. Santos, C. Ventura, R. Elvas-Leitão, L. Santos, S. Vitorino, M. Reis, V. Miranda, H. F. Correia, J. Aires-de-Sousa, V. Kovalishyn, D. A. R. S. Latino, J. Ramos and M. Viveiros, Eur. J. Med. Chem., 2014, 81, 119;(c) R. Maccari, R. OttanaandM. G. Vigorita, Bioorg.Med.Chem.Lett., 2005, 15, P. H. Buu-Hoi, D. Xuong, H. Nam, F. Binon and R. Royer, J. Chem. Soc., 1953, 75, C. Vanucci-Bacqué, C. Carayon, C. Bernis, C. Camare, A. Nègre-Salvayre, F. Bedos-Belval and M. Baltas, Bioorg. Med. Chem., 2014, 22, F.A.R.Rodrigues,A.C.A.Oliveira,B.C.Cavalcanti,C.Pessoa, A. C. Pinheiro and M. V. N. de Souza, Sci. Pharm., 2014, 82,21. 6 (a) D. L. Prohotsky and F. Zhao, J. Pharm. Sci., 2012, 101, 1; (b) A. M. Thayer, Chem. Eng. News, 2007, 85, M. Pudipeddi, A. T. M. Serajuddin, D. J. W. Grant and P. H. Stahl, in Handbook of pharmaceutical salts, properties, selection and use, ed. P. H. Stahl and C. G. Wermuth, Wiley- VCH, Weinheim, Germany, 2002, pp C. L. CookeandR. J. Davey, Cryst. Growth Des., 2008, 8, R. Banerjee, P. M. Bhatt, N. V. Ravindra and G. R. Desiraju, Cryst. Growth Des., 2005, 5, P. M. Bhatt, N. V. Ravindra, R. Banerjee and G. R. Desiraju, Chem. Commun., 2005, G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008, 64, G. M. Sheldrick, SADABS, Program for scaling and correction of area detector data, University of Göttingen, Germany, R. R. Schartman, Int. J. Pharm., 2009, 365, T. Rager and R. Hilfiker, Z. Phys. Chem., 2009, 223, R. Dovesi, R. Orlando, A. Erba, C. M. Zicovich-Wilson, B. Civalleri, S. Casassa, L. Maschio, M. Ferrabone, M. de la Pierre, P. D Arco, Y. Noel, M. Causa, M. Rerat and B. Kirtman, Int. J. Quantum Chem., 2014, 114, (a) B. Civalleri, C. M. Zicovich-Wilson, L. Valenzano and P. Ugliengo, CrystEngComm, 2008, 10, 405; (b) S. Grimme, J. Comput. Chem., 2006, 27, (a)r.f.w.bader,atoms in Molecules A Quantum Theory,Oxford University Press, Oxford, 1990; (b) V. G. Tsirelson, in The Quantum Theory of Atoms in Molecules: From Solid State to DNA and Drug Design,ed.C.MattaandR.Boyd,Wiley-VCH,Berlin,2007,ch C. Gatti and S. Casassa, TOPOND User s Manual, CNR-ISTM of Milano, Milano, (a) L. Bertini, F. Cargnoni and C. Gatti, Theor. Chem. Acc., 2007, 117, 847;(b) A. V. Churakov, P. V. Prikhodchenko, O. Lev, A.G.Medvedev,T.A.Tripol skayaandm.v.vener,j. Chem. Phys., 2010, 133, ; (c) M. V. Vener, A. G. Medvedev, A.V.Churakov,P.V.Prikhodchenko,T.A.Tripol skayaand O. Lev, J. Phys. Chem. A, 2011, 115, I. Mata, I. Alkorta, E. Espinosa and E. Molins, Chem. Phys. Lett., 2011, 507, (a) M. V. Vener, A. N. Egorova, A. V. Churakov and V. G. Tsirelson, J. Comput. Chem., 2012, 33, 2303; (b) A. V. Shishkina, V.V.Zhurov,A.I.Stash,M.V.Vener,A.A.Pinkertonand V. G. Tsirelson, Cryst. Growth Des., 2013, 13, 816; (c) M. V. Vener, A. V. Shishkina, A. A. Rykounov and V. G. Tsirelson, J. Phys. Chem. A, 2013, 117, 8459; (d) A.N.Manin,A.P.Voronin,N.G. Manin, M. V. Vener, A. V. Shishkina, A. S. Lermontov and G. L. Perlovich, J. Phys. Chem. B, 2014, 118, 6803; (e) M.V. Vener,E.O.Levina,O.A.Koloskov,A.A.Rykounov,A.P.Voronin and V. G. Tsirelson, Cryst. Growth Des., 2014, 14, P. M. Dominiak, E. Espinosa and J. Angyan, in Modern Charge Density Analysis, ed. C. Gatti and P. Macchi, Springer, Heidelberg, London, New York, 2012, pp (a) A. J. Pertsin and A. I. Kitaigorodsky, The Atom Atom Potential Method. Application to Organic Molecular Solids, Springer-Verlag, New York, 1987; (b) A. Gavezzotti, New J. Chem., 2011, 35, (a) A. Gavezzotti, J. Phys. Chem., 2003, B107, 2344; (b) A. Gavezzotti, Mol. Phys., 2008, 106, V. R. Hathwar, R. Pal and T. N. G. Row, Cryst. Growth Des., 2010, 10, Advanced Chemistry Development (ACD/Laboratories) Software V (a) K.-S. Huang, D. Britton, M. C. Etter and S. R. Byrn, J. Mater. Chem., 1997, 7, 713; (b) H. StahlandC. G. Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, Zürich, 2002; (c) B. G. Bhogala, S. Basavojuand A. Nangia, CrystEngComm, 2005, 7, 551; (d) S. L. Childs, G. P. Stahly and A. Park, Mol. Pharmaceutics, 2007, 4, A. J. Cruz-Cabeza, CrystEngComm, 2012, 14, (a) A. C. Kathalikkattil, S. Damodaran, K. K. Bisht and E. Suresh, J. Mol. Struct., 2011, 985, 361; (b) C. C. P. da Silva, R. de Oliveira, J. C. Tenorio, S. B. Honorato, A. P. Ayala and J. Ellena, Cryst. Growth Des., 2013, 13, 4315; (c) P. Sanphui, S. Tothadi, S. Ganguly and G. R. Desiraju, Mol. Pharmaceutics, 2013, 10, 4687; (d) E. Elacqua, D.-K. Bučar, R. F. Henry, G. G. Z. Zhang and L. R. MacGillivray, Cryst. Growth Des., 2013, 13, 393; (e) L. H. Thomas, A. R. Klapwijk, C. Wales and C. C. Wilson, CrystEngComm, 2014, 16, F. H. Allen, Acta Crystallogr., Sect. B: Struct. Sci., 2002, 58, (a) M.C.Etter,Acc. Chem. Res., 1990, 23, 120; (b) J.Bernstein, R. E. Davis, L. Shimoni and N.-L. Chang, Angew. Chem., Int. Ed. Engl., 1995, 34, M. V. Vener, A. N. Egorova, D. P. Fomin and V. G. Tsirelson, J. Phys. Org. Chem., 2009, 22, M. V. Vener, A. V. Manaev, A. N. Egorova and V. G. Tsirelson, J. Phys. Chem. A, 2007, 111, C. Gatti, Z. Kristallogr., 2005, 220, (a) M. A. Oliveira, M. L. Peterson and R. J. Davey, Cryst. Growth Des., 2011, 11, 449; (b) S. ZhangandA. C. Rasmuson, Cryst- EngComm, 2012, 14, 4644; (c) S. Zhang and A. C. Rasmuson, Cryst. Growth Des., 2013, 13, 1153; (d) K. Ma, Y. Zhang, H. Kan, L. Cheng, L. Luo, Q. Su, J. Gao, Y. Gao and J. Zhang, Pharm. Res., 2014, 31, Y. Gao, H. Zu and J. Zhang, J. Pharm. Pharmacol., 2011, 63, New J. Chem., 2015, 39, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 View publication stats