Packing incentives and a reliable N-H•••N-pyridine synthon in co-crystallization of bipyridines with two agrochemical actives

The co-crystallization of agrochemical actives thiophanate-methyl and thiophanate-ethyl with 2,2’-bipyridine, 4,4’-bipyridine and 1,2-bis(4-pyridyl)ethane was investigated with conventional crystallization, the slurry method and liquid-assisted grinding. Co-crystals of both thiophanates with all bipyridines were found and the structures solved with single crystal X-ray diffraction. Whereas the 2,2’-bipyridine co-crystals seem to form because of a combination of weak interactions, and in the case of the 10 thiophanate-methyl, partly because of close packing incentives, the 4,4’-bipyridine and 1,2-bis(4-pyridyl)ethane co-crystals form mainly because of a favourable N-H•••N-pyridine hydrogen bonding synthon.


Introduction
The phenomenon of co-crystallization 1,2 is important for pharmaceutical [3][4][5] , agrochemical 6,7 and other industrial applications because of the different and sometimes improved properties of co-crystals of active ingredients compared to those of the parent molecules.For pharmaceutical applications the goal of co-crystal forms is usually to increase the solubility and bioavailability of the active ingredient while maintaining the physical stability of the dosage form, but for agrochemical actives, which are often administered as slurry formulations in water, the goal is to lower the solubility of the active to make a more stable formulation and stop the active being washed away too soon once administered.
In a simplified model co-crystallization can be seen to arise for two different reasons, either close packing or favorable hydrogen bonding interactions 1,8 .The strategy to find co-crystals that form because of close packing requires a trial and error approach since it is very hard to predict, if a pair of molecules can pack tightly.For co-crystals caused by favorable hydrogen bonding interactions one can make educated guesses about possible cocrystal formers based on knowledge about hydrogen bonding synthons 9 with, for example, the help of the CCDC database.In practice, however, packing in crystals is influenced by an intricate combination of close packing incentives, hydrogen bonding and other interactions and therefore there is as yet no reliable way to design co-crystals.
Previously, we have been investigating the polymorphism and versatile solvate formation of two agrochemical actives, thiophanate-methyl (TM) 10 and thiophanate-ethyl (TE) 11 (Scheme 1), and decided now to examine co-crystallization with common co-crystal formers, namely 4,4'-bipyridine (44bp) [12][13][14][15][16][17] and 2,2'bipyridine (22bp) 18 (Scheme 1) as pyridine solvates of TM and TE are also known.Due to the success with the first bipyridines, crystallizations with the similar 1,2-bis(4-pyridyl)ethane (44bpe) (Scheme 1) were also attempted.An important goal was to see how much the difference of one carbon at the end of the chains in TE, in comparison to TM, affects the crystallization behavior and 50 packing of the molecules.

Crystallizations
Equimolar (1:1) amounts (< 40 mg in total) of the compounds were weighed and dissolved in acetonitrile, the solutions were transferred to clean crystallization test tubes, the test tubes covered with parafilm with a few holes and the solvent was let evaporate in room temperature.Crystallization of TM with 22bp and TE with 44bp was also attempted in ethanol with and without seeding by the grinded samples, but the crystallizations produced single crystals of the ethanol solvate of TM 10 and form I of TE 11 , respectively.

Grinding
Liquid-assisted grinding was carried out on a Retsch MM 200 ball mill in 10 ml milling vessels for 20 min with an oscillation frequency of 20 s -1 .50 mg of TM/TE and a molar amount of 22bp or 44bp, according to the ratio in the co-crystal structures, were weighed and a few drops of 1:1 water:ethanol solution were added to the milling vessels.All but the TE -44bp mixture produced PXRD patterns similar to the calculated patterns from the structures.For the TE -44bp mixture and the TM -22bp mixture, milling was also done with 3 drops of 25% MeCN in water to confirm the results.

Thermomicroscopy
The behavior of the 22bp and 44bp co-crystals during heating was studied under polarized light with a Mettler FP82HT hot stage connected to a Mettler FP90 central processor with an Olympus BH-2 microscope.The primary heating rate used was 10°C/min from 30°C to melting/decomposition of the sample.

PXRD
For powder X-ray diffraction analyses the samples were pressed to a zero background silicon plate sample holder and measured on a PANalytical X'Pert Pro system in reflection mode with nonmonochromated Cu-radiation.A 2θ-angle range of 5-35° and a step time of 38 s were used with step resolution of 0.0167°.Figures were drawn with X'Pert HighScore Plus 19 .

Single crystal X-ray diffraction
The X-ray diffraction data were collected on a Nonius Kappa 40 CCD diffractometer with an Apex II detector, using graphitemonochromated CuKα radiation (λ = 1.54178Å) at 173 K.The structures were solved by direct methods, refined, and expanded by Fourier techniques with the SHELX-97 software package 20 .
Absorption correction was performed with Denzo 1997 21 .All 45 non-hydrogen atoms were refined anisotropically.Hydrogen atoms were placed in idealized positions, or in the case of hydrogen bonding hydrogens, found from the electron density map and included in structure factor calculations.The N-H hydrogen atoms found in the electron density map were 50 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 22 .Crystal data and collection parameters of the structures are presented in Table 1.

C CP/MAS NMR
The 13 C CP/MAS NMR spectra were measured of the slurried TM samples with a Bruker Avance 400 FT NMR spectrometer 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, 60 time domain 2 K, which was zero filled to 8 K in frequency domain.Exponential window function with 5 Hz line broadening was used.20 000 scans were acquired. 13C shifts are referenced to the CO-signal of glycine (176.03ppm) measured before the TM sample. 65

Results and discussion
The formation of the TM co-crystals with 4,4'-bipyridine and 2,2'-bipyridine was first observed in slurry experiments, with the results confirmed by PXRD and 13 C CP/MAS NMR, after which they were also found to crystallize from solution.Co-crystals for TE were acquired directly with solution crystallization and used also in the liquid-assisted grinding and thermomicroscopy experiments.The 1,2-bis(4-pyridyl)ethane co-crystals were later 5 acquired by solution crystallization for structural comparison.

C CP/MAS NMR results
High resolution solid state NMR and especially 13 C CP/MAS is nowadays often used as an aid or complementary technique in polymorph, solvate and co-crystal characterization. 10,23In this 10 work 13 C CP/MAS spectra were measured for the TM-22bp and TM-44bp co-crystals, acquired by the slurry method, as well as for pure TM alone (Fig. 1).The assignment of the spectra are based on the comparison with liquid state NMR spectra.where those are at 183.3 and 175.9 ppm, respectively.Taking into account the X-ray data (see below) it is quite clear to assign the TM-22bp resonance at 175.9 ppm to that one involved in hydrogen bonding.According to X-ray data C=S groups do not form hydrogen bonds in TM-44bp co-crystal, which explains the 40 small shift difference.In pure TM sample the C=S shift difference is very close to that of TM-22bp suggesting similar hydrogen bonding behavior.
Surprisingly no trace from MeCN is visible in TM-22bp spectrum although the powder X-ray pattern of the sample is 45 similar to the calculad pattern of a structure that contains also MeCN.This, together with the ability of the co-crystal to form also in ethanol solution, suggests that the co-crystal is still intact even without the MeCN.

Co-crystal structures with 2,2'-bipyridine 50
The MeCN solvate of the TM co-crystal with 2,2'-bipyridine crystallized in space group P-1 with a TM:22bp:MeCN ratio of 4:3:2, containing half of this in the asymmetric unit.The TM molecules build up double chains composed of rings of four TM molecules hydrogen bonded to each other (Fig. 2a).The rings are 55 made with two hydrogen bonding arrangements, one of which is a R2,2(8) motif 24

Co-crystal structures with 4,4'-bipyridine
In the TM•4,4'-bipyridine co-crystal the TM to 44bp ratio is 2:1, while in the TE 4,4'-bipyridine co-crystal the TE to 44bp ratio is 1:1.However, there are similarities in the hydrogen bonding patterns of these co-crystals (Fig. 3a and b).The similar hydrogen 20 bonded unit consist of two molecules of TM/TE connected with a R2,2(12) motif of two N-H•••O=C hydrogen bonds, and two molecules of 44bp which are hydrogen bonded to this pair.The difference of the structures lies in the hydrogen bonding of these units into chains.
The TM co-crystal with 4,4'-bipyridine crystallized in space group P-1 with one molecule of TM and half a molecule of 44bp in the asymmetric unit.The R2,2 (12) hydrogen bonded pairs are connected with hydrogen bonds to the 44bp molecules making up infinite parallel chains where a pair of TM molecules and a 44bp 30 molecule alternate (Fig. 3a).Interestingly, in this structure the sulfurs are not involved in hydrogen bonding and one N-H hydrogen is also left without a hydrogen bond acceptor.The R2,2 (12) hydrogen bonded pairs are connected with a R2,2(8) motif of two N-H•••S hydrogen bonds to neighboring pairs forming hydrogen bonded chains of TE molecules in the structure (vertical direction in Fig. 3b).These chains pack parallel to each other with the 44bp molecules of the neighboring chains 55 located partly in between the arms of the TE molecules.Unlike in the TM co-crystal, the other pyridine acceptor in the 44bp does not take part in hydrogen bonding.
In the The TM co-crystal with 1,2-bis(4-pyridyl)ethane there 65 are two unconnected domains in the structure consisting of the symmetry unequivalent TM molecules and half molecules of 44bpe on an inversion center.In one case the pairs are only connected with hydrogen bonds to the bipyridine, similarly to the TM co-crystal with 44bp, making up chains, but in the other case the hydrogen bonded pairs are connected with a D(2) N-H•••S motif to other TE molecules to build up double chains which then connect into sheets via the bipyridine molecules (Fig. 4a).The two domains make up sheets that stack up on each other in the crystal.In the TE co-crystal with 1,2-bis(4-pyridyl)ethane the 10 hydrogen bonding in the between the TE molecules is identical to that in the TE co-crystal with 44bp, but additionally the TE chains are connected via the 44bpe molecules like in the TM cocrystal with 44bp (Fig. 4b).In the chains of TE pairs of the two symmetry unequivalent TE molecules connected with the 15 R2,2 (12) motif alternate.

Packing incentives
The packing coefficients 25 (Fig. 5) for co-crystal structures were calculated using the formula C(k) = Z*V(mol)/V(cell), where Z is the number of molecules in the unit cell, V(mol) is the molecular volume of the molecules 26 , and V(cell) the volume of the unit cell.When compared to the packing coeffficients of the two polymorphs of TM and the three polymorphs of TE 11 , the results indicate that the packing of the co-crystals is in general more loose-fitting than that of the polymorphs with the TM-22bp co-25 crystal being the exception.Form III of TE is left out of consideration as the structure has voids and is thus very loosely packed.

Grinding
Having acquired such a group of similar co-crystals, we decided to use test whether the known co-crystals could be produced with liquid-assisted grinding.Compounds were weighed in the ratios of the known co-crystals and water:ethanol was used as solvent.

65
The TM-22bp co-crystal is known to contain MeCN, but this was disregarded partly to see if a co-crystal that does not contain acetonitrile exists.
The TM-44bp and TE-22bp mixtures made the known cocrystals when grinded with water:ethanol, but the TE-44bp and does not have peaks from pure 44bp or any of the known TE polymorphs.This could indicate another co-crystal that has better packing than the original co-crystal.The crystallization of such a co-crystal was attempted from ethanol without success.The TM-22bp PXRD is similar to the calculated pattern from the MeCN solvate of the co-crystal, indicating that the formation of the cocrystal may not depend on the presence of MeCN, and that there may be a co-crystal in which ethanol takes the place of MeCN.Attempts to crystallize this were also unsuccessful.New grinding experiments of TE-44bp and TM-22bp with 3 drops of 25% MeCN in H 2 O were done to confirm the formation of the original co-crystals.Both the samples gave the pattern of the known cocrystal structure.

Thermomicroscopy
Thermomicroscopy was used to investigate the melting behavior of the co-crystals.As both TM and TE decompose upon 20 melting 10,11 , we were unable to use melt film methods to produce the co-crystals.We wanted to know if the formation of a cocrystal could be determined with the thermomicroscope.The melting ranges for the co-crystals in Table 2 are very broad due to poor repeatability of the melting observations caused by 25 interference from the unco-crystallized pure compounds and the changes in crystallite size when the co-crystals were produced with different methods.The melting points of the TM-22bp co-crystal and both the TE co-crystals, however, do show a clear difference to the pure compounds, all melting at temperatures in between the melting points of the pure compounds, and the formation of these cocrystals can thus be recognized with thermomicroscopic observations.The TM-44bp co-crystal, however, melts only at the temperature where TM starts to decompose and can not be recognized in this manner from pure TM.If single crystals were not obtained to confirm the co-crystal formation, thermomicroscopy, which requires very little material, could still have been used together with powder diffraction to help verify at least three of the co-crystals.

40
The melting points of the found co-crystals are mostly lower than those of the pure actives, which is the opposite of what is generally preferred for agrochemical co-crystals.For TM and TE, making the melting point higher with co-crystals is unlikely because of the thermal decomposition of the actives upon melting.The co-crystals could still be viable forms for processing of formulations because of possible solubility differences and crystallite shape preferences.

Conclusions
With the discovery of these six co-crystals, we have acquired further proof that TM and TE can include a number of guest molecules in their crystal lattices.The reasons for this behavior 55 seem to be the large amount of hydrogen bond donors coupled to weak acceptors, the flexibility of the molecules, as well as the possibility for aromatic interactions.The two arms of the molecule being ortho-substituted in the benzene ring may also make the packing of the molecules somewhat difficult via forcing 60 the sulfur and oxygen atoms quite close to each other unless guest molecules are included between the arms.
The N-H•••N-pyridine hydrogen bond was found to be a reliable synthon for co-crystal design with TM and TE.The inherent weakness of the N-H•••S=C hydrogen bond commonly 65 used in the polymorphs and solvates of TM and TE is likely the cause, as was found for a more simple dithiooxamide by Piotrkowska et al. 27 An important goal was to see how much the size difference of TM and TE affects the crystallization behavior and packing of the 70 molecules.For the hydrogen bonded co-crystals of TM and TE the size difference of the molecules was found to effect the structures in a subtle manner.The main hydrogen bond pairing was always the same, but the size of the molecule influenced the usage of the rest of the donors and acceptors.In the TM co-75 crystals with 4,4'-bipyridine and 1,2-bis(4-pyridyl)ethane one N-H donor and all or most the C=S acceptors are unused, while in the TE co-crystal with 4,4'-bipyridine only a pyridine acceptor is unused and in the TE co-crystal with 1,2-bis(4-pyridyl)ethane all donors and acceptors are in use.This is because the larger 80 molecules have more conformational freedom and can pack well even with more hydrogen bonds in use.For the 2,2'-bipyridine co-crystals the difference is not clear while TE makes a channel structure known to also fit other molecules and TM bouilds a clathrate type structure templated by the 2,2'-bipyridine.

85
The stoichiometric diversity in the co-crystals with the same bipyridines raises the question of whether there could be more cocrystals with different molar ratios like in the work of Trask et al. 28 on caffeine co-crystals.The TE-4,4'-bipyridine sample grinded with EtOH could be an example of this, but further 90 investigation is required.We believe there are also numerous other possibilities for co-crystals as well as solvates of TM and TE.Similar molecules with flexible chains attached ortho on a benzene ring could also be reliable co-crystal formers and we plan on further investigating these.

Fig. 1
Fig. 1 CPMAS NMR of the TM-22bp co-crystal (top), TM form I (middle) and the TM-44bp co-crystal (bottom).In the TM spectrum its C=O resonance is at 153.8 ppm and the composed of two N-H•••S hydrogen bonds and the other a second order R2,2(8) motif with one N-H•••S and one N-H•••O-C hydrogen bond.There is also a very weak C-H•••O-C bond supporting the connection with this motif.The rings of TM 60 arrange into chains with a R2,2(12) motif of two N-H•••O=C hydrogen bonds.One of the 22bp molecules resides right inside the ring of TM molecules while the other 22bp molecule along with the MeCN molecules is located between the TM double chains.There are 65 edge-to-face aromatic interactions between the benzene rings of the TM molecules and the 22bp that are located outside the rings with ring centroid-C distances of 3.71 and 3.94 Å, and subsequent ring centroid-H distances of 2.78 and 3.03 Å.The TE co-crystal with 2,2'-bipyridine crystallized in space 70 group P2 1 /c with a molecule of TE and half a molecule of 22bp in the asymmetric unit, giving the ratio of 2:1.The TE molecules build up hydrogen bonded chains (Fig. 2b) with a R2,2(8) motif consisting of two N-H•••S hydrogen bonds.There are additionally only intramolecular N-H•••O=C hydrogen bonds in π-π stacking interactions to neighboring chains with ring distances of approximately 3.4 Å.The 22bp molecules are located in between the arms of the TE molecules in channels running through the structure in the direction of the crystallographic a-axis (Fig. 2b).There are weak hydrogen bonds between the 22bp nitrogens and the ethyl hydrogens of TE molecules in neighboring chains.The arrangement of TE molecules in the structure of this co-crystal is interestingly the same as that in the previously obtained isomorphous acetone, DCM, chloroform and dioxane solvates of TE 11 with the 22bp 10 taking up the space of two solvent molecules.

Fig. 2
Fig. 2 (a) Rings of TM with 22bp in the MeCN solvate of the TM cocrystal with 22bp and (b) the channels of 22bp in the TE co-crystal with 22bp.MeCN molecules and C-H hydrogen atoms removed for clarity.

Fig. 3
Fig. 3 Hydrogen bonding and packing in the (a) TM and (b) TE-44bp co-35

Fig. 5
Fig. 5 Packing coefficients of the polymorphs of TM and TE and the30

Fig. 6
Fig. 6 PXRD patterns of the EtOH and MeCN grinding experiments of 15

Table 1
Crystal data and collection parameters for the co-crystal structures

Table 2
Approximate melting points (MP) of the compounds and cocrystals as determined by hot stage microscopy