Comparison of the polymorphs and solvates of two analogous fungicides - a case study of the applicability of a supramolecular synthon approach in crystal engineering

The polymorphism and solvate formation of thiophanate-ethyl (TE), a fungicidal active, was investigated by solvent crystallization and compared to a close analogue, thiophanate-methyl (TM). Four polymorphs and seven solvates of TE were found and structurally compared with the previously found two polymorphs and fourteen solvates of TM by analyzing the hydrogen bonding patterns and using 10 fingerprint plots, packing coefficients and lattice energies. TE and TM have the same functional groups that can build identical supramolecular synthons. Despite the strong similarities, the polymorphs and solvates of the two actives show significant differences in hydrogen bonding and packing. The results demonstrate the challenges in using a supramolecular synthon approach, and promote the importance in finding methods to also make use of packing effects and lipophilic interactions in crystal engineering. 15 In a crystallization process, the molecules arrange in an energetically favorable way. The energetic incentive of favorable intermolecular interactions, the strongest of which are hydrogen bonds for neutral molecules, and reduction of void space, negotiate for the best arrangement of molecules in the crystal. For 20 most of organic molecules, there are several ways to arrange, causing polymorphs to appear. 1 In a solvent based polymorph screening, it is typical to also find solvates, that are often more stable than the polymorphs of a compound

In a crystallization process, the molecules arrange in an energetically favorable way.The energetic incentive of favorable intermolecular interactions, the strongest of which are hydrogen bonds for neutral molecules, and reduction of void space, negotiate for the best arrangement of molecules in the crystal.For most of organic molecules, there are several ways to arrange, causing polymorphs to appear. 1 In a solvent based polymorph screening, it is typical to also find solvates, that are often more stable than the polymorphs of a compound in the solvate forming solvent. 2 The crystallization outcome then depends on the kinetics of crystallization and the presence of other contributing additives, either the solvent or other species, in the crystallization medium.
The aim in modern crystal engineering is the targetted discovery of multicomponent crystals, i.e. cocrystals.As solvates can be considered to belong to a subgroup of cocrystals, the study of solvate formation can give an insight for designing cocrystals.Suitable cocrystals can enable the tuning of the crucial physical and chemical parameters such as the solubility, vapour pressure, crystal habit etc., of a an active ingredient in pharmaceutical, agrochemical or other areas. 3The prevailing model for cocrystal engineering is using a supramolecular synthon approach, where supramolecular synthons, which consist of intermolecular interactions (especially hydrogen bonds), are viewed as bonds for the construction of supermolecules, i.e. crystals. 4tal structures give the most definite understanding of the crystal packing and intramolecular interactions, and help give strategies for crystal engineering.The conformation of the molecule, hydrogen bonding and other intermolecular interactions as well as the packing can be assessed with the help of a number of tools.The packing coefficients 5 , fingerprint plots 6 and lattice energies 7 can be calculated to ensure not missing vital features when assessing the structure visually.The use of graph set symbols 8 for hydrogen bonding facilitate the easy comparison of hydrogen bonding in similar structures.50   We have previously investigated thoroughly the polymorphism and solvate formation 9 , as well as cocrystal formation 10 , of the fungicidal active thiophanate-methyl (TM, dimethyl 4,4'-(ophenylene)bis(3-thioallophanate), Scheme 1.According to our findings, TM has two conformational polymorphs and at least 55 fourteen solvates.TM is an excellent example of a small molecule with possibilities to serve as a versatile supramolecular building block, that has conformational flexibility and is able to form host-guest frameworks.To get an understanding of the applicability of a supramolecular synthon approach, we 60 investigated the polymorphism of a close analogue of TM that differs only with two CH 2 groups.Thiophanate-ethyl (TE, diethyl 4,4'-(o-phenylene)bis(3-thioallophanate), Scheme 1) is a fungicide that has been withdrawn from the European market 11 because of safety issues and the availability of an efficient 65 alternative, mainly TM, which is identical in fungicidal function and from a supramolecular synthon perspective.This paper reports the four found polymorphs of TE, as well as the analysis of the achieved single crystal structures of three polymorphs and seven solvates, as comparison with the previously reported TM

Crystallizations
Approximately 20 mg of TE was dissolved in 5 ml of solvent (acetonitrile, 1:1 acetonitrile:water, acetone, ethanol, methanol, DCM, chloroform, pyridine, dioxane and toluene:methanol).The solution was transferred to a clean test tube, which was covered with parafilm with a few holes.The solvent was left to evaporate in room temperature.

PXRD
For powder X-ray diffraction analysis the original TE was hand grinded, pressed to a zero background silicon plate and measured on a PANalytical X'Pert Pro system in reflection mode with CuKα1-radiation.A 2θ-angle range of 5-40° and a step time of 60 s were used with step resolution of 0.0167°.Figures were drawn with X'Pert HighScore Plus 12 .

Single crystal X-ray diffraction
The X-ray diffraction data was collected on Nonius Kappa CCDdiffractometer with Apex II detector at 173 K, using graphitemonochromated CuKα radiation (λ = 1.54178Å), or in the case of the pyridine solvate graphite-monochromated MoKα radiation (λ = 0.71073 Å).Absorption correction was performed with Denzo-SMN 1997 13 .The structures were solved using direct methods, refined, and expanded by using Fourier techniques with the SHELX-97 software package 14 .All non-hydrogen atoms, except for the disordered toluene molecule, were refined anisotropically.Hydrogen atoms were placed in idealized positions or found from the electron density map (hydrogen bonding N-H hydrogens), and included in structure factor calculations.The N-H hydrogen atoms found in the electron density map were restrained to a distance of 0.91 Å to give the best fit to the X-ray data and to ensure stable refinement.Pictures of the structures were drawn with Mercury. 15Crystal data and collection parameters are presented in Table 1 and Table 3.
In the dioxane I, DCM and chloroform solvates the solvent molecules are disordered over a symmetry axis in two positions.There are voids of 42 Å 3 in the toluene solvate structure that are surrounded by the ethyl groups of TE and the toluene molecules 45 making the inclusion of water unlikely due to hydrophobic surroundings.The toluene molecule is on a general position, but disordered over two positions that are coplanar and in a 180° angle relative to each other.In Form III there are voids of around 45 Å 3 between the arms of one of the symmetry unequivalent 50 molecules with no significant residual electron density.

Lattice energy calculations
The lattice energies (E latt ) of the polymorphs of TE and TM were calculated using the Cerius² program package. 16All computations were performed with the default settings of the program and 55 following the previously published procedure. 7Atomic charges were assigned by QEq method and cross checked with AM1 which yielded essentially similar results.The E latt values were calculated with a Dreiding II force field for the energetically optimized structures.During structure optimization the molecules 60 were treated as rigid objects and only the unit cell dimensions were allowed to change.The optimized unit cells diverged less than 3 % of the original values.
The approximated modeling yielded generally similar energetic values for the polymorphs in a meaningfull energy 65 window.However, due to the inaccurate nature of the method (e.g.lack of polarizability), the absolute values are not reliable and only the relative energies should be judged.Precise E latt calculations would need expansive DFT calculations and/or extensive calibration of the force fields to reach the accuracy that 70 is needed to judge the total energies which often diverge 1-2 kcal/mol between the polymorphs. 17

Results and discussion
75 Four polymorphs and seven solvates of thiophanate-ethyl were found during the solvent screening experiments.The polymorphs were named in order of discovery as forms I-IV.Samples crystallized from acetonitrile and methanol produced crystals of the benzene rings and dipole-dipole interactions between the carbonyls in adjacent chains.The parallel chains make up sheetlike structures that stack up on each other in a manner similar to that in form I with benzene and ethyl groups of TE protruding to both sides of the sheets.Solution NMR evidence suggest that there is some interaction between the N-H hydrogens and the acetate present in the 20 crystallization, which could have influenced the aggregation of TE molecules initiating the crystallization of this form.The Z' of 2 and the presence of small voids suggests that this could be a case of crystallization "on the way". 18

TE Form IV
Form IV was found to be a desolvation product of the isomorphous solvates and can be identified by the PXRD pattern.The original sample from ChemService was found to be a combination of form I and form IV (PXRD in Fig. 2).This can be most clearly seen in the peaks caused by form IV at 9.5, 16.3 and 30 23.7°2θ.
The unit cell of this polymorph was determined from the powder pattern using X'Pert HighScore Plus and the most likely candidate is a monoclinic cell with a possible spacegroup of C2/c with a= 11.50, b= 18.76, c= 9.22, β= 112.24° and V= 1846.5 Å 3 .

35
A structure in this cell can easily be imagined from the isomorphous solvate structures by removing the solvent and moving the TE molecules to fill the formed voids.Efforts to solve the structure from powder data are currently underway.

Packing efficiency of the polymorphs 40
The fingerprint plots in Fig. 1 show a graphical representation of the packing in forms I to III.The packing coefficients (Fig. 3) of the structures were also determined 19 in order to get a numerical estimate of the packing efficiency.According to the results, form II packs the most tightly with form I in second place.The packing 45 coefficient and the hydrogen bonding seem to explain the similar stability of forms I and II since form I has more and stronger intermolecular hydrogen bonds than form II, but form II packs more tightly.Form III, on the other hand, packs very loosely and is expected to be less stable even though there is a lot of 50 hydrogen bonding.
The packing coefficients were calculated also for the previously reported polymorphs of TM.TM form I is clearly more densely packed than form II.This can also be seen in the fingerprint plots (

Experimental observations on the stability
Thermal methods to determine the stabilities of the forms could not be used because TE decomposes when melting or heated for prolonged periods.Very few experiments were done to find out the stability order of the polymorphs, because the forms could not 65 be reproduced in large quantities.The occasional concomitant crystallization suggests that form I and form II are quite similar in stability, but attempts to crystallize form II in larger amounts resulted only in form I. The kinetics of the crystallizations are likely the cause of this behavior.When the original sample, a 70 mixture of form I and form IV, was slurried in MeCN-water mixtures, it changed to pure form I.

Lattice energies of the polymorphs
The lattice energies (E latt ) of the polymorphs (Table 2) were calculated to get understanding of the energies governing the 75 packing of the forms.
The computed E latt of different TE polymorphs are inside 3 kcal/mol and indicate a stability order of I > II > III which is supported by the available experimental observations.The Van der Waals energy (E vdw ) component, which describes the nonbonding attractive and repulsive interactions, is typically most accurately modeled by the force field calculations.This is considerably larger, indicating denser packing, for form II compared to forms I and III, which is in-line with the crystallographical densities of the polymorphs.Also the fingerprint plots (Fig. 1) show average shorter intermolecular distances for form II. The calculated coulombic energies (E C ), which describe the electrostatic interactions in the crystal, show a discrepancy in that the energy value for TE form II is positive, indicating an unfavorable interaction.The cause for this is likely the short intra-and intermolecular C=O•••O=C distances which are in the range of 3.05 to 3.11 Å.The E c calculations, however, are problematic due to inaccurate determination of the electronic charges and should be judged with some caution. 20Calculated hydrogen bond energies (E H ) 21 gave similar energies for forms I and II, but clearly lower for form III. The result is somewhat surprising as the fingerprint plots indicate that form I has some substantially stronger intermolecular hydrogen bonds than the two other polymorphs.However, the high E H term of form II can be explained by the intramolecular N-H•••O=C bonds which are most linear and shortest in form II.
The energy calculations of TM polymorphs gave significantly higher E vdw for form I as also indicated by the packing coefficient and the fingerprint plot Fig. 4, but also indicate stronger total hydrogen bonding (E H ) in the crystals of form II. In any case, the total E latt shows that form I is clearly more stable of the two polymorphs, though, the relatively high E C of form I might be overestimated and thus exaggerate the total energy difference.

Structural comparison to TM
A hydrogen bonding diagram and fingerprint plots of TM forms I and II were drawn for comparison (Fig 4).The polymorphs of TE and TM do not have matching hydrogen bonding arrangements.TE form I and TM form II are very similar on a quick glance but the hydrogen bonds are different.The structure of the hydrogen bonded sheets in TE form I is, however, identical to those of TM in the 1,2-dichlorobenzene solvate 9 (fingerprint plot in Fig. 4).In the TM solvate the solvent molecules are located between the sheets that are consequently further away from each other than in the TE polymorph.The extra methyl groups of TE possibly enables closer packing of the sheets without the need for guests.A conformation where the sulfur atoms are on the same side of the benzene ring is interestingly only seen in these two structures of all the 26 solved structures for TM 9  The hydrogen bonded chains of TE and packing in form III is most similar to that in the very stable ethanol and methanol solvates of TM, which also have a Z' of 2. The D1,1(2) motif and the unused C=S acceptor in one of the TE molecules in form III is replaced in the TM solvents with hydrogen bonds to the solvent molecules.

TE solvates
Structures of four isomorphous solvates (acetone, dioxane I, DCM and chloroform), a toluene solvate, a dioxane solvate polymorph and a pyridine solvate of TE were acquired.

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In all the solvates the hydrogen bonding consists of N-H•••S hydrogen bonds in the R2,2(8) motif with no intermolecular N-H•••O=C bonds.In the isomorphous solvates the R2,2(8) motifs connect into chains (Fig. 5a) that pack parallel to each other with π-π stacking interactions to neighboring parallel chains with ring 80 distances of approximately 3.4 Å.The solvent molecules are located between the arms of the TE molecules in channels running through the structure in the direction of the crystallographic c-axis (Fig. 5b).
In the pyridine and dioxane II solvates the N-H ) motifs to the solvent, respectively, block the formation of chains of TE molecules leaving only hydrogen bonded pairs of TE molecules (Fig. 6a-b) connected with the R2,2(8) motif.
In the toluene solvate the R2,2(8) motifs connect into spiraling chains, causing the chirality of the structure (Fig. 7).The parallel 90 spirals intertwine making up stacks of benzene rings running through the structure.The disordered toluene molecules are located in channels running in the same direction as the elongation of the spirals.According to the packing coefficients (Fig. 3) the hydrogen bonded pyridine and dioxane solvates pack more tighly than the isomorphous and toluene solvates, the only exception being dioxane I, where the dioxane molecule more effectively fills the space available for it in the solvent channels.The isomorphous and toluene solvate crystals are very unstable when out of solution, desolvating in seconds/minutes, whereas the hydrogen bonded solvates, especially pyridine, are stable for hours.

Comparison to TM solvates
Both TE and TM have a large amount of solvates (Table 4), Interestingly, no methanol and ethanol solvates of TE were found, whereas in the case of TM these solvates crystallize easily and are very stable.The methanol and ethanol solvates of TM are isomorphous with each other and also very similar in hydrogen 25 bonding to the acetone and cyclohexanone solvates.Likely, the formation of similar solvates of TE is hindered by packing problems regarding the larger size and more lipophilic nature of TE.The same kind of hydrogen bonding is, however, exhibited by TE form III, which has voids in the structure and likely if the 30 correct solvent and crystallization conditions could be found, a solvate with similar hydrogen bonding could crystallize.TE, unlike TM, is able to make evident solvent channels that run in between the two arms of the molecule.In addition to acetone, dioxane, dichloromethane and chloroform other solvent 35 molecules of similar size (molecular volume of around 55 to 90 Å 3 , see Table 4) are suspected to fit in these channels and also form isomorphous solvates.Ethanol is perhaps too small, but propanol could be ideal and 1,2-dichloroethane and DMSO possible solvate formers.The molecular volume of pyridine (~80 40 Å 3 ) is also such that it could fit in the solvent channels.The strong hydrogen bonding interactions between the solvent and the TE molecules, however, seem to determine the structure of the solvate in this case.Toluene is likely too large or unsuitably shaped to fit in the solvent channels and thus makes another type 45 of structure.

Conclusions
Four polymorphs and seven solvates of thiophanate-ethyl were found and the crystal structures of all but polymorph IV solved.Thiophanate-ethyl and thiophanate-methyl are an example of when the applicability of a supramolecular synthon approach alone is limited.The behavior of TE is quite similar to thiophanate-methyl in that both willingly form solvates and polymorphs, but with varying combinations of hydrogen bonding The challenge is that even though there are clearly a few best hydrogen bonding motifs, the conformational possibilities enable these to be used in a variety of combinations, which can not be predicted.Another reason is the possibility for other weaker and 30 not so directional, but combinatorially strong interactions like ππ-stacking and lipophilic effects.These can outweigh the propensity for certain kinds of hydrogen bonding synthons between molecules, like in the isomorphous solvates of TE.The reduction of void space, is another factor since the energetic 35 advantages in a certain kind of packing of molecules are difficult to predict.
We expect a supramolecular synthon approach to be most applicable with conformationally rigid molecules that have a limited number of clear hydrogen bonding possibilities.Another 40 case where a supramolecular approach could be used successfully, even with more complicated systems, would involve using reoccurring hydrogen bonds, like the hydrogen bond from TE/TM to the pyridine nitrogen.A further strategy for cocrystal design with TE could be in fitting small cocrystallizing molecules 45 in the channels of the isomorphous solvates.⁄ � 4 , where θ is the angle between the donor, hydrogen and acceptor and C and D are empirical parameters.Accurate determination of the hydrogen bond energy is, however, problematic for several reasons of which one is the power of 10 in the attractive term which makes the function sensitive for the separation of the hydrogen and acceptor atom as the energy drops rapidly with the increasing distance.The function is also sensitive for the bonding angle due to the power of 4 in the cosine θ term.

Fig. 1
Fig. 1 Hydrogen bonding in the sheets of TE form I (top), in the chains of TE form II (middle) and the double chains of TE form III (bottom) with nonhydrogen bonding hydrogen atoms removed for clarity The simplified diagrams of the hydrogen bonding and the fingerprint plots of forms I-III.TE form I and a sample crystallized from ethanol produced crystals of form II. Samples crystallized from 1:1 acetonitrile:water concomitantly produced crystals of form I and form II. Form III was crystallized from a methanol solution that also contained sodium acetate.The pyridine, toluene, acetone, dioxane, DCM and chloroform samples produced solvate crystals.Two polymorphic solvates (dioxane I and II) were crystallized from dioxane in the course of trying to get better crystals of the first one.The fourth polymorph of TE was identified by powder diffraction, but no single crystal structure was obtained.Form IV appears upon desolvation of the isomorphous solvates (acetone, dioxane I, DCM and chloroform).TE Form I In TE form I as in all the structures of TE there is an intramolecular N-H•••O=C hydrogen bond in a S(6) motif in both the arms of the molecule that restricts the conformation of the arms.The molecules of TE in form I are connected to each other by three kinds of hydrogen bonding motifs, of which one consists of two N-H•••S hydrogen bonds R2,2(8), another of two N-

Fig. 2
Fig. 2 From top to bottom calculated PXRD patterns of TE form I-III at 173 K and form I at RT, the PXRD pattern of the original TE sample and of form IV from desolvation of the chloroform solvate.

Fig. 3
Fig. 3 Packing coefficients of the single crystal structures of TE and the polymorphs of TM TE Form III Unlike in forms I and II, the Z' of form III is 2. The molecules build hydrogen bonded chains of the two symmetrically 10 Fig 4).From the fingerprint plots one can also 55 say that the N-H•••S hydrogen bonds in form II are shorter, indicating a stronger nature.The stronger N-H•••O hydrogen bonds are, however, somewhat shorter for form I.

Fig. 4
Fig. 4 Hydrogen bonding diagram and fingerprint plots of TM form I, 60

Fig. 5
Fig. 5 (a) Hydrogen bonded chains of TE acetone solvate and (b) the channels of acetone in the structure with non-hydrogen bonding hydrogen atoms removed for clarity though neither has been found to have a pure hydrate.Even though TM and TE are very similar it is difficult to find clearly similarly hydrogen bonded structures between them.The closest analogues are the pyridine solvates, in which the pyridines are N-20 H•••N hydrogen bonded to the thiophanates.

Fig. 6 Fig. 7
Fig. 6 Two hydrogen bonded double pairs of (a) the TE pyridine solvate and (b) the TE dioxane solvate with non-hydrogen bonding hydrogen atoms removed for clarity

20 motifs
and conformations of molecules.The N-H•••O=C bond is the strongest possible hydrogen bond for TM and TE so one would expect it to show up more frequently than the weaker N-H•••S=C hydrogen bond.This, however, is not the case, especially for TE.Moreover, the solvate structures of the two actives vary 25 greatly giving no general strategies for the design of cocrystals.

Table 1
Crystal data and collection parameters of the polymorphs of TE

Table 2
Lattice energies of the polymorphs of TE and TM in kcal/mol

Table 3
Crystal data and collection parameters of the solvates of TE a Low data completeness (83%) and new crystallizations produced TE-dioxane II.b Contains one TE in a general position and half a TE on a twofold axis.

Table 4
Molecular volumes a of some common solvents and whether TM or TE solvates have been found with these.
a Volumes from molinspiration.com.b a MeCN/H2O combination solvate -none found or --not tested.