Apr. 30, 2024
Chemicals
The same study was carried out for the polymorphic form 6MU_II . Being more stable than the polymorph 6MU_I , these crystals sublimated at 230 °C. As a result, coarse crystals of 6MU_IV were formed without any impurity of the polymorph 6MU_III . In contrast to the sublimation of the polymorphic form 6MU_I , heating of 6MU_II was not accompanied by the appearance of a white haze.
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The additional study of the 6MU_I heating process was performed by the powder diffraction method. A sample of polymorph 6MU_I was heated up to various temperatures and analyzed using the Rietveld method ( Table 2 ). The data obtained have confirmed the formation of polymorph 6MU_III at 200 °C and polymorph 6MU_IV at 245 °C and above. In addition, the presence of the polymorphic form 6MU_II as an impurity within the temperature range of 210–245 °C may explain some of the problems with an ointment containing 6MU due to the appearance of grains.
To separate new polymorphic forms 6MU_III and 6MU_IV as a pure crystal phase, DSC thermograms of the polymorphs 6MU_I and 6MU_II ( ) have been studied thoroughly. Polymorphic crystals 6MU_I have been heated in a double Petri dish up to a temperature below the endothermic effect found in the DSC thermogram ( , at the top). It was found that partial sublimation of the sample (white haze can be seen) occurs at ∼200 °C, and the product was crystallized on the cover dish as very thin needles of polymorph 6MU_III . Heating up to a temperature of 250 °C led to re-crystallizing of the powder mass in the bottom dish giving elongated prismatic crystals of the polymorph 6MU_IV . It should be noted that crystals of 6MU_IV obtained at this temperature contained crystals of 6MU_III as an impurity and were purified due to further heating up to ∼270 °C.
To exclude the solvation effects, an attempt was made to crystallize 6MU from the melt. A sample of polymorph 6MU_I was dissolved in water, boiled, and dried. Then the obtained powder was heated up to the sintering temperature (heating up to the melting point was not performed to avoid compound decomposition). The analysis of the obtained sample by powder diffraction has revealed that the main crystal phase is the polymorph 6MU_II but an impurity of a new phase can be found. Indeed, a more thorough analysis has revealed needle-like crystals of a new polymorph 6MU_III and prism-like crystals of a new polymorph 6MU_IV in the pre-melting sample. Both new polymorphs of 6MU were identified primarily by the single crystal X-ray analysis ( Table 1 ). It should be noted that unit cell parameters of form 6MU_III are very close to the ones determined for polymorph 6MU_I . Therefore, additional arguments are needed to prove the existence of form 6MU_III as a new polymorph.
Our attempts to crystallize 6MU from various solvents have always led to the formation of needle-like crystals of the polymorphic form II . Even crystallization from water used in the technological process did not give the polymorph 6MU_I in the laboratory. The main difference between crystallization in the technological process and in the laboratory is not the solvent used, but the volume of the crystallizing mixture and the stirring of the solution during the crystallization process. Thus, it can be presumed that the gradient of temperature decrease and the inertness of the reaction mixture can play a key role in the formation of the polymorph 6MU_I during the technological process.
Being a very effective drug, 6MU is manufactured by the pharmaceutical industry in the tablet form or as an ointment. Therefore, the control of its polymorphic form is of great importance for the quality of the final product. The analysis of the pharmaceutical substance (API) has revealed that 6MU exists rather in polymorphic form I ( 6MU_I ) that has been determined only by powder diffraction data which have low enough quality. 20 So these data proved to be not very good for a reliable analysis of API and need to be improved.
The pure polymorphic forms of 6MU have been characterized by infrared spectroscopy ( Figures S2–S4 and Table S5 ) in the wavenumber range of 500–4000 cm –1 at room temperature. All of these spectra were measured with a resolution of 1 cm –1 . The analysis of infrared (IR) spectra has revealed the characteristic vibrations of the N–H bond in the regions of 3090–3040 and 2990–2938 cm –1 , vibrations of the CH 3 group in the region of 2850–2800 cm –1 . The carbonyl groups give vibrational lines in the regions of 1747–1704 and 1651–1644 cm –1 while the C=C bond is characterized by vibrations in the region of 1615–1614 cm –1 . Any vibrations corresponding to enol tautomers of 6MU were not detected. The most significant changes are observed in the vibrational frequencies of the N–H bond (the region of 2850–2800 cm –1 ) and carbonyl groups ( Table S5 ), indicating different participation of the N1H and both carbonyl groups in hydrogen bonding. The vibrations of the methyl group and endocyclic double bond are very close in all polymorphic forms under study.
The pure polymorphic forms of 6MU have been characterized by the DSC method ( ). The 6MU_I and 6MU_III samples demonstrate the same melting process. The blue curve corresponded to polymorph 6MU_II and has a wider melting band. The curve measured for the polymorphic form 6MU_IV is very similar to the ones found for the samples of 6MU_I and 6MU_III . The similarity in DSC of 6MU_I and 6MU_III forms may be due to the similarity in the mutual arrangement of molecules and close energies of intermolecular interactions in their crystal structures.
All of the separated individual polymorphic forms of 6MU were characterized by the powder diffraction method ( ). Rietveld refinement proved a nice agreement between the experimental diffractograms and the theoretical ones obtained from the single crystal study ( Figure S1 ). The powder X-ray diffraction patterns of the polymorphs 6MU_I and 6MU_III with close unit cell parameters ( Table 1 ) proved to be very similar ( ). But the thorough analysis of these patterns has revealed some differences in the 2Θ region from 20 to 30° ( , and Tables S1 and S3 ). This fact allows to consider structure 6MU_III as a new polymorphic form.
The contribution of two zwitterionic structures to molecular geometry should slightly lengthen the C5–C6 bond and shorten the N3–C4, C4–C5, and N1–C6 bonds in 6MU molecules found in structures 6MU_I , 6MU_III , and 6MU_IV . The tendency for such a redistribution of electron density has been found in structures 6MU_I and 6MU_III and is absent in structure 6MU_IV ( Table 3 ). Therefore, the difference in bond lengths is caused mainly by the difference in the participation of carbonyl groups in intermolecular interactions. The higher ability of the C4–O2 carbonyl bond to be elongated can also be due to the stronger conjugation between the endocyclic C=C and exocyclic C=O double bonds compared to the conjugation between the electron lone pair of the nitrogen atom and the C=O bond. 28
The bond length analysis has revealed the diketo form of 6MU. T he C2–O1 and C4–O2 bonds have the same lengths ( Table 3 ) in the polymorphic crystals 6MU_II . This polymorph has been obtained from various solvents as a result of slow evaporation. The C=O bonds are not equivalent in 6MU molecules found in all other structures ( Table 3 ). Such a difference can be explained by two main reasons:
The positions of hydrogen atoms are usually determined not very reliably in an X-ray diffraction study. Consequently, the tautomeric form of a molecule in a crystal phase can be discussed mainly based on the analysis of bond lengths. Such an analysis was performed for 6MU molecules in the polymorphic crystals 6MU_I – 6MU_III . An asymmetric part of the unit cell contains two molecules ( A and B ) in the 6MU_IV structure, where molecule B is disordered over two positions with equal populations due to rotation around the virtual axis passing through the N3B···C6B atoms ( ). To refine this disorder, the bond lengths in this molecule were restricted (see the Experimental Section ). As a result, only bond lengths of molecule A may be analyzed in the 6MU_IV polymorphic crystals.
The 6MU molecule can theoretically exist in six tautomeric forms ( Scheme 1 ). According to the quantum chemical calculations using the MP2 electron correlated method, the diketo tautomeric form A proved to be the most stable. 22 Calculations by the m06-2x/cc-PVTZ method 26 , 27 coincide in general with the earlier data ( Scheme 1 ) indicating that tautomer A is the most energetically preferable and tautomer F is the least expected. However, all possible tautomeric forms are expected to be in equilibrium in a liquid or gas phase, and transitions between them can occur due to the transfer of a hydrogen atom. Which of these tautomeric forms can be found in a crystal phase depends on the crystallization conditions. It may be presumed that the kinetically controlled crystallization (rapid cooling of a supersaturated solution, rapid evaporation, sublimation, etc.) can lead to the formation of metastable polymorphic crystals containing a less energetically preferable tautomeric form or a mixture of several tautomers. Thermodynamically controlled crystallization should result in the formation of stable polymorphic crystals containing molecules in the tautomeric form with the lowest energy.
The analysis of intermolecular interactions in the polymorphic crystals under study has revealed different patterns formed by N–H···O hydrogen bonds in structures 6MU_I, 6MU_III, 6MU_IV, and 6MU_II ( ). According to Etter’s rules,29,30 all strong proton donors and acceptors must participate in the formation of hydrogen bonds. The 6MU molecule contains two NH groups and two carbonyl groups, which can form two strong centrosymmetric N–H···O hydrogen bonds. However, this rule is realized only in structure 6MU_II while the N3H donor and the C=O2 acceptor form a centrosymmetric dimer of the same type in all other crystals studied ( , Table 4).
Another NH group is also involved in intermolecular hydrogen bonding but with different acceptors in different polymorphic structures. The centrosymmetric dimer formed by the N1H and C=O1 groups is found only in the 6MU_II structure. The centrosymmetric dimers of two types (formed by the N3–H···O2 or N1–H···O1 hydrogen bonds) form a ribbon as the main structural motif ( ). The neighboring ribbons are bound by the weaker C–H···π hydrogen bonds (Table 4).
In structures 6MU_I, 6MU_III, and 6MU_IV, the N1H group forms the linear intermolecular hydrogen bond with the C=O2 carbonyl group acting as a bifurcated proton acceptor. In addition, four centrosymmetric dimers bound by the N1–H···O2 hydrogen bonds form a cyclic fragment that can be recognized as a repeating part of the corrugated layer parallel to the (100) crystallographic plane in the structures 6MU_I and 6MU_III ( ). The neighboring layers are bound by the weaker C5–H···O1 intermolecular hydrogen bonds.
In the 6MU_IV polymorphic form, the centrosymmetric dimers formed due to the N3A–H···O2B and N3B–H···O2A hydrogen bonds between molecules A and B are bound by the linear N1A–H···O2B hydrogen bonds. So, a zigzag chain in the [010] crystallographic direction can be recognized as a structural motif in the polymorph 6MU_IV ( ). The disorder of molecule B in structure 6MU_IV means the formation of hydrogen bonds of two types in one direction (N1C–H···O1A/C5B–H···O1A or N1B–H···O2A/C5C–H···O2A hydrogen bonds) linking the neighboring chains.
The energies of the hydrogen bonds revealed in four polymorphic crystals were estimated within Bader’s “Atoms in Molecules” (AIM) theory.31 The wave function for each of the hydrogen-bonded dimers was recorded by the m06-2x functional26 and the standard cc-pVTZ basis set27 (m06-2x/cc-pVTZ) and was analyzed using the AIM2000 program32 with all default options. The energy of a hydrogen bond can be calculated using the correlation between the hydrogen bond energy and the pressure exerted on the electrons around the (3, −1) bond critical point revealed by Espinosa and co-workers.33 The N3–H···O2′ intermolecular hydrogen bond proved to have the highest energy in all of the studied structures as compared to the N1–H···O1/O2 hydrogen bonds. This ratio persists even in the 6MU_II polymorph (Table 4), where the N1–H···O1′ hydrogen bonds form a centrosymmetric dimer. The higher energy of the N3–H···O2 hydrogen bond is assumed to be a result of the higher ability of the C4=O2 bond to be delocalized. It should be noted that the smaller energy of the N3–H···O2′ hydrogen bond in 6MU_I compared to 6MU_II can be explained by the O2 participation in two hydrogen bonds simultaneously. The bifurcated character of N1–H···O2′ and N3–H···O2′ hydrogen bonds in 6MU_I weakens both interactions.
The 6MU molecule contains the conjugated system that creates pre-conditions for the formation of stacking interactions. The analysis of short contacts in the polymorphic structures 6MU_I – 6MU_IV has revealed the distances and overlapping degree between neighboring 6MU molecules, which are characteristics of stacking. However, intermolecular interaction of this type is very complicated to be characterized.34,35
The analysis of pairwise interaction energies between neighboring molecules takes into account the contribution not only of hydrogen bonds but also stacking and non-specific interactions. Being more analytical and objective, such an analysis allows understanding the role of intermolecular interactions of different types in the crystal packing formation.36 The first coordination sphere of the 6MU molecule contains a different number of neighboring molecules in four polymorphic structures (Table 5). Furthermore, the highest interaction energy between a basic molecule and its first coordination sphere is found in polymorphic structure 6MU_II, where the number of neighbors is the smallest. This may be explained by the presence of stronger hydrogen bonds between molecules forming a centrosymmetric dimer in the polymorphic form 6MU_II (Table 5).
Based on our earlier data,37,38 such interactions of a basic molecule with its environment allow presuming that the polymorphic form 6MU_II is the most stable in comparison with other studied forms. The calculations of the lattice energies in periodic approximation for all of the polymorphic forms under study have revealed that polymorph 6MU_II has the lowest lattice energy. The polymorphic structures 6MU_I, 6MU_III, and 6MU_IV have lattice energies which are greater than 1.55, 1.53, and 1.67 kcal/mol, respectively. These data allow considering form 6MU_II as stable while the forms 6MU_I, 6MU_III, and 6MU_IV have to be recognized as metastable.
The basic molecule of 6MU forms only one centrosymmetric dimer with the strongest interaction energy, but this dimer differs in the polymorphic structures 6MU_I, 6MU_III, 6MU_IV, and 6MU_II (Tables 5, and S6–S9). It is bound by the N3–H···O2 hydrogen bond in the structures 6MU_I, 6MU_III, and 6MU_IV or by the N1–H···O1 hydrogen bond in structure 6MU_II. The question of which centrosymmetric dimer is the strongest in the structure 6MU_II turned out to be somewhat controversial (Table 6).
According to the geometric characteristics, the N3–H···O2′ hydrogen bond is stronger than the N1–H···O1′ one. This is confirmed by the estimation of the hydrogen bond energy using the characteristics of the (3, −1) bond critical point within AIM analysis (Table 6). However, the interaction energy between two molecules in a centrosymmetric dimer bound by the N3–H···O2 bonds turned out to be smaller as compared to a similar dimer bound by the weaker N1–H···O1 bonds (Table S7). To resolve this contradiction, the interaction energies for two dimers bound by these hydrogen bonds were calculated using the m06-2x functional26 with the TZVp basis set,39 and their components were analyzed using the LMOEDA method40 implemented in the GAMESS-US software package.41 This analysis has shown that a stronger hydrogen bonding leads to an increase in energy of the electrostatic, exchange, polarization, and dispersion components of the total interaction energy (Table 6). In addition to the energy of the repulsive component increases to a greater extent due to an approach of molecules bound by the stronger N3–H···O2 hydrogen bonds. This results in a decrease in the total interaction energy between two molecules. This fact provides arguments in a long-standing controversy about what is the nature of the energy estimated from the characteristics of the (3, −1) bond critical point between two molecules bound by a hydrogen bond.42,43 Is this the energy of interaction between contacting atoms or between molecules? Comparison of the energies of the N3–H···O2 and N1–H···O1 hydrogen bonds estimated from the (3, −1) bond critical points and the energies of interaction between molecules in the dimers bound by these hydrogen bonds (Table 6) suggests that the AIM theory provides the interaction energy between two contacting atoms rather than between two molecules.
The centrosymmetric dimer with the strongest interaction energy should be considered as a complex dimeric building unit (DBU0) in all of the polymorphic 6MU structures. The first coordination sphere of the DBU0 contains the least amount of neighboring DBUi in the 6MU_II structure (Table 7). In addition, the total interaction energy with all surrounding is also the smallest in the 6MU_II structure.
Structures 6MU_I and 6MU_III are very close ( ). A basic DBU0 forms four equal interactions (N1–H···O2 hydrogen bonds) in each of them (Table 8). As a result, the layer parallel to the (100) crystallographic plane may be recognized as BSM1. The interaction energy of the DBU0 with all neighboring DBUi within the layer is −56.36 kcal in the 6MU_I structure and −51.24 kcal/mol in the 6MU_III structure. Neighboring layers are bound mainly by stacking interactions and C–H···O hydrogen bonds, and interaction energy between molecules belonging to neighboring layers is less than two times lower than the interaction energy within the layer (Table 7).
In the 6MU_II structure, a DBU0 also forms four dimers with neighboring DBUi with very close energies (Table 8). But these dimers are not equivalent: two of them are bound by the N–H···O hydrogen bonds, and two more are bound by stacking interactions. Thus, the layer parallel to the (001) crystallographic plane and formed by interactions of two types, hydrogen bonding and stacking, can be recognized as BSM1. The interaction energy of DBU0 within the layer is −54.7 kcal/mol, while the interaction energy between neighboring layers is only −14.67 kcal/mol. It should be concluded that the 6MU_II structure is also layered, similar to the 6MU_I and 6MU_III structures, but this polymorph has a much more anisotropic structure in terms of the interaction energies between molecules ( and Table 7).
In the 6MU_IV structure, the strongest interaction between dimeric BUs is the stacking, which proved to be slightly stronger than the N–H···O hydrogen bonds (Table 8). The formation of three interactions with comparable energies results in the packing that is much more isotropic as compared to the polymorphic forms 6MU_I – 6MU_III. A very corrugated layer parallel to the (100) crystallographic plane may be recognized as a BSM1 in the 6MU_IV structure ( ), but the interaction energies of DBU0 with its neighbors within this layer and with DBUi belonging to neighboring layers are very close (Table 7).
Comparison of the structures and crystallization conditions of all 6MU polymorphs showed some regularities observed earlier for crystals obtained as a result of a kinetically or thermodynamically controlled crystallization process.38,44 Crystallization under nonequilibrium conditions (stirring a solution or sublimation from a solid) results in crystals with lower density and more isotropic energies of interactions between molecules (structures 6MU_I, 6MU_III, and 6MU_IV). Slow crystallization from a solution under ambient conditions leads to a crystal structure with a higher density and more anisotropic energies of interactions (the 6MU_II structure).
It should be noted that the metastable polymorph 6MU_I is used in the pharmaceutical industry, and two new metastable forms, 6MU_III and 6MU_IV, can be formed during the technological process due to temperature violations. Therefore, the question of their behavior under mechanical stress or pressure is of great practical importance. To estimate the possibility of a polymorphic transition under external influence in these metastable polymorphic forms, the method proposed earlier45−47 was applied. The results of the study of pairwise interaction energies in metastable polymorphic forms can be used to determine a possible way of the crystal structure deformation. All of these structures are layered where interaction energies between DBUs within the layer are high and interactions between layers are weak. Therefore, it may be assumed that one layer can be shifted in relation to the neighboring one as a result of mechanical stress or pressure exerted on a crystal. The displacement of the neighboring layers can be modeled using the model system where the fragment of a layer plays the role of a fixed part and one DBU belonging to the neighboring layer acts as a mobile part. The molecule of 6MU is conformationally rigid, which allows us to use the rigid body model. The displacement of the mobile part in relation to the fixed part with some step models a possible structure deformation. To evaluate the probability of this process, it is sufficient at the first stage to measure at each point the closest distances between atoms belonging to the fixed and mobile parts of the model system. These distances should be compared with the sums of the van der Waals radii of the corresponding atom in order to take into account the nature of the closest atoms. The parameter δ calculated as the difference between the shortest distance and the corresponding van der Waals radius sum during the displacement of the mobile part relative to the fixed part can be used to construct two-dimensional (2D) maps ( ).
Such 2D maps constructed for the displacements along the (100) crystallographic plane in the metastable structures 6MU_I, 6MU_III, and 6MU_IV ( ) clearly show that any deformation of the studied crystal structures leads to a significant shortening of the distances between the closest atoms belonging to molecules of the mobile and fixed parts of the model system that causes the appearance of strong repulsion. As a result, any polymorphic transition without the loss of crystallinity looks impossible. Thus, it can be concluded that the metastable polymorphic forms of 6MU have to be resistant to any external influences during the technological process.
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