Crystal Structures and Physicochemical Properties of Solvated Estradiol①

WANG Hui-Ping XU Juan NING Li-Feng LI Peng Chen Xiao-Feng

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Crystal Structures and Physicochemical Properties of Solvated Estradiol①

WANG Hui-Ping XU Juan NING Li-Feng LI Peng Chen Xiao-Feng②

(100081)

Polymorphscreening is currently one of the most important tasks for innovators and for generic companies from both pharmaceutical and intellectual property rights aspects. The hemihydrate form (Form I) and formamide solvate (Form II) of estradiol are isolated and prepared via systemic crystallization screening in this paper, and the formamide solvate form is reported for the first time. Both polymorphic forms were characterized by single-crystal X-ray structure analysis (SXRD), powder X-ray diffraction (PXRD), and thermal analysis (TGA and DSC). The PXRD experiments indicate that the samples in this study are the pure polymorphic forms via comparing the patterns with the simulated ones. The stability and equilibrium solubility data of the solid-state phase were also examined in order to check the impact of the differences observed in their crystalline structures. It has been found that Forms I and II are of conformational polymorph and Form II is the more thermodynamically stable solid form, while Form I possesses higher solubility, indicating its possibility as an alternate solid form for its further solid formulation development if necessary.

estradiol, crystalline structure, solubility, stability;

1 INTRODUCTION

Steroids are naturally found in animals, microor- ganisms and plants and possess a construction of three cyclohexane carbon rings in companion with one pentagonal carbon ring (arranged in a 6-6-6-5 structure), which is attached to various functional groups and side chains. All steroidal compounds are derived from cholesterol. Estrone (E1), 17b-estradiol (E2) and estriol (E3) (main natural estrogens) are C18 steroids that have different oxidation states of their rings. These compounds induce female second- dary sexual characteristics and reproductive structures, which was widespread used in hormonal replacement therapy for the treatment of postme- nopausal symptoms and the protection against long-term consequences of estrogen deficiency[1-3]. E2 (Estra-1,3,5(10)-triene-3,17-diol, Fig. 1) appears as white crystals or pale yellow crystalline powder, which is a member of the estrogen family of hormones. In recent years, interest in these mole- cules has focused primarily on understanding their biological role in initiating breast cancer. It is well known that their ability to form hydrogen bonds in the active site of estrogen receptor (ER) influences biological activity. This ability to form hydrogen bonds has been clearly demonstrated in an array of crystal structures, especially in that of E2, containing different solvent molecules or other hydrogen-bond acceptors[4-9].

Fig. 1. Schematic representation of the estradiol molecule

Estradiol has a poor water solubility, and indicates lower oral bioavailability[10].And the dissolution rate is slow, leading to low bioavailability (about 10% of the body). Due to its excellent pharma- cological activity and good clinical application value, estradiol ought to be further developed. The bioavailability of most insoluble drugs is associated with polymorphs. Most compounds can exist one or more crystalline forms[11-15]. The different crystalline forms of the same compound are called polymorphs or modifications[16-19]. Different polymorphs of a drug can show different physicochemical properties, including stability, dissolution rate, bioavailability and solubility, which can affect pharmacokinetics and pharmacodynamics[20-25]. Different crystals of drugs show different chemical stability, such as amisulpride[26], carbamezepine[27]and enalapril maleate[28]. A lot of researches on polymorph-de- pendent bioavailability or absorption rate had been reported, for example chiortetracycline[29], chloram- phenicol palmitate[30]and cimetidine[31]. Even a polymorph may be ineffective, as occurring with polymorph II of ritonavir[32]. Therefore, it is important to pay attention to the solid-state forms of estradiol to improve the physicochemical properties.

Many studies have been made on the polymer- phism of estradiol in the anhydrous solvents,such as ethyl acetate, chloroform, absolute ethanol[33-35]. However, no emphasis has been put on the pharma- ceutical relevant parameters (physicochemical pro- perties) of these reported polymorphs. This insti- gated us to investigate the polymorphic potential of estradiol and to further investigate its biopharma- ceutical profile.

The most common methods for the charac- terization of various polymorphs are powder X-ray diffractomerty (XRPD), single-crystal X-ray diffrac- ttometry (SC-XRD), differential scanning calorime- try (DSC), optical and electron microscopy, infrared (IR), Raman, and more recently solid-state nuclear magnetic resonance spectroscopy (ssNMR)[36-42]. Following our ongoing researches to determine the crystal structures of estradiol, we report here two crystalline structures of estradiol. The first estradiol crystal structure, which was determined by single-crystal X-ray diffraction, shows the estradiol molecule in an orthorhombic form. Because the crystal is a hemihydrate, this structure is termed Form I. Form I was characterized by various analytical technics, such as thermal analysis (TG/DSC), infrared spectroscopy, and powder X-ray diffraction. A second crystalline (Form II), the Estradiol formamide solvate (1:1), was firstly discovered through comprehensive polymorph screening, and then the crystal structures and physic- cochemical properties of Forms I and II were comparatively studied.

2 EXPERIMENTAL

2. 1 Reagents and instruments

Single-crystal X-ray diffraction data were collected by Rigaku AFC-10/Saturn 724+CCD dif- fractometer equipped with a graphite-monochro- matized Moradiation (0.71073 ?) up to a 2H limit of 50.0° at room temperature (25 ℃). Indexing and scaling of the data were performed using DENZO and SCALEPACK. The structure was solved by direct methods and expanded by difference Fourier techniques with Shelxs-97 and refined on2by successive full-matrix least-squares techniques for the non-hydrogen atoms.

XRPD spectra were recorded with a BRUKER D8 Advance diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) system with Curadiation (= 1.5406 ?) over the interval 5～90°/2. The measurement conditions were as follows: target, Cu; filter, Ni; voltage, 40 kV; current, 40 mA; time constant, 0.1 s; angular step 0.016°. Detecor: NaI (l) scintillation detector.

A Spectrum RX-FTIR spectrometer (Perkin Elmer, UK) was employed in the KBr diffuse-reflectance mode (sample concentration 2 mg in 20 mg of KBr) for collecting the IR spectra of samples. Dry KBr (50 mg) was finely ground in mortar and sample (1～2 mg) was subsequently added and gently mixed in order to avoid trituration of the crystals. A manual press was used to form the pellet. The spectra were measured over the range of 4000～450 cm-1. Data were analyzed using Spectrum software.

Thermo gravimetric analysis (TGA) was conduc- ted on a Netzsch TG 209F3 equipment (Netzsch, Selb, Germany) under a flow of nitrogen (20 mL×min-1) at a scan rate of 10 ℃×min-1from 25～300 ℃. Differential scanning calorimetry (DSC) was performed with a PerkinElmer DSC 8500 instrument (Perkin Elmer Co., Schelton, USA) at a heating rate 10 ℃×min-1from 25～300 ℃. For TGA and DSC, typical samples of weighing 2～20 mg were used.

A Jeol JSM-6100 scanning electron microscope (SEM) was used to obtain photomicrographs. Samples were mounted on a metal stub with an adhesive tape and coated under vacuum with gold.

For the study on stability, the powder samples of Forms I and II were exposed to three sets of stress conditions with 60 ℃ in a temperature controlled oven, 90 ± 5% RH at 25 ℃in a sealed humidity container and light exposure of 5000 lx at 25 ℃ in a light chamber for 10 days, respectively. The stress samples were characterized by PXRD for the form confirmation and further by the HPLC method purity assay.

2. 2 Materials and measurements

Estradiol raw material was purchased from ZIZU Pharmaceuticals Co., Ltd. (Beijing, China, batch number: 20170517). The chemical purity of this lot is higher than 99.0% mass fractions, whichis determined by high-performance liquid chromato- graphy (HPLC). All of the solvents used for recrystallization were of analytical reagent grade.

2. 3 Preparation of two crystal forms of estradiol

Different techniques were used for the preparation of polymorphs. Hemihydrate was obtained by dis- solving estradiol completely in the mixing solution (THF:water = 2:1, volume), then recrystallized at the constant temperature of 10 ℃ for about 1 day. The formamide solvate form was prepared by dissolving estradiolcompletely in formamide, then recrystalli- zed at the constant temperature of 10 ℃ for about 2 days. For convenience of description, hemihydrate form and formamide solvate of estradiol are represented by Forms I and II, respectively.

2. 4 Methods

We studied the dissolution curves of two crystal forms of estradiol by pharmacopoeia method. Solid samples were sieved using a Gilson mesh sieve (No. 80) to obtain uniform particle size.The two forms were added in the following media: pH = 1 buffer (0.01 mol·L-1), pH = 4.5 acetate buffer (0.050 mol·L-1), pH = 5.8 potassium phosphate buffer (0.054 mol·L-1), pH = 6.8 potassium phosphate buffer (0.072 mol·L-1),0.2% sodium dodecyl sulfate (SDS) buffer and 0.5% sodium dodecyl sulfate buffer.

The dissolution of experiment was performed on the dissolution apparatus (FADT-800RC) with the rotation speed set at 100 rpm, and the samples were taken at 10, 20, 30, 45, 60, 90, 120, 180, 240 and 300 min, respectively. Then filtered through 0.45 μm membrane filters and the solution concentrations were measured on HPLC (Agilent-1100) with UV/Vis detector set at 275 nm. A Discovery C18 HPLC column (Octadecyl silane, 4.6mm × 250mm, 5m) was used with column oven kept at 40 ℃. The mobile phase consisted of acetonitrile-phos- phate buffer (30 mM, pH 6.4) (20:80,/). The flow rate of mobile phase was 1 mL/min, and the injection volume was 10L. For data acquisition and processing, the LC solution software was used.

3 RESULTS AND DISCUSSION

3. 1 Structure determination by single-crystal X-ray diffraction (SXRD)

In order to obtain more details of the polymorphic structure information at the atomic level, high quality singlecrystals of polymorphic forms I and II were submitted to single-crystal X-ray diffraction analysis.

Fig. 2. Molecular view of the compound, showing 50%probability displacement ellipsoids and atom labeling scheme

The molecular structure of the title compound with atom labeling scheme drawn at 50% probability displacement ellipsoid is depicted in Fig. 2, and the corresponding packing diagram is shown in Fig. 3. For a better comparison, a summary of the conditions for data collection and structure refinement parameters is given as follows. Form Icrys- tallizes in the orthorhombic system, space group21212 with= 2,= 12.061(2),= 19.260(4),= 6.5490(13) ?,= 1521.3(5) ?3,D= 1.229 g·cm-3, formula C36H5O5,(000) = 612,= 0.080 mm-1, the final= 0.0659 and= 0.1439 with> 2(). Another polymorphic Form II belongs to the orthorhombic system, space group212121with= 8,= 8.1564(16),= 16.937(3),= 24.686(5) ?,= 3410.3(12) ?3,D= 1.236 g·cm3, formula C19H27NO3,(000) = 1376,= 0.083 mm-1, the final= 0.0651 and= 0.1298 with> 2(). We can see that the two polymorphs are of the same crystal system, but with different unit cell parameters. Both Forms I and II adopt a head-to-tail packing configuration and form a one-dimensional infinity chain along theoraxis with the intermolecular hydrogen bonding interaction. The two independents have the same configuration but different conformations. The conformational dif- ferences from the rings and the rotating of the single bond of the side-chain substituents are the main causes of the bimolecular phenol- mena. Although ring A is planar, rings B, C and D adopt envelope, chair and envelope conformations in the two polymorphs, respectively, the twist degrees of the rings are rather not the same. The dihedral angles between C(4)–C(5)–C(7) and C(8)–C(9)– C(10) of Forms I and II are 129.24° and 146.52° respectively. A significant difference was observed in the O(2)–C(17)–C(16) bond angles (Table 1). The key parameters that describe doubtlessly the different confor- mations are the torsional angles of the mole- cular backbone. The most relevant torsion angles responsible for this polymorphic conformation of E2 are compared in Table 2. The differences of these angles confirmed the existence of two conformational polymorphs. The number of molecules in the asymmetric unit of the two polymorphs is different, too (Fig. 3).

Fig. 3. Packing diagram of E2 molecules in the unit cell of Forms I (left) and II (right)

Table 1. Selected Bond Angles (°) of Form II in Comparison to That of Form I

Molecular packing diagrams with main hydrogen bonds viewed down the-axis are shown in Fig. 4. Detailed parameters of main hydrogen-bonding interactions with symmetry codes are listed in Table 3. The inclusion of solvents plays a vital role in forming different crystalline forms. The adduction of a particular solvent, which acts as intermolecular hydrogen-bonding acceptors and/or donors, were manifested to explicitly or implicitly affect the intermolecular arrangements and therefore stabilize crystal lattices by fostering stronger interact ions. In form I, the red spheres representing the water molecules (Fig. 4) coincide with the tetramer linkage points[43]. In Form II, an asymmetric unit consists of two E2 and two formamide molecules. Hydrogen- bonding interactions in Form II gives rise to two-dimensional networks by fostering two ind- ependent chains of O–H···O and N–H···O hydrogen bonds, and there are some non-classical hydrogen bonds in Form II, such as C(6)–H(6)···O(6) (–1/2+, 3/2–, 1–), C(37)–H(37)···O(3)(1–, 1/2+, 3/2–) and C(38)–H(38)···O(3)(1–, 1/2+, 3/2–), and such interactions are likely to strengthen the stability of the crystal.

Table 2. Relevant Torsion Angles (°) of Form II in Comparison with Corresponding Torsion Angles (°) of Form I

Fig. 4. Hydrogen bonds of E2 molecules in the unit cell of Forms I (left) and II (right)

3. 2 Powder X-ray diffraction (PXRD)

PXRD is always the definitive method for the identification of polymorphs. The PXRD results of the two novel polymorphs are illustrated in Fig. 5. Form I shows the characteristic peaks at 2= 11.71, 13.35, 15.65, 18.42, 20.56 and 26.85° while Form II presents the characteristic peaks at 2= 12.5, 15.0, 20.82 and 21.65°, indicating the presence of twodistinctive polymorphs. The PXRD patterns measured from powder samples are also in goodagreement with those patterns calculated from thesingle-crystal structures.

3. 3 Thermal analysis (TG-DSC)

It is observed there is noticeable weight lossoccurring before its decomposition appears, whichindicates Forms I and II are solvated crystallineforms. In form I, the dehydration (0.5 mol of waterper mol of E2) of the sample was shown to be a hemihydrate by the diffraction techniques (= 175℃; Δm= 3.158%/Δm= 3.2%) (Fig. 6). The melting of Form I is observed as a pronounced endothermic peak with an extrapolated onset temperature of 181.94 ℃ and an associated heat of absorption of –79.42 J×g-1. Therefore, the molar ratio of 1:2 for water:E2 was confirmed in the raw material by TG/DTA analysis. In form II, the dehydration (1 mol of formamide per mol of E2) of the sample thatwas shown to be a solvate form by the diffraction techniques (= 145.94 ℃; Δm= 14.05%/Δm= 14.2%) in Fig. 6. The melting of Form II is observed as a pronounced endothermic peak with an extrapolated onset temperature of 179.72 ℃ and an associated heat of absorption of –57.67 J×g-1.

Table 3. Hydrogen Bonds for Estradiol Forms (?), where D = Donor and A = Acceptor

Symmetry codes as in Forms I and II : (a) 1–, 1–,; (b) –1+,, –1+; (c) 1+,,; (d) –1+,,; (e) 1/2–, 1–, 1/2+; (f) –1/2+, 1/2-, 1–; (g) 1/2+, 1/2–, 1–; (h) –1/2+, 3/2–, 1–; (i) 1–, 1/2+, 3/2–

Fig. 5. Experimental and calculated PXRD patterns of E2

3. 4 Infrared spectroscopy with attenuated total reflectance by Fourier transform (FTIR-ATR)

Both polymorphs of estradiol can be easily identified and assigned by their IR spectra which are shown in Fig. 7. Prominent differences in the IR spectra can be found in the region above 3000 cm-1which reflects the X–H stretching vibrations. Form I is a Hemihydrate modification and its molecule of crystalline water formed hydrogen bonds with nitrogen and oxygen atoms in E2 molecule. There are two broadened bands at 3426 and 3228 cm-1, contributed to the O–H stretching asymmetrical deformation vibration of the intro and intermolecular hydrogen bonding. Form II is a formamide solvate form. There is a broadened band at 3444 cm-1which is contributed to the N–H stretching asymmetrical deformation vibration. The pinnacles at 1654 and 1463 cm-1are contributed to C=O and C–N stretching asymmetrical deformation vibration, respectively.

Fig. 6. TGA-DSC profiles of Forms I and II

Fig. 7. IR spectra for polymorphs of E2 with scanning region of 4000～450 cm-1

3. 5 Scanning electron microscopy (SEM)

The SEM pictures (Fig. 8) reveal that the crystals of the polymorphs present differences in size, morphology and surface. Form I tends to be scattered and crystal nucleus growth more evenly. However, Form II tends to clump together and presents an irregular block structure. These outcomes indicated that Forms I and II displayed the obvious differences due to the different molecular arrangement.

Fig. 8. SEM pictures of Forms I and II

3. 6 Study on stability

In order to evaluate the potential physicochemical stability of the estradiol polymorphs, the powder samples of Forms I and II were exposed to stress conditions of high temperature of 60 ℃, high humidity of 90 ± 5% RH and light exposure of 5000 lx, respectively. After 10 days under the stress conditions, the powder samples were subjected to PXRD characterization to confirm the crystal phase and it was found that the stress samples of Forms I and II kept consistence with the original forms (data not shown herewith). Furthermore, the purity of the samples was measured by the HPLC method and no obvious degradation occurred (Table 4).

Table 4. Purity Assay (%) of Forms I and II before Stress Testing and after 10 Days under Stress Conditions

Fig. 9. Dissolution profiles in different buffer of Forms I and II

3.7 Studies on the solubility of hemihydrated and solvated form of estradiol

The dissolution profiles for Forms I and II were obtained from the dissolution experiments perfor- med in different buffer at 25 ℃. The samples collected at pre-set time were filtered prior to HPLC analysis and the surplus solids after dissolution experiments were identified by PXRD. The PXRD patterns measured from the surplus solids are in good agreement with those of the original forms, indicating the consistence of polymorphs during the dissolution experiments process. The contents of estradiol were determined using the external standard method. As shown in Fig. 9, the con- centrations of the two forms in buffer increase rapidly at first, then approach equilibrium slowly with the increasing time. The equilibrium solubility of Form I is found to be approximately higher than that of Form II in all the buffers, which further indicates Form II is the more thermodynamically stable form.

4 CONCLUSION

Two crystal forms of estradiol are found and pre- pared during the polymorph screening via changing the different solvents, temperature, speed and other parameters of recrystallization, and one is reported firstly. The solid-state properties of two polymorphs are studied by various analytical technics. SXRD analysis shows that one polymorph is a hemihydrate while the other one is a formamide solvate form, and they crystallize in different symmetric lattices. Besides that, two forms exhibit different conforma- tions and hydrogen bonds. The PXRD experiments indicated that the samples in this study are the pure polymorphic forms via comparing the patterns with the simulated ones, and furthermore, these PXRD patterns can be considered as the standard reference maps of estradiol polymorphs. This article also provides that IR characterization can be usedfor polymorphic identification because of the obvious differences at the regions of hydrogen bonds, functional groups and fingerprint. In addition to the solid-state properties, the stability properties are also studied. It has been found that Forms I and II are of conformational polymorph and Form II is the more thermodynamically stable solid form, while Form I possesses higher solubility, indicating its possibility as an alternate solid form for its further solid formulations development if necessary.

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18 January 2017;

16 March 2018 (CCDC 1576901 for Form I and 1561687 for Form II)

①This research was supported by National Key R&D Program of China (No. 2016YFC1000901), and thePostdoctoral Innovation Fund of National Research Institute for Family Planning (No. KYS [2017] BSHCX001)

. Associate professor, majoring in medical chemistry. E-mail: saintcxf2017@163.com

10.14102/j.cnki.0254-5861.2011-1946