Elsevier

Journal of Molecular Structure

Volume 1178, 15 February 2019, Pages 702-710
Journal of Molecular Structure

Novel anhydrous solid-state form of Azathioprine: The assessing of crystal structure by powder X-Ray diffraction, Infrared Absorption Spectroscopy and Raman scattering

https://doi.org/10.1016/j.molstruc.2018.10.065Get rights and content

Highlights

  • Crystal structure of a new anhydrous solid-state form of Azathioprine, determined directly form powder X-Ray diffraction data.

  • The IR and Raman spectra confirmed the results obtained from X-Ray diffraction.

  • Rietveld refinement and structure determination are explained.

  • The new anhydrous polymorph (Form II) of Azathioprine is accessible only by a solid-state.

  • Dehydration process.

Abstract

Azathioprine (Imuran), one of the oldest immunosuppressants, having been used in transplantation since the early 1960`s, is known to have only two crystal forms: an anhydrous form and a dihydrate phase. We report the crystal structure of a new anhydrous solid-state form of Azathioprine, determined directly form powder X-Ray diffraction data, employing the direct-space genetic algorithm technique for structure solution, followed by Rietveld refinement. The new anhydrous polymorph is accessible only by a solid-state dehydration process of the readily obtained monohydrate form of Azathioprine, the form for which a crystal structure has not previously been reported. The IR and Raman spectra confirmed the results obtained from X-Ray diffraction indicating the presence of all functional groups involved in intermolecular hydrogen bonding which dictates different arrangement of molecules in the structural packing.

Introduction

Polymorphism is the property of a substance to arrange in two or more crystalline structures while retaining the same chemical composition. It is the equivalent of allotropy which is a property of chemical elements, as in the case of carbon - graphite, graphene, fullerene, diamond etc. Lattice energy differences between pairs of polymorphs are generally small. A computational study of 1061 experimentally determined crystal structures of 508 polymorphic organic molecules showed that over 50% of pairs are separated by less than 2 kJ/mol [1].

Even though the lattice energy differences are small, polymorphs may exhibit large differences in physical properties [[2], [3], [4]] such as density [5], hardness [6], crystal shape [[7], [8], [9]], melting point, optical and electrical properties and dissolution rate (which consequently may influence bioavailability) [10]. Due to these differences, the phenomenon of polymorphism has to be taken into account in various industry fields, including in the pharmaceutical industry [9]. As more than 40% of new chemical entities developed by the pharmaceutical industry are practically insoluble in water [11], finding polymorphs which present a better dissolution profile is one route of solving formulation issues.

Active pharmaceutical ingredients, like many other organic and inorganic compounds, may have a tendency to incorporate water or other solvents in their crystal lattice, leading to the formation of molecular adducts called hydrates or solvates [12], in a “host-guest” relationship [[13], [14], [15]]. Water or other solvents influence the interaction between host molecules, affecting the internal energy and enthalpy and consequently the free energy, thermodynamic activity, solubility, dissolution rate, stability, and bioavailability of the chemical entity [16,17] (see Scheme 2).

Azathioprine (Scheme 1) is an immunosuppressive drug mainly used to prevent rejection of transplanted organs [18]. It is also used for treating a variety of autoimmune diseases such as inflammatory bowel disease [19] and a wide array of skin diseases [20].

To date, only two crystal forms of Azathioprine have been described in the literature: an anhydrous form (namely Form I, CIPWUT entry code from Cambridge Structural Database) and a dihydrate (AZTHPN entry code from Cambridge Structural Database). The crystal structure from single-crystal X-Ray diffraction studies of the dihydrate form has been first reported in 1975 [21] while the structure of anhydrous Azathioprine was reported in 1984. Azathioprine comprises two moieties, 6-mercaptopurine and imidazole, connected via the sulphur atom [22].

The focus of the present study is on the application of modern powder X-ray diffraction data processing techniques [23] combined with analysis of solid-state IR and Raman spectroscopy for structure determination of pharmaceutical materials that can only be prepared by desolvation processes [24,25], related on the specific example of Azathioprine. We demonstrate here that azathioprine forms a new anhydrous polymorph (Form II) accessible only by a solid-state dehydration process [23] of the monohydrate form (Hy) obtained by reverse anti-solvent addition in water. Determination of the crystal structure directly from powder XRPD data is an essential tool for quantitatively understanding structural properties of pharmaceutical materials prepared by processes that intrinsically generate polycrystalline product phases, including (as is the case in the present work) those accessed by solid-state dehydration procedures [[23], [24], [25], [26]].

Following the crystal structure determination of novel anhydrous Form II of azathioprine from powder XRPD data by means of “direct-space” strategy and Rietveld refinement, complementary experimental techniques such as IR spectroscopy [27] and Raman scattering were also used to identify the functional groups involved in distinct hydrogen-bonded arrays which differ substantially related to conformational arrangements of the structures. The key feature of this combined strategy is that the quality of the crystal structure determined from the powder X-ray data is assessed both against the observed XRPD pattern and against the data obtained by infrared absorption spectroscopy and Raman scattering, representing a stringent and robust assessment of the quality and validity of the crystal structure.

Section snippets

Materials

Azathioprine and all solvents (reagent grade) were purchased from several commercial suppliers and used as received.

Characterization of the anhydrous and monohydrate polymorphs of azathioprine

TGA and DSC data recorded for Form I, form 30 °C–350 °C are shown in Fig. 1. The DSC curve shows only one endothermic peak centered at 260.5 °C, related to the melting of the sample (melting ΔH = 46 J g-1). No mass loss is recorded prior to the melting event (associated with sample decomposition), suggesting the anhydrous character and a notable stability of this form even at high temperature.

The TGA and DSC traces of the monohydrate form crystallized from 2,2,2-trifluoroethanol in water at

Conclusions

Using the Rietveld refinement, the crystal structure of a new anhydrous solid-state form of Azathioprine, determined directly from powder X-ray diffraction data, employing the direct-space genetic algorithm technique for structure solution was reported. The new anhydrous polymorph is accessible only by a solid-state dehydration process of the readily obtained monohydrate form of Azathioprine. Structural characterization of the new anhydrous and monohydrate polymorphs was provided on the basis

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgment

This work was funded in the frame of project co-funded by the European Regional Development Fund under the Competitiveness Operational Program 2014–2020 entitled “Physico-chemical analysis, nanostructured materials and devices for applications in the pharmaceutical field and medical in Romania”, the financing contract no. 58/05.09.2016 signed of National Institute of Materials Physics with National Authority for Scientific Research and Innovation as an Intermediate Body, on behalf of the

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