Polymorphism and versatile solvate formation of thiophanate-methyl

The polymorphism of a fungicide, thiophanate-methyl (TM), was investigated with conventional solvent screening methods. Two polymorphs, the thermodynamically most stable form I and the less stable form II, were found. TM was also found to crystallize as plethora of different solvates which produced mostly form II upon desolvation. The structures of form I and form II and the fourteen discovered solvates were solved by single crystal X-ray diffraction. The most stable forms 10 were further characterized by powder diffraction, thermoanalytical (TG/DTA, DSC and thermomicroscopy) and spectroscopic (IR, Raman, 13 C CP/MAS NMR) methods.


Introduction
The growing field of crystal engineering deals with designing and synthesizing molecular solid state structures with desired 15 properties. 1 One approach to the subject is using supramolecular synthons 2 composed of molecular fragments and the interactions between them to approximate the possible structural outcome of a crystal. Polymorphism 3 , the ability of a compound to crystallize in 20 more than one distinct crystal form, can be seen as a challenge in crystal engineering, but also as a means to investigate the principles of crystal formation as nature does it. In addition to polymorphs, a number of solvate forms with solvent molecules included in the crystal lattice are commonly formed 25 with the solvent of crystallization. 4 These can further complicate the crystallization of compounds, but can also resolve some unanswered questions and be, for instance, routes to other crystal forms, not easily reached otherwise. 5 The solvent molecules in the crystal structure may either be 30 included to decrease void space in the crystal or be for example hydrogen bonded to the molecules of the compound to better satisfy the possible intermolecular interactions. 6 The occurance of polymorphs and solvates is especially high amongst molecules with flexible torsions and several low 35 energy conformers. 7 The literature reflects the importance of polymorphism in pharmaceutical substances. 3 The basic questions in agrochemical actives 8 are essentially the same; how many forms can be found and what are properties and 40 thermodynamical relationships of the forms. We carried out conventional solvent screening investigating the polymorphism of a pesticide active, thiophanate-methyl (TM) (Scheme 1), dimethyl 4,4'-(o-phenylene)bis (3thioallophanate), which is relatively flexible and capable of 45 forming multiple hydrogen bonds as well as aromatic interactions. The motivation was to investigate whether the variation in melting points reported in the literature (135ºC to 200ºC 9 ) is due to the existance of different polymorphs of TM, and on the other hand whether these forms exhibit 50 varying hydrogen bonding arrangements. Recently, multi component crystals, co-crystals, of TM have been reported with different agrochemical actives. 10 TM is a fungicide and wound protectant and has been used, for example, in protecting citrus fruits against post harvest 55 decay in packing houses. 11 TM belongs to a group of fungicides that transform into benzimidazoles during use with TM specifically transforming into carbendazim. 11,12 Benzimidazoles work by impairing microtubule growth in fungal cells, which consequently prevents correct cell 60 division, as microtubules are needed in forming the spindle that guides the movement of chromosomes during cell division. 13 The  crystallizations from acetonitrile, DMA and methyl isobutyl  ketone produced form I, while all other crystallizations   80   produced solvates. Form II was acquired by fast  crystallization from acetone under reduced pressure and a  single crystal was acquired from a 1:1 mixture of acetonitrile  and  were placed in idealized positions (C-H hydrogens) or found from the electron density map (most N-H hydrogens) and included in structure factor calculations. The N-H distances of the hydrogen bonding hydrogens were restrained to 0.91 Å to give the best fit to the X-ray data and to ensure a stable 120 refinement. The WinGX program system 17 and the Shelxtl program package 18 were used. Residual electron density in the DMSO solvate that could be assigned to severely disordered DMSO molecules was removed with the program SQUEEZE 19 . The quality of the ethanol solvate structure 125 solution is poor and thus only preliminary data is given. The methanol in the methanol solvate is disordered over two postions and could only be refined isotropically without hydrogen atoms. The poor quality of several of the structures is due to insufficient data collection as the structures were 130 measured for industrial purposes. Pictures of the structures were drawn with Mercury 20 . Crystal data and collection parameters of the structures are presented in Tables 1 and 2. PXRD. Powder X-ray diffraction patterns were measured with a Siemens D5000 X-ray diffractometer with a Cu anode (λ = 135 1.5406 Å; 45 kV, 25 mA). The measurement temperature was 25°C (RT) and a 2θ-angle range of 5-35° and a step resolution of 0.020° was used with a step time of 4.5 s. 13 C CP/MAS NMR . The 13 C CP/MAS NMR spectra were  measured with a Bruker Avance 400 FT NMR spectrometer   140 with a dual 4 mm CP/MAS probehead. The sample was packed in a 4 mm diameter ZrO 2 rotor, which was spun at 10 KHz rate at 296 or 373 K. Contact time for CP was 4 ms, pulse interval 4 s, time domain 2 K, which was zero filled to 8 K in frequency domain. Exponential window function with 5 145 Hz line broadening was used. 20 000 scans were acquired. IR and Raman spectroscopy. The IR spectra were measured from KBr tablets on a Thermo Nicolet Nexus 470 IR spectrometer with a DTGS KBr detector. The Raman spectra were measured with a Nicolet 950 FT-Raman spectrometer.

Results and discussion
Polymorphs Two polymorphs of TM were found and characterized. Form I, of which the original commercially available sample was 180 composed of, crystallized from acetonitrile solution (also from DMA, methyl isobutyl ketone and 1,2-propanediol solutions) and form II, or a mixture of form I and form II, was acquired mostly by desolvation of the found solvates. Pure powdered samples of form II were obtained by reduced pressure 185 evaporation from warm acetone solutions of TM followed by heating at 80°C in a vacuum oven for one hour. Form II was also found to crystallize from acetonitrile:water (1:1, V:V) solution, though not consistently. The next paragraphs describe the crystal structures of both polymorphs. The 190 differences in the conformations of TM are discussed later in the chapter "conformations".
Crystal structure of form I Block crystals of form I crystallized from acetonitrile solution in the monoclinic space group P2 1 /n with one TM molecule in 195 the asymmetric unit (Fig. 1a). In the crystal structure one molecule of TM is hydrogen bonded with 8 hydrogen bonds in all to three adjacent molecules of TM. In addition, there are  bonding within the arms of the molecule. The TM molecules are connected with two different hydrogen bonding arrangements -one with two N-H•••S=C and one with two N-H•••O=C hydrogen bonds. These arrangements produce infinite two dimensonal sheets of TM 230 molecules ( Fig. 1f) that then stack up on each other like those in form I with hydrophobic interactions between the methyl and benzene groups.

Further characterization
The polymorphs were also characterized with PXRD, DSC, 235 TG/DTA, thermomicroscopy, CP/MAS NMR, IR and Raman methods. In the DSCs (ESI) of both forms decomposition started at around 165°C with peak maxima at 174.2°C and 175.9°C for form I and form II, respectively. In the DSC of form I there is additionally a very small endothermic peak at 240 around 115°C, which showed up at all heating rates and is possibly explained by impurities as at that temperature there is no change observed with the thermomicroscope with which form I and fom II could not be distinguished. The TG/DTA (ESI) curves of the two polymorphs were also practically 245 indistinguishable.
The PXRD patterns (Fig. 2a) of the two polymorphs can be clearly distinguished. PXRD patterns were thus used for form identification in further experiments. (Comparisons between experimental and calculated PXRD patterns in ESI). 250 Forms I and II can also be distinguished from their 13 C CP/MAS NMR spectra (Fig. 2b). The methyl group peaks are at 55.1 ppm for form I and 52.2 ppm for form II with a small peak at 55.0 ppm, indicating that the methyl groups are in somewhat different environments in the two forms. The peaks 255 of the ester carbon atoms are at 153.2 ppm and 153.1 ppm for form I and form II, respectively, and thus are in very similar environments.The benzene carbon peaks are different for the two forms and point to 3 types of environments for form I and two types of environments for form II. By comparing the 260 chemical shifts of the corresponding C=S carbon atoms, it can be concluded that the C=S carbons with peaks at around 180 ppm are in more similar surroundings in form II than in form I  30 There are differences in the IR and Raman spectra (Fig. 2c  and d) of the two forms, but these were not investigated further due to PXRD being such a good method to differentiate the two forms. The IR rule 31 can be used to 275 determine the stability order of polymorphs. The first absorption band of carbonyl oxygen atoms in the fingerprint region for form I is at 1711 cm -1 and for form II at 1714 cm -1 .
The difference is small and inconsistent with other data as it indicates form II being more stable. This difference of 3 cm -1 280 could also indicate greater involvement of the carbonyl oxygen atoms of form I in intermolecular interactions.

Transformation and stability
According to thermal analysis the relationship between the two polymorphs is monotropic, as no endothermic (or 285 exothermic) transition is observed for either form. Temperature variable 13 C CP/MAS NMR (form II, RT to 100°C) and PXRD (form I, RT to 130°C) analyses also indicate no transformations. However, no melting temperature could be determined and no heat-of-fusion could be measured 290 because of the decomposition of TM and thus the heat-offusion rule 31 could not be used to back up our interpretation.
Form I has a calculated density of 1.51 g/cm³ and form II 1.46 g/cm³. According to the density rule 31 the denser polymorph, in this case form I, is more stable at absolute zero. 295 As the relationship between the polymorphs is monotropic, form I is also more stable at all temperatures. The energies of dissolution, measured by solution microcalorimetry, were -4.011 kJ/mol for form I and -6.724 kJ/mol for form II. According to the results, form I is approximately 2.7 kJ/mol 300 lower in energy than form II. Form II also converts to form I when mixed in a suspension of water and in water-glycerol mixtures, giving further evidence of the stability of form I.
The solution-mediated transformation from form I to form II occurred faster at elevated temperatures (80°C) than at room 305 temperature indicating form I to be more stable also at higher temperatures.

Solvates
Fourteen solvates of TM (acetonitrile/water, methanol, ethanol, acetone, DMSO, cyclohexanone, dichloromethane, 310 1,2-dichloroethane, dioxane, pyridine, 1,2-dichlorobenzene, THF, chloroform and benzene) were encountered during the investigations (Table 3). All but the acetonitrile solvate monohydrate crystallized quite consistently from solutions of the corresponding solvents. Crystal structures of all the found 315 solvates were determined. The hydrogen bonding networks and packing of the solvates is complex, as can be expected by the various hydrogen bonding possibilities and the vast amount of low energy conformers offered by TM. We do not feel that a detailed analysis is necessary here. However, we 320 try to bring up some common features and categorize the structures where applicable.

Stability and desolvation behavior
When taken out of solution, the solvates desolvate at temperatures from room temperature (RT) to around 130°C, as 325 determined by thermomicroscopy (HS) and TG/DTA ( Table  3). Most of the solvates also desolvate quite rapidly at room temperature when left out of solution, though the methanol and ethanol solvates seem to be rather stable and stay solvated for weeks even when out of solution. Upon desolvation (in a 330 vacuum oven at temperatures of 80-130°C depending on the desolvation temperature of the solvate) the tested solvates produce form II or a mixture of form I and form II, as determined with PXRD. According to the experiments, a correlation could not be recognized between the structure of 335 the solvated or the desolvation temperature and the preferred desolvation product, even though the acetone, DCM, THF and chloroform solvates, which desolvate even at room temperature, appear to produce preferentially only form II. The reason for the emergence of mixtures may be solvent-340 mediated transformation occurring when the solvent does not leave from around the crystals as they desolvate.   (Fig. 3a). This arrangement of hydrogen bonds causes 1-dimensional parallel chains of TM molecules. The solvent molecules are situated in channels between chains of TM molecules (Fig. 3b). The distance between the 355 closest aromatic H atoms and the Cl atoms in the DCM structure is approximately 3.07 Å, which indicates a weak interaction. The C-H hydrogen atoms of the solvent molecules in the DCM structure are also weakly hydrogen bonded to the sulfur atoms of TM with C•••S distances of 3.68 Å and 3.93 Å 360 and angles of 156° and 173°, respectively.

One-armed chains
The methanol, ethanol, acetone and THF solvates crystallize in the triclinic space group P-1 with two TM molecules in the asymmetric unit. The cyclohexanone solvate also crystallizes 365 in the spacegroup P-1, but with four TM molecules in the asymmetric unit, whereas the DMSO solvate crystallizes in the monoclinic space group C2/c with one TM molecule in the asymmetric unit. In these solvates the main arrangement of hydrogen bonding is one where the molecules of TM arrange 370 in chains, which include hydrogen bonds mainly to one arm of the molecules (Fig. 4 and ESI   In the methanol, ethanol, acetone and cyclohexanone solvates the connected chains build up two-dimensional sheets 400 composed of solvent molecules in between two layers of TM molecules (Fig. 5). These sheets then stack up on each other, being the cause for the plate-like habit of the crystals.
The THF and chloroform solvates are isomorphic with each other (Fig. 6a and b). The difference between the two is the 405 ability of the THF molecule to form hydrogen bonds with TM and in the chloroform solvate the parallel one-armed chains are connected through hydrogen bonding via an arrangement of two N-H•••S=C hydrogen bonds but in the THF solvate they are not. These two solvates have a 1 to 1 ratio of TM to 410 solvate unlike the other solvates with the same one-armed   chain hydrogen bonding arrangement of TM molecules.
In the DMSO solvate the one-armed chains, which pack parallel to each other, are not connected through hydrogen bonding. The severely disordered DMSO molecules in the 415 DMSO solvate are placed in cavities that are lined up to form small tubular channels running through the crystal. These channels can be seen between the parallel chains when viewed from the side (Fig. 7). No hydrogen bond donors of the TM molecules point into these cavities and it is likely that the 420 disordered DMSO molecules are merely co-crystallized to fill the empty space. The analysis of the removed electron density supports the hypothesis of having eight disordered DMSO molecules in the unit cell making the TM to solvent ratio 1:2. Two-armed chains 425 The dioxane and pyridine solvates crystallize in the monoclinic space group C2/c and the triclinic space group P-1, respectively, but the hydrogen bonding pattern of the TM molecules in these solvates is the same. The molecules of TM build up chains with arrangements of two N-H•••S=C 430 hydrogen bonds and two N-H•••O=C hydrogen bonds involving both the arms of the molecule (Fig. 8a and b). Again as for the one-armed chains, the N-H•••S=C hydrogen bonds show more variability in angles than the N-H•••O=C hydrogen bonds with the hydrogen bonds in the pyridine solvate being 435 somewhat longer than those in the dioxane solvate.
In the pyridine solvate the chains arrange parallel to each other and in the dioxane solvate they cross each other (Fig. 8c  and d). In the pyridine solvate the pyridine molecules are located in channels running down the crystallographic a-axis 440 (Fig. 8d). In the dioxane solvate there are no specific channels where the dioxane molecules reside, but they are located   (Fig. 11a). The distance between the sulfur, that is not hydrogen bonded to an amine hydrogen, and the methyl carbon is 3.732 Å which points toward possible weak C-H•••S=C hydrogen 495 bonding interactions. The chains pack parallel to each other with the benzene molecules in channels running through the structure (Fig. 11b).

Summary of the structures
There are interesting similarities and differences in the 500 hydrogen bonding arrangements and the conformations of the TM molecules in the sixteen described structures. These will be summarized here and the calculated gas phase conformers will also be looked at briefly.

Conformations
The arms of the TM molecules are fairly planar in all 525 structures due to intramolecular N-H•••O=C hydrogen bonds between N2 and O1, and N3 and O3 (the numbering of atoms is in Fig. 1).The conformations of the molecules are thus described by comparing the position of the arms of the molecules in respect to the plane of the benzene ring. 530 As can be seen in Figure 12 a-d, the two polymorphs, form I (Fig. 12a) and form II (Fig. 12b), represent not only different hydrogen bonding, but also conformational polymorphism and, moreover, different conformers of TM are also observed in the solvate structures. The most remarkable difference in 535 between form I and form II is that, unlike form II, form I has an intermolecular N-H•••S=C hydrogen bond connecting the two arms of the molecule in addition to the above mentioned N-H•••O=C hydrogen bonds within the arms of the molecule. Interestingly, the acetonitrile/water solvate, with Z´=3, has  Calculations of the gas phase conformers of TM were done in order to get insight about the relative energies of the found conformers. Relative to the conformer with the lowest calculated energy, there are six conformers with a ΔE less than 20 kJ/mol and a total of 214 with a ΔE less than 100 550 kJ/mol. The six lowest energy conformers thus lie in an energy range comparative to one moderately strong hydrogen bond. The four most stable conformers (ΔE less than 8 kJ/mol, Fig.12e-h) all have the intramolecular N-H•••O=C hydrogen bond that is seen in all of the crystal structures. In 555 conformations with higher energy this bond can be changed into a N-H•••O-C hydrogen bond, and in even higher energy conformers an arm of the TM molecules can be twisted into a non-planar conformation with no intra-molecular hydrogen bonding, making these higher energy conformations unlikely 560 in crystals structures.
The conformers observed in the crystal structures have varying torsion angles between the plane of the benzene ring and the arms of the molecules with the gas-phase minimized state and direct comparison is not feasible. For example, the 565 most stable gas phase conformer (Fig. 12e) has no good matches in the determined crystal structures, though one of the TM molecules in the MeCN/H 2 O solvate comes close. The second conformer (Fig. 12f) resembles that in the benzene solvate (Fig. 12d). The third gas-phase conformer (Fig. 12g) is 570 quite like that in many of the solvate crystal structures and form I, but most closely resembles the TM molecules in the THF and cyclohexanone solvates. The fourth conformer (Fig.  12h) resembles most closely that in form II of TM (Fig. 12b).
The aromatic solvates have conformations of TM most 575 unlike those in the other structures. In the benzene solvate one arm of the molecule is more in the plane of the benzene ring whereas the other arm is, conversely, less in the plane of the benzene ring than in form I. Further, the conformation of TM in the 1,2-DCB solvate (with sulfur atoms on the same side of 580 the aromatic plane) was not found among the gas phase conformers. In this structure the planes of the arms of the TM molecules are almost parallel to each other, which is also the case in form II, and enables the formation of the similar hydrogen bonded sheet arrangements in the structures. 585 A lattice energy analysis and a constrained energy optimization of the observed crystal structure conformers is left out of this study as the authors do not expect that such an analysis would bring more essential information. It is, however, clear that the variability of the conformations of TM 590 in the solved crystal structures is paralleled by a number of gas phase conformers with small energy differences and this flexibility probably accounts for the large number of solvates of TM.

595
TM was found out to exists in two polymorphic forms which have very similar melting points. The original and known form I is the thermodynamically most stable form, which is monotropically related to Form II. Form II can be accessed via desolvation of various different solvate forms. 600 The discovery of a series of fourteen solvates and the structure determination of these via single crystal X-ray measurement is the highlight of this work. Solvent molecules capable of forming hydrogen bonds (acting either as hydrogen bond acceptors or both acceptor and donor) presented the 605 majority, but also other, such as 1,2-dichlorobenzene and benzene were among solvate forming solvents. The isostructural methanol and ethanol solvates, with desolvation points around 140°C, were remarkably stable against desolvation, whereas the non-hydrogen bonded solvates 610 solvated already at ambient conditions. The single crystal structures reported here represent a variety of interaction possibilities of TM varying from hydrogen bonding to aromatic and lipophilic interactions. No clear patterns in packing or formation could be drawn from the 615 structures. It is however noteworthy that TM has a large amount of low energy conformers and several possibilities of forming hydrogen bonds. These two facts can as tradeoff, lead to new hydrogen bonding and close packing modes and can reduce the total energy difference between alternate crystal 620 structures. The search in the CSD by van de Streek 32 shows that there are only a few compounds, many of which are not organic neutral compounds of the size of TM, with ten or more solvate structures reported. It is very likely that, similar to sulfathiazole with its over one hundred solvates 33 , several 625 new solvates and also co-crystals with a variety of different functional groups could be found for TM.
As shown in this paper, crystallographical methods play a key role in studying polymorphism and solid state structures. However, also all the used spectroscopical methods ( 13 C 630 CP/MAS NMR, IR and Raman) are usefull in identifying the polymorphic forms of TM from each other and can be valuable in cases where crystallographical methods are not available or can not be applied. Thermoanalytical methods are also especially helpful in determining the stability of a new 635 modification and resolving whether it is a solvate or not.