Concomitant polymorphs of methoxyflavone (5-methyl-7-methoxyflavone)

Abstract

A novel metastable polymorph and an unstable amorphous phase of methoxyflavone were discovered after a decade since the first report of the X-ray crystal structure of this bioactive compound. The new polymorph (form B) was crystallized from a single solvent that produced two different polymorphs concomitantly, which is different from the reported structure of form A (CCDC code: FATYOP). The two polymorphs crystallized in the same P2₁/c space group, the asymmetric units in form A contain one and form B contains two molecules with different conformations. The conformational differences and the weak C–H⋯O intermolecular interactions played a major role in generating the methoxyflavone polymorphs. The polymorphs were characterized by X-ray diffraction, differential scanning calorimetry and FT-IR spectroscopy. Thermodynamic properties were unambiguously established using room-temperature competitive slurry experiments, solid-state milling and heating. Form A was more stable than form B, the amorphous phase was unstable and easily converted to form A at room temperature in 30 minutes. Form A had higher absorption area, the C_max and AUC of form A were approximately two times those of form B.

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Introduction

Polymorphism is frequently defined as the ability of a substance to exist as two or more crystalline forms that have different arrangements and/or conformations of the molecules in the crystal lattice.^1,2 In some cases, one crystallization experiment yields two or more polymorphs within the same crystal batch known as concomitant polymorphism, which makes controlling the formation of a specific polymorph challenging in crystal engineering.³ Polymorphism is very common amongst drugs.^4–7 Substances that exist in a non-crystalline solid state are said to be amorphous. The identity of chemical composition implies that all crystalline and amorphous phase of a given species have the same chemical behavior in solution or in melted state. However, their physical characteristics (solubility, hardness, compressibility, density, melting point, etc.), and their reactivity and bioavailability may be different at solid state,⁸ which in turn have a impact on its therapeutic efficacy.

Isoflavones are phytoestrogens, which are structurally similarly to endogenous estrogen and have a high binding affinity to the primary estrogen receptor in the vascular wall.⁹ In recent years, phytoestrogen supplements have become attractive as safer alternatives to endogenous estrogen. Their efficacy has been investigated in experimental and clinical trials. Due to these observations, isoflavone-rich food products or nutritional supplements could play a significant role in prostate cancer chemoprevention,¹⁰ decrease risk of breast cancer,^11,12 protecte against cardiovascular disease,^13,14 and decrease bone mineral loss.^15,16

Methoxyflavone (5-methyl-7-methoxyisoflavone, C₁₆H₁₂O₃, Fig. 1) is a sensational, non-steroidal anabolic isoflavone. It shows 3 times more potent than ipriflavone for increasing muscle mass and endurance. It is regarded as the perfect anabolic agent that offers benefits such as increasing lean mass without the side-effects of steroids.¹⁷ It is believed to considerably increase the levels of potassium, nitrogen, calcium and phosphorous retention making it an unmatched and superior anabolic horsepower supplement. It can be used for fat loss, increase vitality and muscle gain besides the maintenance of low cholesterol level and strengthen bones. Furthermore, it acts as an estrogen sensitizer on several body tissues, supporting its claims of reduced bone resorption and bone loss.^18,19 Currently, the systematic explorations on polymorphism of methoxyflavone were not reported much, only one crystal structure of methoxyflavone was reported earlier.²⁰

Fig. 1 The chemical structure of methoxyflavone.

In the screening and evaluation process the proper polymorphs should be used to avoid the unwanted other polymorphs to change the bioavailability, solubility and stability of the pharmaceutical reagents.^21–23 In this work, two conformation polymorphs of methoxyflavone were isolated and identified (the original structure was designated form A), and an amorphous phase was also acquired. Polymorphs of methoxyflavone were characterized by single crystal X-ray diffraction, powder X-ray diffraction, infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis methods. The essential reasons leading to the polymorphism of methoxyflavone were discussed. At the same time, the in vivo biological activity, stability and transformation were evaluated and studied.

Materials and methods

Methoxyflavone raw material was purchased from Jiaxing Junkang Trade Co., Ltd. (Jiangsu Province, China). The chemical purity was higher than 99.0%, which was determined by high-performance liquid chromatography (HPLC). All of solvents used for recrystallization were of analytical reagent grade. Male wistar rats weighing 190–198 g were purchased from Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College.

Sample preparation and crystallization

The form A and B were crystallized from a single solvent by evaporative crystallization and produces two different polymorphs concomitantly. Samples were dissolved in methanol at 60 °C and stirred for 30 minutes to obtain a saturated solution. The solution was then filtered through filter paper, and the solvent was evaporate slowly at 10 °C. After a few days, crystals with two different morphologies were observed (Fig. 2). Amorphous phase was obtained by heating the form A or form B to melt at 125 °C for 15 minutes then cooling to 0 °C sharply.

Fig. 2 Two concomitant different morphology crystals of methoxyflavone.

X-ray crystallography

Single crystal X-ray diffraction data of the form A and B of methoxyflavone were collected by on a MicroMax 002 + diffractometer equipped with a Cu fine-focus sealed tube and a 0.3 mm MonoCap collimator, the structures were solved by direct methods using SHELXS-97 and refined by full-matrix least-squares refinement on F² with anisotropic displacement parameters for non-H atoms using SHELXL-97. All H atoms were refined isotropically and were placed in calculated positions using riding models.

Powder X-ray diffraction analysis (XPRD) of polymorphs of methoxyflavone was performed on a D/max-2550 (Rigaku, Japan) X-ray diffractometer with graphite monochromatized CuKα (λ = 1.54187 Å) radiation at room temperature. Powders for XPRD measurement were obtained by grinding crystalline materials or amorphous sample in an agate mortar, particle size around 5 μm. The data were recorded in the angular range of 3° to 40° (2θ) with a step size of 0.02° and scanning speed of 8° min⁻¹.

The simulated powder patterns were calculated from the single crystal data using Mercury 3.3. Energy calculations were carried out using the Gaussian09 program.²⁴ Starting from crystallographic data, the single point energy calculations for the molecule from form A and isolated molecules from form B were performed using the DFT/B3LYP method with the 6-31G(d) basis set, without any symmetry restriction.²⁵

Thermal analysis

The differential scanning calorimetry (DSC) and thermogravimetric analyses (TGA) were carried out on a Mettler-Toledo TGA/DSC STARe system (Mettler, Switzerland) at a heating rate of 10 °C min⁻¹ under an atmosphere of dry N₂ flowing at 50 cm³ min⁻¹. For DSC, the measurement range extended from 50 °C to 150 °C; for TGA, the measurement range extended from 50 °C to 500 °C. The TGA/DSC data were analyzed by using STARe software.

Fourier transform infrared spectroscopy (FT-IR)

The FT-IR absorption spectra were recorded on a PerkinElmer Spectrum 400 FT-IR spectrophotometer (PerkinElmer, U.S.) to analyze the presence of functional groups in the polymorphs of methoxyflavone. The spectra were collected in the range from 650 to 4000 cm⁻¹ with a 4 cm⁻¹ resolution. An attenuated total reflectance (ATR) sampling accessory with a diamond window was used for measurements.

Solid-state milling

Solid samples were milled using a Pulverisette 6 ball mill (Fritsch, German), using 250 mL agate containers with 20 agate ball (Ø 15 mm), shaken at 400 rpm for 20 min. The ground solids were analyzed immediately by XPRD.

In vivo-absorption studies

All rats had free access to tap water and deprived of food 12 h before the experiment. Each polymorphic sample for in vivo-absorption test was prepared to suspension 17 mg mL⁻¹ (calculated on the dried basis) with saline. Each rat received a 200 mg kg⁻¹ oral dose of the suspension. Blood samples were collected about 3–5 mL by orbital vein, 5, 15, 30, 60 minutes post-dosing, then centrifuged at 5000 rpm for 15 min. Rat plasma (150 μL) and ethyl acetate (1 mL) were mixed together, then the solution was firstly oscillated for 3 min and centrifuged at 13 400 rpm for 5 min. The upper organic phase was collected and blow-dried with nitrogen. After the residue was dissolved in methanol (75 μL), the solution was oscillated for 0.5 min and centrifuged at 13 400 rpm for 1 min. Then the supernatant liquid (20 μL) was analyzed with HPLC. Chromatographic conditions: the samples were separated by chromatography using an Aligent XDB-C18 5 μm (150 mm × 4.6 mm) column. The mobile phase was methanol–water (75 : 25, v/v) and the pump flow rate was 1.0 mL min⁻¹. The column temperature was 30 °C and the injection volume was 20 μL. The methoxyflavone profiles were detected at 254 nm.

Results

Single crystal X-ray diffraction (SXRD)

A crystallization process for a specific compound will be governed by the compound's inherent properties for nucleation and growth. The polymorph formation will also depend on the properties of the surrounding environment such as solvents and temperature and the rate of generation of supersaturation. Even concomitant crystallized in the same solvent, the shape of the crystal of the form A and B were different, the form A was bulk and form B was needle, they were easily to distinguish from the appearance. The different morphology crystals were selected for single crystal X-ray diffraction. Crystallographic data and refinement details of the form A to B of methoxyflavone were listed in Table 1. Especially, the form B of methoxyflavone was reported firstly.

Table 1 Crystal parameters of polymorphs of methoxyflavone

	Form A (literature)	Form A (this work)	Form B (this work)
Color/shape	Colorless/prism	Colorless/bulk	Colorless/needle
Crystal size (mm)	0.20 × 0.35 × 0.55	0.25 × 0.31 × 0.56	0.08 × 0.19 × 0.56
Empirical formula	C17H14O3	C17H14O3	C17H14O3
Molecular weight (g mol^−1)	266.28	266.28	266.28
Crystal system	Monoclinic	Monoclinic	Monoclinic
Space group	P21/c	P21/c	P21/c
a (Å)	10.321(3)	10.330(6)	12.610(4)
b (Å)	11.290(5)	11.296(3)	7.764(5)
c (Å)	11.979(8)	11.992(4)	28.134(6)
a (°)	90.00	90.000	90.000
b (°)	107.00(5)	107.195(7)	104.02(3)
g (°)	90.00	90.000	90.000
Volume (Å^3)	1334.9(11)	1336.8(10)	2672.3(9)
Z	4	4	8
Density (g cm^−3)	1.325	1.323	1.324
F(000)	—	560	1120
Theta range for data	2.06 < q < 27.17	4.48 < q < 72.04	3.24 < q < 72.44
	−13 ≤ h ≤ 12	−11 ≤ h ≤ 10	−15 ≤ h ≤ 14
h/k/l ranges	−1 ≤ k ≤ 14	−13 ≤ k ≤ 13	−8 ≤ k ≤ 3
	0 ≤ l ≤ 15	−14 ≤ l ≤ 14	−32 ≤ l ≤ 34
Reflections collected	3430	2557	5313
Independent reflections	2967	2207	4246
Completeness (%)	—	97.1	96.0
Final R, wR(F^2) values [I > 2σ(I)]	0.0409, 0.1038	0.0475, 0.1493	0.0520, 0.1446
Final R, wR(F^2) values (all)	0.0967, 0.1254	0.0538, 0.1778	0.0606, 0.1574
Goof	1.013	1.059	1.071
CCDC number	207247 (FATYOP)	—	1412292

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Polymorphs of methoxyflavone were crystallized in the same monoclinic and P2₁/c space group. The asymmetric units in the form A contains only one molecule and form B contains two molecules with different conformations, the conformational differences from the rings and the rotating of the single bond of the side-chain substituents were the main causes of the bimolecular phenomena. In the molecules, the ring 1 (O₁, C₂, C₃, C₄, C₁₀, C₉) and the ring 2 (C₅, C₆, C₇, C₈, C₉, C₁₀) were co-planar, the ring 3 (C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈) was planar, the dihedral angles of ring 1 and 3 were 59.39 (7)° (form A), 33.51 (9)° and 141.90 (9)°(form B), respectively. The torsion angle of the side-chain methoxy substituents also had the conformational differences in polymorphs. In the form A, the torsion angle of C₁₂–O₃–C₇–C₆ was 170.17 (13)°, but in the form B, the torsion angles were −176.03 (18)° and −0.1 (3)° respectively. The molecule overlay was shown in Fig. 3.

Fig. 3 Overlay of molecular conformations (green, form A; yellow, form B, molecule 2a; blue, form B, molecule 2b).

There are no conventional hydrogen bonds due to the lack of donors in the form A and B. A weak C–H⋯O intermolecular interaction joins the molecules related by the screw axis and the glide-planes. In form A, four neighboring molecules form a ring via two pairs of C₂–H⋯O₂, C₁₈–H⋯O₃ hydrogen bonds constructed a circular network extend into a layer along the b–c plane. In form B, seven neighboring molecules form a helical along the b axis via two pairs of C_2B–H⋯O_3A, C_2A–H⋯O_2B hydrogen bonds and four C_8B–H⋯O_3B hydrogen bonds. The lengths and angles of C–H⋯O in the form A and B were listed in Table 2. The packing of polymorphous of methoxyflavone were shown in Fig. 4. The weak π⋯π stacking interactions had been found between ring 2, and the centroid···centroid distance were 3.626 Å (form A) and 3.749 Å (form B), respectively. Form above, the results showed that the conformational differences and the C–H⋯O type of intermolecular interactions played a major role in generating of methoxyflavone polymorphs.

Table 2 Hydrogen bonds geometry (in Å and deg)

Form	Donor–H⋯Acceptor	D–H (Å)	H⋯A (Å)	D⋯A (Å)	∠DHA (°)	Symmetry code
A	C(2)–H(2A)⋯O(2)	0.93	2.46	3.247(3)	143	x, 1/2 − y, 1/2 + z;
	C(18)–H(18A)⋯O(3)	0.93	2.57	3.413(3)	151	1 − x, −1/2 + y, 3/2 − z
B	C(2A)–H(2A)⋯O(2B)	0.93	2.54	3.268(2)	135	2 − x, 1/2 + y, 3/2 − z
	C(2B)–H(2B)⋯O(3A)	0.93	2.50	3.366(2)	155	2 − x, 1 − y, 1 − z
	C(18A)–H(18A)⋯O(2A)	0.93	2.50	2.942(2)	110
	C(18B)–H(18B)⋯O(2B)	0.93	2.51	2.955(2)	110

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Fig. 4 The packing of polymorphous of methoxyflavone.

The density functional calculation revealed that the single point energy of form A was −803.28321367 a.u, the energy of molecular 2a and 2b in form B were −803.28321365 a.u and −803.28324127 a.u, respectively. Theoretically, the packing of a molecule can be arranged in many ways during the recrystallization process and reflected the alternative ways in which molecules in a crystal strive towards a free energy minimum. The negligible free energy difference implies the different polymorphs can be concomitant in the same condition.^26,27

Powder X-ray diffraction

Polymorphic purity can be determined by XPRD, the simulated powder X-ray diffraction pattern calculated by the data of SXRD could be regarded as 100% polymorphic purity. Powder X-ray diffraction patterns of two forms of methoxyflavone were distinguishable, hence they could be easily analyzed in a mixture of crystals of the two forms. The calculated patterns of the form A and B were compared with experimental patterns in Fig. 5, and they had good consistency. Peak positions and relative intensities of ten most intense peaks were listed in Table S1 (ESI, Table S1†). The form C was an amorphous, and showed a brand blunt peak in the powder X-ray diffraction pattern, which differented from the form A and B significantly. The form C was unstable form, so it only characterizated by XPRD.

Fig. 5 The XPRD patterns of methoxyflavone polymorphs and amorphous phase.

Thermal analysis method

Thermal analysis could provide valuable information that was characteristic of different polymorphs. The crystalline samples of methoxyflavone polymorphs were investigated by DSC, TGA. In the DSC experiments, both polymorphs were heated from 30 to 150 °C (Fig. 6). The form A showed a single endothermic peak at 119.47 °C which was ascribed to the melting point of the methoxyflavone. The form B showed a small endothermic peak was observed at 110.5 °C, then a small exothermic peak at 111.22 °C, followed by a major endothermic peak at 119.61 °C excluded the interfere of inert gas fluctuations. A small endothermic peak before the melting endotherm in the form B corresponded to the phase transition from form B to A, and the particular temperature was the thermodynamic transition point. This had been verified by XPRD and DSC by heating the form B at 115.0 °C for 15 min. In the TGA experiments, there was no weight loss before the melting temperature of 120 °C (ESI, Fig. S1†), therefore it could be concluded that the crystals obtained were free from solvent inclusion. Further heating resulted in decomposition of both the crystals.

Fig. 6 The DSC thermogram of methoxyflavone polymorphs.

Infrared spectroscopy (FT-IR)

Both polymorphs of methoxyflavone could be easily identified and assigned by their FT-IR spectra which were shown in Fig. 7. There were two aromatic rings in the structure of methoxyflavone. For the form A, the C=O stretching vibrations presented at 1634 cm⁻¹, the C=C stretching vibrations presented at 1600 cm⁻¹, 1563 cm⁻¹, 1453 cm⁻¹. For the form B, the C=O stretching vibrations presented at 1632 cm⁻¹, 1622 cm⁻¹, the C=C stretching vibrations presented at 1600 cm⁻¹, 1566 cm⁻¹, 1451 cm⁻¹. The main vibrational data with tentative assignments for polymorphs of methoxyflavone were shown in Table S2 (ESI, Table S2†). Even the vibration peak at the similar position, the relatively intension were different significantly, which indicated the different interaction in form A and B.

Fig. 7 The FT-IR patterns of methoxyflavone polymorphs.

Stability and transformation

Once the polymorphs were established, it becomes critical to determine the boundaries of stability for the different forms and how they might be interconvert. The tendency for stability and transformation between the polymorphs were examined by competitive slurring experiments, heating experiments (115.0 °C, 15 min), and mechanical milling experiments. For samples of the individual polymorphs slurred in methanol or ethanol solutions at ambient conditions, the form A and B remained stable in all solvents. But for a 1 : 1 mixture of the form A and B in methanol, however, only the form A was present after several days, establishing the form A to be thermodynamically more stable under these conditions. The form A was stable at heating experiments, after heating 15 min at 115.0 °C, but the form B transformed to the form A, and the thermally induced B → A phase transition is irreversible. The amorphous phase was unstable and easily transforms to form A at room temperature in 30 minutes (ESI, Fig. S2†). During solid-state milling, the amorphous phase transformed to form A, while form A and B remained unchanged.

In vivo absorption studies

Different polymorphs of the drug substance may have different solubility, dissolution rate, bioavailability and toxicity. Since these properties are directly related to the effectiveness and safety of treatment with the human body, so for drugs, studies of polymorphism are much more concerned. The solubility measurement and intrinsic dissolution rate (IDR) of the drug are often measured by many researchers to predict the in vivo effect of different crystal forms, it is undeniable that differences of solubility and dissolution rate of the pharmaceutical polymorphs in vitro may reflect its absorption in vivo to a certain extent. But in fact, we usually find that IDR prediction of drug polymorphs in vitro is differ from the practical effect in vivo. The reasons should be ascribed to the impact of multiple factors in vivo cannot be fully mimicked in the in vitro experiments. Therefore, the overall evaluation by the whole animal is essential to study the in vivo biological differences of pharmaceutical polymorphs.

Bioavailability tests of methoxyflavone polymorphs were carried out by giving different forms orally to rats, and monitoring for a period of change of the plasma concentration, and then the differences between the polymorphs were observed. Fig. 8 showed the plasma concentration–time curves of two crystal forms of methoxyflavone after oral administration. Two forms of methoxyflavone were rapidly absorbed after administration, and their plasma concentrations began to increase in 5 min. The form B had the faster absorption rate, it reached the maximum absorption at 5 min while the form A at 29 min. But the form A had the higher absorption area among in two polymorphs. After the peak, plasma concentrations of two forms began to decline, the form A was metabolized faster than the form B, they metabolized totally in 60 min. Overall, the C_max and AUC of the form A were approximately 2 times than that of the form B.

Fig. 8 Plasma concentration–time curves of methoxyflavone polymorphs after oral administration to rats.

Conclusions

We had described a novel concomitant polymorph and an unstable amorphous phase of methoxyflavone, and compared it with that of the published crystal form. The form B and amorphous phase were reported firstly in this paper. The solid-state properties of the two polymorphs and amorphous phase were characterized by various analytical techniques. Although crystallized in the same space group, the form A and B had the different molecules in the asymmetry unit. They also had the different C–H⋯O bond contacts. The form A molecules extend in a network arrangement and the form B molecules form a screw arrangement. The form A was more stable crystal form, form B was metastable crystal form and could transform directly by the solid-state phase transition to form A by heating at 115.0 °C for 15 min. The amorphous phase was unstable, and could easily transform to form A at ambient temperature in 30 min after prepared. The XPRD, DSC, FT-IR methods were all used to characterize the two polymorphs. The XPRD was the best method suited for differentiated them from each other. It should be noticed in formulation process that polymorphs existed during the preparation process, and the different absorption effect. The results of polymorphic characterization, stability, transformation of polymorphs and bioavailability would be the basic scientific data benefited to improve the efficacy and controlling the quality of the drug.

Acknowledgements

The authors would like to acknowledge the financial support from the Ministry of Science and Technology of the People's Republic of China for the National Science and Technology Major Projects (Grant 2013ZX09102110) and Graduate education reform project of Peking Union Medical College (Grant PUMC-GS-2015020). We also thank professor li Li for the energy calculations.

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Footnote

† Electronic supplementary information (ESI) available: The d-spacings (Å), 2θ values (°) and relative intensities (%) of the ten most intense peaks in XPRD patterns and the main vibrational data with assignment of methoxyflavone polymorphs (Tables S1 and S2). The TGA diagram of polymorphs of methoxyflavone and the polymorph transformation of methoxyflavone amorphous phase (Fig. S1 and S2). CCDC 1412292. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra05995c

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