Tautomeric preference in polymorphs and pseudopolymorphs of succinylsulfathiazole: fast evaporation screening and thermal studies†

Succinylsulfathiazole (SST) is known to exist in seven different crystalline forms, namely two unsolvated polymorphic forms (SST-I, SST-II), a monohydrate (SST/H2O), dihydrate (SST/2H2O) and solvates of butanol (SST/But), pentanol (SST/Pnt) and dioxane (SST/Diox). Most of these forms have been characterised only by IR and PXRD, while the single crystal structures have been determined for SST/But, SST/Pnt, SST/H2O and SST/Diox solvates. Previous studies also noted a lack of reproducibility in preparation of these different forms. Here, we employed a fast evaporation (FE) crystallization method and identified two new solvates from acetone (SST/AcMe) and tetrahydrofuran (SST/THF), as well as determined the single crystal structures for SST-I, SST-II, SST/AcMe and SST/THF. This revealed that SST exclusively adopts the imidine tautomeric form in all its solid form structures, but never the amidine form. The succinyl group of SST shows a conformational flexibility and adopts either anti- or syn-geometry to facilitate hydrogen bonding in the different structures. The study also allowed us to rationalize the hydrogen bonding preferences of various functional groups in all the forms. Notably the neat grinding and liquid assisted grinding methods resulted in only SST/H2O from various solvents, while the FE method produced polymorphs or pseudopolymorphs from different solvents.

Succinylsulfathiazole (SST), also known as sulfasuxidine, is a sulfonamide drug and classified as an ultra long acting drug.According to BCS 1 it belongs to either the class II or class IV drug molecules. 2SST is used for the prevention and treatment of gastrointestinal infections.This drug has solubility problems and shows unfavourable absorption. 3,4ccording to Moustafa et al., 5 aqueous suspensions of succinylsulfathiazole exhibit physical instability which causes caking, formation of cement-like precipitates and difficult resuspendability.Hence, SST has been a topic of interest to solid-state pharmaceutical chemists since the 1940s and many attempts have been made to identify its alternative solid forms with superior properties.SST is known to exist in several solid forms, I-VI, as named in previous studies, along with an amorphous form. 5Among them, reportedly, forms I and IV are true polymorphs, forms II and III contain two and one moles of water, respectively, form V contains one mole of butanol and form VI contains one mole of pentanol.5a In due course the single crystal structures of the monohydrate and solvates, butanol, pentanol along with a new form 1,4-dioxan were reported, 6 while the rest have been characterized only by IR and powder XRD.The lack of reproducibility in the preparation and confusion over the solvent ratios, as noted in some previous studies, makes the structural determination of these forms significant. 5As the naming of the forms (forms I-VI) in previous studies was also somewhat inconsistent, here we rename them as SST/H 2 O (monohydrate), SST/2H 2 O (dihydrate), SST/AcMe (acetone), SST/THF (tetrahydrofuran), SST/But (butanol), SST/Pnt (pentanol), SST/Diox (dioxane) and the true polymorphs of SST, SST-I and SST-II.
This study has also allowed us to examine the tautomerism and hydrogen bonding preferences in the SST solid forms and their selective preparation by using the fast evaporation (FE) method, which we have exploited recently in the context of polymorphs and co-crystal screening. 7The solid forms of succinylsulfathiazole have been characterized by powder and single crystal X-ray diffraction techniques, FTIR, differential scanning calorimetry and thermogravimetric analysis.

Materials
Succinylsulfathiazole drug (monohydrate form) was purchased from Sigma-Aldrich.Commercially available solvents were used as received without further purification.

Fast evaporation method
A sufficient amount of SST was dissolved in various boiling solvents, taken in separate conical flasks.The solutions were filtered into round bottom flasks (rbf) and warmed once again gently to achieve dilute and clear solutions with no solid particles, thus to prevent self-seeding of the original form. 6The clear solutions in the rbf were dried rapidly using a rotovapor by setting an appropriate reduced pressure, water bath temperature (50 °C) and revolution speed of the rbf (130 rpm).Upon the completion of the solvent evaporation, a continuous vacuum was applied, in order to reach the minimum possible pressure, (9-10 mbar) and held there for about 5 min before collecting the dry solids for characterization.As the solvates can convert to other forms over time, the characterization was done immediately after their preparation without much delay.The phase purity of the solids was established by DSC and comparing the experimental PXRD patterns with those calculated from the corresponding single crystal data (Fig. S3 †).IR spectroscopy was also performed using the same batches of samples (Fig. S1 †).

Single crystal preparation
Succinylsulfathiazole drug was dissolved in the boiling solvents acetonitrile (MeCN), ethyl acetate (EA), water (H 2 O), acetone (AcMe) or tetrahydrofuran (THF).The resulting clear solutions were boiled for 10 min before being filtered into a fresh conical flask.The filtrate was left to evaporate slowly at ambient conditions.Single crystals suitable for X-ray diffraction studies were obtained in 4-6 days.

Powder X-ray diffraction (PXRD)
The PXRD patterns were collected on a Rigaku SmartLab with Cu Kα radiation (λ = 1.540Å).The tube voltage and amperage were set at 40 kV and 50 mA, respectively.Each sample was scanned between 5 and 70°2θ with a step size of 0.02°.The instrument was previously calibrated using a silicon standard.

Crystallography
Crystals of the SST forms were individually mounted on a glass pip.Intensity data were collected on a Bruker's KAPPA APEX II CCD Duo system with graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å).All the data were collected at 100 K. Data reduction was performed using Bruker SAINT software.8a The crystal structures were solved by direct methods using SHELXL-97 and refined by full-matrix leastsquares on F 2 with anisotropic displacement parameters for non-H atoms using SHELXL-97.8b Hydrogen atoms associated with carbon atoms were fixed in geometrically constrained positions.Hydrogen atoms associated with oxygen and nitrogen atoms were included in the located positions.Structure graphics shown in the figures were created using the X-Seed software package version 2.0. 9

Differential scanning calorimetry (DSC)
DSC was conducted on a Mettler-Toledo DSI1 STAR e instrument.Accurately weighed samples (2-3 mg) were placed in hermetically sealed aluminium crucibles (40 μL) and scanned from 30 to 300 °C at a heating rate of 5 °C min −1 under a dry nitrogen atmosphere (flow rate 80 mL min −1 ).The data were analyzed using STAR e software.

Thermogravimetric analysis (TGA)
TGA was performed on a Mettler-Toledo TGA/SDTA 851 e instrument.Approximately 10-15 mg of the sample was added to an aluminium crucible and heated from 30 to 500 °C at a rate of 10 °C min −1 under continuous nitrogen purging.

IR spectroscopy
Transmission infrared spectra of the solids were obtained using a Fourier-transform infrared spectrometer (PerkinElmer 502).KBr samples (2 mg in 20 mg of KBr) were prepared and 6 scans were collected at 4 cm −1 resolution for each sample.The spectra were measured over the range of 4000-400 cm −1 .

Computational details
Geometry optimization of the SST tautomers was performed with Gaussian 03 10 using the B3LYP method 11 with the 6-31G(d) basis set, 12 followed by single point energy calculations at the 6-311++G(2df, 2p) level, in a density functional theory (DFT) type calculation.The initial atomic coordinates for the molecules were always taken from the crystal structures.

Screening by fast evaporation technique
Fast evaporation method was employed to conduct a screening for identifying possible new solid forms and to prepare all the known forms of SST from suitable solvents (Table S2 †).In our recent reports we established the efficiency of the FE method for screening of co-crystals, co-crystal polymorphs 7a and single component polymorphic systems.7b Here our reinvestigation of the old drug, succinylsulfathiazole, allowed us to test the utility of the FE method for identification and preparation of the API solvates (Table 1).Characterization of FE products by PXRD, IR spectroscopy, DSC and TGA confirmed the formation of pure solids of previously known polymorphic forms, SST-I and SST-II, a monohydrate and two new solvates, SST/AcMe and SST/THF.
Commercial SST sample contained the monohydrate form (SST/H 2 O).The FE product obtained from water had a mixture of two hydrated forms (Fig. S2a †) and also a possible unidentified form (in the PXRD two new peaks were observed at 27°and 29°which did not match with any known form).The PXRD pattern of SST/2H 2 O obtained by a slow evaporation method showed a good agreement with the previously identified dihydrate form (Fig. S2a †).As reported in previous studies SST formed cake like particles in water. 13

Despite several
This journal is © The Royal Society of Chemistry 2014 attempts, we could not obtain single crystals suitable for SCXRD.It was also observed that all the forms of SST converted to SST/H 2 O on long exposure to the atmosphere (Fig. S4 †), but not to SST/2H 2 O which is contrary to findings in previous reports.However, SST-II converted to SST/2H 2 O powder.
In the case of polymorphs, the solvent used was different for the slow and fast evaporation methods.The single crystals of SST-I and SST-II were obtained from MeCN and EA, respectively, by slow evaporation but for the same, EA and EtOH were used in the FE method.In case of the solvates, the same solvent was used for both slow and fast evaporation methods.The FE product from MeCN, a mixture of MeCN-MeOH, and MeOH resulted in only SST/H 2 O (Fig. S3b †).The two new solvate forms, SST/AcMe and SST/THF (see Fig. S1 †), obtained by the FE method, could not be prepared by the liquid assisted grinding (LAG) method (Fig. S5 †).LAG 14 always resulted in SST/H 2 O. Probably, the intake of moisture during the LAG promoted the exclusive formation of SST/H 2 O.The results of the FE method suggest that a closed environment and faster kinetics during the evaporation of the solvent probably helped to prevent water intake, thus resulting in the solvates, instead of hydrates.This study proves the unique advantage of the FE method for quick screening of solvates.Hence the FE method, which is complementary to the existing screening techniques, has potential to become a regular screening tool in solid state pharmaceutical laboratories.

Crystal structure analysis
Single crystal structures were determined for the two polymorphic forms, SST-I and SST-II, and two solvates, SST/AcMe and SST/THF.The structure of the monohydrate (SST/H 2 O) is a redetermination.Crystallographic data are listed in Table 2.
A hydrogen bond table (Table S1 †) and the ORTEP diagrams (Fig. S6-S10 †) for all the solid forms are included in the ESI.† Examination of all the structures revealed a tautomeric preference in the polymorphs and pseudopolymorphs where the SST molecule adopts the imidine tautomeric form.The structure determination also allowed us to study the hydrogen bonding or synthon 15 competition among various functional groups in the different forms.
Succinylsulfathiazole/tetrahydrofuran solvate (SST/THF) SST/THF also crystallizes in the triclinic P1 ¯space group with two molecules of SST and one molecule of THF in the asymmetric unit.Indeed, this is isostructural 16 to SST/AcMe, hence the synthons formed are the same, except some minor differences in the solvent THF interactions with the neighbouring molecules (Fig. 5b).8][19][20][21] In this series of succinylsulfathiazole polymorphs and pseudopolymorphs, the API exists only in the imidine tautomeric form, in that the proton transfer occurs from the sulphonamide N-atom to the thiazole N-atom (Scheme 1).Hence, the amidine tautomeric form of SST is not seen in any of the structures, including in the earlier reported solvates of butanol and pentanol. 6A search in the Cambridge Structural Database (CSD) (V 5.34) for sulfonamide drugs, with at least >3 hits (exclusively) of the imidine tautomeric form, revealed that such a preference is present only in two other cases, sulfathiazole and sulfapyridine (Scheme S1 †).To rationalize the tautomeric preference in the solid forms of SST, we performed DFT calculations using Gaussian software.In all cases, the molecules were taken from their corresponding crystal structures and a geometry optimization was performed, before proceeding to the single point energy calculations.The calculated energy difference between the amidine and corresponding imidine tautomeric forms revealed that the imidine tautomeric form is more stable than the amidine tautomeric form by 0.1816 kcal mol −1 , which is consistent with the observed tautomeric preference of the former.It is to be noted that this energy difference between the two tautomeric forms corresponds to the isolated gaseous state molecules, but in the crystalline environment, with stabilizing hydrogen bonds, the difference could increase further to promote the imidine form.A more detailed computational study may unravel the fundamental reasons behind the imidine tautomeric preference in the solid forms of SST and other sulphonamide drugs.

Thermal properties of the SST solid forms
The thermal behaviour of all the polymorphs and pseudopolymorphs of SST, were studied by DSC and TGA experiments (Fig. 6 and 7).The DSC thermograms for SST-I and SST-II show a single endothermic transition peak each at 207.6 °C and 192.5 °C, respectively, corresponding to melting.The DSC thermogram of SST/H 2 O showed two endotherms; a minor peak at 148.33 °C, corresponding to the loss of a water molecule, and a major peak at 193.02 °C, corresponding to melting.The two solvates, SST/AcMe and SST/THF, also showed two endotherms each.Major endotherms corresponding to melting were observed at 191.7 °C and 189.5 °C for the SST/AcMe and SST/THF solvates, respectively, while minor endotherms appeared at 169.08 °C (for the loss of AcMe) and 173.94 °C (for the loss of THF).The analyses of the DSC results suggest that SST-I is thermodynamically more stable compared to all the other forms.
The TGA experiments of the two polymorphic forms showed a single weight loss associated with decomposition after melting.In the case of hydrate and solvates, two weight losses were observed.For the SST monohydrate, the first small endotherm corresponding to loss of water (∼4.99% of the total weight) matched approximately to one mol of water.The slurry product of SST/H 2 O in water also showed a similar weight loss (4.82%), suggesting that there is no conversion of SST/H 2 O to SST/2H 2 O.The weight loss of the solid obtained from the slow evaporation method is (9.12%), equivalent to two moles of water (Fig. S2b †).For the SST/THF and SST/AcMe solvates, the first broad weight loss corresponding to the loss of solvent molecules (∼17.49% and ∼12.0% of the total weight, respectively) is equivalent to one mol of THF and AcMe, respectively.

Synthon competition study
SST has a total of three strong hydrogen bond donors, from its acid (-OH), amide (-NH) and imidine (-NH) groups, and five strong acceptors, namely from its acid (CO), amide (CO), SO 2 (two SO) and imidine (N-atom) groups.Hence, there is an imbalance of acceptors and donors, which can potentially lead to multiple solid forms.Analysis of the synthons formed in the current and previously reported SST/But, SST/Pnt and SST/H 2 O suggests the following.The  This journal is © The Royal Society of Chemistry 2014 most popular interaction is 5, homodimer between the imidine groups, which is observed in all of the five solvates (or pseudopolymorphs), but absent in both the polymorphs.The second most popular is synthon 2, which is formed between the acid (CO) and amide (NH) groups when there is at least one SST conformer in the anti geometry.As can be expected, the most dominating groups are the imidine site and the carboxylic acid group.But synthon 3, formed between these two groups, is observed only once in SST-II.The remaining synthons 1, 4 and 6, which are seen occasionally, involve the less effective SO 2 and amide (CO/NH) groups.However, notably, the most stable form, SST-I, with synthon 1 and 2, and anti geometry at the -CH 2 -CH 2groups, has the highest density of crystal packing.The balance among the large number of competing functional groups in a conformationally flexible molecule can easily be influenced by different solvent conditions.Hence the formation of several solid forms of SST is in line with general observations in the solid state pharmaceutical chemistry and crystal engineering.

Conclusions
Screening of succinylsulfathiazole by a fast evaporation (FE) method for possible new solid forms and to prepare all the known forms of SST from various solvents, demonstrated the efficiency of the technique for the identification and preparation of API solvates.Two new solvate forms, SST/AcMe and SST/THF were identified successfully by the FE method, which could not be obtained by the liquid assisted grinding (LAG) method.Probably, the intake of moisture during the LAG promotes the exclusive formation of SST/H 2 O. Hence the study demonstrates the complementary nature of the FE method to existing screening techniques and its potential to become a good screening tool in solid state pharmaceutical laboratories.In contrast to previous studies, we observed that the SST/H 2 O is more stable under ambient conditions than SST/2H 2 O.The DSC study revealed that SST-I is thermodynamically the more stable form compared to any other form.In this study, all the solid forms of SST were seen exclusively in the imidine form which was rationalized by performing DFT calculations.The energy difference between the two tautomeric forms is consistent with the imidine preference in the structures.Crystal structure analysis revealed that the homodimer formed by the imidine sites is the most popular synthon, which is observed in all of the SST solvates.

Fig. 1
Scheme 1 Schematic representation of the tautomerism and conformational flexibility in succinylsulfamethazole (SST).Notice the alternate conformations, anti (black) or syn (grey) geometries, which can be adopted by the succinyl group.Fig. 1 Polymorph I of succinylsulfathiazole (SST-I).(a) L-shape molecular geometry of two independent SST molecules with the anti -CH 2 -CH 2conformation.(b) Ladders formed by the combination of synthons 1 and 2 (see Scheme 2).

Fig. 2
Fig. 2 Polymorph II of SST (SST-II).(a) Syn geometry of the SST at the -CH 2 -CH 2group.(b) Formation of wave like chains.(c) Interlocked 3D packing viewed along the a-axis.

Fig. 3
Fig. 3 SST/monohydrate (SST/H 2 O).(a) Syn geometry adopted at -CH 2 -CH 2group of SST.(b) Packing of two adjacent chains, showing the interactions formed by bridging water molecules between two SST molecules.

Fig. 4
Fig. 4 Packing of SST/AcMe solvate.(a) Syn and anti geometries of two independent SST molecules at -CH 2 -CH 2 -.(b) Crystal packing showing the different types of interactions.(c) Occupation of solvent molecules, AcMe, in channels formed by host SST molecules.

Fig. 5
Fig. 5 Packing of SST/THF solvate.(a) Syn and anti geometries of two independent SST molecules at -CH 2 -CH 2 -.(b) Crystal packing showing the different types of interactions present in the structure.(c) Occupation of solvent molecules, THF, in channels formed by host SST molecules.

Fig. 6
Fig. 6 Differential scanning calorimetry plots of different forms of SST.

Fig. 7
Fig. 7 Thermogravimetric analysis plots of different forms of SST.

Table 1
A comparison table of the present and previous crystallization studies employed for preparing various solid forms of succinylsulfathiazole CrystEngComm, 2014, 16, 4706-4714 | 4709 This journal is © The Royal Society of Chemistry 2014

Table 2
Crystallographic data and structure refinement parameters of different forms of SST Chemical formula C 13 H 13 N 3 O 5 S 2 C 13 H 13 N 3 O 5 S 2 C 13 H 13 N 3 O 5 S 2 , H 2 O 2(C 13 H 13 N 3 O 5 S 2 ), C 3 H 6 O 2(C 13 H 13 N 3 O 5 S 2 ), C 4 H 8 O Scheme 1 Schematic representation of the tautomerism and conformational flexibility in succinylsulfamethazole (SST).Notice the alternate conformations, anti (black) or syn (grey) geometries, which can be adopted by the succinyl group.